Interactive Fly, Drosophila



The zinc finger protein Zn72D and DEAD box helicase Belle interact and control maleless mRNA and protein levels

The Male Specific Lethal (MSL) complex is enriched on the single X chromosome in male Drosophila cells and functions to upregulate X-linked gene expression and equalize X-linked gene dosage with XX females. The zinc finger protein Zn72D is required for productive splicing of the maleless (mle) transcript, which encodes an essential subunit of the MSL complex. In the absence of Zn72D, MLE levels are decreased, and as a result, the MSL complex no longer localizes to the X chromosome and dosage compensation is disrupted. To understand the molecular basis of Zn72D function, proteins were identified that interact with Zn72D. Among several proteins that associate with Zn72D, the DEAD box helicase Belle (Bel) was found. Simultaneous knockdown of Zn72D and bel restored MSL complex localization to the X chromosome and dosage compensation. MLE protein was restored to 70% of wild-type levels, although the level of productively spliced mle transcript was still four-fold lower than in wild-type cells. The increase in production of MLE protein relative to the amount of correctly spliced mle mRNA could not be attributed to an alteration in MLE stability. These data indicate that Zn72D and Bel work together to control mle splicing and protein levels. Thus Zn72D and Bel may be factors that coordinate splicing and translational regulation (Worringer, 2009).

Why is it important to regulate MLE protein levels? MLE localizes to all chromosomes and throughout the nucleus when overexpressed. Incorrect MLE expression is detrimental to the development of the fly, because heat shock over-expression of transgenic MLE protein results in male and female lethality. It is possible that translational repression by Zn72D and Bel is one mechanism by which levels of MLE protein are tightly controlled. Expression of a transgenic mle cDNA in S2 cells resulted in overproduction of MLE protein; however, inclusion of the first two introns in the same transgene reduced the amount of MLE protein produced from the transgene. This suggests that perhaps recruitment of Zn72D to the mle transcript has the effect of not only productively splicing the transcript but also targeting it for translational regulation (Worringer, 2009).

Like Zn72D, its human homologue ZFR is also found mainly in the nucleus, with a subset in the cytoplasm. Cytoplasmic ZFR colocalizes in neuronal granules with Staufen2, a protein involved in mRNA transport and localization. ZFR interacts with and is required for cytoplasmic localization of the Staufen262 isoform. As neuronal granules are involved in translational regulation and localization of mRNAs, these data suggest that ZFR may have a role regulating cytoplasmic localization of mRNAs. If Zn72D has a similar function in flies, it has the potential to regulate gene expression at two steps. Zn72D may first promote productive splicing of mRNAs and then later affect their cytoplasmic localization, which in turn may impact translation (Worringer, 2009).

This study has identified several proteins that interact with Zn72D, including the DEAD box helicase Bel. Co-knockdown of both bel and Zn72D restores the MSL complex localization to the X chromosome and dosage compensation of X-linked genes that was lost in the absence of Zn72D. In addition, it was found that co-knockdown of Zn72D and bel resulted in restoration of MLE protein levels to 70% of wild-type levels, despite a four-fold reduction in properly spliced mle mRNA. These data implicate Zn72D and bel as being factors that target spliced mRNAs for localized, regulated translation in the cytoplasm (Worringer, 2009).

MLE functions as a transcriptional regulator of the roX2 gene

Dosage compensation is a process that equalizes transcription activity between the sexes. In Drosophila, two non-coding RNA, roX1 and roX2, and at least six protein regulators, MSL-1, MSL-2, MSL-3, MLE, MOF, and JIL-1, have been identified as essential for dosage compensation. Although there is accumulating evidence of the intricate functional and physical interactions between protein and RNA regulators, little is known about how roX RNA expression and function are modulated in coordination with protein regulators. A relatively short (about 350 bp) upstream genomic region of the roX2 gene, Prox2, harbors an activity that drives transcription of the downstream gene. This study has shown that MLE can stimulate the transcription activity of Prox2 and that MLE associates with Prox2 through direct interaction with a newly identified 54-bp repeat, Prox. These observations suggest a novel mechanism by which roX2 RNA is regulated at the transcriptional level (Lee, 2004).

When ectopically expressed, MSL2 induces dosage compensation even in female flies. Because roX RNAs are critical for dosage compensation, transcription of the corresponding genes, which are suppressed in female flies, must be activated in these transgenic female flies expressing MSL2. To understand the mechanism underlying transcription activation of the roX2 gene, attempts were made to identify an upstream genomic region potentially important for transcriptional regulation of the roX2 gene. Five genes, including roX2 and nod, are identified in the sense template upstream of rox3, and two genes, are in the antisense template. A 350-bp intergenic region, which has been titled Prox2, is present between CG11695 and the roX2 gene and contains a T-rich region at position -331 to -150 and a 54-bp repeated sequence at position -261 to -154 (Lee, 2004).

At present, the molecular mechanisms underlying roX gene transcription remain unknown. It has been shown that mutant embryos, lacking MLE, normally synthesize roX1 RNA, but roX1 RNA appears to be concentrated at its site of synthesis. Based on this observation, it has been proposed that MLE is required for the stability of roX1 RNA and its movement from the transcription site, but not for its synthesis. MLE has also been implicated in stable maintenance of the steady-state level of roX2 RNA. Expression of mleGET in place of the wild type MLE results in drastic reduction in the roX2 RNA level and formation of the MSL complex devoid of roX2 RNA. In addition to this post-transcriptional function, the present study provides two pieces of evidence of a direct involvement of MLE in transcription regulation of the roX2 gene: (1) MLE interacts with the upstream promoter region (i.e. Prox2) of the roX2 gene through association with a 54-bp repeat, Prox; (2) overexpression of MLE activates transcription driven by Prox2 either in a reporter construct or a chromosomal context (Lee, 2004).

This study has shown that ATP is not essential for the interaction of MLE with Prox. In addition, MLE ATPase activity is dispensable for transcriptional activation supported by Prox2. These results are consistent with the findings that MLE retains X chromosome binding ability despite various mutations introduced in the ATPase motifs and that ATPase activity is dispensable for transcriptional activation of the X-linked genes. Because mutations in the ATPase motifs of MLE affect the viability of male flies, the ATPase activity seems to be required for normal development of male flies. Then, by what mechanism does the ATPase activity of MLE influence dosage compensation? Based on poor binding of roX1 RNA to the X chromosome in flies expressing mleGET, it has been proposed that MLE ATPase activity plays an early role, perhaps in packaging roX2 RNA into growing MSL complexes. In support of this hypothesis, a recent study shows that in the absence of an ATP-dependent function of MLE, MSL complex can be assembled but is devoid of roX RNA. Thus, it is likely that in addition to transcriptional regulation by an ATP-independent function of MLE, roX2 RNA is post-transcriptionally regulated through association with MLS proteins, which require an ATP-dependent function of MLE (Lee, 2004).

Splicing of mle

The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also (see U2 small nuclear riboprotein auxiliary factor 50) required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point (Kennedy, 1997).

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. A 59-nucleotide Drosophila melanogaster intron from the Drosophila mle gene contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. It is suggested that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly (Kennedy, 1998).

The Drosophila dosage compensation complex binds to polytene chromosomes independently of developmental changes in transcription

In Drosophila, the dosage compensation complex (DCC) mediates upregulation of transcription from the single male X chromosome. Despite coating the polytene male X, the DCC pattern looks discontinuous and probably reflects DCC dynamic associations with genes active at a given moment of development in a salivary gland. To test this hypothesis, binding patterns of the DCC and of the elongating form of RNA polymerase II (PolIIo) were compared. Unlike PolIIo, the DCC demonstrates a stable banded pattern throughout larval development and escapes binding to a subset of transcriptionally active areas, including developmental puffs. Moreover, these proteins are not completely colocalized at the electron microscopy level. These data combined imply that simple recognition of PolII machinery or of general features of active chromatin is either insufficient or not involved in DCC recruitment to its targets. It is proposed that DCC-mediated site-specific upregulation of transcription is not the fate of all active X-linked genes in males. Additionally, it was found that DCC subunit MLE associates dynamically with developmental and heat-shock-induced puffs and, surprisingly, with those developing within DCC-devoid regions of the male X, thus resembling the PolIIo pattern. These data imply that, independently of other MSL proteins, the RNA-helicase MLE might participate in general transcriptional regulation or RNA processing (Kotlikova, 2006).

The nature of targets for the DCC is a long-standing problem. Originally, it was proposed that specific enhancer-like sequences might reside close to individual X-linked genes, serving as targets for DCC. In contrast, the 'spreading' model postulates that the male X is marked by a quite limited number of DNA sequences (35) that recruit the DCC and accumulate locally at high levels, which in turn results in association of the complex with numerous sites of low affinity. Nevertheless, the modern view of the problem assumes that there might be many more DNA sequences required both for the initial recruitment/assembly of DCC (CES) and for the association of a functional complex with additional sites (non-CES). In contrast to these postulated DNA sequences, most sites on the X, which are targets for functional DCC, are thought to mark genes actively transcribed in a given tissue and time of development. This idea implies that DCC mediates transcription enhancement via direct involvement in transcription regulation of each active gene. In this article, an effort was made to test further this model by precisely investigating the relative localization of DCC and PolIIo along the male X in the course of larval development (Kotlikova, 2006).

Previously it was demonstrated that in vivo PolIIo and various elongation factors, as well as the H3.3 histone variant, dynamically associate with active genes, accompany their expression, and look colocalized in Drosophila polytene chromosomes. This overlap is most obvious as diffuse labeling of developmental and heat-shock-induced puffs. Intriguingly, it was found that DCC demonstrates striking stability in both the number and intensity of binding sites along the X throughout larval development. Moreover, despite being targets for MLE, the sites of the most intensive gene expression both on the male X and within an autosomal DCC-spreading area appear to not be targets for DCC at all. DCC gaps were demonstrated in regions associated with active genes. Additionally, DCC skips over a number of transcriptionally active regions when it inappropriately spreads in cis from an autosomal roX1 transgene. It is therefore suggested that active transcriptional status of the chromosomal region or association with MLE is not sufficient for DCC targeting and that, if DCC binds to actively transcribed regions, it does so very selectively (Kotlikova, 2006).

The findings on MLE localization on the polytene chromosomes might reflect dual functioning of MLE on the male X. In addition to being a subunit of DCC, MLE probably accomplishes some unrelated functions, which are neither X nor sex specific. On the basis of the observed MLE association with a large number of sites of active transcription in both sexes, it is believed that this RNA-helicase might be important not only for splicing certain genes, as was shown for the gene para, but also for playing some general role in the transcription process. In support of this idea, the mammalian MLE homolog, RHA, was demonstrated to contribute to various steps of transcription -- from initiation to processing of nascent transcripts (Zhang, 2004). Assuming that MLE apparently is able to bridge DCC with transcriptionally active regions, this might occur only if some additional requirements for DCC binding are realized (Kotlikova, 2006).

Earlier, it was reported that a partial MSL complex lacking MSL2 protein is present in normal female nuclei. Accordingly, mutations in various msl genes except mof disassociate all MSLs from the chromosomes in females. Nevertheless, the data on MLE distribution in polytene chromosomes of females homozygous for msl1 or msl3 null alleles indicate that even if MSLs form a partial complex in females, MLE binds the chromatin in an MSL-independent manner (Kotlikova, 2006).

These findings raise questions as to what are the reasons for exclusion of DCC from some active X-linked regions and whether dosage compensation does take place there. One can speculate that highly active chromatin in puffing regions turns into a poor substrate for the DCC due to drastic changes in packaging, possibly, up to nucleosome removal. However, a cluster of CESs bound by DCC is detected in the puffed 2B region throughout larval development. Moreover, strong transcription induced in EP transposons on the male X sometimes results in ectopic DCC recruitment, suggesting that the complex is able to recognize very active chromatin (Kotlikova, 2006).

Revealing active genes within each of the cytologically extensive DCC gaps provides yet another puzzle. In the neo X chromosome of D. miranda, the blocks of chromatin escaping dosage compensation do alternate with other blocks that are dosage compensated and therefore bind DCC. However, no data indicate that such clustering takes place in D. melanogaster. If X-linked genes actually possess still unknown features needed for DCC targeting, then DCC gaps might reflect evolutionary incompleteness of this process in D. melanogaster. It should be noted that, in contrast, 70 autosomal regions are competent to recruit functional DCC in wild-type males. Alternatively, the active genes located within the DCC gaps might serve as targets for the complex but cannot realize this ability probably due to the chromatin environment (Kotlikova, 2006).

Whether the genes within puffs and DCC gaps undergo dosage compensation remains to be answered. It seems plausible to suggest that transcription upregulation could be not so essential for a subset of highly expressed genes. Nevertheless, whatever the reasons for highly expressed loci to escape association with DCC, these genes are probably dosage compensated, which was shown at least for Sgs4 and the Broad-Complex. If many active genes lack DCC-binding sites in the immediate vicinity, they might achieve dosage compensation by a yet unknown pathway. It is very possible that upregulation of active genes within DCC gaps and puffing regions might be achieved, at least to some extent, via DCC-mediated establishment of a more open chromatin structure of the whole male X, suggesting that DCC affects transcription indirectly. Site-specific localization of H4Ac16 probably initiates a cascade of molecular remodeling events resulting in diffuse appearance of the whole male X chromosome. Generally, such a chromatin state would facilitate the access of various transcription and replication factors. Accordingly, in females having ectopic dosage compensation induced, the DCC gap corresponding to the intercalary heterochromatin region on the polytene X demonstrated a greater extent of both polytenization and replication than in the wild type. This clearly correlated with the higher local concentrations of the DCC in neighboring areas. Thus, despite the fact that the DCC-mediated site-specific histone acetylation pattern correlates with an increase in transcription of the underlying sequences, it would be more accurate to suggest that there is no common scenario of dosage compensation for all the X-linked genes. Also, the DCC pattern appears essentially permanent and displays only negligible variations, both in the course of larval development and in different tissues, which might point to the contribution of yet unidentified epigenetic factors in the establishment and maintenance of DCC binding. For example, the transcriptional activity of X-linked genes could govern DCC settling on the male X in early embryogenesis, and this pattern might be subsequently reproduced epigenetically. Hence, this scenario would imply high stability of the DCC pattern at least for the housekeeping genes, rather than dramatic changes in DCC distribution resulting from fine-tuned transcriptional programs further in development (Kotlikova, 2006).

Future molecular studies of the dosage compensation status of active genes mapping to the DCC gaps could determine whether dosage compensation can also utilize some unknown mechanisms other than site-specific acetylation of H4 at lysine 16 leading to site-specific transcription enhancement. Alternatively, there may be many more X-linked genes whose expression does not require dosage compensation than was expected to date. Regardless, the important question remains how functional DCC recognizes its targets among the active genes on the male X chromosome (Kotlikova, 2006).

Protein Interactions

Mle, while present in the nuclei of both male and female cells, differs in its association with polytene X chromosomes in the two sexes. Mle is associated with hundreds of discrete sites along the length of the X chromosome in males but not in females. The predominant localization of Mle to the X chromosome in males makes it a strong candidate to be a direct regulator of dosage compensation (Kuroda, 1991).

To investigate how dosage compensation is regulated, it has been determined whether Sex lethal and the other msls are required for Mle X chromosome binding. In females, Sxl functions to prevent Mle from binding to the two X chromosomes. Additionally, Mle X chromosome binding requires wild-type msl1, msl2, and msl3 functions. These data support a model whereby the activity of the Mle protein is regulated through its association with one or more of the other msl proteins (Gorman, 1993).

The Mle protein sequence contains motifs common to members of a family of RNA-dependent ATPases. Association of Mle with the male X chromosome is RNase sensitive, and mutations in the ATPase motifs affect MLE function. Overexpression of MLE or its carboxyl terminus, which includes glycine-rich repeats, reveals an RNase-sensitive affinity for all chromosome arms. These results suggest that nascent transcripts or a hypothetical RNA component of chromatin play a critical role in the biochemical mechanism of dosage compensation. The potential relationship between interaction with RNA and transcriptional control of the X chromosome suggests that the mechanism of dosage compensation is distinct from classical models for transcriptional activation (Richter, 1996).

Dosage compensation in Drosophila occurs by an increase in transcription of genes on the X chromosome in males. This elevated expression requires the function of at least four loci, known collectively as the male-specific lethal (msl) genes. The proteins encoded by two of these genes, maleless (mle) and male-specific lethal-1 (msl-1), are found associated with the X chromosome in males, suggesting that they act as positive regulators of dosage compensation. A specific acetylated isoform of Histone H4, H4Ac16, is also detected predominantly on the male X chromosome. Mle and Msl-1 bind to the X chromosome in an identical pattern and the pattern of H4Ac16 on the X chromosome is largely coincident with that of Mle/Msl-1. H4Ac16 was not detected on the X chromosome in homozygous msl mutant males, correlating with the lack of dosage compensation in these mutants. Conversely, in Sxl mutants, H4Ac16 is detected on the female X chromosomes, coincident with an inappropriate increase in X chromosome transcription. These data suggest that synthesis or localization of H4Ac16 is controlled by the dosage compensation regulatory hierarchy. Dosage compensation may involve H4Ac16 function, potentially through interaction with the product of the msl genes (Bone, 1994).

Dosage compensation ensures that males with a single X chromosome have the same amount of most X-linked gene products as females with two X chromosomes. In Drosophila, this equalization is achieved by a twofold enhancement of the level of X chromosome transcription in males, relative to each X chromosome in females. The products of at least five genes, maleless (mle), male-specific lethal 1, 2, and 3 (msl-1, msl-2, msl-3) and males absent on the first (mof), are necessary for dosage compensation. MOF transcript is found in larvae and adults of both sexes. The proteins produced by these genes form a complex that is preferentially associated with numerous sites on the X chromosome in the somatic cells of males, but not females. Binding of the dosage compensation complex to the X chromosome correlates with a significant increase in the presence of a specific histone isoform (histone 4 acetylated at lysine 16), on the chromosome. Experimental results and sequence analysis suggest that the mof gene encodes an acetyl transferase that plays a direct role in the specific histone acetylation associated with dosage compensation. The predicted amino acid sequence of MOF exhibits a significant level of similarity to several other proteins, including the human HIV-1 Tat interactive protein Tip60 (dee Drosophila Tip60), the human monocytic leukemia zinc finger protein MOZ and the yeast silencing proteins SAS3 and SAS2. Also studied has been the role played by the various components of the complex in the targeting of MOF to the X chromosome. To this end, indirect cytoimmunofluorescence was used to monitor the binding of these components in males carrying either complete or partial loss-of-function mutations as well as in XX individuals in which formation of the dosage compensation complex has been induced by genetic means (Gu, 1998 and Hilfiker, 1997).

Tbp-related factor (Trf) is present as a constituent of larval salivary gland polytene chrmosomes, revealing information about potential Trf target gene selectivity. Trf is found in 17 out of up to 600 resolvable sites, suggesting that Trf is localized in a highly gene-specific pattern at the same time that Tbp is present almost ubiquitously. The identified sites have neural or gonadal functions. For example, 63AB contains the shaker cognate b (shab) gene that encodes a potassium channel. This finding is consistent with the observation that trf mutant flies display a leg shaking phenotype, generally attributed to abnormalities in potassium channel function. Trf also associates with the location of quiver, which is also associated with a shaker phenotype. The genes maleless/no action potential exhibit Trf association as well as a number of chromosomal sites that contain one or more tRNA genes. These data suggests that Trf, like Tbp, may also play a role in RNA polymerase III transcription, at least in the salivary gland (Hansen, 1997).

Ordered assembly of roX RNAs into MSL complexes on the dosage-compensated X chromosome in Drosophila

In the male Drosophila, the X chromosome is transcriptionally upregulated to achieve dosage compensation, in a process that depends on association of the MSL proteins with the X chromosome. A role for non-coding RNAs has been suggested in recent studies. The roX1 and roX2 RNAs are male-specific, non-coding RNAs that are produced by, and also found associated with, the dosage-compensated male X chromosome. Whether roX RNAs are physically part of the MSL complex has not been resolved. roX RNAs are found to colocalize with the MSL proteins and are highly unstable unless the MSL complex is coexpressed, suggesting a physical interaction. roX2 RNA could be precipitated from male tissue-culture cells with antibodies to the proteins Msl1 and Mle, consistent with an integral association with MSL complexes. Localization of roX1 and roX2 RNAs in mutants indicates an order of MSL-complex assembly in which roX2 RNA is incorporated early in a process requiring the Mle helicase. It was also found that the roX2 gene, like roX1, is a nucleation site for MSL complex spreading into flanking chromatin in cis. These results support a model in which MSL proteins assemble at specific chromatin entry sites (including the roX1 and roX2 genes); the roX RNAs join the complex at their sites of synthesis; and complete complexes spread in cis to dosage compensate most genes on the X chromosome (Meller, 2000).

Wild-type males have complete MSL complexes bound to hundreds of bands along the X chromosome in a highly reproducible pattern where they mediate hypertranscription. Genetic analysis of debilitated MSL complexes lacking either Mle, Msl3 or Mof subunits, or containing enzymatically dead Mof acetyltransferase or Mle helicase, has shown that partial MSL complexes can bind to only ~35 bands that have been termed chromatin entry sites (formerly called high-affinity sites). It has been proposed that these sites direct dosage compensation to the X chromosome by serving as sites of assembly and subsequent spreading of the MSL complex into flanking chromatin in cis. roX2 has been identified as a chromatin entry site based on the ability of roX2 transgenes to attract the MSL complex to autosomal insertion sites, even in the absence of Msl3. To test whether roX2 could also support spreading of MSL complexes into flanking autosomes, multiple lines of transgenic larvae were created carrying genomic roX2 transgenes. It was found that the roX2 chromatin entry site also supports variable spreading of MSL complexes into flanking chromatin. The spreading displays the same characteristics as has been reported for a roX1 transgene: bidirectionality, dependence on insertion site, and substantial variability even from nucleus to nucleus in the same animal. These results strongly support the idea that most or all chromatin entry sites will act as nucleation points for MSL spreading in cis (Meller, 2000).

To determine whether the roX RNAs and MSL proteins are associated in the same binding pattern on the X chromosome, the immunostaining protocol was adapted to allow detection of roX RNAs by in situ hybridization to spread polytene chromosomes. It was found that the MSL proteins, roX1 and roX2 RNAs are precisely colocalized along the male polytene X chromosome. In addition, a subtle enrichment for roX1 RNA has been consistently detected in several bands surrounding its site of synthesis (Meller, 2000).

MSL proteins are found at decreased levels when they are unable to assemble into a wild-type complex. Therefore, it was asked whether roX RNAs might exhibit the same behavior, indicative of a requirement for complex assembly for their integrity. roX1 RNA was expressed constitutively from an H83roX1 transgene and it was determined whether roX1 RNA could accumulate and perhaps even associate with the X chromosomes in females, which lack MSL complexes. It was found that ectopic roX1 RNA fails to bind the X chromosome in females as assayed by in situ hybridization. To determine whether the roX1 RNA produced from the H83roX1 transgene could be stabilized by the presence of the MSL complex, its behavior was examined in males lacking endogenous roX1, or females that form MSL complexes as a result of ectopic expression of Msl2. It was found that transgenic roX1 RNA associates with the X chromosome in males and in Msl2-expressing females. Thus, roX1 RNA is dependent on the MSL complex for its stability (Meller, 2000).

The idea that the MSL complex assembles at chromatin entry sites rests on the observation that these are the only places partial MSL complexes bind when one subunit is removed by mutation. Therefore, it was determined whether roX RNAs might also be associated with these partial complexes in the absence of spreading into flanking chromatin. To visualize chromatin entry sites, females were used that ectopically express Msl2 but are mutant for msl3 or mle. They assemble partial MSL complexes at the same sites as dying mutant males, but are viable and produce excellent polytene chromosomes. Surprisingly, a difference was found in the localization of roX1 and roX2 RNAs in msl3 mutants. Although roX1 RNA is detected only at cytological position 3F, the site of its synthesis, roX2 is consistently seen at many chromatin entry sites including 3F. This result suggests that roX2 may assemble first into partial MSL complexes and then be exported to other chromatin entry sites. These roX2-containing complexes may subsequently acquire a second roX RNA species (such as roX1) or be present in distinct complexes from roX1 (Meller, 2000).

mle mutants were examined for localization of roX RNAs. It was found that roX2 RNA is no longer able to move to other chromatin entry sites, but is only detected at its site of synthesis, like roX1. This demonstrates that the Mle helicase plays an early role, perhaps in packaging roX2 RNA into growing MSL complexes. Blocking assembly at this point prevents roX2 RNA from reaching other chromatin entry sites (Meller, 2000).

To further test the idea that roX2 assembly may occur at an earlier step than roX1, it was determined whether roX2 RNA assembles correctly in the absence of roX1. It was found that roX2 RNA is localized to the male X chromosome in the absence of roX1, with no apparent perturbation of its staining pattern. It is concluded that roX2 RNA, like the MSL protein complex, is independent of roX1 for localization on the X chromosome (Meller, 2000).

MSL complexes can be immunoprecipitated from Schneider SL2 tissue-culture cells, which exhibit male-like X chromosomal MSL complex and histone H4Ac16 by immunostaining. Therefore, reverse transcription (RT)-PCR analyses was performed to determine whether roX RNA could be coprecipitated with MSL complexes from SL2 cell extracts. Focus was placed on roX2 because the genetic experiments had predicted that it may be included at an early step in MSL complex assembly. RT-PCR products were first confirmed from male larval total RNA, SL2 cell total RNA, and SL2 cell protein lysates, using primers flanking an intron of 141 bp. Surprisingly, a smaller RNA with a 270 bp intron removed is the predominant product detected in both male larvae and SL2 cell total RNA. The intron encompasses the 141 bp intron and utilizes the same 3' splice site. The spliced roX2 RNA is detected in immunoprecipitates from SL2 cells using anti-Msl1 and anti-Mle antibodies. It is concluded that the MSL proteins are physically associated with the spliced form of roX2 RNA (Meller, 2000).

Until recently, it had been assumed that X-chromosome-specific cis-acting sequences would be associated with most genes bound by the MSL proteins. However, studies of roX1 have suggested that, if provided with a chromatin entry site (a roX1 transgene), the MSL complex could be attracted in cis to genes on autosomes that were never before dosage-compensated. The complex could not normally gain access to the autosomes in wild-type males because the MSL chromatin entry sites are found only on the X chromosome. Here, it has been shown that roX2 transgene can also function as a chromatin entry site for spreading of MSL complexes into autosomal chromatin (Meller, 2000).

In addition to roX genes functioning as chromatin entry sites, it is thought that the RNAs themselves may play roles in MSL complex assembly or function on the X chromosome. Although large ribonucleoprotein machines are well known in the form of ribosomes, spliceosomes and telomerases, RNAs have not been considered important factors in transcriptional regulation. There have been recent reports, however, of transcriptional control by non-coding RNAs in several diverse systems. A prominent example is the Xist RNA, which has been shown to play a central role in silencing most of the genes along one of the two X chromosomes in mammals. Another example is SRA, a novel non-coding RNA discovered as a coactivator of steroid hormone receptors. Individually, roX RNAs do not appear essential for MSL complex assembly on the X chromosome, but the phenotype of mutant embryos that lack roX1, roX2 and an unknown number of neighboring genes suggest that assembly of the MSL complex is delayed or abolished when both RNAs are removed (Meller, 2000 and references therein).

These data are consistent with assembly of roX RNAs into MSL complexes: (1) roX2 RNA can be immunoprecipitated from male tissue-culture cells, using anti-Msl1 or anti-Mle antibodies; (2) the roX RNAs are highly unstable in the absence of MSL proteins even if their synthesis is driven by a strong constitutive or inducible promoter; (3) movement of roX2 RNA from its site of synthesis to other chromatin entry sites requires Mle helicase. These observations are most easily understood if the MSL proteins physically contact the roX RNAs, protecting them from cytoplasmic export or degradation and targeting them instead to the X chromosome (Meller, 2000).

One clear difference between roX1 and roX2 RNAs is that roX2 RNA can travel to ~35 other chromatin entry sites along the X chromosome in the absence of Msl3. Maturation of the complex is thought to take place at these sites and then to initiate spreading into flanking chromatin. In the same nuclei lacking Msl3 protein, roX1 RNA is found only at its site of transcription. This suggests that the protein and RNA components of the MSL complex may assemble in a stepwise manner at several sites, and that roX2 may play a role in early events (Meller, 2000).

These results support three interrelated roles for roX genes in dosage compensation: (1) to produce RNA components of the MSL complex; (2) to provide sites for complex assembly and, (3) to mark the X chromosome for dosage compensation by acting as nucleation sites for spreading the MSL complex in cis to surrounding genes. A model has been presented for an ordered pathway of MSL-roX assembly, in which roX2 RNA joins partial MSL complexes at an early step and is exported to other chromatin entry sites in a process dependent on the Mle helicase. The roX2-containing complexes may complete assembly at other chromatin entry sites, perhaps by acquiring a second roX RNA species (such as roX1). Only mature complexes are functional to spread and dosage compensate flanking genes on the X chromosome (Meller, 2000).

Targeting the chromatin-remodeling MSL complex of Drosophila to its sites of action on the X chromosome requires both acetyl transferase and ATPase activities

Dosage compensation in Drosophila is mediated by a multiprotein, RNA-containing complex that associates with the X chromosome at multiple sites. Investigated were the roles that the enzymatic activities of two complex components, the histone acetyltransferase activity of Mof and the ATPase activity of Mle, may have in the targeting and association of the complex with the X chromosome. Mle and Mof activities are necessary for complexes to access the various X chromosome sites. The role that histone H4 acetylation plays in this process is supported by the observations that Mof overexpression leads to the ectopic association of the complex with autosomal sites (Gu, 2000).

The normal association of the MSL complex at hundreds of sites along the X chromosome appears to be a process with at least three major steps. The first is the formation of functional complexes at the two entry sites where the roX RNAs are transcribed. It should be noted that although the Msl1 and Msl2 proteins are able to access the X chromosome at the entry sites and to recruit Mle, further complex assembly can only occur in the presence of the roX RNAs. This contention is supported by the observation that, in the absence of the two roX genes, no complex is seen to form in embryonic stages where it is normally evident. A caveat is that removal of roX2 was accomplished by using a deletion of such size that other roX-like genes or other unidentified components of the complex or genes whose product is required for complex stability, closely linked to roX2, may have been deleted as well. In any event, since the roX RNAs are unstable unless they are associated with the complex, the process of assembly can proceed only at the entry sites containing the roX genes. Once complexes are formed, they access the X chromosome through all of the entry sites, presumably via the affinity of their Msl1/Msl2 components for these sites. Finally, the complexes spread from the entry sites to the many other sites along the X chromosome where they are normally found. This last step requires the histone acetyltransferase activity of Mof. It is suggested that the spreading process involves the acetylation of neighboring nucleosomes, thereby altering the conformation of adjacent chromatin and rendering it more accessible to the entry of additional MSL complexes. The latter may require the presence of acetylated histone H4 tails in order to stabilize their chromatin association. This conclusion is consistent with the observation that, in S2 cells overexpressing Mof, the resulting abnormal ectopic acetylation of histone H4 at Lys16 leads to the association of the MSL complex along autosomal chromatin. This may mirror the normal situation in vivo where complexes, initially attracted to the entry sites, acetylate histone H4 at Lys16 and thereby make adjacent chromatin regions accessible to more complexes. The affinity of the MSL complex for histone H4 tails implied in this model is reminiscent of a similar role played by histone tails in the spreading of complexes containing SIR2, 3 and 4 during the silencing of mating type loci and telomeric heterochromatin formation in yeast. Although critical to the spreading process, the role played by the ATPase function of Mle, either directly or in conjunction with roX RNA, is not sufficiently understood to be incorporated in the model (Gu, 2000).

It is thought that the process just described can provide the following explanations for the gaps in Msl binding that occur along the X chromosome, or at ectopic autosomal sites where the complex has been caused to form at the site of a roX transgene. It is possible that the spread of H4 acetylation and complex association may be stopped by some insulator or some as yet uncharacterized boundary elements. This would not necessarily require that the entry sites be entirely responsible for the pattern seen along the X chromosome. The interphase chromosome is believed to consist of a series of rosettes formed by loops of the chromatid fiber anchored to a central core by dispersed regions that have affinity for one another. In such an arrangement, a cluster of complexes that have been stopped by some boundary element could acetylate the nucleosomes on a neighboring loop, initiating a spreading process on the other side of a gap (Gu, 2000).

The above considerations raise a number of questions that remain to be resolved. Is the pattern of complex association on the X chromosome tissue specific? Is it dependent on a tissue-specific distribution of the entry sites (other than those containing the roX loci, which must remain invariant in all tissues)? Is the tissue-specific distribution established when the complex first forms in early embryogenesis and is the pattern perpetuated through the mitotic divisions that give rise to a particular tissue? To answer these questions will require a thorough melding of cytological and biochemical approaches (Gu, 2000).

MSL1 plays a central role in assembly of the MSL complex, essential for dosage compensation in Drosophila: The association of MOF and MSL3 with the MSL1-MSL2 complex is MLE dependent

In male Drosophila, histone H4 acetylated at Lys16 is enriched on the X chromosome, and most X-linked genes are transcribed at a higher rate than in females (thus achieving dosage compensation). Five proteins, collectively called the MSLs, are required for dosage compensation and male viability. Here it has been shown that one of these proteins, MSL1, interacts with three others, MSL2, MSL3 and MOF. The latter is a putative histone acetyl transferase. Overexpression of either the N- or C-terminal domain of MSL1 has dominant-negative effects, i.e. causes male-specific lethality. The lethality due to expression of the N-terminal domain is reduced if msl2 is co-overexpressed. MSL2 co-purifies over a FLAG affinity column with the tagged region of MSL1, and both MSL3 and MOF co-purify with the FLAG-tagged MSL1 C-terminal domain. Furthermore, the MSL1 C-terminal domain binds specifically to a GST-MOF fusion protein and co-immunoprecipitates with HA-tagged MSL3. The MSL1 C-terminal domain shows similarity to a region of mouse CBP, a transcription co-activator. It is concluded that a main role of MSL1 is to serve as the backbone for assembly of the MSL complex (Scott, 2000).

In general, the amino acid sequences of the MSLs suggest regions or domains within the proteins that could be important for function in vivo. Indeed, this has been confirmed by mapping loss-of-function mutations to the domain, such as the helicase domain of MLE, the putative acetylase domain of MOF and the RING finger region of MSL2. The amino acid sequence of MSL1 is the least informative, containing no recognizable domains, although regions rich in acidic amino acids and possible PEST sequences have been identified. To identify regions within MSL1 that are important for function in vivo, it was determined which regions have dominant-negative effects when overexpressed. Two regions of MSL1, one near the N-terminus and the other at the C-terminus, are likely to be important for assembly of the MSL complex in vivo, because overexpression of either region causes male-specific lethality. Genetic evidence, decreased male viability of msl2 heterozygotes and increased male viability by co-overexpression of MSL2, suggests that the region of MSL1 at the N-terminus interacts with MSL2. This has been confirmed by co-purification of MSL2 with FLAG-tagged versions of MSL1 over FLAG affinity columns. Similarly, the C-terminal region of MSL1 interacts with both MOF and MSL3. Furthermore, expression of the C-terminal domain results in significant loss of MOF from the male X chromosome (Scott, 2000).

The N-terminal FN region of MSL1 that binds to MSL2 was chosen originally for expression in flies because it was predicted that almost half of FN (amino acids 96-172) would form a two-stranded, alpha-helical, coiled-coil structure. Coiled-coil structures are comprised of a heptad repeat (abcdefg)n where hydrophobic residues occupy positions a and d on the same side of the alpha-helix. The coiled-coil motif of GCN4 mediates dimerization. If a similar structure mediates the formation of the MSL1-MSL2 heterodimer, then part of the region of MSL2 that interacts with MSL1 should form a coiled-coil structure. The Ring finger domain region of MSL2 interacts with MSL1. It is predicted that the region immediately preceding the RING finger could form a coiled-coil structure. It is particularly significant that several of the mutations that disrupt the interaction with MSL1 in yeast introduce amino acid changes that either significantly disrupt the alpha-helix (leucine to proline) or introduce a charged amino acid into the predicted hydrophobic face of the alpha-helix. The RING domain is found in a number of proteins, including the V(D)J recombination-activating protein RAG1. The crystal structure of the RAG1 dimerization domain, which includes the RING finger, reveals that dimerization is stabilized by interaction between alpha-helices that form a hydrophobic core. The RING finger is thought to form the structural scaffold upon which the dimer interface is formed. It is tempting to speculate, by analogy with RAG1, that the association of MSL1 and MSL2 involves the interaction of amphipathic alpha-helices that depend on the RING finger domain. This could best be addressed by determining the crystal structure of the MSL1-MSL2 complex (Scott, 2000).

In vitro translated MSL1 C-terminal domain co-immunoprecipitates with in vitro translated HA·MSL3 but not HA·MOF. Thus, C interacts directly with MSL3 but the interaction with MOF requires either another factor present in fly extracts or post-translational modification of MSL1 or MOF. While the possibility of a nucleic acid component of the FC-MOF complex cannot be ruled out, the possiblity (post-translational modification of MSL1 or MOF) is favored since a silver stain of FLAG affinity-purified FC-MOF complex separated by SDS-PAGE shows only two main bands corresponding to the sizes expected for FC and MOF. The C-terminal domain of MSL1 is rich in serine and threonine residues, and contains several potential phosphorylation sites and a predicted PEST sequence. PEST sequences have been suggested to contribute to the instability of the MSL1 protein. However, the role of these sequences in MSL1 has not been determined. Indeed, an alternative function for the PEST sequences is suggested by the observations that the PEST domains of PU.1 and IB are required for their respective interactions with Pip and c-Rel. In both cases, phosphorylation of a serine residue within the PEST sequence is required for the respective protein-protein interactions. The recent finding that a serine/threonine kinase is associated preferentially with the male X chromosome raises the possibility that MSL1 or another MSL is phosphorylated by this enzyme (Scott, 2000).

In the sequential model for assembly of the MSL complex, the first step involves the binding of the MSL1-MSL2 complex to several 'high affinity' sites on the male X chromosome. Since the localization of both MOF and MSL3 to the X chromosome requires mle+ function, this suggests that the association of MOF and MSL3 with the MSL1-MSL2 complex is MLE dependent. MLE could either bind directly to MOF and/or MSL3, or somehow stabilize the MSL complex together with roX RNA. In support of the latter model, MOF and MSL3 bind directly to the C-terminal domain of MSL1. Furthermore, MLE did not co-purify with an FC-MOF-MSL3 complex over an affinity column. However, the affinity chromatography experiments were designed to maximize the likelihood of detecting protein-protein association and are not quantitative. It is possible that MOF and MSL3 may have a higher affinity for the C-terminal domain of MSL1 than full-length MSL1. Thus, one possible mechanism is that in vivo the C-terminal domain of MSL1 is not freely available to bind to MOF and/or MSL3, and that the binding of MLE to the MSL1-MSL2 complex causes a conformational change in MSL1, such that the C-terminal domain becomes more accessible (Scott, 2000).

Previous searches of the protein sequence database with the complete MSL1 sequence have failed to identify any significant similarities. However, when a search is carried out with just the C-terminal domain sequence, some similarity is found to a 254 amino acid region of mouse CBP. Although the similarity is not high, given that the similarity extends across almost the entire C-terminal domain of MSL1, and that both CBP and the MSL1 C-terminal domain bind to histone acetyl transferases (or putative histone acetyl transferases), it is thought that this homology may be significant. If this similarity reflects a conserved function, then it would be predicted that the MSL1-similar region of CBP, which has no known function, would associate with either an MOF-like histone acetyl transferase or an MSL3-like protein in mammalian cells (Scott, 2000).

It is not known how the MSL complex binds to the male X chromosome. None of the MSLs contain a recognizable DNA-binding motif. The F84 version of MSL1, lacking the first 84 amino acids, binds to MSL2, MSL3 and MOF but does not bind preferentially to the male X chromosome. This suggests that the male lethality that results from overexpression of F84 is due to this protein being able to bind to three MSLs, but not being able to bind to the X chromosome because the first 84 amino acids of MSL1 are required for recognition of the X chromosome. Alternatively, the lack of binding of F84 to the male X chromosome could be because the beginning of MSL1 is required for assembly of the MSL complex in vivo. However, if so, then it would be expected that F84 would have bound to the 'high affinity' sites since F84 does bind to MSL2. Assuming that MSL1 and MSL2 are the only components of the high affinity complex, it would then appear more likely that the first 84 amino acids of MSL1 are required for X chromosome binding rather than complex formation. However, there are several lines of evidence that suggest that the roX RNAs are part of the MSL complex, which raises the possibility that one or both of the roX RNAs could be part of the high affinity complex. Thus it will be of interest to determine if the MSL complex containing the F84 protein binds to roX RNA with a lower affinity than the complex containing full-length MSL1 (Scott, 2000).

Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the male X chromosome where it acts to increase its expression ~2-fold. A model for the targeted assembly of the MSL complex has been proposed in which initial binding occurs at ~35 dispersed chromatin entry sites, followed by spreading in cis into flanking regions. Here, one of the chromatin entry sites, the roX1 gene, was analyzed to determine which sequences are sufficient to recruit the MSL complex. Association and spreading of the MSL complex from roX1 transgenes was found in the absence of detectable roX1 RNA synthesis from the transgene. The recruitment activity was mapped to a 217 bp roX1 fragment that shows male-specific DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is sufficient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions (Kageymama, 2001).

MSL1 and MSL2 are thought to form a core complex that binds to chromatin entry sites, while MLE and other components including roX RNAs may be added sequentially. To assess whether all the MSL components are required to bind to the roX1 exons, MSL1 localization on the roX1c3.4 transgene was analyzed in msl mutant animals. Since msl mutant males show poor morphology of polytene chromosomes, females carrying a Hsp83-MSL2 transgene were analyzed. Females normally lack MSL2 and can not form MSL complexes, but when MSL2 is constitutively expressed in females, the complex forms and is localized to both X chromosomes. The roX1c3.4 transgene recruits the MSL complex in the absence of functional mof as well as in cells lacking msl3, as expected for an authentic chromatin entry site. However, in mle mutant animals, surprisingly, the transgene does not bind detectable partial complexes. By careful re-examination, it was found that the endogenous roX1 locus at 3F shows two strong MSL binding sites as a tight doublet in the absence of msl3, but shows a faint, single MSL signal in mle mutants. This suggests that there are two distinct MSL binding sites located at 3F, one site within the roX1 gene that requires MLE, and an additional site, similar to the majority of entry sites, that can attract partial MSL complexes in the absence of MLE. It should be noted that even at entry sites that attract partial complexes independent of MLE, the strength or stability of binding to those sites is weak. Since the requirement for MLE is also a characteristic of the endogenous roX2 locus (at 10C), it is possible that this is a common feature of entry sites that encode roX RNAs (Kageymama, 2001).

Local spreading of MSL complexes from roX genes on the Drosophila X chromosome

MSL proteins and noncoding roX RNAs form complexes to up-regulate hundreds of genes on the Drosophila male X chromosome, and make X-linked gene expression equal in males and females. Altering the ratio of MSL proteins to roX RNA dramatically changes X-chromosome morphology. In protein excess, the MSL complex concentrates near sites of roX transcription and is depleted elsewhere. These results support a model for distribution of MSL complexes, in which local spreading in cis from roX genes is balanced with diffusion of soluble complexes in trans. When overexpressed, MSL proteins can recognize the X chromosome, modify histones, and partially restore male viability even in the absence of roX RNAs. Thus, the protein components can carry out all essential functions of dosage compensation, but roX RNAs facilitate the correct targeting of MSL complexes, in part by nucleation of spreading from their sites of synthesis (Oh, 2003).

To date, all evidence for cis spreading comes from autosomal roX transgenes. MSL complexes do spread locally from the endogenous roX genes on the X chromosome, the natural target of dosage compensation. Wild-type males require a balance of MSL proteins and roX RNAs to evenly distribute MSL complexes both locally and at a distance along the X chromosome. When the amounts of MSL1 and MSL2, thought to be the limiting proteins, are artificially increased, MSL complexes spread predominantly over a local segment of the X chromosome surrounding a roX gene. More remote regions bind little MSL complex. This dramatically alters the morphology of polytene X chromosomes. Surprisingly, overexpressing MSL1 and MSL2 partially restores viability to males lacking roX RNA. This indicates that the MSL proteins have intrinsic affinity for the X chromosome that is enhanced or stabilized in wild-type males by the roX RNAs (Oh, 2003).

Earlier observations that the MSL complex could spread in cis from an autosomal roX transgene have lead to speculation that complexes normally spread from the endogenous roX loci on the X chromosome to paint the entire chromosome. However, the initial characterization of roX1 clearly demonstrates that soluble MSL complexes could diffuse between chromosomes. More recent work reveals that the ability of the MSL complex to spread from a site of roX transcription, or diffuse away, is highly sensitive to the balance between MSL proteins and roX transcripts in the nucleus. This study demonstrates that the wild-type pattern of MSL complexes along the male X chromosome is the result of a delicate interplay between two targeting strategies. Local spreading from roX loci operates in parallel with a second route where soluble MSL complexes diffuse and reattach to distant segments of the X chromosome. The proportion of MSL complexes entering each pathway can be altered by manipulating the amount of MSL proteins or roX RNAs present. It is speculated that the underlying mechanism controlling these two outcomes rests on how efficiently MSL subunits can assemble into functional complexes (Oh, 2003).

ATP-dependent roX RNA remodeling by the helicase maleless enables specific association of MSL proteins

Dosage compensation in Drosophila involves a global activation of genes on the male X chromosome. The activating complex (MSL-DCC) consists of male-specific-lethal (MSL) proteins and two long, noncoding roX RNAs. The roX RNAs are essential for X-chromosomal targeting, but their contributions to MSL-DCC structure and function are enigmatic. Conceivably, the RNA helicase MLE, itself an MSL subunit, is actively involved in incorporating roX into functional DCC. This study determined the secondary structure of roX2 and mapped specific interaction sites for MLE in vitro. Upon addition of ATP, MLE disrupted a functionally important stem loop in roX2. This RNA remodeling enhanced specific ATP-dependent association of MSL2, the core subunit of the MSL-DCC, providing a link between roX and MSL subunits. Probing the conformation of roX in vivo revealed a remodeled stem loop in chromatin-bound roX2. The active remodeling of a stable secondary structure by MLE may constitute a rate-limiting step for MSL-DCC assembly (Maenner, 2013).

Since the discovery of Xist RNA as a crucial epigenetic regulator involved in mammalian X inactivation about 20 years ago, an increasing number of long, noncoding (lnc) RNAs have been found associated with chromatin-modifying complexes. Their hypothetical functions include roles as versatile scaffolds for protein complexes and in targeting associated regulators to genomic loci, either as nascent RNA or through R-loop or triple helix formation. Considering the conformational flexibility and structural diversity of RNA, its regulatory potential is enormous. However, a detailed mechanism of action has only been resolved in a few cases (Maenner, 2013 and references therein).

The highly elaborate dosage compensation systems that evolved to compensate for sex chromosome monosomy provide instructive examples for regulatory functions of lnc RNAs. The inactivation of one X chromosome in female mammals is orchestrated by the interplay of at least three lnc RNAs. In Drosophila melanogaster, dosage compensation involves doubling the transcription of many X-linked genes. This activation is achieved by a ribonucleoprotein dosage compensation complex (DCC) consisting of five male-specific-lethal (MSL) proteins and two lnc RNAs, roX1 and roX2 (for 'RNA-on-the-X'). These RNAs are considered scaffolds for the proper assembly of the MSL proteins. The MSL-DCC only forms in male flies due to the male-specific expression of two key components: the core subunit MSL2 and the roX RNAs. The remaining protein subunits-MSL1, MSL3, the acetyltransferase MOF (Males-absent-on-the-first), and the RNA helicase MLE (Maleless)-are also expressed in female cells and have additional functions outside of the MSL-DCC (Maenner, 2013).

Genome-wide mapping of the chromosomal interactions revealed that most of the MSL-DCC interacts with the bodies of actively transcribed genes on the X chromosome. According to a popular model, the X-specific targeting of the MSL-DCC involves an initial recognition of a limited number of 'chromosomal entry' or 'high-affinity' sites ('CES' or 'HAS,' respectively), which are distributed along the X. Accordingly, the complex transfers from these sites to active genes in the nuclear vicinity. Once tethered to the active chromatin, the MSL-DCC activates transcription through acetylation of lysine 16 of histone H4 (H4K16ac) by MOF, which is thought to unfold the chromatin fiber and facilitate the production of mature mRNAs (Maenner, 2013).

RoX1 and roX2 RNAs differ greatly in size (3.7 kb versus 600 nt) and show very little sequence similarity. Despite these differences, each alone can support the assembly of a functional MSL-DCC. Inactivation of both roX genes abolishes dosage compensation and is lethal for males. Exploring the common denominator between the two RNAs by phylogenetic comparison and functional analyses, Kuroda, Park, and colleagues discovered a series of conserved sequence motifs (GUUNUNCG), the 'roX-boxes,' which reside in the 3' ends of the roX RNAs. In silico analysis of structural motifs predicts that prominent roX-boxes participate in the formation of stable stem-loop structures (SLroX). The integrity of SLroX structure is essential for roX function. Furthermore, expressing an RNA consisting solely of six tandem repeats of SLroX2 suffices to recruit the MSL proteins to the X chromosome and acetylate H4K16 on the X in the absence of endogenous roX RNA. These data highlight the importance of the SL for roX RNA function, but the underlying molecular mechanism remains unclear. Although four of the five MSL proteins are able to bind RNA in vitro, so far no specific interaction of any protein with SLroX has been demonstrated (Maenner, 2013).

Several lines of evidence suggest a primary role for the RNA helicase MLE in incorporating roX into the MSL-DCC. RoX1 is initially expressed in preblastoderm embryos of both sexes, where it is stabilized by maternal MLE until dosage compensation is established. At later developmental stages, MLE is required for steady-state assembly and targeting the MSL-DCC to the X chromosome. Inactivating the ATPase activity of MLE by point mutation (MLEGET) impairs X-chromosomal targeting of the DCC. A mutation that selectively inactivates the helicase function greatly limits the spreading of the MSL-DCC onto the active gene bodies (Maenner, 2013).

It is unclear where in the nucleus roX RNAs and MSL proteins assemble to form a functional DCC. This may happen at the site of roX gene transcription. Another possibility is that HASs are sites of MSL-DCC formation. It was recently found in a genome-wide study of chromosomal interactions that MLE strongly enriched at some 240 HAS sites on the X, in close proximity to MSL2. Since the specific chromatin immunoprecipitation (ChIP) methodology applied in this study emphasizes direct chromatin interactions, it was consider that MLE may affect the assembly or reorganization of the MSL-DCC at HAS. In line with this argument, the recent ChIRP (chromatin isolation by RNA purification) profiling of chromatin interactions also found roX2 RNA enriched at HAS (Maenner, 2013).

The current study suggests that the assembly of the ribonucleoprotein MSL-DCC is initiated by an obligatory, energy-consuming RNA remodeling step, during which MLE recognizes the SLroX structure and converts it into a more extended form. Accordingly, the stable, low-energy SL conformation that roX2 adopts in vitro represents an inactive, closed conformation, which needs to be actively opened to expose binding sites for MSL proteins (Maenner, 2013).

This study has found that the specific interaction of MSL2, the key identifier of the MSL dosage compensation complex, with roX RNA depends on the prior remodeling of a prominent stem-loop structure in the 3' end of roX2. This hairpin, together with a related structure in roX1, is indispensable for roX function in flies. Previous failures to detect specific MSL interactions with SLroX2 are now at least in part explained by the observation that SLroX2 itself does not serve as a binding site but has to be converted into an alternative, open conformation through the ATP-dependent remodeling action of MLE. The data describe a function for a nuclear helicase in lncRNA conformation. The occurrence and functional importance of the proposed conformational switch during organismal development remains to be tested in future studies (Maenner, 2013).

A role for MLE in resolving long dsRNA stems was already suggested more than a decade ago. A mutation in MLE, the mlenapts allele, causes the failure to express the para-encoded Na+ channel. Para transcripts are subject to consecutive RNA editing and splicing. Adenosine-to-inosine editing is guided by the formation of a stable dsRNA secondary structure that needs to be resolved for subsequent splicing. The mutated helicase binds to and stabilizes the stem structure rather than unwinding it, as it would normally do to allow access of the splicing machinery. Interestingly, the ds editing structure of the para transcript also contains two A-U motifs in the vicinity of the edited sequence, which resemble the roX-box motif. It is suggested that MLE functions more generally to unwind long base-paired RNA structures to expose ssRNA sequences for downstream interactions (Maenner, 2013).

The data show that MLE preferentially binds to RNA containing an SLroX even in the absence of ATP and that this interaction is improved by increasing the stability of the SL. In the presence of ATP, the specificity of interaction with this particular binding site is reinforced. Because the MLEGET mutant that binds nucleotides with reduced affinity does not show the effect, it is assumed that the ATP-dependent disruption of the SL strengthens the MLE interaction with roX. The SLroX structures share a conserved sequence, the roX-box, which contributes to MLE affinity. The four consecutive A-U pairs may facilitate the unwinding of the stem and/or provide a binding site for MLE and/or other MSL subunits in their single-stranded conformation (Maenner, 2013).

It is hypothesized that RNA remodeling by MLE physically connects the two male-specific principles that are key to the formation of the MSL-DCC: the male-specific MSL2 protein and the roX RNAs. Numerous observations already suggested a close interdependency of their functions. In the absence of MSL2, roX RNAs are unstable and, conversely, any MSL2 that is not in a complex with roX will be degraded. The enrichment of MLE on the X chromosome depends on MSL2 and MSL1. MSL2 may physically connect the MLE-roX module to the MSL1-MSL3-MOF module. If MSL1 is mutated to abolish the MSL2 interaction, roX RNAs are no longer incorporated into the MSL-DCC. It is currently unclear whether MLE would remain part of a stable MSL-DCC complex once it has remodeled the roX RNA. Earlier findings that MLE readily dissociates during purification of MSL complexes indicate that MLE was rather loosely bound. The helicase may only fulfill a transient function during the biogenesis of the complex. The striking enrichment of MLE at 240 HAS on the X chromosome in direct neighborhood of MSL2 identifies these loci as primary sites of MLE action (Maenner, 2013).

In summary, it is proposed that the active remodeling of roX RNA by the ubiquitous RNA helicase MLE constitutes an obligatory, regulated step in the biogenesis of the MSL-dosage compensation complex. This study paves the way for a mechanistic dissection of this fundamental conformational switch. Lessons learned from the dosage compensation system will undoubtedly enhance the understanding of other lncRNA-containing chromatin regulators (Maenner, 2013).

Tandem stem-loops in roX RNAs act together to mediate X chromosome dosage compensation in Drosophila

Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. By using UV crosslinking followed by deep sequencing, this study shows that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem-loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, different roX RNA domains were shown to have overlapping function, since only combinatorial mutations in the tandem stem-loops result in severe loss of dosage compensation and consequently male-specific lethality. It is proposed that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes (Ilik, 2014).

Identification of functional domains in lncRNAs is an important step toward understanding how they work in vivo. This study characterizes roX1 and roX2 as the most significant RNAs associated with MLE and MSL2. roX1 and roX2 contain common, conserved, and distinct structural domains, which form the binding platform for these proteins. Interestingly, regions of lncRNAs that lie outside of these MLE/MSL2 interaction domains appear to be unstructured and not conserved. These data also provide evidence on how roX1 and roX2 RNAs can be functionally redundant by showing that MLE-MSL2-interacting regions are present in multiple copies in both RNAs (Ilik, 2014).

Earlier work on roX RNAs has identified short stretches of RNA that are shared between these lncRNAs that are conserved throughout evolution, called the roX boxes. This study shows that all three roX boxes at the 3' end of roX2 RNA and one of the three roX boxes in roX1 form stable helices in vitro. Moreover, these elements represent binding sites for MLE and MSL2 proteins in vivo. Detailed analysis of MLE individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) data revealed that there are other RNA elements in both roX1 and roX2 that resemble the roX box sequence, and also serve as binding sites for MLE. These elements were named roX box-like sequences. Together roX box and roX box-like sequences uncover a consensus binding site for MLE in roX RNAs. No other RNAs were identified in the Drosophila transcriptome that contained the roX box/roX box-like motif other than the roX RNAs, adding to the evidence that it is likely that there are no more roX-like RNAs that can function in Drosophila dosage compensation (Ilik, 2014).

The results expose the logic of a roX RNA: stem-loops containing RBL elements at the 5' end and roX boxe (RB) containing helical structures joined by a flexible, single-stranded spacer region. In roX2, these elements are repeated on a very small scale with perfect copies of RBL/RB elements. In contrast, in roX1, these sequence motifs deviate from the ideal consensus more than roX2, but roX1 probably compensates for this by containing multiple, autonomous interaction domains that form a much larger RNA, which is about six times as big as roX2 (Ilik, 2014).

Endogenously expressed MLE and affinity-tagged MLE proteins have the ability to differentiate between the two stem-loop clusters in the two halves of roX2 exon-3 (1–280 versus 281–504). Interestingly, addition of ATP led to the specific interaction of endogenously expressed MLE with the second stem-loop cluster of roX2 exon-3 that contains roX box-containing helical structures. Furthermore, it was observed that the N-terminal dsRBDs are not only required for the ATP-independent interactions with the 5' end of roX2 exon-3 but are also required for the ATP-dependent interactions of MLE with the 3' end of roX2 exon-3. A similar dichotomy was observed in High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) and iCLIP experiments. In HITS-CLIP, the reads accumulated around the first stem-loop cluster of roX2 exon-3, concentrating on R2H1, whereas in iCLIP the balance was shifted toward the second cluster (Ilik, 2014).

MLE is a member of the RHA/DEAH family of RNA helicases, which can remodel RNA and RNPs. Interestingly, biochemical work on the closely related DEAD box RNA helicases shows that these enzymes have varying degrees of affinity toward their RNA substrates during their ATPase cycles. Since the GRNA chromatography experiments reveal that the second half of roX2 gains MLE binding when supplemented with ATP in vitro, it is possible that the quaternary complex between MLE, roX2, and ADP-Pi is being scored for in these experiments, and enzyme is being caught on its way to eventually remodeling of this part of the RNA (Ilik, 2014).

The ATP-independent binding of MLE to the first half of roX2 RNA via its N-terminal dsRBDs could be an initial regulatory step as roX RNAs constitute important tethers for MLE within the MSL complex. However, the in vivo analysis revealed that at high expression levels this cluster 'A' is dispensable for dosage compensation. The second hairpin cluster appears to recruit MLE through the helicase domain in a way that still requires N-terminal dsRBDs, thus providing a dynamic platform for rerecruitment and spreading of MLE along the roX RNA. The function of this cluster 'B' can only be partially compensated by higher expression, suggesting that it is functionally distinct from 'A.' It is proposed that the dynamic interaction of MLE with roX RNAs may ensure that different regions of roX RNAs are exposed such that they can be used in a redundant or cooperative manner for the interaction with the MSL complex members providing plasticity, as clearly detected for MSL2 binding on roX RNAs in the iCLIP data in vivo. However, it can be excluded that integration of roX RNAs with the 'core' MSL complex may also follow a more direct route involving independent MSL-RNA interactions that need not require MLE as a mediator (Ilik, 2014).

roX genes have a dual function in Drosophila dosage compensation: they are sites of roX transcription, but they also contain two high-affinity sites (HASs) that can recruit the MSL complex independent of the roX RNAs. Moreover, roX RNAs can travel from their sites of synthesis to the X chromosome when placed as a transgene to an autosomal site. This suggests that the holo- MSL complex, containing the core components (MSL1-3 and MOF) and MLE together with roX RNAs, can form on chromatin at roX transcription sites and spread in cis on the X chromosome, but it may also form in solution and be targeted to X-chromosomal sites in trans. It has been recently shown that MSL2 interacts with a dimer of MSL1 and is itself present as a dimer within the MSL complex. Interestingly, this study found that roX RNAs do not interact with each other in vivo, suggesting that there could be one roX RNA species per holo-MSL complex (Ilik, 2014).

Notably, iCLIP methodology utilizing UV crosslinking provides a snapshot of a pool of interactions that are present at the instant of irradiation. Although iCLIP data clearly show that MLE and MSL2 bind to the same domains, it is possible that they occupy different stem-loops on different molecules of roX RNAs rather than occupying the same structure at the same time. roX RNAs, by evolving multiple interaction platforms, can indeed support such combinatorial binding events, thus facilitating spreading along the X chromosome. It is proposed that roX RNAs, by virtue of being able to interact with MLE and the MSL complex, play a central role in the assembly of the holo- MSL complex containing the 'core' (MSL1, MSL2, MSL3, MOF) and MLE. These complexes may form in solution or on chromatin such as on sites of roX transcription. Such configuration thus brings different enzymatic activities together (ATPase/ helicase activity of MLE; acetyltransferase activity of MOF and ubiquitin-ligase activity of MSL2) for the X chromosome-specific dosage compensation process. Directing the assembly of such a complex and by providing plasticity through its multiple stem-loops, roX RNAs could thus facilitate local spreading of the MSL complex from HAS into neighboring chromatin including low-affinity sites (LASs) (Ilik, 2014).

Domains identified in roX1 and roX2 could be defined as 'information units' within these lncRNAs, which organizationally resemble stem-loops strung together like 'beads on a string'. It is tempting to hypothesize that one could compare the 'information unit' within roX lncRNAs to be the counterpart of codons which provide the basic information units in mRNAs. For roX lncRNAs, the information unit is a bit larger (11–15 nt) and involves both primary sequence and secondary structure. Another important aspect is that the domains are repeated in the lncRNA but do not appear to have a strict requirement of spacing or order relative to each other (i.e., no polarity and reading frame), so functional versions may be relatively easy to evolve during evolution (Ilik, 2014).

In summary, these data suggest that lncRNAs may utilize discrete repetitive motifs for distinct protein-RNA or even RNADNA interactions to achieve functional specificity in vivo. Such a mechanism may also be used for other global epigenetic phenomenon such as the X-inactivation in mammalian cells. Future analysis of other lncRNAs that combine CLIP methods and RNA structural analysis will be crucial in identifying structural domains within these RNAs and understanding their precise roles in the regulation of gene expression (Ilik, 2014).

UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation

Dosage compensation is a regulatory process that balances the expression of X-chromosomal genes between males (XY) and females (XX). In Drosophila, this requires non-coding RNAs and RNA-binding proteins (RBPs) whose specific functions remain elusive. This study shows that the Drosophila RBP UNR promotes the targeting of the activating male-specific-lethal complex to the X-chromosome by facilitating the interaction of two crucial subunits: the RNA helicase MLE and the long non-coding RNA roX2 (Militti, 2014).

In Drosophila, dosage compensation involves the binding of the MSL dosage compensation complex (MSL-DCC) to hundreds of sites on the single male X-chromosome and the subsequent twofold hypertranscription of active genes. The MSL-DCC contains five proteins: MSL1, MSL2, MSL3, Maleless (MLE) and Males-absent-on-first (MOF), and two long non-coding RNAs, RNA on X (roX) 1 and 2, which differ in size and sequence but display redundant functions. MSL2 is the limiting subunit of the MSL-DCC and, together with MSL1, nucleates complex formation at specific X-chromosomal sites known as high-affinity sites (HAS). The RNA helicase MLE colocalizes with MSL2 at HAS and facilitates the incorporation of roX into the complex. The RNA is important for the distribution of the MSL-DCC along the X-chromosome, where it activates target genes (Militti, 2014).

Upstream of N-Ras (UNR) is a conserved RNA-binding protein (RBP) containing five cold-shock domains (CSD) that regulates mRNA translation and stability by interacting with single-stranded RNA. It has been shown previously that Drosophila UNR performs sex-specific opposing roles in dosage compensation. In females, UNR inhibits MSL-DCC assembly by repressing the synthesis of MSL2. In males, UNR promotes the targeting of the MSL-DCC to the X-chromosome by a poorly understood mechanism that does not involve translational regulation of MSL proteins . UNR-dependent regulation can be recapitulated in male S2 cells, which express only roX2. This work describes efforts to determine whether UNR interacts with the DCC assembly and targeting machinery in more specific ways. Primed by the finding of a preferred RNA-binding element for UNR close to the known roX remodelling site of the helicase MLE, this study systematically explored the relationship between UNR and MLE. UNR was found to facilitates the binding of MLE to its target at limiting MLE concentrations. These biochemical analyses approximate the physiological conditions to a good extent, since depletion of UNR also diminishes the MLE-roXinteraction and reduces the association of MLE with HAS in dosage-compensating cells. These results identify UNR as a general RBP with specific roles in dosage compensation (Militti, 2014).

To assess whether recombinant UNR could directly bind to roX2, an electrophoretic mobility shift assay (EMSA) was used. UNR binds full-length roX2 RNA and several roX2-derived fragments. UNR interacted most strongly with a roX2 fragment spanning nucleotides 316-379. Interestingly, this purine-rich region is located within stem-loop 6 (SL6) just upstream of a prominent, conserved stem-loop structure (SLroX2 or SL7) that, when multimerized, is sufficient to restore the X-chromosomal targeting defects of a roX null mutant. Pull-down assays using MS2-tagged RNA as bait confirmed that UNR bound with highest affinity to roX2 fragments containing SL6 (Militti, 2014).

Because of the roles of UNR in RNA metabolism, whether UNR binding affected roX2 levels, nucleocytoplasmic distribution or splicing of its major isoforms in S2 cells was assessed. UNR depletion showed no effect on any of these features. To gain insight into UNR-roX2 interactions, enzymatic and chemical footprinting was performed. Addition of UNR protected unpaired nucleotides of SL6, in particular the terminal loop (nts 367-374) and the internal bulge (nts 352-357), suggesting UNR binding to these regions. UNR also protected to a lower extent the terminal loop of SL7 and the single-stranded region between SL7 and SL8 but did not protect efficiently the terminal loop of SL8. These results confirmed that UNR interacts preferentially with SL6. Mutational analysis showed that UNR recognizes the purine-rich stretches in the loops. Interestingly, nucleotides 417-419 and 497 were rendered more reactive upon the addition of UNR. These positions form a 7bp-extended version of SL7, which maybe disrupted by UNR. These results suggest a role for UNR as a roX2 chaperone (Militti, 2014).

In bacteria, CSD-containing RNA chaperones associate with RNA helicases to promote RNA remodelling . It was thus, asked whether UNR interacts with the RNA helicase MLE. Co-immunoprecipitation experiments with recombinant proteins demonstrated a weak interaction between MLE and UNR. Interestingly, this interaction was strongly stimulated by roX2 but not by an unrelated control RNA of similar length. The interaction persisted after efficient RNase treatment, monitored by measuring the presence of trace-labelled roX2 in the pellet, indicating that roX2 promotes strong direct interactions between MLE and UNR, or that any connecting RNA within the complex is protected from RNase digestion. To test whether these interactions occurred in vivo, UNR was immunoprecipitated from nuclear extracts of S2 cells. Although UNR is primarily cytoplasmic, a small amount can be found in the nucleus that interacts with MLE, but not with MSL3, indicating that the interaction is specific. Consistent with the in vitro data and with the presence of roX2 in the nucleus, the interaction of endogenous MLE and UNR is resistant to RNase treatment after formaldehyde crosslinking, suggesting that interactions involve protein-protein crosslinks and not merely protein-RNA crosslinks. Altogether, the data suggest that UNR, MLEand roX2 form a complex. Indeed, a ternary complex is detected by EMSA when roX2 or its 3' half fragment (SL678) is incubated with recombinant UNR and MLE (Militti, 2014).

Intriguingly, the base of the extended SL7 of roX2 that is potentially remodelled by UNR serves as a binding site for MLE, which disrupts SL7 thereby promoting subsequent interactions with MSL2. Thus, melting of the base of SL7 by UNR and exposure of the corresponding nucleotides could facilitate MLE interaction with roX2. To test this hypothesis, binding of recombinant UNR and MLE to roX2 derivatives was monotored using RNA pull-down assays. MLE on SL67 was titrated in the presence or absence of UNR. At high concentrations, MLE was able to bind SL67. MLE, however, was unable to efficiently bind the target RNA at reduced concentrations. Remarkably, under those conditions the interaction of MLE with SL67 was strongly facilitated by adjacent UNR binding. To test the correlation between MLE and UNR binding, the roX2 derivatives SL2345 and SL67 were employed. UNR bound to both RNAs in the absence of MLE and, consistent with the EMSA, it bound with higher affinity to SL67. MLE bound to roX2 fragments after UNR addition in a manner that correlated with the strength of UNR binding. In addition, MLE bound strongly to full-length roX2 in the presence of UNR, and this binding was reduced upon deletion of either SL6 or SL7. These results indicate that UNR promotes the association of MLE with roX2 in vitro (Militti, 2014).

To test whether UNR promotes the association of MLE with roX2 in vivo, UNR from was depleted from S2 cells. Ablation of UNR diminished the amounts of roX2 associated with the endogenous helicase. This effect could neither be attributed to variations in the amounts of MLE or roX2 upon UNR depletion nor to differences in the efficiency of MLE immunoprecipitation. To explore whether the reduced MLE-roX2 interaction affects the association of MLE with its chromosomal targets, the HAS, chromatin immunoprecipitation (ChIP) was used. Notably, it was found that UNR depletion decreased the MLE association to four different HAS. As controls, UNR depletion did not affect the association of MLE to promoters within the same genes, or the association of MOF to HAS or to an autosomal gene. Altogether, these results demonstrate a role for UNR in facilitating the binding of the RNA helicase MLE with its target sites on roX2 RNA in vitro and in vivo (Militti, 2014).

It is concluded that in the absence of MLE (and roX), MSL2 and MSL1 can associate with HAS but no functional DCC is assembled that distributes the activating histone acetylation to the target genes. ChIP experiments show prominent, specific interaction of MLE at HAS in S2 cells. The current data now suggest that the general RBP UNR facilitates this interaction. Remarkably, UNR also binds and regulates the activity of msl2 mRNA together with the sex determination switch SXL , suggesting that sex determination and dosage compensation have co-evolved to employ similar factors from the large portfolio of RBPs. UNR, thus, extends the small list of RBPs involved in dosage compensation. Such factors include hnRNPU and YY1, which tether the lncRNA Xist to the inactive X-chromosome during mammalian dosage compensation. Rather than a molecular tether, however, the role of UNR seems more transient and directed to facilitate initial steps of MSL-DCC assembly through the modulation of RNA conformation. UNR, therefore, acts as a 'catalyst' of the MLE-roX interaction. RNA structural transitions are at the basis of many fundamental post-transcriptional processes . The current results illustrate the emerging concept that lncRNA structural dynamics may contribute to chromatin organization, and indicate that general RBPs, such as UNR, can be harnessed to contribute key molecular events in the assembly of specialized machineries (Militti, 2014).

maleless: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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