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

Gene name - maleless

Synonyms - male-lethal

Cytological map position - 42A1--42A4

Function - RNA/DNA helicase

Keywords - sex determination hierarchy, chromatin associated proteins

Symbol - mle

FlyBase ID: FBgn0002774

Genetic map position - 2-55.2

Classification - DEAH-box subfamily ATP-dependent helicase

Cellular location - nuclear

NCBI link: Entrez Gene
mle orthologs: Biolitmine
Recent literature
Prabu, J. R., Muller, M., Thomae, A. W., Schussler, S., Bonneau, F., Becker, P. B. and Conti, E. (2015). Structure of the RNA helicase MLE reveals the molecular mechanisms for uridine specificity and RNA-ATP coupling. Mol Cell 60: 487-499. PubMed ID: 26545078
The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. This study identified a stable MLE core comprising the DExH helicase module and two auxiliary domains: a dsRBD and an OB-like fold. MLEcore is an unusual DExH helicase that can unwind blunt-ended RNA duplexes and has specificity for uridine nucleotides.The 2.1 A resolution structure of MLEcore was determined bound to a U10 RNA and ADP-AlF4. The OB-like and dsRBD folds bind the DExH module and contribute to form the entrance of the helicase channel. Four uridine nucleotides engage in base-specific interactions, rationalizing the conservation of uridine-rich sequences in critical roX substrates. roX2 binding is orchestrated by MLE's auxiliary domains, which is prerequisite for MLE localization to the male X chromosome. The structure visualizes a transition-state mimic of the reaction and suggests how eukaryotic DEAH/RHA helicases couple ATP hydrolysis to RNA translocation.

Cugusi, S., Li, Y., Jin, P. and Lucchesi, J.C. (2016). The Drosophila helicase MLE targets hairpin structures in genomic transcripts. PLoS Genet 12: e1005761. PubMed ID: 26752049
RNA hairpins are a common type of secondary structure that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE has been shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. This study reports that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, it was shown that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. The study uses two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

Ilik, I. A., Maticzka, D., Georgiev, P., Gutierrez, N. M., Backofen, R. and Akhtar, A. (2017). A mutually exclusive stem-loop arrangement in roX2 RNA is essential for X-chromosome regulation in Drosophila. Genes Dev 31(19): 1973-1987. PubMed ID: 29066499
The X chromosome provides an ideal model system to study the contribution of RNA-protein interactions in epigenetic regulation. In male flies, roX long noncoding RNAs (lncRNAs) harbor several redundant domains to interact with the ubiquitin ligase male-specific lethal 2 (MSL2) and the RNA helicase Maleless (MLE) for X-chromosomal regulation. However, how these interactions provide the mechanics of spreading remains unknown. By using the uvCLAP (UV cross-linking and affinity purification) methodology, which provides unprecedented information about RNA secondary structures in vivo, the minimal functional unit of roX2 RNA was identified. By using wild-type and various MLE mutant derivatives, including a catalytically inactive MLE derivative, MLE(GET), this study showed that the minimal roX RNA contains two mutually exclusive stem-loops that exist in a peculiar structural arrangement: When one stem-loop is unwound by MLE, an alternate structure can form, likely trapping MLE in this perpetually structured region. This functional unit is necessary for dosage compensation, as mutations that disrupt this formation lead to male lethality. Thus, it is proposed that roX2 lncRNA contains an MLE-dependent affinity switch to enable reversible interactions of the MSL complex to allow dosage compensation of the X chromosome.
Lv, M., Yao, Y., Li, F., Xu, L., Yang, L., Gong, Q., Xu, Y. Z., Shi, Y., Fan, Y. J. and Tang, Y. (2019). Structural insights reveal the specific recognition of roX RNA by the dsRNA-binding domains of the RNA helicase MLE and its indispensable role in dosage compensation in Drosophila. Nucleic Acids Res. PubMed ID: 30649456
In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). This study reports a 2.90A crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, this research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.
Ankush Jagtap, P. K., Muller, M., Masiewicz, P., von Bulow, S., Hollmann, N. M., Chen, P. C., Simon, B., Thomae, A. W., Becker, P. B. and Hennig, J. (2019). Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless. Nucleic Acids Res. PubMed ID: 30805612
Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. This study determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. The NMR and SAXS data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAXK RNA binding motifs. NMR titrations combined with filter binding experiments and isothermal titration calorimetry (ITC) document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.
Lv, M., Yao, Y., Li, F., Xu, L., Yang, L., Gong, Q., Xu, Y. Z., Shi, Y., Fan, Y. J. and Tang, Y. (2019). Structural insights reveal the specific recognition of roX RNA by the dsRNA-binding domains of the RNA helicase MLE and its indispensable role in dosage compensation in Drosophila. Nucleic Acids Res 47(6): 3142-3157. PubMed ID: 30649456
In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). This paper reports a 2.90A crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, this research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.
Bhardwaj, V., Semplicio, G., Erdogdu, N. U., Manke, T. and Akhtar, A. (2019). MAPCap allows high-resolution detection and differential expression analysis of transcription start sites. Nat Commun 10(1): 3219. PubMed ID: 31363093
The position, shape and number of transcription start sites (TSS) are critical determinants of gene regulation. Most methods developed to detect TSSs and study promoter usage are, however, of limited use in studies that demand quantification of expression changes between two or more groups. This study combined high-resolution detection of transcription start sites and differential expression analysis using a simplified TSS quantification protocol, MAPCap (Multiplexed Affinity Purification of Capped RNA) along with the software icetea. Applying MAPCap on developing Drosophila melanogaster embryos and larvae, stage and sex-specific promoter and enhancer activity was detected, and the effect of mutants of maleless (MLE) helicase at X-chromosomal promoters was quantified. It was observed that MLE mutation leads to a median 1.9 fold drop in expression of X-chromosome promoters and affects the expression of several TSSs with a sexually dimorphic expression on autosomes. These results provide quantitative insights into promoter activity during dosage compensation.
Muller, M., Schauer, T., Krause, S., Villa, R., Thomae, A. W. and Becker, P. B. (2020). Two-step mechanism for selective incorporation of lncRNA into a chromatin modifier. Nucleic Acids Res. PubMed ID: 32510132
The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, vitRIP, an unbiased, transcriptome-wide in vitro assay was developed that reveals RNA binding specificity. MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.

Dosage compensation (compensation for having different numbers of X chromosomes) is a crucial developmental process: without it, females would have twice the amount of X-linked gene product as males, because females have two X chromosomes to a single X chromosome for males. In Drosophila equalization of the amounts of gene product produced by X-linked genes in the two sexes is achieved by hypertranscription of the single male X chromosome. This process is controlled by a set of male-specific lethal (msl) genes that appear to act at the level of chromatin structure. Four proteins (encoded by the male-specific lethal genes) are required for dosage compensation; they associate with the X chromosome in males but not in females. The sequences of the MSL proteins contain several motifs potentially associated with transcriptional control.

Mle is highly homologous to human RNA helicase A and the bovine counterpart of RNA helicase A, nuclear helicase II, proteins implicated in the unwinding of RNA (Lee, 1997).

Clues to an understanding of the role of Mle in dosage compensation come from three sources: biochemical analysis, genetic analysis, and information about physical interaction of Mle with polytene chromosomes. Mle protein has been shown to possess RNA/DNA helicase activity, adenosine triphosphatase (ATPase) activity and single-stranded (ss) RNA/ssDNA binding activities. Helicases are DNA and RNA unwinding proteins. The helicase activity demonstrates a degree of substrate specificity. Mle displaces substrates containing single stranded RNA regions more efficiently than substrates containing single stranded DNA regions. A mutant of mle (mle-GET) was created that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis shows that this mutation abolishes both ATPase and helicase activities of MLE but affects the ability of MLE to bind to single stranded DNA and RNA less severly. In vivo, Mle-GET protein can still localize to the male X chromosome but fails to complement mle1 mutant males. These results indicate that the ATPase/helicase activities are essential functions of Mle for dosage compensation but that the interaction of Mle with chromosomes is largely independent of the helicase activity. In other words, the role of Mle in dosage compensation requires at least two functions: (1) the binding to chromosomes, and (2) a helicase activity (Lee, 1997).

How does Mle protein bind to chromosomes? Association of MLE with the male X chromosome is ribonuclease sensitive. 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 messenger RNA transcripts or a hypothetical RNA component of chromatin plays a critical role in the biochemical mechanism of dosage compensation (Richter, 1996).

Part of the Mle protein structure is a conserved RNA binding motif. One RNA binding domain, termed the double-stranded RNA-binding domain (dsRBD), was first identified as 70 residue repeat motifs in three proteins: dsRNA-dependent (DAI) protein kinase, Xenopus RNA-binding protein xlrbpa and Drosophila maternal effect protein Staufen. Two copies of the domain have been detected in helicases: in mammalian RNA helicase A and in Drosophila Maleless. The helicase domain appears to be insufficient on its own to promote helicase activity and additional RNA-binding capacity must be supplied either as domains adjacent to the helicase domain, as in Mle, or by bound RNA recognizing partners (Gibson, 1994).

What is the hierarchy of gene and protein function in dosage compensation? At the top of the hierarchy is Sex lethal, the master regulator of sex determination in Drosophila. Sex lethal regulates the splicing of Msl-2 producing a functional Msl-2 protein in males only (Kelley, 1995 and 1997). The other three male specific lethals are not differentially spliced, although putative SXL-binding sites are also found in the 3' UTR of a subset of MSL-1 transcripts. Msl-2, spliced into a functional form in males, serves to heighten transcriptional activation of the solitary X chromosome. MSL-2 acts in concert with three partners in this task: Maleless, Male-specific lethal-1 and Male specific lethal-3. It is thought that Msl-2 acts in some manner to promote the binding of the other three factors to chromatin. However, Mle might not interact specifically with chromatin but rather with RNA. Is chromosomal RNA the direct target of Mle in dosage compensation, or does RNA have an unknown role in assembling the male specific lethal gene activation complex to the chromosome? There is no reason to assume these possible roles for Mle are mutually exclusive, and both may be involved.

Chromatin is thought to be the target of the MSL proteins. 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 male specific lethal mutant males, correlating with the lack of dosage compensation in these mutants. 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).

The role of Maleless in the chromosomal activation hierarchy remains unclear. Its DNA helicase activity may contribute to destabilizing chromatin structure, analgous to ATP-dependent nucleosomal disruption by the SWI/SNF and Drosophila NURF complexes (see Imitation SWI and Brahma). Its RNA helicase activity could facilitate transcription by altering the structure of nascent RNA, a process that can stimulate reinitiation and/or elongation. Alternatively, Mle may interact with a hypothetical structural RNA component of the chromosome. There is precedence for such RNA in the existence of X chromosome associated RNA on the X. Interestingly, while Mle is released following RNase treatment, the other male specific lethal proteins remain associated with the chromosome (Richter, 1996). This suggests that the several distinct known biochemical functions of male specific lethal proteins will lend themselves to distinct independent functions in dosage compensation (Lee, 1997).

Modulation of heterochromatin by male specific lethal proteins and roX RNA in Drosophila melanogaster males

The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. MLE, but not Jil-1 kinase, was found to contribute to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, a panel of roX1 transgenes and deletions was tested; the X chromosome and heterochromatin functions were found to be separable by some mutations. Widespread autosomal binding of MSL3 occurs before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. It is postulated that this binding may contribute to the sex-specific differences in heterochromatin that have been noted (Koya, 2015).

A central question raised by this study is how factors known for their role in X chromosome dosage compensation also modulate autosomal heterochromatin. Although the MSL proteins were first identified by their role in X chromosome compensation, homologues of these proteins participate in chromatin organization, DNA repair, gene expression, cell metabolism and neural function throughout the eukaryotes. Furthermore, flies contain a distinct complex, the Non-Sex specific Lethal (NSL) complex, containing MOF and the MSL orthologs NSL1, NSL2 and NSL3. The essential NSL complex is broadly associated with promoters throughout the fly genome, where it acetylates multiple H4 residues. In light of the discovery that the MSL proteins represent an ancient lineage of chromatin regulators, it is unsurprising that members of this complex fulfill additional functions (Koya, 2015).

An alternative hypothesis for the dosage compensation of male X-linked genes proposes that the MSL proteins are general transcription regulators, and recruitment of these factors to the male X chromosome reduces autosomal gene expression, thus equalizing the X:A expression ratio. Arguing against this idea are ChIP studies finding that the MSL complex, and engaged RNA polymerase II, are increased within the bodies of compensated X-linked genes. In agreement with this, a study that normalized expression to genomic DNA concluded that compensation increases the expression of male X-linked genes. The current study now reveals that autosomal heterochromatic genes are indeed dependent on a subset of MSL proteins for full expression. However, native heterochromatic genes make up only 4% of autosomal genes, and their misregulation is not expected to compromise genome-wide expression studies normalized to autosomal expression (Koya, 2015).

Expression of heterochromatic genes is thought to involve mechanisms to overcome the repressive chromatin environment. It is possible that a complex composed of roX RNA and a subset of MSL proteins participates in this process. This would explain why heterochromatic genes are particularly sensitive to the loss of these factors. Alternatively, it is possible that roX and MSL proteins participate in heterochromatin assembly. This would explain the simultaneous disruption of heterochromatic gene expression and suppression of PEV at transgene insertions (Koya, 2015).

Heterochromatin assembly is first detected at 3-4 h AEL, a time when MSL3 is bound throughout the genome. Intriguingly, studies from yeast identify a role for H3K4 and H4K16 acetylation in formation of heterochromatin. Active deacetylation of H4K16ac is necessary for spreading of chromatin-based silencing in yeast, demonstrating the need for a sequential and ordered series of histone modifications (Koya, 2015).

As MOF is responsible for the majority of H4K16ac in the fly, a MOF-containing complex could fulfill a similar role during heterochromatin formation. While this study found a significant effect of MOF in expression only on the X and 4th chromosomes, it is possible that examination of a larger number of genes would reveal a more widespread autosomal effect (Koya, 2015).

In roX1 roX2 males the 4th chromosome displays stronger suppression of PEV and more profound gene misregulation than do other heterochromatic regions. This is consistent with the observation that heterochromatin on the 4th chromosome is genetically and biochemically different from that on other chromosomes. Loss of roX RNA leads to misregulation of genes in distinct genomic regions, the dosage compensated X chromosome and autosomal heterochromatin. This study found that the regulation of these two groups is, to some extent, genetically separable. MSL2, which binds roX1 RNA and is an essential member of the dosage compensation complex, is not required for full expression of heterochromatic genes in males. Ectopic expression of MSL2 in females induces formation of MSL complexes that localize to both X chromosomes, inducing inappropriate dosage compensation. As would be expected from the lack of a role for MSL2 in autosomal heterochromatin in males, ectopic expression of this protein in females has no effect on PEV (Koya, 2015).

Elegant, high-resolution studies reveal that MLE and MSL2 bind essentially indistinguishable regions of roX1. Three prominent regions of MLE/MSL2 binding have been identified, one overlapping the 3' stem loop. This stem loop incorporates a short 'roX box' consensus sequence that is present in D. melanogaster roX1 and roX2, and conserved in roX RNAs in related species (Koya, 2015).

An experimentally supported explanation for the concurrence of MLE and MSL2 binding at the 3' stem loop is that MLE, an ATP-dependent RNA/DNA helicase, remodels this structure to permit MSL2 binding. The finding that disruption of this stem blocks dosage compensation but does not influence heterochromatic integrity is consistent with participation of roX1 in two processes that differ in MSL2 involvement. However, a region surrounding the stem loop is required for the heterochromatic function of roX1, as roX1Δ10, removing the stem loop and upstream regions, is deficient in both dosage compensation and heterochromatic silencing. Further differentiating these processes is the finding that low levels of roX RNA from a repressed transgene fully rescue heterochromatic silencing, but not dosage compensation. An intriguing question raised by this study is why the sexes display differences in autosomal heterochromatin (Koya, 2015).

The chromatin content of males and females are substantially different as XY males have a single X and a large, heterochromatic Y chromosome. It is speculated that this has driven changes in how heterochromatin is established or maintained in one sex. A search for the genetic regulators of the sex difference in autosomal heterochromatin eliminated the Y chromosome and the conventional sex determination pathway, suggesting that the number of X chromosomes determines the sensitivity of autosomal heterochromatin to loss of roX activity. Interestingly, the amount of pericentromeric X heterochromatin, rather than the euchromatic 'numerator' elements, appears to be the critical factor. The recognition that heterochromatin displays differences in the sexes, and that a specific set of proteins are required for normal function of autosomal heterochromatin in males suggests a useful paradigm for the evolution of chromatin in response to genomic content (Koya, 2015).

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).


Bases in 5' UTR - 77

Exons - 5

Bases in 3' UTR - 450


Amino Acids - 1293

Structural Domains

Maleless contains seven short segments that define a superfamily of known and putative RNA and DNA helicases. This group includes proteins from E. coli, yeast, Drosophila, mammals, and DNA and RNA viruses. Several have been shown to have DNA or RNA helicase activity, or nucleic acid-dependent ATPase activity. The members of the superfamily share seven distinct conserved segments. Two segments contain the "A" and "B" sites of an NTP binding domain motif; no activity has been assigned individually to the others. Mle contains similarity to each of the conserved segments, including the six amino acids that are invariant throughout the superfamily. The location of similarity within the Mle protein falls in the central portion and spans approximately 360 amino acids, from amino acids 400 to 760 (Kuroda, 1991).

Within the superfamily, several subfamilies of related proteins can be distinguished by two criteria: particular signature motifs [e.g., the sequence DEAD (Asp, Glu, Ala, Asp) in segment II] and a higher overall sequence identity throughout the entire helicase domain. On the basis of these criteria, Mle is a member of a subfamily of putative helicase proteins, the DEAH family. The first described members of this family are three proteins involved in pre-mRNA processing in yeast (PRP2, PRP16 and PRP22). The similarity between Mle and PRP2, PRP16 and PRP22 is extensive throughout the putative helicase domain. Similarity between Mle and DEAH family members extends approximately 100 amino acids beyond the last conserved segment (VI) of the large superfamily. Despite the high overall similarity of Mle to DEAH family members, Mle carries a difference in the proposed signature sequence, with the sequence DEIH rather than DEAH in segment II of the putative ATP-binding motif. Outside of the putative helicase domain, Mle does not show significant sequence similarity to proteins in current data bases. The C-terminal region contains nine imperfect repeats of a glycine-rich sequence. The posterior embryonic determinant Vasa, a member of the superfamily of putative helicases, encodes six copies of a glycine-rich heptad repeat (Kuroda, 1991 and references).

Structure of the RNA helicase MLE reveals the molecular mechanisms for uridine specificity and RNA-ATP coupling

The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. This study identified a stable MLE core comprising the DExH helicase module and two auxiliary domains: a dsRBD and an OB-like fold. MLEcore is an unusual DExH helicase that can unwind blunt-ended RNA duplexes and has specificity for uridine nucleotides. The 2.1 Å resolution structure of MLEcore bound to a U10 RNA and ADP-AlF4 was determined. The OB-like and dsRBD folds bind the DExH module and contribute to form the entrance of the helicase channel. Four uridine nucleotides engage in base-specific interactions, rationalizing the conservation of uridine-rich sequences in critical roX substrates. roX2 binding is orchestrated by MLE's auxiliary domains, which is prerequisite for MLE localization to the male X chromosome. The structure visualizes a transition-state mimic of the reaction and suggests how eukaryotic DEAH/RHA helicases couple ATP hydrolysis to RNA translocation (Prabu, 2015).

It is concluded that the roX RNAs are a paradigm for how lncRNAs function to assemble ribonucleoprotein complexes. The results provide a structural underpinning to the observation of interdependence between MLE and roX RNA for X chromosome targeting and lay the foundation for a future mechanistic analysis of roX RNA remodeling by MLE. It is envisioned that MLE may load on the helical regions of the roX RNAs in an ATP-independent manner via the exposed surface of dsRBD2 and then thread the 3' end of the RNA into the helicase core, rationalizing why the roX boxes are found near the 3' end of the roX RNAs. The finding that MLE also binds uridine-rich sequences at the 5' end of the roX RNAs might be due to the accumulation of the helicase as it progresses from the 3' to the 5' end of the transcript or due to direct loading by a partial opening of the DExH ring. In addition to the canonical helicase interactions with the sugar-phosphate backbone of the RNA, the OB-like fold of MLE can recognize uridine-rich sequences by base-specific interactions. The pattern of uridine-specific interactions observed in the structure (UxUUU) does not exactly match the sequence motif of the roX boxes (UUUU). Imperfectly matching pockets might tune RNA binding strength and the efficiency of translocation, which are inversely correlated. Along these lines, slowed unwinding as MLE interacts with the uridine-rich sequences could provide a window of opportunity for recruiting MSL proteins near the base of helicase, where the 3' end of the unwound strand of the roX RNA emerges, thus contributing to subsequent steps of complex assembly (Prabu, 2015).

Besides revealing the roles of the auxiliary domains in imparting specific uridine-binding and dsRNA-unwinding properties to MLE, the structure reported in this study provides the snapshot of a eukaryotic DExH helicase in the transient 'on' state, showing how the nucleotide and RNA ligands are coupled. The structure of the Ski2-like protein Hel308 showed that unwinding is achieved by a β-hairpin in the RecA2 domain and suggested that translocation is achieved by a ratchet helix in the HA2 domain. MLE operates with different mechanisms, because it has different auxiliary domains and different features that line the unwinding entrance of the helicase channel. The interpretation from the structural data on MLE is that global and local changes in RecA2 propel the movement in the 3'-5' direction, with a loop jointed on RecA2 acting as the pawl in the ratcheting mechanism. It is proposed that a conceptually similar mechanism for 3'-5' directional translocation might be at play in other eukaryotic RHA/DEAH helicases (Prabu, 2015).

maleless: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 July 97  

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