The Interactive Fly

Zygotically transcribed genes

Splicing factors for processing pre-messenger RNA

The architecture of pre-mRNAs affects mechanisms of splice-site pairing

Transportin-Serine/Arginine rich: an importin-ß family member responsible for transporting SR protein splice factors into the nucleus

>SR proteins control a complex network of RNA-processing events

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

Drosophila Nmnat functions as a switch to enhance neuroprotection under stress

The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development

m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination

Alternative splicing within and between Drosophila species, sexes, tissues, and developmental stages

U7 snRNP is recruited to histone pre-mRNA in a FLASH-dependent manner by two separate regions of the Stem-Loop Binding Protein

Sex-specific transcript diversity in the fly head Is established during pupal stages and adulthood and is largely independent of the mating process and the germline

Splicing factors


The architecture of pre-mRNAs affects mechanisms of splice-site pairing

The exon/intron architecture of genes determines whether components of the spliceosome recognize splice sites across the intron or across the exon. Using in vitro splicing assays, this study demonstrates that splice-site recognition across introns ceases when intron size is between 200 and 250 nucleotides. Beyond this threshold, splice sites are recognized across the exon. Splice-site recognition across the intron is significantly more efficient than splice-site recognition across the exon, resulting in enhanced inclusion of exons with weak splice sites. Thus, intron size can profoundly influence the likelihood that an exon is constitutively or alternatively spliced. An EST-based alternative-splicing database was used to determine whether the exon/intron architecture influences the probability of alternative splicing in the Drosophila and human genomes. Drosophila exons flanked by long introns display an up to 90-fold-higher probability of being alternatively spliced compared with exons flanked by two short introns, demonstrating that the exon/intron architecture in Drosophila is a major determinant in governing the frequency of alternative splicing. Exon skipping is also more likely to occur when exons are flanked by long introns in the human genome. Interestingly, experimental and computational analyses show that the length of the upstream intron is more influential in inducing alternative splicing than is the length of the downstream intron. It is concluded that the size and location of the flanking introns control the mechanism of splice-site recognition and influence the frequency and the type of alternative splicing that a pre-mRNA transcript undergoes (Fox-Walsh, 2005).

Pre-mRNA splicing is an essential process that accounts for many aspects of regulated gene expression. Of the ~25,000 genes encoded by the human genome, >60% are believed to produce transcripts that are alternatively spliced. Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein isoforms from a single pre-mRNA, exponentially enriching the proteomic diversity of higher eukaryotic organisms. Because regulation of this process can determine when and where a particular protein isoform is produced, changes in alternative-splicing patterns modulate many cellular activities (Fox-Walsh, 2005).

The spliceosome assembles onto the pre-mRNA in a coordinated manner by binding to sequences located at the 5' and 3' ends of introns. Spliceosome assembly is initiated by the stable associations of the U1 small nuclear ribonucleoprotein particle with the 5' splice site, branch-point-binding protein/SF1 with the branch point, and U2 snRNP auxiliary factor with the pyrimidine tract. ATP hydrolysis then leads to the stable association of U2 snRNP at the branch-point and functional splice-site pairing (Fox-Walsh, 2005).

Intron size has been correlated with rates of evolution and the regulation of genome size. The exon/intron architecture has also been shown to influence splice-site recognition. For example, increasing the size of mammalian exons results in exon skipping. However, the same enlarged exons are included when the flanking introns are small. Thus, splice-site recognition is more efficient when introns or exons are small. Because, in the human genome, the majority of exons are short and introns are long, it is expected that the vast majority of splice sites in the human genome are recognized across the exon. Lower eukaryotes have a genomic architecture that is typified by small introns and flanking exons with variable length, suggesting that splice-site recognition occurs across the intron. Consistent with this model, expansion of small introns in yeast or Drosophila causes loss of splicing, cryptic splicing, or intron retention. Taken together, these observations suggest that splice sites are recognized across an optimal nucleotide length (Fox-Walsh, 2005).

It is unknown whether splice-site recognition across the intron or across the exon results in similar efficiencies of spliceosomal assembly and/or splice-site pairing. This study demonstrates that splice-site recognition across the intron ceases when the intron reaches a length between 200 and 250 nt. Because splice-site recognition is more efficient across the intron, alternative splicing is less likely for exons flanked by short introns. This influence is supported experimentally and by computational analyses of Drosophila and human alternative-splicing databases. It is concluded that the size and location of the flanking introns control the mechanism of splice-site recognition and influence the frequency and the type of alternative pre-mRNA splicing (Fox-Walsh, 2005).

Previous studies have suggested that genes with small introns tend to be recognized across the intron, and genes with large introns are recognized across the exon. To determine the distance at which recognition of splice sites switches from cross-intron interactions to cross-exon interactions occurs, advantage was taken of an in vitro kinetic splicing assay that was originally used to demonstrate that exonic splicing enhancers (ESEs), discrete sequences within exons that promote both constitutive and regulated splicing, activate both splice sites of an exon simultaneously (Lam, 2002). A number of pre-mRNAs were designed with intron lengths ranging from 120 to 425 nt. Within each set, the pre-mRNAs differ only in the presence or absence of a well characterized 13-nt ESE derived from the Drosophila doublesex and Drosophila fruitless pre-mRNAs. Each pre-mRNA harbors the same weak 5' and 3' splice sites that require the activities of ESEs for recognition in their natural context (Tian, 1992; Lam, 2003). Because splicing factors present in HeLa cell nuclear extracts activate the ESEs used (Lam, 2003), the presence of functional or mutant enhancer elements within each test substrate determine its splicing efficiency. If the splice sites are recognized across the exon, it is expected that the activation of the splice sites on each exon constitutes a different step during spliceosomal assembly, because the ESE located on each exon will only aid in the recognition of its weak splice site. Thus, the activities of the separate ESEs are expected to display synergistic kinetics, because the activation of each ESE accelerates an independent step during spliceosomal assembly. However, if the splice sites are recognized across the intron, the ESE located on each exon will aid in the recognition of both weak splice sites, because the recruited spliceosomal components define the entire intron within one step. In this scenario, the activities of the separate ESEs are expected to display additive kinetics, because the activation of each ESE accelerates the same rate-limiting step during spliceosomal assembly (Fox-Walsh, 2005).

In vitro splicing assays were performed with each of the four pre-mRNA sets over a 3-h time course to determine the apparent rates of splicing. Pre-mRNAs with an intron size of 120 nt display additive kinetics. Using Drosophila nuclear extract (Kc), it was possible to demonstrate additive kinetics for substrates containing the 120-nt intron; however, it was not possible to detect sufficient splicing for the substrates containing longer introns. These results are consistent with in vitro studies demonstrating that splicing of pre-mRNAs with long introns is supported in HeLa nuclear extract but not in Kc extract. The kinetics of pre-mRNAs containing an intron 200 nt or less in length are additive. This behavior indicates that the spliceosomal components required for the recognition of both splice sites are recruited to the intron simultaneously. However, constructs with introns >200 nt demonstrate synergistic kinetics. It is concluded that the change from splice-site recognition across the intron to splice-site recognition across the exon occurs when the intronic length is between 200 and 250 nt (Fox-Walsh, 2005).

The kinetic analysis demonstrates that the upstream 5' splice site and the downstream 3' splice site are recognized simultaneously across introns <200 nt. Significantly, in the absence of ESEs, splice-site recognition across the intron is a much more efficient process than splice-site recognition across the exon. Thus, splice-site recognition across the intron may be able to rescue the inclusion of internal exons harboring weak splice sites. To test this hypothesis, a series of pre-mRNA substrates containing three exons was designed for in vitro splicing analysis in which the internal exon contains splice sites that are insufficiently recognized in the absence of ESEs. The four substrates generated differed only in their ability to be recognized across each intron by changing the length of the intron from <200 to >250 nt, thus permitting or discouraging splice-site recognition across the intron. As expected, the internal exon is predominantly excluded when flanked by two long introns. However, significant inclusion of the internal exon is observed if one of the flanking introns is short enough to support splice-site recognition across the intron. In fact, two short introns increase exon inclusion ~30 times greater than two long introns (Fox-Walsh, 2005).

To estimate the fractions of splice sites that may be recognized through cross-intron interactions, the flanking-intron lengths were recored for every internal exon within the human and Drosophila genomes. Genome information was obtained from the Alternative Splicing Database (ASD), which contains information about the exon/intron structure and EST-verified alternative-splicing events of several thousand genes. Within the human genome, many exons are flanked by at least one short intron, creating two separate populations, separated roughly by the intron length that is proposed to represent the transition of splice-site recognition from across the intron to across the exon. As expected from previous intron-length analyses, a very different distribution is seen in the Drosophila genome, where ~85% of exons are flanked by at least one short intron. An overlay of the Drosophila and human genomes demonstrates that the minimum intron length in the human genome is at the same location that demarcates the maximum intron length of the major Drosophila exon population. This difference in genome constraint may reflect specific compositional variations between the Drosophila and human spliceosomes (Fox-Walsh, 2005).

Because splice-site recognition across the intron rescues exon inclusion, how intron length influences alternative splicing within the Drosophila and human genomes was investigated. To do so, the flanking-intron information of each exon was correlated with exon-skipping and alternative-splice-site-activation events reported in the ASD to compute the probability that an exon is involved in alternative splicing, without taking into consideration the contributions made by splice-site signal strength and splicing enhancers or silencers. Thus, the correlation simply tests whether the influence of the exon/intron architecture on alternative splicing is significant enough to be detectable amid all other splicing determinants. Computational analysis of the Drosophila genome supports a significant role for intron length in defining the likelihood of alternative splicing. A striking influence of the exon/intron architecture is observed for simple exon-skipping events. Exons flanked by very long introns are up to 90-fold more likely to be skipped than exons that are flanked by two short introns. Significantly, the most drastic increase in the probability of alternative splicing (>10-fold) was observed when the length of flanking introns increased from 225 to 525 nt. In agreement with the experimental results, a greater probability that an exon is alternatively spliced was observed when the upstream intron is long. This polarity could be the consequence of coupling pre-mRNA splicing to transcription by RNA polymerase II. Even in the category of alternative 5' or 3' splice-site activation, alternative splicing is up to 10-fold more likely for exons that are flanked by long introns. It is concluded that, in Drosophila, exon skipping is a rare event for exons flanked by short introns and that the length of the upstream intron is of greater importance than the length of the downstream intron in determining whether an exon will be involved in exon skipping (Fox-Walsh, 2005).

Within the human genome, a similar correlation between the exon/intron architecture and the probability of exon skipping is observed; however, the ~5-fold maximal variance calculated is significantly lower than that observed for Drosophila. As for Drosophila, the length of the upstream intron is more important in determining the frequency of alternative splicing. In the case of alternative 5' or 3' splice-site usage, the opposite distribution of alternative splicing is seen in the human genome. The activation of alternative splice sites is less likely if the flanking introns are long. It is concluded that exon/intron architecture influences the frequency and type of alternative splicing that an exon may undergo in the Drosophila and human genomes (Fox-Walsh, 2005).

These experiments support the existence of two different mechanisms for splice-site recognition, splice-site recognition across the intron, and splice-site recognition across the exon. Splice-site recognition across the intron ceases when the intron size reaches the threshold length of >200 nt. Importantly, splice-site recognition across the intron is more efficient and increases the inclusion of exons with weak splice sites. These results demonstrate that the distance between splice sites affects efficient spliceosomal assembly. Presumably, the pairing of cross-exon-defined splice sites requires the interaction between two sets of pre-spliceosomes across an intron of variable length. In contrast, splice-site recognition across the intron already identifies the splice sites that will be paired. It is also possible that the kinetics of splice-site pairing are slowed because longer introns associate with an increased number of hnRNP proteins. HnRNP proteins coat nascent pre-mRNAs and are thought to interfere with the splicing reaction. Therefore, larger introns may reduce splicing by decreasing the relative concentration of splicing components through competition with hnRNPs (Fox-Walsh, 2005).

Additive kinetics of splice-site activation demonstrate that splice-site recognition across the intron is achieved through the recruitment of a multicomponent complex that contains components of the splicing machinery required for 5' and 3' splice-site definition. Interestingly, the activation of a single ESE results in a significant increase in splicing activity, suggesting that ESEs influence splice-site activation of adjacent exons. As anticipated from ESE distance/activity correlations, this effect depends on intron length. Given the unique combination of splice sites and cis-acting elements, it is possible that the precise transition from splice-site recognition across the intron to splice-site recognition across the exon may vary for different substrates. The presence of strong splice sites and enhancers or silencers could modulate the cross-intron recognition by increasing or decreasing the strength of interaction between spliceosomal components and the pre-mRNA (Fox-Walsh, 2005).

The observation that increasing exon length decreases exon inclusion suggests that similar distance limitations exist for splice-site recognition across the exon. Approximately 80% of human exons are <200 bp in length, the average being 170 bp. Importantly, exon length is tightly distributed when compared with intron length. These results demonstrate that maintaining exon size in the human genome is more important to the architecture and evolution of a gene than is maintaining intron size. In contrast to the human genome, exon size varies much more than intron size in yeast. The maximum intron length of 182 nt lies well within the size limitations of splice-site recognition across the intron. Taken together, these considerations support the notion that the majority of splice sites in higher eukaryotes are recognized across the exon, whereas lower eukaryotes employ splice-site recognition across the intron (Fox-Walsh, 2005).

It is well established that several types of exon and intron elements influence splice-site choice. The most prominent include the exon/intron junction signals and splicing enhancers and silencers. The results show that the exon/intron architecture is an additional parameter that affects the efficiency of splice-site recognition and alternative pre-mRNA splicing. When compared in otherwise isogenic test substrates, splice-site recognition across the intron rescues the inclusion of a weak internal exon by >10-fold. Even though the computational analysis ignores the contributions made by variable splice sites, enhancers, and silencers, a striking increase in the probability of alternative splicing is observed for Drosophila exons, whose splice sites are recognized across the exon. Thus, the exon/intron architecture in Drosophila is a major determinant in governing the probability of alternative splicing. Within the human genome, a qualitatively similar trend was observed for exon-skipping events but with a reduced magnitude. One major difference between the Drosophila and human gene architecture is intron length. Human genes are dominated by long introns (87% of introns are >250 nt), whereas short introns are much more common in Drosophila (66% are <250 nt). One possible explanation for the small intron size in Drosophila could be the pressure to maintain a constrained genome size in these fast-replicating organisms (Fox-Walsh, 2005).

Alternative splicing is extensive in both species, supporting the argument that both species benefit from expanded proteomes generated from alternative splicing. However, genome analysis suggests that there are significant differences in the weight of the mechanisms by which alternative splicing can be induced. In Drosophila, intron length is a major determinant in promoting alternative splicing patterns. In the human, additional mechanisms of controlling alternative splicing may have gained more influence on intron expansion to maintain balanced levels of alternative splicing (Fox-Walsh, 2005).

SR proteins control a complex network of RNA-processing events

SR proteins are a well-conserved class of RNA-binding proteins that are essential for regulation of splice-site selection, and have also been implicated as key regulators during other stages of RNA metabolism. For many SR proteins, the complexity of the RNA targets and specificity of RNA-binding location are poorly understood. It is also unclear if general rules governing SR protein alternative pre-mRNA splicing (AS) regulation uncovered for individual SR proteins on few model genes, apply to the activity of all SR proteins on endogenous targets. Using RNA-seq, this study characterized the global AS regulation of the eight Drosophila SR protein family members. A majority of AS events are regulated by multiple SR proteins, and that all SR proteins can promote exon inclusion, but also exon skipping. Most coregulated targets exhibit cooperative regulation, but some AS events are antagonistically regulated. Additionally, it was found that SR protein levels can affect alternative promoter choices and polyadenylation site selection, as well as overall transcript levels. Cross-linking and immunoprecipitation coupled with high-throughput sequencing (iCLIP-seq), reveals that SR proteins bind a distinct and functionally diverse class of RNAs, which includes several classes of noncoding RNAs, uncovering possible novel functions of the SR protein family. Finally, it was found that SR proteins exhibit positional RNA binding around regulated AS events. Therefore, regulation of AS by the SR proteins is the result of combinatorial regulation by multiple SR protein family members on most endogenous targets, and SR proteins have a broader role in integrating multiple layers of gene expression regulation (Bradley, 2014).

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. Twenty distinct RNA binding proteins (RBPs) were immunopurified from cultured Drosophila melanogaster cells under native conditions, and both the RNA and protein compositions of these RNP complexes were determined. "High occupancy target" (HOT) RNAs were identified that interact with the majority of the RBPs surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery as well as RNA binding and translation initiation proteins. The RNP complexes contain proteins and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultra-complex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). This data is consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. From the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs this study identified extensive cross-regulatory and hierarchical interactions in post-transcriptional control (Stoiber, 2015).

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. This study, using extensive mutagenesis of expression plasmids and RNAi screening, revealed that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, base-pairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (approximately 400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3' end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, it was found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes (Kramer, 2015).

It was long assumed that eukaryotic pre-mRNAs are always canonically spliced to generate a linear mRNA that is subsequently translated to produce a protein. However, it is now becoming increasingly clear that many genes can be noncanonically spliced to produce circular RNAs with covalently linked ends. These transcripts are almost exclusively derived from exons, accumulate in the cytoplasm, and are thought to be products of alternative splicing events known as 'backsplicing.' In contrast to canonical splicing, which joins the exons in a linear order (joining exon 1 to exon 2 to exon 3, etc.), backsplicing joins a splice donor to an upstream splice acceptor (e.g., joining the 3' end of exon 2 to the 5' end of exon 2). A handful of RNAs generated in this manner were identified in the 1990s, and recent deep sequencing studies have expanded this observation to thousands of circular RNAs expressed across eukaryotes, including humans, Caenorhabditis elegans, Drosophila (Salzman. 2013; Ashwal-Fluss, 2014; Westholm, 2014), Schizosaccharomyces pombe, and plants. Perhaps surprisingly, for some genes, the abundance of the circular RNA exceeds that of the associated linear mRNA by a factor of 10, suggesting that the major function of some protein-coding genes may be to generate circular RNAs (Kramer, 2015).

Most exons in eukaryotic genomes have splicing signals at both ends and theoretically can circularize. However, only certain exons are observed in circular RNAs, and these backsplicing events often occur in a tissue-specific manner. This suggests that circular RNA biogenesis is tightly regulated. As splicing generally occurs cotranscriptionally, most introns, along with their upstream splice acceptors (which are needed for backsplicing), are rapidly removed. Therefore, for circular RNAs to be produced, canonical splicing likely must occur more slowly around these exons, and/or exon skipping events may be coupled to circular RNA biogenesis. In the latter, the circular RNA is derived from an exon-containing lariat, allowing a pre-mRNA to yield both a linear mRNA and a circular RNA comprised of the skipped exons (Kramer, 2015).

There is little known about the splicing factors that regulate these events. In some cases, the Muscleblind (Mbl) and Quaking proteins appear to facilitate backsplicing by bridging between two introns and causing the splice sites from the intervening exons to be brought into close proximity (Ashwal-Fluss, 2014; Conn, 2015). For example, circular RNA production from the Drosophila mbl gene is triggered when the Mbl splicing factor binds to its own introns (Ashwal-Fluss, 2014). However, in humans, mice, and C. elegans, the predominant determinants of whether a pre-mRNA is subjected to backsplicing are intronic repetitive elements, such as sequences derived from transposons. Almost 90% of human circular RNAs have complementary Alu elements in their flanking introns, and, analogous to the protein-bridging mechanism, base-pairing between complementary sequences allows the intervening splice sites to be brought close together. Interestingly, repeats <40 nucleotides (nt) can drive circular RNA production in human cells, but it is clear that more than simple thermodynamics regulates circularization. For example, base-pairing interactions can be disrupted by ADAR (adenosine deaminase acting on RNA), which converts adenosines in double-stranded regions to inosines. In addition, most mammalian pre-mRNAs contain multiple intronic repeats, allowing distinct circular (or linear) RNAs to be produced depending on which repeats base-pair to one another. Therefore, other factors likely help dictate splicing outcomes by regulating these exon circularization events (Kramer, 2015).

Despite key regulatory roles for intronic repeats in multiple eukaryotes, it has been suggested that circular RNA biogenesis in Drosophila melanogaster is not driven by base-pairing interactions (Westholm, 2014). Instead, a positive correlation between the length of the flanking introns and circular RNA abundance was identified in Drosophila (Westholm, 2014). However, the effect of modulating intron lengths on backsplicing has not yet been directly addressed. It is also completely unknown how Drosophila circular RNAs besides Mbl, of which there are >2500 annotated circular RNAs derived from other genomic loci, are generated or post-transcriptionally regulated. Therefore, it is still unclear whether circular RNA biogenesis strategies are conserved across eukaryotes or whether species such as Drosophila use unique mechanisms to determine which exons should be backspliced (Kramer, 2015).

Once produced, circular RNAs are stable transcripts that are naturally resistant to degradation by exonucleases. Two circular RNAs (ciRS7/CDR1as and Sry) modulate the activity of specific microRNAs (Hansen, 2013; Memczak, 2013), but most other RNA circles (in species other than Drosophila) contain few microRNA-binding sites and likely function differently. For example, it has been proposed that many circular RNAs may regulate neuronal functions, and artificial circular RNAs containing an IRES (internal ribosome entry site) can be translated. However, the lack of efficient methods for modulating circular RNA levels or ectopically expressing circular RNAs has limited the ability to define functions for these transcripts (Kramer, 2015).

This study focused on the Drosophila laccase2 gene, as it produces an abundant circular RNA in vitro and in vivo. Evidence is provided that intronic repeats collaborate with trans-acting splicing factors to regulate circularization in flies. Mechanistically, it was found that miniature introns (<150 nt) containing the splice sites and inverted repeats were sufficient to support Laccase2 circular RNA production. The intronic repeats must base-pair to one another for circularization to occur, as has been observed in other eukaryotes. Furthermore, it was found that the strength of these base-pairing interactions dictates whether backsplicing occurs co- or post-transcriptionally: Long flanking repeats appear to allow cotranscriptional processing. Screening a panel of genes, this study found that multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine‚Äďarginine) family proteins regulate Laccase2 circular RNA levels in a combinatorial manner. Comparisons with the mbl locus suggest that the circularization mechanisms are distinct, as the Laccase2 circular RNA was not regulated by the Mbl or Laccase2 gene products. Additional circular RNAs were identified that are regulated by unique combinations of hnRNP and SR proteins, suggesting that combinatorial control may be a common regulatory strategy that modulates circular RNA levels. This led to a test of whether this biogenesis mechanism is active in human cells, and it was found that the laccase2 introns can indeed robustly generate circular RNAs. It is thus now possible to efficiently generate "designer" circular RNAs in cells with minimal linear RNA production. In total, the results reveal new insights into how trans-acting factors and intronic repeats collaborate to regulate circular RNA biogenesis across eukaryotes as well as provide new tools for exploring the functions of circular RNAs (Kramer, 2015).

This study demonstrates that intronic repeats and trans-acting hnRNPs and SR proteins combinatorially regulate circularization of the Drosophila laccase2 gene. Base-pairing between transposable elements in the flanking introns facilitates circularization, and the strength of these interactions likely dictates whether backsplicing occurs co- or post-transcriptionally. This mechanism is distinct from the one that regulates Drosophila Mbl circular RNA production (Ashwal-Fluss, 2014) but is similar to that used to generate many circles in humans, mice, and C. elegans. This suggests that base-pairing between intronic repeats may be a major mechanism promoting exon circularization across eukaryotes. Moreover, this study found that the laccase2 exon is dispensable, allowing the laccase2 introns to be used to efficiently generate 'designer' circular RNAs from plasmids in diverse organisms. Altogether, the results suggest that circular RNA biogenesis strategies are conserved across eukaryotes and provide new tools for exploring the functions of circular RNAs (Kramer, 2015).

The current results on the laccase2 locus indicate that base-pairing between complementary intronic sequences efficiently promotes RNA circularization in flies. As the DNAREP1_DM repeats closely flank exon 2 of the laccase2 gene, a model is proposed in which the repeats base-pair to one another, bringing the intervening splice sites into close proximity and facilitating catalysis. The Laccase2 circular RNA then accumulates as one of the most abundant circular RNAs in Drosophila (fifth most abundant across >100 Drosophila RNA sequencing libraries). At the endogenous laccase2 gene locus, the long introns that flank this exon likely slow the overall speed of cotranscriptional splicing, thereby allowing the backsplicing reaction to effectively compete with canonical splicing. Indeed, it was found that the strength of the base-pairing interactions between the flanking introns dictates how quickly backsplicing can occur. When very stable interactions are present, it is possible that exon definition is improved, allowing the rapid and cotranscriptional generation of a circular RNA. Nevertheless, further studies are still required to clarify the exact role that long flanking introns may play in regulating circularization (Kramer, 2015).

Upon examining the introns that flank other abundant Drosophila circular RNAs, this study identified other examples in which complementary regions >60 nt in length flank circularizing exons, including CaMKI, CG11155, CG2052, Parp, and PlexA (which are among the top 25 most abundant Drosophila circular RNAs). Interestingly, the Semaphorin-2b (CG33960) circular RNA (39th most abundant circular RNA) is flanked by introns containing short (CA)n simple repeats that are complementary to each other over a <30-nt region. Upon cloning a 980-nt region of the Semaphorin-2b pre-mRNA downstream from the pMT, circular RNA production from the plasmid was observed in DL1 cells. Removal of either of the (CA)n simple repeats, however, strongly reduced circularization. This suggests that diverse inverted repeat sequences, including short simple repeats, may play a general role in facilitating circularization in Drosophila (Kramer, 2015).

Complementary repeats, however, are not observed at all Drosophila loci that generate circular RNAs. Furthermore, many exons that do not circularize are flanked by complementary repeats, so there must be other mechanisms that regulate circularization. This has been most notably demonstrated at the Drosophila mbl locus, which requires the Mbl splicing factor for its circularization. When Mbl protein is in excess, an intricate feedback mechanism is induced: The Mbl protein decreases the production of its own mRNA by binding its pre-mRNA. This blocks canonical splicing and promotes the biogenesis of the Mbl circular RNA, which further functions as a sponge that binds and sequesters the excess Mbl protein. However, this Mbl-driven mechanism appears to be specific for the mbl locus, as this study found that knockdown of the Mbl linear mRNA had no effect on Laccase2, PlexA, or a panel of other circular RNAs. Knockdown of the Laccase2 linear mRNA likewise did not affect Laccase2 circular RNA levels, indicating that the laccase2 locus is not subjected to a similar direct cis-acting feedback mechanism. Instead, it was found that other splicing factors, including hnRNPs and SR proteins, regulate Laccase2 RNA levels (Kramer, 2015).

At the laccase2 locus, it is proposed that hnRNPs (e.g., Hrb27C and Hrb87F) and SR proteins (e.g., SF2 [SRSF1], SRp54 [SRSF11], and B52 [SRSF6]) add an additional layer of control on top of the DNAREP1_DM intronic repeats. Base-pairing between the intronic repeats promotes circularization, but protein binding likely helps ensure that the appropriate ratio of linear to circular Laccase2 RNA is produced. Depletion of any one of these splicing factors alters Laccase2 circle levels, and additive effects were observed when multiple factors were depleted. This suggests combinatorial control, with each protein playing a nonredundant role. Furthermore, Laccase2 circular RNA production does not appear to be linked to exon skipping, and thus these proteins may specifically modulate spliceosome assembly, the speed of splicing, and/or the stability of the mature circular RNA. Notably, it does not seem that Hrb27F, SF2, SRp54, or B52 affects Laccase2 circular RNA stability, as depletion of these factors did not cause the expression of a plasmid-derived Laccase2 circular RNA to increase. It is thus instead proposed that these hnRNPs and SR proteins regulate Laccase2 circular RNA biogenesis (e.g., by binding to the flanking introns or exons), but further studies are required to understand exactly how the intronic repeats and trans-acting factors collaboratively dictate the splicing outcome. Nevertheless, the same SR proteins that regulate the laccase2 locus also regulate the PlexA circular RNA but not the Mbl circular RNA. Since the laccase2 and PlexA exons are both flanked by inverted repeats, it is hypothesized that intronic repeats may generally provide the opportunity for circularization to occur. This is then further regulated by trans-acting factors that combinatorially fine-tune the amount of each circular RNA that the cell ultimately produces (Kramer, 2015).

Catalogs of circular RNAs expressed in various species and cell types have been reported, but the functions for nearly all of these transcripts, including Laccase2, are currently unknown. This is due in part to the current lack of methods for efficiently generating circular RNAs in cells. For example, the circular RNA expression plasmids that have been described all generally produce circular transcripts at a low efficiency (often 20% or less). These plasmids instead generate abundant amounts of linear RNA, which limits their utility for defining circular RNA functions. Using the Drosophila laccase2 and human ZKSCAN1 introns, this study largely overcame this hurdle and generated circular RNAs (ranging in size from 300 to 1500 nt) at a high efficiency in human and fly cells. These transcripts accumulate in the cytoplasm, are resistant to RNase R treatment, and are likely translated when an IRES is present. Furthermore, easy-to-use restriction sites are present in the plasmids, allowing any desired sequence to be queried. Beyond allowing ectopic expression of circular RNAs, these plasmids can be designed to sponge microRNAs or proteins as well as identify novel IRES sequences (Kramer, 2015).

In summary, the current findings provide key insights into how trans-acting factors and intronic repeats regulate circular RNA biogenesis as well as provide new tools for exploring the functions of circular RNAs across eukaryotes. From humans to flies, repetitive elements in introns can act to facilitate backsplicing, but it is still largely unclear why circular RNAs accumulate only in certain tissues. It is hypothesized that base-pairing between repeats is only one part of the "splicing code", and it is ultimately a combination of cis-acting elements and trans-acting splicing factors, including hnRNPs and SR proteins, that dictates whether canonical splicing or backsplicing occurs. Nevertheless, this study has defined a minimal set of elements that is sufficient for promoting efficient exon circularization, which should facilitate the prediction of circular RNAs as well as enable the functions of many circular RNAs to be revealed. Considering that a surprisingly large number of protein-coding genes generates circular RNAs, these previously overlooked transcripts likely represent key ways that gene functions are expanded and modulated (Kramer, 2015).

Drosophila Nmnat functions as a switch to enhance neuroprotection under stress

Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a conserved enzyme in the NAD synthetic pathway. It has also been identified as an effective and versatile neuroprotective factor. However, it remains unclear how healthy neurons regulate the dual functions of NMNAT and achieve self-protection under stress. This study shows that Drosophila Nmnat (DmNmnat) is alternatively spliced into two mRNA variants, RA and RB, which translate to protein isoforms with divergent neuroprotective capacities against spinocerebellar ataxia 1-induced neurodegeneration. Isoform PA/PC translated from RA is nuclear-localized with minimal neuroprotective ability, and isoform PB/PD translated from RB is cytoplasmic and has robust neuroprotective capacity. Under stress, RB is preferably spliced in neurons to produce the neuroprotective PB/PD isoforms. These results indicate that alternative splicing functions as a switch that regulates the expression of functionally distinct DmNmnat variants. Neurons respond to stress by driving the splicing switch to produce the neuroprotective variant and therefore achieve self-protection (Ruan, 2015).

The exon junction complex regulates the splicing of cell polarity gene dlg1 to control Wingless signaling in development

Wingless (Wg)/Wnt signaling is conserved in all metazoan animals and plays critical roles in development. The Wg/Wnt morphogen reception is essential for signal activation, whose activity is mediated through the receptor complex and a scaffold protein Dishevelled (Dsh). This study reports that the exon junction complex (EJC) activity is indispensable for Wg signaling by maintaining an appropriate level of Dsh protein for Wg ligand reception in Drosophila. Transcriptome analyses in Drosophila wing imaginal discs indicate that the EJC controls the splicing of the cell polarity gene discs large 1 (dlg1), whose coding protein directly interacts with Dsh. Genetic and biochemical experiments demonstrate that Dlg1 protein acts independently from its role in cell polarity to protect Dsh protein from lysosomal degradation. More importantly, human orthologous Dlg protein is sufficient to promote Dvl protein stabilization and Wnt signaling activity, thus revealing a conserved regulatory mechanism of Wg/Wnt signaling by Dlg and EJC (Liu, 2016).

The EJC is known to act in several aspects of posttranscriptional regulation, including mRNA localization, translation and degradation. After transcription, the pre-mRNA associated subunit eIF4AIII is loaded to nascent transcripts about 20-24 bases upstream of each exon junction, resulting in binding of Mago nashi (Mago)/Magoh and Tsunagi (Tsu)/Y14 proteins to form the pre-EJC core complex. The pre-EJC then recruits other proteins including Barentsz (Btz) to facilitate its diverse function). In vertebrates, the EJC is known to ensure translation efficiency as well as to activate nonsense-mediated mRNA decay (NMD). In Drosophila, however, the EJC does not contribute to NMD. It is instead required for the oskar mRNA localization to the posterior pole of the oocyte. Very recently, the pre-EJC has been shown to play an important role in alternative splicing of mRNA in Drosophila. Reduced EJC expression results in two forms of aberrant splicing. One is the exon skipping, which occurs in MAPK and transcripts that contain long introns or are located at heterochromatin (Ashton-Beaucage, 2010; Roignant, 2010). The other is the intron retention on piwi transcripts. Furthermore, transcriptome analyses in cultured cells indicates the role of EJC in alternative splicing is also conserved in vertebrates (Liu, 2016).

This study has utilized the developing Drosophila wing as an in vivo model system to investigate new mode of regulation of Wg signaling. The pre-EJC was found to positively regulate Wg signaling through its effect on facilitating Wg morphogen reception. Further studies reveal that the basolateral cell polarity gene discs large 1 (dlg1) is an in vivo target of the pre-EJC in Wg signaling. Dlg1 acts independently from its role on cell polarity to stabilize Dsh protein, thus allowing Wg protein internalization required for signaling activation. Furthermore, it was demonstrated that human Dlg2 exhibits a similar protective role on Dvl proteins to enhance Wnt signaling in cultured human cells. Taken together, this study unveils a conserved regulatory mechanism of the EJC and Dlg in Wg/Wnt signaling (Liu, 2016).

In summary, this study uncovers a specific role of the RNA binding protein complex EJC in the Drosophila wing morphogenesis. Genetic and biochemical analyses demonstrate that the pre-EJC is necessary for Wg morphogen reception to activate the signal transduction. The identification of the cell polarity determinant dlg1 as one of the pre-EJC targets provides mechanistic basis for the pre-EJC regulation of the Wg signaling. Dlg1 controls the stability of the scaffold protein Dsh, which is the hub of the Wg signaling cascade. Importantly, this mode of regulation of Dvl by Dlg is conserved from flies to vertebrates (Liu, 2016).

The EJC as well as other RNA binding protein complexes are thought to function in a pleiotropic manner. However, the current data together with several recent studies argue that RNA regulatory machineries can act specifically on developmental signaling for pattern formation and organogenesis. It has been increasingly recognized that the production, transport or the location of mRNA are subject to precise regulation in Wg/Wnt signaling. For example, apical localization of wg RNA is essential for signal activation in epithelial cells. The specific role of RNA machineries on cell signaling is not limited to Wg/Wnt signaling. It has been reported that RNA-binding protein Quaking specifically binds to the 3'UTR of transcription factor gli2a mRNA to modulate Hedgehog signaling in zebrafish muscle development. RNA binding protein RBM5/6 and 10 could differentially control alternative splicing of a negative Notch regulator gene NUMB, thus antagonistically regulating the Notch signaling activity for cancer cell proliferation. Therefore, generally believed pleotropic RNA regulatory machineries emerge as important regulatory means to specifically control cell signaling and related developmental processes (Liu, 2016).

The most studied function of the EJC in development is to localize oskar mRNA to the posterior pole of the oocyte for oocyte polarity establishment and germ cell formation in Drosophila. Further study suggests that the proper oskar RNA localization relies on its mRNA splicing. In light of the current study of the EJC activity on dlg1 mRNA as well as the roles of EJC on mapk and piwi splicing, it is suspected that EJC might regulate oskar mRNA splicing to mediate its mRNA localization. RNA-seq analyses identified several hundreds of candidate mRNAs whose expression may be directly or indirectly subjected to EJC regulation. Apart from defects in Wg and MAPK signaling, however, altered wing patterning associated with other developmental signaling systems was not seen in EJC defective flies, arguing that EJC may primarily regulate Wg and MAPK signaling in patterning the developing wing (Liu, 2016).

Wg/Wnt signaling plays a fundamental role in development and tissue homeostasis in both flies and vertebrates. Its activation and maintenance rely on appropriate activity of the ternary receptor complex including Fz family proteins. In Drosophila, polarized localization of Fz and Fz2 proteins is essential for activation of non-canonical and canonical Wg signaling, respectively. Dsh, which acts as a hub mediating both canonical and non-canonical Wg signaling, however, is found at both the apical cell boundary and in the basal side of the cytoplasm. Thus, the polarized activity of Dsh must require distinct regulatory mechanisms at different sub-membrane compartments. The results provide the in vivo evidence suggesting that the basolateral polarity determinant Dlg1 may play a dominant role to control the Dsh abundance/activity in canonical Wg signaling (Liu, 2016).

Altered Dvl production or activity has been linked with several forms of cancer. The stability of Dvl proteins can be controlled through regulated protein degradation both in vertebrates and in Drosophila as reported in this study. In HEK293T cells, Dapper1 induces whilst Myc-interacting zinc-finger protein 1 (MIZ1) antagonizes autophagic degradation of Dvl2 in lysosome. It is also reported that a tumor suppressor CYLD deubiquitinase inhibits the ubiquitination of Dvl. As Dlg1 prevents Dsh from degradation in Drosophila, it is important to investigate if Dlg1 participates in a posttranslational regulatory network of Dvl to integrate endocytosis and autophagy. Furthermore, upregulation of dvl2 and dlg2 expression has been found in various forms of cancer as shown in the COSMIC database. The study of the interaction between Dlg1 and Dsh may aid the development of novel approaches to prevent or treat relevant diseases. (Liu, 2016).

Dlg1 acts together with L(2)gl to form a basolateral complex in polarized epithelium. Dsh is known to interact with L(2)gl. On one hand, Dsh activity is required for correct localization of L(2)gl to establish apical-basal polarity in Xenopus ectoderm and Drosophila follicular epithelium. On the other hand, L(2)gl can regulate Dsh to maintain planar organization of the embryonic epidermis in Drosophila. Despite the complex interaction between L(2)gl and Dsh, not much is known about mutual regulation between Dlg1 and Dsh. A recent report suggests that Dsh binds to Dlg1 to activate Guk Holder-dependent spindle positioning in Drosophila. The current results unveil another side of the relationship in which Dlg1 controls the turnover of Dsh to ensure developmental signal propagation. Apart from its apical localization at the cell boundary, Dsh is also found in the basal side of the cytoplasm. It is likely that the interactions among Dsh, Dlg1 and L(2)gl may be dependent on their localization, and Dsh may serve as a bridge to connect cell signaling and polarity (Liu, 2016).

Developmental signaling and cell polarity intertwine to control a diverse array of cellular events. It is well known that Wg/Wnt signaling controls cell polarity in distinct manner. Non-canonical signaling acts through cytoskeletal regulators to establish planar cell polarity. Canonical signaling may also directly affect apical-basal cell polarity. On the other hand, disruption of epithelial cell polarity has a profound impact on protein endocytosis and recycling, both of which are essential regulatory steps for signal activation and maintenance. The current results add another layer of complexity by which polarity determinants could contribute to cell signaling independent of their conventional roles in polarity establishment and maintenance. Interestingly, this mode of regulation is also observed for other signaling processes. Loss of Dlg5 impairs Sonic hedgehog-induced Gli2 accumulation at the ciliary tip in mouse fibroblast cells that may not rely on cell polarity regulation. Similarly, L(2)gl regulates Notch signaling via endocytosis, independent of its role in cell polarity. It is believed that other cell polarity determinants may similarly participate in polarity-independent processes, however, the exact mechanism of how they cooperate to modulate developmental signaling awaits further investigation (Liu, 2016).

m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination

N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. Drosophila mRNA m6A methylosome consists of Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. This study shows that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. The m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression (Haussmann, 2016).

Alternative splicing within and between Drosophila species, sexes, tissues, and developmental stages

Alternative pre-mRNA splicing ("AS") greatly expands proteome diversity. The transcriptomes from several tissues and developmental stages were studied in males and females from four species across the Drosophila genus. 20-37% of multi-exon genes were found to be alternatively spliced. While males generally express a larger number of genes, AS is more prevalent in females, suggesting that the sexes adopt different expression strategies for their specialized function. The proportion of expressed genes that are alternatively spliced is highest in the very early embryo, before the onset of zygotic transcription. This indicates that females deposit a diversity of isoforms into the egg, consistent with abundant AS found in ovary. Cluster analysis by gene expression levels shows mostly stage-specific clustering in embryonic samples, and tissue-specific clustering in adult tissues. Clustering embryonic stages and adult tissues based on AS profiles results in stronger species-specific clustering, suggesting that diversification of splicing contributes to lineage-specific evolution in Drosophila. Most sex-biased AS found in flies is due to AS in gonads, with little sex-specific splicing in somatic tissues (Gibilisco, 2016).

U7 snRNP is recruited to histone pre-mRNA in a FLASH-dependent manner by two separate regions of the Stem-Loop Binding Protein

Cleavage of histone pre-mRNAs at the 3' end requires Stem-Loop Binding Protein (SLBP) and U7 snRNP that consists of U7 snRNA and a unique Sm ring containing two U7-specific proteins: Lsm10 and Lsm11. Lsm11 interacts with FLASH and together they bring a subset of polyadenylation factors to U7 snRNP, including the CPSF73 endonuclease that cleaves histone pre-mRNA. SLBP binds to a conserved stem-loop structure upstream of the cleavage site and acts by promoting an interaction between the U7 snRNP and a sequence element located downstream of the cleavage site. This study shows that both human and Drosophila SLBPs stabilize U7 snRNP on histone pre-mRNA via two regions that are not directly involved in recognizing the stem-loop structure: helix B of the RNA Binding Domain and the C-terminal region that follows the RNA Binding Domain. Stabilization of U7 snRNP binding to histone pre-mRNA by SLBP requires FLASH but not the polyadenylation factors. Thus, FLASH plays two roles in 3' end processing of histone pre-mRNAs: it interacts with Lsm11 to form a docking platform for the polyadenylation factors and it co-operates with SLBP to recruit U7 snRNP to histone pre-mRNA (Skrajna, 2017).

Sex-specific transcript diversity in the fly head Is established during pupal stages and adulthood and is largely independent of the mating process and the germline

Alternative splicing (AS), the process which generates multiple RNA and protein isoforms from a single pre-mRNA, greatly contributes to transcript diversity and compensates for the fact that the gene number does not scale with organismal complexity. A number of genomic approaches have established that the extent of AS is much higher than previously expected, raising questions on its spatio-temporal regulation and function. The present study addresses AS in the context of sex-specific neuronal development in the model Drosophila melanogaster. At least 47 genes display sex-specific AS in the adult fly head. Unlike targets of the classical Sex lethal-dependent sex determination cascade, sex-specific isoforms of the vast majority of these genes are not present during larval development but start accumulating during metamorphosis or later, indicating the existence of novel mechanisms in the induction of sex-specific AS. It was also established that sex-specific AS in the adult fly head is largely independent of the germline or the mating process. Finally, the role of sex-specific AS of the sulfotransferase Tango13 pre-mRNA was investigated and first evidence is provided that differential expression of certain isoforms of this protein significantly affects courtship and mating behavior in male flies (Mohr, 2017)


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Zygotically transcribed genes

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