Splicing Regulatory Elements Research Articles

Splicing defects caused by exonic mutations in PKD1 as a new mechanism of pathogenesis in autosomal dominant polycystic kidney disease

The correct splicing of precursor-mRNA depends on the actual splice sites plus exonic and intronic regulatory elements recognized by the splicing machinery. Surprisingly, an increasing number of examples reveal that exonic mutations disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. This contradicts the general assumption that missense mutations disrupt protein function and that synonymous mutations are merely polymorphisms. Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused mainly by mutations in the PKD1 gene. Recently, we analyzed a substantial number of PKD1 missense or synonymous mutations to further characterize their consequences on pre-mRNA splicing. Our results showed that one missense and 2 synonymous mutations induce significant defects in pre-mRNA splicing. Thus, it appears that aberrant splicing as a result of exonic mutations is a previously unrecognized cause of ADPKD.

Structural determinants for alternative splicing regulation of the MAPT pre-mRNA

Alternative splicing at the MAPT gene exon 10 yields similar levels of the 3R and 4R tau protein isoforms.1 The presence of mutations, particularly in exon 10 and intron 10–11, changes the quantity of tau isoforms. Domination each of the isoform yields tau protein aggregation and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we report for the first time the secondary structure of the 194/195 nucleotide region for the wild type (WT) and 10 mutants of the MAPT gene pre-mRNA determined using both chemical and microarray mapping. Thermodynamic analyses indicate that single nucleotide mutations in the splicing regulatory element (SRE) that form a hairpin affect its stability by up to 4 and 7 kcal/mol. Moreover, binding the regulatory hairpin of small molecule ligands (neomycin, kanamycin, tobramycin and mitoxantrone) enhance its stability depending on the nature of the ligands and the RNA mutations. Experiments using the cos-7 cell line indicate that the presence of ligands and modified antisense oligonucleotides affect the quantity of 3R and 4R isoforms. This finding correlates with the thermodynamic stability of the regulatory hairpin. An alternative splicing regulation mechanism for exon 10 is postulated based on our experimental data and on published data.

Identifying splicing regulatory elements with de Bruijn graphs.

Splicing regulatory elements (SREs) are short, degenerate sequences on pre-mRNA molecules that enhance or inhibit the splicing process via the binding of splicing factors, proteins that regulate the functioning of the spliceosome. Existing methods for identifying SREs in a genome are either experimental or computational. Here, we propose a formalism based on de Bruijn graphs that combines genomic structure, word count enrichment analysis, and experimental evidence to identify SREs found in exons. In our approach, SREs are not restricted to a fixed length (i.e., k-mers, for a fixed k). As a result, we identify 2001 putative exonic enhancers and 3080 putative exonic silencers for human genes, with lengths varying from 6 to 15 nucleotides. Many of the predicted SREs overlap with experimentally verified binding sites. Our model provides a novel method to predict variable length putative regulatory elements computationally for further experimental investigation.

Abstract SS02-02: A long walk from FGFR2 alternative splicing to cancer progression

Abstract Previously, our group developed fluorescence-based alternative splicing reporters of epithelial plasticity to visualize phenotypic transitions in real time in vivo (Oltean et al., 2008; Oltean et al., 2006; Somarelli et al., 2013). We reasoned that our mesenchymal-epithelial transition (MET) reporters could be extremely useful to test the hypothesis that MET is a modulator of metastatic colonization in undifferentiated, sarcomatoid-like cancers. Along these lines, we generated a lineage-tracing reporter based on combined transcription and alternative splicing regulatory elements to measure the frequency of MET-like events during tumor growth and metastasis in the post-EMT Dunning rat AT3 model of prostate cancer. In a parallel set of experiments, we also used the combinatorial control strategy to drive a suicide reporter that kills cells undergoing MET to test the hypothesis that MET is required for metastatic colonization in undifferentiated, mesenchymal-like cancers. The combinatorial use of both transcription and alternative splicing regulation provided exquisite cell-type discrimination in the expression of enzymes, such as the Cre recombinase and Diphtheria A (DipA) toxin. Using the lineage tracing reporter system, we were able to quantify, for the first time, overall frequencies of MET during primary tumor and metastatic growth. Remarkably, we observed that the frequency of MET within primary tumors and metastatic nodules was not significantly different, with very low rates of MET taking place during the growth of tumors and metastases. Moreover, targeted killing of cells undergoing MET did not reduce the number of macrometastatic colonies in the lungs. This work demonstrates the ability of tumors to metastasize efficiently without the need for MET, and we suggest that this may be similar to the behavior of highly aggressive prostate carcinosarcomas or undifferentiated carcinomas. Using this system we will describe new experiments that will address differential behavior in different types of prostate cancer. Citation Format: Jason A. Somarelli, Jason A. Somarelli, Daneen Schaeffer, Daneen Schaeffer, Mathew S. Marengo, Mathew S. Marengo, Tristan Bepler, Tristan Bepler, Douglas Rouse, Anne F. Buckley, Jonathan I. Epstein, Andrew J. Armstrong, Mariano A. Garcia-Blanco, Mariano A. Garcia-Blanco. A long walk from FGFR2 alternative splicing to cancer progression. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr SS02-02. doi:10.1158/1538-7755.DISP13-SS02-02

Genomic HEXploring allows landscaping of novel potential splicing regulatory elements.

Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based ‘HEXplorer score’ as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide.

Dual Masking of Specific Negative Splicing Regulatory Elements Resulted in Maximal Exon 7 Inclusion of SMN2 Gene

Spinal muscular atrophy (SMA) is a fatal autosomal recessive disease caused by survival motor neuron (SMN) protein insufficiency due to SMN1 mutations. Boosting SMN2 expression is a potential therapy for SMA. SMN2 has identical coding sequence as SMN1 except for a silent C-to-T transition at the 6th nucleotide of exon 7, converting a splicing enhancer to a silencer motif. Consequently, most SMN2 transcripts lack exon 7. More than ten putative splicing regulatory elements (SREs) were reported to regulate exon 7 splicing. To investigate the relative strength of each negative SRE in inhibiting exon 7 inclusion, antisense oligonucleotides (AONs) were used to mask each element, and the fold increase of full-length SMN transcripts containing exon 7 were compared. The most potent negative SREs are at intron 7 (in descending order): ISS-N1, 3' splice site of exon 8 (ex8 3'ss) and ISS+100. Dual-targeting AONs were subsequently used to mask two nonadjacent SREs simultaneously. Notably, masking of both ISS-N1 and ex8 3'ss induced the highest fold increase of full-length SMN transcripts and proteins. Therefore, efforts should be directed towards the two elements simultaneously for the development of optimal AONs for SMA therapy.

Novel Mutations in the GH Gene (GH1) Uncover Putative Splicing Regulatory Elements

Mutations affecting exon 3 splicing are the main cause of autosomal dominant Isolated GH Deficiency II (IGHDII) by increasing the level of exon 3-skipped mRNA encoding the functionally inactive dominant-negative 17.5-kDa isoform. The exons and introns of the gene encoding GH (GH1) were screened for the presence of mutations in 103 sporadic isolated GH deficiency cases. Four different variations within exon 3 were identified in 3 patients. One carried c.261C>T (p.Pro87Pro) and c.272A>T (p.Glu91Val), the second c.255G>A (p.Pro85Pro) and c.261 C>T, and the third c.246G>C (p.Glu82Asp). All the variants were likely generated by gene conversion from an homologous gene in the GH1 cluster. In silico analysis predicted that positions c.255 and c.272 were included within 2 putative novel exon splicing enhancers (ESEs). Their effect on splicing was confirmed in vitro. Constructs bearing these 2 variants induced consistently higher levels both of transcript and protein corresponding to the 17.5-kDa isoform. When c.255 and c.272 were combined in cis with the c.261 variant, as in our patients, their effect was weaker. In conclusion, we identified 2 variations, c.255G>A and c.272A>T, located in 2 novel putative exon splicing enhancers and affecting GH1 splicing in vitro by increasing the production of alternatively spliced isoforms. The amount of aberrant isoforms is further regulated by the presence in cis of the c.261 variant. Thus, our results evidenced novel putative splicing regulatory elements within exon 3, confirming the crucial role of this exon in mRNA processing.

The multiplicity of alternative splicing decisions in Caenorhabditis elegans is linked to specific intronic regulatory motifs and minisatellites.

BackgroundAlternative splicing diversifies the pool of messenger RNA molecules encoded by individual genes. This diversity is particularly high when multiple splicing decisions cause a combinatorial arrangement of several alternate exons. We know very little on how the multiple decisions occurring during the maturation of single transcripts are coordinated and whether specific sequence elements might be involved.ResultsHere, the Caenorhabditis elegans genome was surveyed in order to identify sequence elements that might play a specific role in the regulation of multiple splicing decisions. The introns flanking alternate exons in transcripts whose maturation involves multiple alternative splicing decisions were compared to those whose maturation involves a single decision. Fifty-eight penta-, hexa-, and hepta-meric elements, clustered in 17 groups, were significantly over-represented in genes subject to multiple alternative splicing decisions. Most of these motifs relate to known splicing regulatory elements and appear to be well conserved in the related species Caenorhabditis briggsae. The usage of specific motifs is not linked to the gene product function, but rather depends on the gene structure, since it is influenced by the distance separating the multiple splicing decision sites. Two of these motifs are part of the CeRep25B minisatellite, which is also over-represented at the vicinity of alternative splicing regions. Most of the remaining motifs are not part of repeated sequence elements, but tend to occur in specific heterologous pairs in genes subject to multiple alternative splicing decisions.ConclusionsThe existence of specific intronic sequence elements linked to multiple alternative splicing decisions is intriguing and suggests that these elements might have some specialized regulatory role during splicing.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-364) contains supplementary material, which is available to authorized users.

Pressure-overload cardiac hypertrophy is associated with distinct alternative splicing due to altered expression of splicing factors.

Chronic pressure-overload cardiac hypertrophy is associated with an increased risk of morbidity/mortality, largely due to maladaptive remodeling and dilatation that progresses to dilated cardiomyopathy. Alternative splicing is an important biological mechanism that generates proteomic complexity and diversity. The recent development of next-generation RNA sequencing has improved our understanding of the qualitative signatures associated with alternative splicing in various biological conditions. However, the role of alternative splicing in cardiac hypertrophy is yet unknown. The present study employed RNA-Seq and a bioinformatic approach to detect the RNA splicing regulatory elements involved in alternative splicing during pressure-overload cardiac hypertrophy. We found GC-rich exonic motifs that regulate intron retention in 5′ UTRs and AT-rich exonic motifs that are involved in exclusion of the AT-rich elements that cause mRNA instability in 3′ UTRs. We also identified motifs in the intronic regions involved in exon exclusion and inclusion, which predicted splicing factors that bind to these motifs. We found, through Western blotting, that the expression levels of three splicing factors, ESRP1, PTB and SF2/ASF, were significantly altered during cardiac hypertrophy. Collectively, the present results suggest that chronic pressure-overload hypertrophy is closely associated with distinct alternative splicing due to altered expression of splicing factors.

Functional Analysis of a Large set of BRCA2 exon 7 Variants Highlights the Predictive Value of Hexamer Scores in Detecting Alterations of Exonic Splicing Regulatory Elements

Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n=36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing.

HEXploring of the HIV-1 genome allows landscaping of new potential splicing regulatory elements

Effective selection between true and decoy splice sites is critically controlled by flanking splicing regulatory elements (SREs), which can enhance or repress splice site use. Recent experimental evidence suggests that the entire regional context of SREs rather than a single enhancer/silencer hexamer jointly contribute to splicing. Extending the hexamer score concept [Fairbrother et al.: Science 2002, 297:1007-13], we represent the splicing regulatory property of an entire 5’ss neighborhood by a weighted average of normalized Z-scores for all hexamers overlapping with the target region. These novel “HEXplorer” scores describe the degrees of exon- or intron-likeness (ZEI) and enhancer-likeness (ZWS) for a given region upstream of a 5’ss. These scores can be graphically represented by positive or negative oriented areas along the sequence. Mutation effects on an entire 5’ss neighborhood are then captured by comparing the HEXplorer areas of wild type and mutant sequences upstream of a 5’ss. The fundamental datasets of weak and strong 5’ss used in the definition of these HEXplorer scores were derived based on the HBond score that measures the 5’ss complementarity to U1 snRNA. In a first test, we scanned the small non-coding HIV-1 leader exon 3 for regions enriched in SREs. Here, HEXplorer scores correctly indicated both the well-known exonic splicing silencer ESSV and the recently discovered exonic splicing enhancer ESEvpr upstream of 5’ss D3. Next, we tested the HEXplorer’s capability to predict mutations’ potency to modify 5’ss D3 usage. We systematically examined this ESE region using various single and double mutations predicted to either alter 5’ss usage or act neutrally. In 20 tested mutations, the HEXplorer prediction correlated well with the experimentally detected level of exon inclusion. Extending the HEXplorer approach to all HIV-1 exons, we were able to identify three novel exonic splicing enhancers that contribute to the inclusion of the viral exons 2, 2b and exon 4. All three novel ESEs were experimentally confirmed by HEXplorer predicted point-mutations. Beyond application to HIV-1 5’ss usage, the HEXplorer may also prove particularly useful as a method for assessing pathogenic human exonic mutations.

Systematical identification of splicing regulatory cis-elements and cognate trans-factors

The majority of human genes undergo alternative splicing to generate multiple isoforms with distinct functions. This process is generally controlled by cis-acting splicing regulatory elements (SREs) that recruit trans-acting factors to promote or inhibit the use of nearby splice sites. The growing interest in understanding the regulatory rules of splicing necessitates the systematic identification of these SREs and their cognate protein factors using experimental and computational approaches. Here we describe a strategy to identify and analyze both cis-acting SREs and trans-acting splicing factors. This strategy involves a cell-based screen to identify SREs from a random sequences library and a modified RNA affinity purification approach to unbiasedly identify the splicing factors. These methods can be adopted to identify splicing enhancers or silencers in both exons and introns, and can be extended to different cultured cells. The resulting SREs and splicing factors can be further analyzed with a series of computational and experimental approaches. This approach will help us to collect a molecular part-list for splicing regulation, providing a rich data source that enables a better understanding of the “splicing code”.

Half Pint/Puf68 is required for negative regulation of splicing by the SR factor Transformer2

The SR family of proteins plays important regulatory roles in the control of alternative splicing in a wide range of organisms. These factors affect splicing through both positive and negative controls of splice site recognition by pre-spliceosomal factors. Recent studies indicate that the Drosophila SR factor Transformer 2 (Tra2) activates and represses splicing through distinct and separable effector regions of the protein. While the interactions of its Arg-Ser-rich activator region have been well studied, cofactors involved in splicing repression have yet to be found. Here we use a luciferase-based splicing reporter assay to screen for novel proteins necessary for Tra2-dependent repression of splicing. This approach identified Half pint, also known as Puf68, as a co-repressor required for Tra2-mediated autoregulation of the M1 intron. In vivo, Half pint is required for Tra2-dependent repression of M1 splicing but is not necessary for Tra2-dependent activation of doublesex splicing. Further experiments indicate that the effect of Hfp is sequence-specific and that it associates with these target transcripts in cells. Importantly, known M1 splicing regulatory elements are sufficient to sensitize a heterologous intron to Hfp regulation. Two alternative proteins deriving from Hfp transcripts, Hfp68, and Hfp58, were found to be expressed in vivo but differed dramatically in their effect on M1 splicing. Comparison of the cellular localization of these forms in S2 cells revealed that Hfp68 is predominantly localized to the nucleus while Hfp58 is distributed across both the nucleus and cytoplasm. This accords with their observed effects on splicing and suggests that differential compartmentalization may contribute to the specificity of these isoforms. Together, these studies reveal a function for Half pint in splicing repression and demonstrate it to be specifically required for Tra2-dependent intron inclusion.

A synonymous (c.3390C>T) or a splice‐site (c.3380‐2A>G) mutation causes exon 26 skipping in four patients with von Willebrand disease (2A/IIE)

We characterized four unrelated patients with von Willebrand disease type2A/IIE, sharing the same von Willebrand factor (VWF) in-frame deletion (p.[P1127_G1180delinsR];[=]) resulting from exon26 skipping (Δ26). To identify the VWF mutations and how they caused the mRNA splicing alteration, to evaluate the deletion by invitro expression studies, and to assess whether or not the heterogeneity of the patients' phenotype might be related to a different degree of expression of the deleted subunit in patient plasma VWF. Sequence analysis was performed with patient genomic DNA and platelet mRNA. Semiquantitative RT-PCR was also carried out to compare the expression of the wild-type (WT) and Δ26 alleles in the four patients. Insilico analysis was performed with prediction splicing programs. Expression studies were performed to evaluate mutant recombinant VWF (rVWF) (Δ26 and Δ26/WT) as compared with WT rVWF. Three patients shared the synonymous single-nucleotide substitution (SSS) c.[3390C>T];[=], whereas the novel mutation c.[3380-2A>G];[=] was present in the fourth patient. Semiquantitative RT-PCR of platelet mRNA revealed a different ratio of the WT and Δ26 alleles in the patients, consistent with the different VWF:FVIIIB values present in patient plasma. Expression studies confirmed reduced VWF-FVIII binding of rVWF-Δ26/WT. SSS can induce alternative splicing, and those like c.3390C>T, which impact on the poorly conserved splicing regulatory elements, are difficult to predict, so that their role can be evaluated only by mRNA analysis. Moreover, these mutations seem to have different effects on the efficiency of alternative splicing, producing heterogeneous VWF variants among the four patients.

A highly conserved GC‐rich element regulates alternative splicing of mRNA for the variant thyroid hormone receptor, TRα2

In mammals the THRA gene is alternatively spliced and encodes two proteins. TRα1 is the α‐thyroid hormone receptor, which is widely expressed in all vertebrates. TRα2 is a non‐hormone‐binding variant, present only in eutherian mammals. Coding sequences unique to TRα2 share an antisense overlap with those of Rev‐erbα, a nuclear receptor and core component of the mammalian circadian clock. Intronic and exonic splicing enhancers for TRα2 have evolved in the context of sequences required expression of Rev‐erbα mRNA. Of particular interest is a GC‐rich region (G30) within the C‐terminal sequence of TRα2 mRNA and antisense to the 3'UTR of Rev‐erbα. Closely spaced deletions and substitutions within G30 have dramatically different effects on TRα2 splicing depending on their precise structure and position. For example, deletion of 12 nucleotides can lead to >;95% inhibition or >;2.5‐fold enhancement of TRα2 splicing. Substitutions of 5–8 bases within G30 also display a range of effects. Our results suggest that G30 is a highly structured sequence, possibly a G‐quadruplex, and part of a larger complex splicing regulatory element that exerts both positive and negative effects on TRα2 expression.

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.

The orthologue of the fruitfly sex behaviour gene fruitless in the mosquito Aedes aegypti: evolution of genomic organisation and alternative splicing.

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.

A Biophysical Model for Identifying Splicing Regulatory Elements and Their Interactions

Alternative splicing (AS) of precursor mRNA (pre-mRNA) is a crucial step in the expression of most eukaryotic genes. Splicing factors (SFs) play an important role in AS regulation by binding to the cis-regulatory elements on the pre-mRNA. Although many splicing factors (SFs) and their binding sites have been identified, their combinatorial regulatory effects remain to be elucidated. In this paper, we derive a biophysical model for AS regulation that integrates combinatorial signals of cis-acting splicing regulatory elements (SREs) and their interactions. We also develop a systematic framework for model inference. Applying the biophysical model to a human RNA-Seq data set, we demonstrate that our model can explain 49.1%–66.5% variance of the data, which is comparable to the best result achieved by biophysical models for transcription. In total, we identified 119 SRE pairs between different regions of cassette exons that may regulate exon or intron definition in splicing, and 77 SRE pairs from the same region that may arise from a long motif or two different SREs bound by different SFs. Particularly, putative binding sites of polypyrimidine tract-binding protein (PTB), heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and E/K are identified as interacting SRE pairs, and have been shown to be consistent with the interaction models proposed in previous experimental results. These results show that our biophysical model and inference method provide a means of quantitative modeling of splicing regulation and is a useful tool for identifying SREs and their interactions. The software package for model inference is available under an open source license.

An Intronic G Run within HIV-1 Intron 2 Is Critical for Splicing Regulation of vif mRNA

Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.