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Mutations that affect phenotypes have been identified primarily as those that directly alter amino acid sequences or disrupt splice sites. However, some mutations not located in functionally important sites can also affect phenotypes, such as splice-site-creating mutations (SCMs). To investigate how frequent exon extension/shrinkage events induced by SCMs occur in normal individuals, we used personal genome sequencing data and transcriptome data of the corresponding individuals and identified 371 exon extension/shrinkage events in normal individuals. This number was about three times higher than the number of pseudo-exon activation events identified in the previous study. The average numbers of exon extension and exon shrinkage events in each sample were 3.3 and 11.2, respectively. We also evaluated the impact of exon extension/shrinkage events on the resulting transcripts and their protein products and found that 40.2% of the identified events may have possible functional impacts by either generating premature termination codons in transcripts or affecting protein domains. Our results indicated that a certain fraction of SCMs identified in this study can be pathogenic mutations by creating novel splice sites.

In Eukarya, immature mRNA transcripts (pre-mRNA) often contain coding sequences, or exons, interleaved by non-coding sequences, or introns. Introns are removed upon

Causative mutations for human genetic disorders have mainly been identified in exonic regions that code for amino acid sequences. Recently, however, it has been reported that mutations in deep intronic regions can also cause certain human genetic disorders by creating novel splice sites, leading to pseudo-exon activation. To investigate how frequently pseudo-exon activation events occur in normal individuals, we conducted in silico identification of such events using personal genome data and corresponding high-quality transcriptome data. With rather stringent conditions, on average, 2.6 pseudo-exon activation events per individual were identified. More pseudo-exon activation events were found in 5′ donor splice sites than in 3′ acceptor splice sites. Although pseudo-exon activation events have sporadically been reported as causative mutations in genetic disorders, it is revealed in this study that such events can be observed in normal individuals at a certain frequency. We estimate that human genomes typically contain on average at least 10 pseudo-exon activation events. The actual number should be higher than this, because we used stringent criteria to identify pseudo-exon activation events. This suggests that it is worth considering the possibility of pseudo-exon activation when searching for causative mutations of genetic disorders if candidate mutations are not identified in coding regions or RNA splice sites.

Latent 5' splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of ini-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.

Mutations of splice sites, auxiliary splicing elements and the splicing machinery cause a wide range of genetic disease. Here we report that many of the complex effects of splicing mutations can be predicted from background splicing information, with emphasis on BRCA1, BRCA2 and DMD. Background splicing arises from very low level splicing between rarely used background splice sites and from low-level exon skipping between intron splice sites. We show how this information can be downloaded from the Snaptron database of spliced RNA, which we then compared with databases of human splice site mutations. We report that inactivating mutations of intron splice sites typically caused the non-mutated partner splice site to splice to a known background splice site in over 90% of cases and to the strongest background splice site in the large majority of cases. Consequently, background splicing information can usefully predict the effects of splice site mutations, which include cryptic splice activation and single or multiple exon skipping. In addition, de novo splice sites and splice sites involved in pseudoexon formation, recursive splicing and aberrant splicing in cancer show a 90% match to background splice sites, so establishing that the enhancement of background splicing causes a wide range of splicing aberrations. We also discuss how background splicing information can identify cryptic splice sites that might be usefully targeted by antisense oligonucleotides (ASOs) and how it might indicate possible multiple exon skipping side effects of ASOs designed to induce single exon skipping.

Temperature-dependent alternative splicing was recently demonstrated for intron 2 of the gene coding for heat shock factor HsfA2 of the tomato plant Solanum lycopersicum, but the molecular mechanism regulating the abundance of such temperature-dependent splice variants is still unknown. We report here on regulatory pre-mRNA structures that could function as regulators by controlling the use of splice sites in a temperature-dependent manner. We investigate pre-mRNA structures at the splice sites of intron 2 of the gene coding for HsfA2 from S. lycopersicum using NMR- and CD-spectroscopy as well as in-line probing. The pre-mRNA undergoes conformational changes between two different secondary structures at the 3�� splice site of the intron in a temperature-dependent manner. Previously, it was shown that three single nucleotide polymorphisms (SNPs) in intron 2 of the HsfA2 pre-mRNA affect the splicing efficiency of its pre-mRNA and are linked to the thermotolerance in different tomato species. By comparing pre-mRNA fragments of the tomato species S. lycopersicum and S. peruvianum, we show that these SNPs result in substantial structural differences between the pre-mRNAs of the two species.

How have the branchpoint motifs evolved in organisms of different complexity? Here we identified and examined the consensus motifs (R(1)C(2)T(3)R(4)A(5)Y(6), R: A or G, Y: C or T) of 898 fungal genomes. In Ascomycota unicellular yeasts, the G(4)/A(4) ratio is mostly (98%) below 0.125 but increases sharply in multicellular species by about 40 times on average, and in the more complex Basidiomycota, it increases further by about 7 times. The global G(4) increase is consistent with A(4) to G(4) transitions in evolution. Of the G(4)/A(4)-interacting amino acids of the branchpoint binding protein MSL5 (SF1) and the HSH155 (SF3B1), as well as the 5ʹ splice sites (SS) and U2 snRNA genes, the 5ʹ SS G(3)/A(3) co-vary with the G(4) to some extent. However, corresponding increase of the G(4)-complementary GCAGTA-U2 gene is rare, suggesting wobble-base pairing between the G(4)-containing branchpoint motif and GTAGTA-U2 in most of these species. Interestingly, the G(4)/A(4) ratio correlates well with the abundance of alternative splicing in the two phyla, and G(4) enriched significantly at the alternative 3' SS of genes in RNA metabolism, kinases and membrane proteins. Similar wobble nucleotides also enriched at the 3' SS of multicellular fungi with only thousands of protein-coding genes. Thus, branchpoint motifs have evolved U2-complementarity in unicellular Ascomycota yeasts, but have gradually gained more wobble base-pairing nucleotides in fungi of higher complexity, likely to destabilize branchpoint motif-U2 interaction and/or branchpoint A protrusion for alternative splicing. This implies an important role of relaxing the branchpoint signals in the multicellularity and further complexity of fungi.

Formed by back splicing or back fusion of linear RNAs, circular RNAs (circRNAs) constitute a new class of non-coding RNAs of eukaryotes. Recent studies reveal a spliceosome-dependent biogenesis of circRNAs where circRNAs arise at the intron-exon junctions of mRNAs. In this study, using a novel de novo identification method, we show that circRNAs can originate from the interior regions of exons, introns, and intergenic transcripts in human, mouse and rice, which were referred to as interior circRNAs (i-circRNAs). Many i-circRNAs have some remarkable characteristics: multiple i-circRNAs may arise from the same genomic locus; their back fusion points may not be associated with the AG/GT splicing sites, but rather a new pair of motif AC/CT, their back fusion points are adjacent to complementary sequences; and they may circulate on short homologous sequences. We validated several i-circRNAs in HeLa cells by Polymerase Chain Reaction followed by Sanger sequencing. Our results combined showed that i-circRNAs are bona fide circRNAs, indicated novel biogenesis pathways independent of the splicing apparatus, and expanded our understanding of the origin, diversity, and complexity of circRNAs.

Circular (circ)RNAs have recently become a subject of great biologic interest. It is now clear that they represent a diverse and abundant class of RNAs with regulated expression and evolutionarily conserved functions. There are several mechanisms by which RNA circularization can occur in vivo. Here, we focus on the biogenesis of tRNA intronic circular RNAs (tricRNAs) in archaea and animals, and we detail their use as research tools for orthogonal, directed circRNA expression in vivo.

Circular RNAs (circRNAs) have been identified in diverse eukaryotic species and are characterized by RNA backsplicing events. Current available methods for circRNA identification are able to determine the start and end locations of circRNAs in the genome but not their full-length sequences. In this study, we developed a method to assemble the full-length sequences of circRNAs using the backsplicing RNA-Seq reads and their corresponding paired-end reads. By applying the method to an rRNA-depleted/RNase R-treated RNA-Seq dataset, we for the first time identified full-length sequences of nearly 3,000 circRNAs in rice. We further showed that alternative circularization of circRNA is a common feature in rice and, surprisingly, found that the junction sites of a large number of rice circRNAs are flanked by diverse non-GT/AG splicing signals while most human exonic circRNAs are flanked by canonical GT/AG splicing signals. Our study provides a method for genome-wide identification of full-length circRNAs and expands our understanding of splicing signals of circRNAs.

A key step in pre-mRNA splicing is the recognition of 3’ splicing sites by the U2AF large and small subunits, a process regulated by numeroustrans-acting splicing factors. How thesetrans-acting factors interact with U2AFin vivois unclear. From a screen for suppressors of the temperature-sensitive (ts) lethality of theC. elegansU2AF large subunit geneuaf-1(n4588)mutants, we identified mutations in theRNAbindingmotif generbm-5, a homolog of the tumor suppressorRBM5.rbm-5mutations can suppressuaf-1(n4588)ts-lethality by loss of function and neuronal expression ofrbm-5was sufficient to rescue the suppression. Transcriptome analyses indicate thatuaf-1(n4588)affected the expression of numerous genes andrbm-5mutations can partially reverse the abnormal gene expression to levels similar to that of wild type. Thoughrbm-5mutations did not obviously affect alternative splicing per se, they can suppress or enhance, in a gene-specific manner, the altered splicing of genes inuaf-1(n4588)mutants. Specifically, the recognition of a weak 3’ splice site was more susceptible to the effect ofrbm-5. Our findings provide novelin vivoevidence that RBM-5 can modulate UAF-1-dependent RNA splicing and suggest that RBM5 might interact with U2AF large subunit to affect tumor formation.Author summaryRNA splicing is a critical regulatory step for eukaryotic gene expression and has been involved in the pathogenesis of multiple diseases. How RNA splicing factors interactin vivoto affect the splicing and expression of genes is unclear. In studying the temperature-sensitive lethal phenotypes of a mutation affecting the splicing factor U2AF large subunit geneuaf-1in the nematodeCaenorhabditis elegans, we isolated suppressive mutations in therbm-5gene, a homolog of the human tumor suppressor geneRBM5.rbm-5is broadly expressed in neurons to enhance the lethality of theuaf-1mutants. We found that the uaf-1 mutation causes aberrant expression of genes in numerous biological pathways, a large portion of which can be corrected byrbm-5mutations. The abnormal splicing of multiple genes caused by theuaf-1mutation is either corrected or enhanced byrbm-5mutations in a gene-specific manner. We propose that RBM-5 interacts with UAF-1 to affect RNA splicing and the tumor suppressor function of RBM5 might involve U2AF-dependent RNA splicing.

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.

Tandem alternative splice sites (TASS) form a defined class of alternative splicing and give rise to mRNA insertion/deletion variants with only small size differences. Previous work has confirmed evolutionary conservation of TASS elements while many cases show only low tissue specificity of isoform ratios. We pinpoint stochasticity and noise as important methodological issues for the dissection of TASS isoform patterns. Resolving such uncertainties, a recent report showed regulation in a cell culture system, with shifts of alternative splicing isoform ratios dependent on cell density. This novel type of regulation affects not only multiple TASS isoforms, but also other alternative splicing classes, in a concerted manner. Here, we discuss how specific regulatory network architectures may be realized through the novel regulation type and highlight the role of differential isoform functions as a key step in order to better understand the functional role of TASS.

BRCA1 exon 11 is one of the biggest human exons, spanning 3426 bases. This gene is potentially involved in DNA repair as well as cell growth and cell cycle control. Exon 11 is regulated at the splicing level producing three main different combinations of BRCA1 mature transcripts; one including the whole of exon 11 (full isoform), one skipping the entire exon (D11 isoform), and one including only 117 base pairs of exon 11 (D11q isoform). Using minigene and deletion analyses, we have previously described important splicing regulatory sequences located at the beginning of this exon (5' end). We have now found additional important sequences located at its 3' end. In particular, we describe the presence of a strong splicing enhancer adjacent to the downstream 5' splice site, which minimizes competition from an upstream 5' splice site and so ensures long exon inclusion. Analyses of the proteins binding these RNA sequences have revealed that Tra2beta and hnRNP L are involved in the regulation of BRCA1 exon 11 by influencing the recognition of donor sites. Interestingly, BRCA1 exon 11 carrying deletion of the regulatory sequences bound by these factors also showed unexpected responses to up- or downregulation of these regulatory proteins, suggesting that they can also bind elsewhere in this large exon and elicit different effects on its recognition. The identification of sequences and proteins relevant for the regulation of BRCA1 exon 11 now provides better knowledge on how this exon is recognized and may represent an important step toward understanding how large exons are regulated.

The gene Down syndrome cell adhesion molecule (Dscam) potentially encodes 38 016 distinct isoforms in Drosophila melanogaster via mutually exclusive splicing. Here we reveal a combinatorial mechanism of regulation of Dscam exon 17 mutually exclusive splicing through steric hindrance in combination with RNA secondary structure. This mutually exclusive behavior is enforced by steric hindrance, due to the close proximity of the exon 17.2 branch point to exon 17.1 in Diptera, and the interval size constraint in non-Dipteran species. Moreover, intron-exon RNA structures are evolutionarily conserved in 36 non-Drosophila species of six distantly related orders (Diptera, Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, and Phthiraptera), which regulates the selection of exon 17 variants via masking the splice site. By contrast, a previously uncharacterized RNA structure specifically activated exon 17.1 by bringing splice sites closer together in Drosophila, while the other moderately suppressed exon 17.1 selection by hindering the accessibility of polypyrimidine sequences. Taken together, these data suggest a phylogeny of increased complexity in regulating alternative splicing of Dscam exon 17 spanning more than 300 million years of insect evolution. These results also provide models of the regulation of alternative splicing through steric hindrance in combination with dynamic structural codes.

The spliceosomal component U1snRNP commits pre-mRNAs to the splicing pathway. Recently, a nuclear RNA surveillance function has been ascribed to U1, namely the suppression of intronic polyadenylation sites. This surveillance holds regulatory potential as it alters the 3′ ends of certain receptor tyrosine kinase mRNAs. However, suppression of 3′ end processing by U1 snRNP is also the cause of a severe genetic disorder. We described a 3′UTR point mutation creating a 5′SS leading to U1-mediated suppression of 3′ end formation. Thus, the inhibitory function of U1 is both beneficial and deleterious where misled. The exact mechanism of how U1 interferes with 3′ end processing remains unclear. According to our data, U1 snRNP already interferes with cleavage or poly(A) site selection instead of directly inhibiting poly(A) polymerase as previously assumed. Here, we present alternative models for U1-mediated poly(A) site suppression and discuss the implications for RNA quality control and disease-related mutations.

In this commentary we briefly summarize early work on circular RNAs derived from spliceosome mediated circularization. We highlight how this early work inspired work on the basic mechanisms of nuclear RNA splicing, the possible function of circular RNAs and the potential uses of circular RNAs as tools in biomedicine. Recent developments in the study of circular RNAs, summarized in this volume, have brought these questions back to the foreground.

The 3' splice site of the influenza A segment 7 transcript is utilized to produce mRNA for the critical M2 ion-channel protein. In solution a 63 nt fragment that includes this region can adopt two conformations: a pseudoknot and a hairpin. In each conformation, the splice site, a binding site for the SF2/ASF exonic splicing enhancer and a polypyrimidine tract, each exists in a different structural context. The most dramatic difference occurs for the splice site. In the hairpin the splice site is between two residues that are involved in a 2 by 2 nucleotide internal loop. In the pseudoknot, however, these bases are canonically paired within one of the pseudoknotted helices. The conformational switching observed in this region has implications for the regulation of splicing of the segment 7 mRNA. A measure of stability of the structures also shows interesting trends with respect to host specificity: avian strains tend to be the most stable, followed by swine and then human.

In higher eukaryotes, the 5' splice site (5'ss) is initially recognized through an RNA-RNA interaction by U1 small nuclear ribonucleoprotein (U1 snRNP). This event represents one of the key steps in initial spliceosomal assembly and many disease-associated mutations in humans often disrupt this process. Beside base pair complementarity, 5'ss recognition can also be modified by additional factors such as RNA secondary structures or the specific binding of other nuclear proteins. In this work, we have focused on investigating a few examples of changes detected within the 5'ss in patients, that would not be immediately considered "disease causing mutations". We show that the splicing outcome of very similar mutations can be very different due to variations in trans-acting factor(s) interactions and specific context influences. Using several NF1 donor sites and SELEX approaches as experimental models, we have examined the binding properties of particular sequence motifs such as GGGU found in donor sites, and how the sequence context can change their interaction with hnRNPs such as H/F and A1/A2. Our results clearly show that even minor differences in local nucleotide context can differentially affect the binding ability of these factors to the GGGU core. Finally, using a previously identified mutation in KCNH2 that resulted in intron retention we show how very similar 5'ss mutations found in patients can have a very different splicing outcome due to the neighbouring sequence context, thus highlighting the general need to approach splicing problems with suitable experimental approaches.

The structural complexity of the vertebrate brain is mirrored by its unparalleled transcriptome complexity. In particular, two post-transcriptional processes, alternative splicing and RNA editing, greatly diversify brain transcriptomes. Here we report a close connection between these two processes: we show A-to-I RNA editing in Nova1, a key brain-specific regulator of alternative splicing. Nova1 editing levels increase during embryonic development in mouse and chicken brains and show significant variation across postnatal brain regions. Evolutionary conservation of both editing and editing-associated RNA secondary structure of the Nova1 mRNA for 300 million years attests to the functional importance of Nova1 editing. Using a combination of different assays in human HEK293T cell lines, we report a novel post-translational role for this RNA editing. Whereas functional assays showed no effect of RNA editing on the regulatory splicing activity of the encoded proteins, we found evidence that edited forms exhibit reduced proteasome targeting and increased protein half-life. In addition, we found evidence for similar regulation of protein half-life by an evolutionarily conserved alternative splicing event in Nova1. These results open new venues of research on the multi-level integration of gene expression by: (1) revealing the novel role of RNA editing in regulating protein stability, and (2) establishing protein stability as a new target of multifaceted regulation.

3'-end cleavage and subsequent polyadenylation are critical steps in mRNA maturation. The precise location where cleavage occurs (referred to as poly(A) site) is determined by a tripartite mechanism in which a A(A/U)UAAA hexamer, GU rich downstream element and UGUA upstream element are recognized by the cleavage and polyadenylation factor (CPSF), cleavage stimulation factor (CstF) and cleavage factor I(m) (CFI(m)), respectively. CFI(m) is composed of a smaller 25 kDa subunit (CFI(m)25) and a larger 59, 68 or 72 kDa subunit. CFI(m)68 interacts with CFI(m)25 through its N-terminal RNA recognition motif (RRM). We recently solved the crystal structures of CFI(m)25 bound to RNA and of a complex of CFI(m)25, the RRM domain of CFI(m)68 and RNA. Our study illustrated the molecular basis for UGUA recognition by the CFI(m) complex, suggested a possible mechanism for CFI(m) mediated alternative polyadenylation, and revealed potential links between CFI(m) and other mRNA processing factors, such as the 20 kDa subunit of the cap binding protein (CBP20), and the splicing regulator U2AF65.

Efficient gene expression requires that, during their lifetime, mRNAs associate with different sets of RNA binding proteins to form messenger ribonucleoprotein particles (mRNPs). The protein components of mRNPs are essential for the correct post-transcriptional function and regulation of mRNAs. mRNPs are constitutively remodeled during the maturation of the mRNA in the nucleus and downstream steps in the cytoplasm, and can also change depending on the cellular environment. Here we review the current understanding of the biochemical and structural aspects of a central mRNP component and regulator, the exon junction complex (EJC).

The discovery of increasing numbers of genes with overlapping sequences highlights the problem of expression in the context of constraining regulatory elements from more than one gene. This study identifies regulatory sequences encompassed within two genes that overlap in an antisense orientation at their 3' ends. The genes encode the alpha-thyroid hormone receptor gene (TRalpha or NR1A1) and Rev-erbalpha (NR1D1). In mammals TRalpha pre-mRNAs are alternatively spliced to yield mRNAs encoding functionally antagonistic proteins: TRalpha1, an authentic thyroid hormone receptor; and TRalpha2, a non-hormone-binding variant that acts as a repressor. TRalpha2-specific splicing requires two regulatory elements that overlap with Rev-erbalpha sequences. Functional mapping of these elements reveals minimal splicing enhancer elements that have evolved within the constraints of the overlapping Rev-erbalpha sequence. These results provide insight into the evolution of regulatory elements within the context of bidirectional coding sequences. They also demonstrate the ability of the genetic code to accommodate multiple layers of information within a given sequence, an important property of the code recently suggested on theoretical grounds.

Group II introns are self-splicing catalytic RNAs found in bacteria and the organelles of fungi and plants. They are thought to share a common ancestor with the spliceosome, which catalyzes the removal of nuclear introns from pre-mRNAs in eukaryotes. Recent structural and biochemical evidence supports the hypothesis that the spliceosome has a catalytic RNA core homologous to that found in group II introns. The crystal structure of a eukaryotic group IIB intron was recently determined and reveals the architecture of a branched lariat RNA that is also formed by the spliceosome. Here we describe the active site components of this intron and propose a model for RNA splicing involving dynamic base triples in the catalytic triad. Based on this structure, we draw analogies to the U2/U6 snRNA pairing and RNA-protein interactions that form in the active site of the spliceosome.

Simian Virus 40 (SV40) is a polyomavirus found in both monkeys and humans, which causes cancer in some animal models. In humans, SV40 has been reported to be associated with cancers but causality has not been proven yet. The transforming activity of SV40 is mainly due to its 94-kD large T antigen, which binds to the retinoblastoma (pRb) and p53 tumor suppressor proteins, and thereby perturbs their functions. Here we describe a 100 kD super T antigen harboring a duplication of the pRB binding domain that was associated with unusual high cell transformation activity and that was generated by a novel mechanism involving homologous RNA trans-splicing of SV40 early transcripts in transformed rodent cells. Enhanced trans-splice activity was observed in clones carrying a single point mutation in the large T antigen 5' donor splice site (ss). This mutation impaired cis-splicing in favor of an alternative trans-splice reaction via a cryptic 5'ss within a second cis-spliced SV40 pre-mRNA molecule and enabled detectable gene expression. Next to the cryptic 5'ss we identified additional trans-splice helper functions, including putative dimerization domains and a splice enhancer sequence. Our findings suggest RNA trans-splicing as a SV40-intrinsic mechanism that supports the diversification of viral RNA and phenotypes.

The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5′ splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome. In our work, we found evidence for a kinetic proofreading mechanism in splicing in which the DEAH-box ATPase Prp16 discriminates against substrates undergoing slow 5′ splice site cleavage. Additionally, our study revealed that discriminated substrates are discarded through a general spliceosome disassembly pathway, mediated by another DEAH-box ATPase Prp43. In their work, Tseng et al. described the underlying molecular events through which Prp16 discriminates against a splicing substrate during 5′ splice site cleavage. Here, we present a synthesis of these two studies and, additionally, provide the first biochemical evidence for discrimination of a suboptimal splicing substrate just prior to 5′ splice site cleavage. Together, these findings support a general mechanism for a ubiquitous superfamily of ATPases in enhancing specificity during RNA-dependent processes in the cell.

Understanding alternative splicing is crucial to elucidate the mechanisms behind several biological phenomena, including diseases. The huge amount of expressed sequences available nowadays represents an opportunity and a challenge to catalog and display alternative splicing events (ASEs). Although several groups have faced this challenge with relative success, we still lack a computational tool that uses a simple and straightforward method to retrieve, name and present ASEs. Here we present SPLOOCE , a portal for the analysis of human splicing variants. SPLOOCE uses a method based on regular expressions for retrieval of ASEs. We propose a simple syntax that is able to capture the complexity of ASEs.

(2010). Editorial on alternative splicing and disease. RNA Biology: Vol. 7, No. 4, pp. 388-389.

Heterogeneous nuclear ribonucleoprotein (hnRNP) L can regulate alternative mRNA splicing in diverse ways, binding to exonic or intronic sites and acting as either an activator or repressor. To investigate the mechanistic basis of hnRNP L-regulated alternative splicing, we focus here on two specific cases of hnRNP L-dependent splice site recognition. First, in the case of TJP1 our microarray data had suggested that exon 20 inclusion is regulated by hnRNP L as a repressor. Here we demonstrate by mutational analysis that exon skipping is mediated by a short silencer sequence consisting of three hnRNP L high-score binding motifs located upstream of the 3' splice site of the regulated exon. UV crosslinking and immunoprecipitation experiments showed that hnRNP L binding interferes with 3' splice site recognition by U2AF65. Second, SLC2A2 contains a CA-repeat sequence close to the 5' splice site of the regulated exon 4. Using psoralen crosslinking, we demonstrate that hnRNP L represses splicing by preventing 5' splice site recognition of the U1 snRNP. In sum, our data provide new insights into the mechanisms of how hnRNP L-bound to intronic sites-regulates exon recognition.

Splicing is a molecular mechanism, by which introns are removed from an mRNA precursor and exons are ligated to form a mature mRNA. Mutations that cause defects in the splicing mechanism are known to be responsible for many diseases, including cystic fibrosis and familial dysautonomia. If mutations that cause defects in splicing are responsible for such severe deleterious phenotypic differences, it is possible that mutations in splicing are also responsible for mildly deleterious phenotypic differences. Although deleterious mutations are rapidly eliminated from the population by purifying selection, the selection against mild deleterious effects is not as strong. Since mildly deleterious mutations have a chance of surviving natural selection, we might be mistakenly referring to these mutations as neutral variation between individuals. Splicing has also been shown to be seriously affected in cancer. Examination of cancerous tissues revealed alterations in expression levels of genes involved in mRNA processing and also a slight reduction in the level of exon skipping--the most common form of alternative splicing in humans. This implies that defects in genes involved in the regulation of splicing in cancerous tissues affect the delicate regulation of the inclusion level of alternatively skipped exons, shifting their mode of splicing back to constitutive. It may be that splicing silencers play a more prominent role in alternative splicing regulation than previously anticipated.

Alignment of the Arabidopsis cDNA to genome DNA sequences revealed that approximately half of the mRNAs were found to contain a 3' nontemplated nucleotide addition prior to the poly(A) sequence. These findings suggest that we can more precisely determine the cleavage sites. Based on the nucleotide downstream of cleavage sites, we are able to derive a hierarchy of cleavage preferences A>>U>C>>G. Interestingly, the completely different hierarchy of preferences was derived to be U>>A>>G>C, based on the nucleotide upstream of cleavage sites. Dinucleotide AU is the most preferred composition for cleavage. The determination of the cleavage sites and systematical analysis can help in the enhancement of 3'-processing site prediction and mechanistic understanding of 3'-end processing in plant.