Intronic Splicing Regulatory Elements Research Articles

An interpretable model of pre-mRNA splicing for animal and plant genes.

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.

Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing

BackgroundThe presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein-coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA remain elusive.ResultsEmploying in silico analysis, we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on the number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach, we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream-located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3′-splice site-associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay.ConclusionsWe propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.

Does conservation account for splicing patterns?

BackgroundAlternative mRNA splicing is critical to proteomic diversity and tissue and species differentiation. Exclusion of cassette exons, also called exon skipping, is the most common type of alternative splicing in mammals.ResultsWe present a computational model that predicts absolute (though not tissue-differential) percent-spliced-in of cassette exons more accurately than previous models, despite not using any ‘hand-crafted’ biological features such as motif counts. We achieve nearly identical performance using only the conservation score (mammalian phastCons) of each splice junction normalized by average conservation over 100 bp of the corresponding flanking intron, demonstrating that conservation is an unexpectedly powerful indicator of alternative splicing patterns. Using this method, we provide evidence that intronic splicing regulation occurs predominantly within 100 bp of the alternative splice sites and that conserved elements in this region are, as expected, functioning as splicing regulators. We show that among conserved cassette exons, increased conservation of flanking introns is associated with reduced inclusion. We also propose a new definition of intronic splicing regulatory elements (ISREs) that is independent of conservation, and show that most ISREs do not match known binding sites or splicing factors despite being predictive of percent-spliced-in.ConclusionsThese findings suggest that one mechanism for the evolutionary transition from constitutive to alternative splicing is the emergence of cis-acting splicing inhibitors. The association of our ISREs with differences in splicing suggests the existence of novel RNA-binding proteins and/or novel splicing roles for known RNA-binding proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3121-4) contains supplementary material, which is available to authorized users.

P.20 - Cryptic splice site activation by a splice donor site mutation of dystrophin intron 64 is determined by intronic splicing regulatory elements

P.20 - Cryptic splice site activation by a splice donor site mutation of dystrophin intron 64 is determined by intronic splicing regulatory elements

Frequent Gain and Loss of Intronic Splicing Regulatory Elements during the Evolution of Vertebrates

Splicing regulatory elements (SREs) are sequences bound by proteins that influence splicing of nearby splice sites. Constitutively spliced introns have evolved to utilize many different splicing factors. The evolutionary processes that influenced which splicing factors are used for splicing of individual introns are generally unclear. We demonstrate that in the lineage that gave rise to mammals, many introns lost U-rich sequences and gained G-rich sequences, both of which resemble known SREs. The apparent conversion of U-rich to G-rich SREs suggests that the associated splicing factors are functionally equivalent. In support of this we demonstrated that U-rich and G-rich SREs are both capable of promoting splicing of an SRE-dependent splicing reporter. Furthermore, we demonstrate, using the heterologous MS2 tethering system (bacterial MS2 coat fusion-protein and its RNA stem-loop binding site), that both the U-rich SRE-binding protein (TIA1) and the G-rich SRE-binding protein (HNRNPF) can promote splicing of the same intron. We also observed that gain of G-rich SREs is significantly associated with G/C-rich genomic isochores, suggesting that gain or loss of SREs was driven by the same processes that ultimately resulted in the formation of mammalian genomic isochores. We propose the following model for the gain and loss of mammalian SREs. Ancestral U-rich SREs located in genomic regions that were experiencing high rates of A/T to G/C conversion would have suffered frequent deleterious mutations. However, this same process resulted in increased formation of functionally equivalent G-rich SREs, and acquisition of new G-rich SREs decreased purifying selection on the U-rich SREs, which were then free to decay.

CAPN3 mRNA processing alteration caused by splicing mutation associated with novel genomic rearrangement of Alu elements

Recessive mutations of CAPN3 gene are reported to be responsible for limb girdle muscular dystrophy type 2A (LGMD2A). In all, 15-25% of intronic nucleotide changes identified in this gene were investigated by in silico analysis, but occasionally supported by experimental data or reported in some cases as a polymorphism. We report here genetic and transcriptional analyses in three Tunisian patients belonging to the same consanguineous family sharing the same mutation c.1194-9 A>G and Alu repeats insertion in intron 7 of CAPN3 gene. Reverse transcriptase-PCR experiments performed on total RNA from the patient's muscle biopsy showed retention of the eight last nucleotides of intron 9 in the CAPN3 transcript lacking the first seven exons. Our results provide evidence regarding the potential involvement of Alu elements in aberrant processing of pre-mRNA owing to the disruption of pre-existing intronic splicing regulatory elements. We also demonstrated variable mRNA alternative splicing among tissues and between LGMD2A patients. A deep intronic variation and rearrangement have been reported in the literature as causing genetic diseases in humans. However, this is the first report on a potential pathogenic CAPN3 gene mutation resulting from an Alu insertion.

Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene

Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation-induced exon skipping.

Functional selection and systematic analysis of intronic splicing elements identify active sequence motifs and associated splicing factors

Despite the critical role of pre-mRNA splicing in generating proteomic diversity and regulating gene expression, the sequence composition and function of intronic splicing regulatory elements (ISREs) have not been well elucidated. Here, we employed a high-throughput in vivo Screening PLatform for Intronic Control Elements (SPLICE) to identify 125 unique ISRE sequences from a random nucleotide library in human cells. Bioinformatic analyses reveal consensus motifs that resemble splicing regulatory elements and binding sites for characterized splicing factors and that are enriched in the introns of naturally occurring spliced genes, supporting their biological relevance. In vivo characterization, including an RNAi silencing study, demonstrate that ISRE sequences can exhibit combinatorial regulatory activity and that multiple trans-acting factors are involved in the regulatory effect of a single ISRE. Our work provides an initial examination into the sequence characteristics and function of ISREs, providing an important contribution to the splicing code.

An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.

Improved identification of conserved cassette exons using Bayesian networks

BackgroundAlternative splicing is a major contributor to the diversity of eukaryotic transcriptomes and proteomes. Currently, large scale detection of alternative splicing using expressed sequence tags (ESTs) or microarrays does not capture all alternative splicing events. Moreover, for many species genomic data is being produced at a far greater rate than corresponding transcript data, hence in silico methods of predicting alternative splicing have to be improved.ResultsHere, we show that the use of Bayesian networks (BNs) allows accurate prediction of evolutionary conserved exon skipping events. At a stringent false positive rate of 0.5%, our BN achieves an improved true positive rate of 61%, compared to a previously reported 50% on the same dataset using support vector machines (SVMs). Incorporating several novel discriminative features such as intronic splicing regulatory elements leads to the improvement. Features related to mRNA secondary structure increase the prediction performance, corroborating previous findings that secondary structures are important for exon recognition. Random labelling tests rule out overfitting. Cross-validation on another dataset confirms the increased performance. When using the same dataset and the same set of features, the BN matches the performance of an SVM in earlier literature. Remarkably, we could show that about half of the exons which are labelled constitutive but receive a high probability of being alternative by the BN, are in fact alternative exons according to the latest EST data. Finally, we predict exon skipping without using conservation-based features, and achieve a true positive rate of 29% at a false positive rate of 0.5%.ConclusionBNs can be used to achieve accurate identification of alternative exons and provide clues about possible dependencies between relevant features. The near-identical performance of the BN and SVM when using the same features shows that good classification depends more on features than on the choice of classifier. Conservation based features continue to be the most informative, and hence distinguishing alternative exons from constitutive ones without using conservation based features remains a challenging problem.

Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing.

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.

Correction: Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements

In PLoS Genetics, volume 3, issue 5: 10.1371/journal.pgen.0030085 Mr. Eric L. Van Nostrand's name was incorrectly listed in the online citation as Nostrand ELV. The correct citation is: Yeo GW, Van Nostrand EL, Liang TY (2007) Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements. PLoS Genet 3(5): e85 doi:10.1371/journal.pgen.0030085.

Combinatorial Regulation of Protein 4.1R Exon 16 Alternative Splicing: Modulation of Fox-2 Activated Splicing by Other Intronic and Exonic Motifs.

During erythroid differentiation, stage-specific activation of protein 4.1R exon 16 splicing is critical for the mechanical stability of the erythrocyte plasma membrane. The molecular mechanism of this erythroid splicing switch involves multiple factors including stimulation by Fox proteins acting at splicing enhancers in the proximal downstream intron, and inhibition by hnRNPA1 protein acting at silencer elements in exon 16. To explore how the dynamic interplay among these and other factors can fine tune splicing efficiency, we created a series of exon 16-containing minigenes in which splicing efficiency was measured as a function of variation in exon and intron regulatory elements. In the context of wild type exon 16 with intact hnRNP A1 silencer elements and a weak 5′ splice site, an enhancerless construct with no Fox binding sites exhibited little or no exon 16 inclusion in transfected HeLa cells, and over-expression of Fox-2 did not significantly promote inclusion. Insertion of two wild type UGCAUG elements enhanced splicing substantially in a Fox-2-dependent manner and four elements gave even stronger inclusion. Since another study identified the pentamer GCAUG as the Fox binding site, we tested binding site sequence as a potential source of variation in splicing efficiency. Mutation of the first U residue in UGCAUG yielded weaker, but still Fox-2 dependent, activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. To investigate whether enhancer activity of Fox binding sites can be modulated by adjacent sequence motifs, we compared exon 16 splicing efficiency in constructs having Fox sites flanked either by neutral sequence or by an A1 silencer element UAGGG. Introduction of the A1 binding site led to a dramatically reduced enhancer activity including its responsiveness to Fox-2 overexpression. These results indicated that efficiency of splicing of Fox-regulated exons is strongly influenced by the number and sequence of intron enhancer elements and by the presence of adjacent antagonistic elements. In further experiments, we demonstrated that the efficiency of splicing is also strongly dependent on exon 16 and its splice sites. Constructs lacking the major exon 16 silencer element for hnRNP A1 binding exhibited partial exon 16 inclusion in the absence, and very strong inclusion in the presence, of a Fox intron enhancer. Finally, strengthening the weak 5′ splice site of exon 16 abrogated many of these regulatory effects and led to strong inclusion of exon 16 independent of other variables. These findings are consistent with previous data showing antagonism between A1 and Fox in their effects on exon 16 splicing, and suggest that Fox proteins primarily function to overcome the weak 5′ splice site and its repression by hnRNP A1 bound at nearby exonic site(s). We propose that the erythroid alternative splicing program can activate splicing a number of alternative exons with variable efficiency based on each exon's individual complement of exonic and intronic splicing regulatory elements. Modulation of splicing factor expression, typified by the stage-specific down-regulation of hnRNP A1 during erythroblast differentiation, can further alter splicing efficiency of these exons in a selective, motif-dependent manner. Future experiments with exon microarrays will be aimed at identifying some of the alternative exons that are regulated by that program, and determine its importance to the erythroid differentiation process.

Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements in Mammalian Genomes

Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements in Mammalian Genomes

Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred by α-Tropomyosin Regulatory Elements in Transgenic Mice

The mutually exclusive exons 2 and 3 of alpha-tropomyosin (alphaTM) have been used as a model system for strictly regulated alternative splicing. Exon 2 inclusion is only observed at high levels in smooth muscle (SM) tissues, whereas striated muscle and non-muscle cells use predominantly exon 3. Experiments in cell culture have shown that exon 2 selection results from repression of exon 3 and that this repression is mediated by regulatory elements flanking exon 3. We have now tested the cell culture-derived model in transgenic mice. We show that by harnessing the intronic splicing regulatory elements, expression of an enhanced green fluorescent protein transgene with a constitutively active promoter can be restricted to SM cells. Splicing of both endogenous alphaTM and a series of transgenes carrying regulatory element mutations was analyzed by reverse transcriptasePCR. These studies indicated that although SM-rich tissues are equipped to regulate splicing of high levels of endogenous or transgene alphaTM RNA, other non-SM tissues such as spleen, which express lower amounts of alphaTM, also splice significant proportions of exon 2, and this splicing pattern can be recapitulated by transgenes expressed at low levels. We confirm the importance in vivo of the negatively acting regulatory elements for regulated skipping of exon 3. Moreover, we provide evidence that some of the regulatory factors responsible for exon 3 skipping appear to be titratable, with loss of regulated splicing sometimes being associated with high transgene expression levels.