Expand+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

Pre-mRNA Secondary Structures Influence Exon Recognition

The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code

Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as “splicing mutations,” but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002–1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5′- and 3′-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002–1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75

Splicing Factors Induce Cystic Fibrosis Transmembrane Regulator Exon 9 Skipping through a Nonevolutionary Conserved Intronic Element

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG(m) and T(n) polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG(m) and T(n) polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG(m) and T(n) polymorphism at the 3' splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibrosis

Control of human papillomavirus gene expression by alternative splicing

Human papillomaviruses possess circular double stranded DNA genomes of around 8 kb in size from which multiple mRNAs are synthesized during an infectious life cycle. Although at least three viral promoters are used to initiate transcription, viral mRNAs are largely the product of processing of pre-mRNAs by alternative splicing and polyadenylation. The HPV life cycle and viral gene expression are tightly linked to differentiation of the epithelium the virus infects: there is an orchestrated production of viral mRNAs and proteins. In this review we describe viral mRNA expression and the roles of the SR and hnRNP proteins that respectively positively and negatively regulate splicing. We discuss HPV regulation of splicing factors and detail the evidence that the papillomavirus E2 protein has splicing-related activities. We highlight the possibility that HPV-mediated control of splicing in differentiating epithelial cells may be necessary to accomplish the viral replication cycle

Computational prediction of splicing regulatory elements shared by Tetrapoda organisms

Background: auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries.Results: a total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks.Conclusion: difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical researc

Identification of a novel TSC2 c.3610G > A, p.G1204R mutation contribute to aberrant splicing in a patient with classical tuberous sclerosis complex: a case report

Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in any organ systems. Mutations in the TSC1 or TSC2 gene lead to the dysfunction of hamartin or tuberin proteins, which cause tuberous sclerosis complex. Case presentation: We describe the clinical characteristics of patients from a Chinese family with tuberous sclerosis complex and analyze the functional consequences of their causal genetic mutations. A novel heterozygous mutation (c.3610G > A) at the last nucleotide of exon 29 in TSC2 was identified. On the protein level, this variant was presumed to be a missense mutation (p.Gly1204Arg). However, the splicing assay revealed that this mutation also leads to the whole TSC2 exon 29 skipping, besides the wild-type transcript. The mutated transcript results in an in-frame deletion of 71 amino acids (p.Gly1133_Thr1203del) and its ratio with the normal splice product is of about 44:56. Conclusions: The novel c.3610G > A TSC2 mutation was identified in association with tuberous sclerosis complex. And it was proven to code both for a missense-carrying transcript (56%), and for an isoform lacking exon 29 (44%)

The Alternative Choice of Constitutive Exons throughout Evolution

Alternative cassette exons are known to originate from two processes exonization of intronic sequences and exon shuffling. Herein, we suggest an additional mechanism by which constitutively spliced exons become alternative cassette exons during evolution. We compiled a dataset of orthologous exons from human and mouse that are constitutively spliced in one species but alternatively spliced in the other. Examination of these exons suggests that the common ancestors were constitutively spliced. We show that relaxation of the 59 splice site during evolution is one of the molecular mechanisms by which exons shift from constitutive to alternative splicing. This shift is associated with the fixation of exonic splicing regulatory sequences (ESRs) that are essential for exon definition and control the inclusion level only after the transition to alternative splicing. The effect of each ESR on splicing and the combinatorial effects between two ESRs are conserved from fish to human. Our results uncover an evolutionary pathway that increases transcriptome diversity by shifting exons from constitutive to alternative splicin

The Expanding Landscape of Alternative Splicing Variation in Human Populations.

Alternative splicing is a tightly regulated biological process by which the number of gene products for any given gene can be greatly expanded. Genomic variants in splicing regulatory sequences can disrupt splicing and cause disease. Recent developments in sequencing technologies and computational biology have allowed researchers to investigate alternative splicing at an unprecedented scale and resolution. Population-scale transcriptome studies have revealed many naturally occurring genetic variants that modulate alternative splicing and consequently influence phenotypic variability and disease susceptibility in human populations. Innovations in experimental and computational tools such as massively parallel reporter assays and deep learning have enabled the rapid screening of genomic variants for their causal impacts on splicing. In this review, we describe technological advances that have greatly increased the speed and scale at which discoveries are made about the genetic variation of alternative splicing. We summarize major findings from population transcriptomic studies of alternative splicing and discuss the implications of these findings for human genetics and medicine