Evolution of NCoR-1 and NCoR-2 corepressor alternative mRNA splicing in placental mammals.

ObjectiveThe NCoR-1 and NCoR-2 corepressors are products of an early gene duplication near the beginning of vertebrate evolution and play both overlapping and divergent roles in development and physiology. Alternative-splicing of NCoR-1 and NCoR-2 further customizes their functions. To better understand the evolutionary basis of this phenomenon we extended our prior study of NCoR-1 and NCoR-2 alternative-splicing to an expanded series of species.ResultsAlternative-splicing of NCoR-2 was observed in all vertebrates examined whereas alternative-splicing of NCoR-1 was largely limited to placental mammals. Notably the most prominent of the NCoR-1 alternative-splicing events specific to the placental lineage, in exon 37 that plays a key role in murine metabolism, mimics in many features an analogous alternative-splicing event that appeared in NCoR-2 much earlier at the beginning of the vertebrate radiation. Detection of additional alternative-splicing events, at exons 28 in NCoR-1 or NCoR-2, was limited to the Rodentia or Primates examined, indicating both corepressor paralogs continued to acquire additional splice variations more recently and independently of one another. Our results suggest that the NCoR-1/NCoR-2 paralogs have been subject to a mix of shared and distinct selective pressures, resulting in the pattern of divergent and convergent alternative-splicing observed in extant species

Predominant contribution of cis-regulatory divergence in the evolution of mouse alternative splicing

Divergence of alternative splicing represents one of the major driving forces to shape phenotypic diversity during evolution. However, the extent to which these divergences could be explained by the evolving cis-regulatory versus trans-acting factors remains unresolved. To globally investigate the relative contributions of the two factors for the first time in mammals, we measured splicing difference between C57BL/6J and SPRET/EiJ mouse strains and allele-specific splicing pattern in their F1 hybrid. Out of 11,818 alternative splicing events expressed in the cultured fibroblast cells, we identified 796 with significant difference between the parental strains. After integrating allele-specific data from F1 hybrid, we demonstrated that these events could be predominately attributed to cis-regulatory variants, including those residing at and beyond canonical splicing sites. Contrary to previous observations in Drosophila, such predominant contribution was consistently observed across different types of alternative splicing. Further analysis of liver tissues from the same mouse strains and reanalysis of published datasets on other strains showed similar trends, implying in general the predominant contribution of cis-regulatory changes in the evolution of mouse alternative splicing

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

Corepressor diversification by alternative mRNA splicing is species specific.

BackgroundSMRT and NCoR are corepressor paralogs that help mediate transcriptional repression by a variety of transcription factors, including the nuclear hormone receptors. The functions of both corepressors are extensively diversified in mice by alternative mRNA splicing, generating a series of protein variants that differ in different tissues and that exert different, even diametrically opposite, biochemical and biological effects from one another.ResultsWe report here that the alternative splicing previously reported for SMRT appears to be a relatively recent evolutionary phenomenon, with only one of these previously identified sites utilized in a teleost fish and a limited additional number of the additional known sites utilized in a bird, reptile, and marsupial. In contrast, extensive SMRT alternative splicing at these sites was detected among the placental mammals. The alternative splicing of NCoR previously identified in mice (and shown to regulate lipid and carbohydrate metabolism) is likely to have arisen separately and after that of SMRT, and includes an example of convergent evolution.ConclusionsWe propose that the functions of both SMRT and NCoR have been diversified by alternative splicing during evolution to allow customization for different purposes in different tissues and different species

A bioinformatic analysis identifies circadian expression of splicing factors and time-dependent alternative splicing events in the HD-MY-Z cell line

The circadian clock regulates key cellular processes and its dysregulation is associated to several pathologies including cancer. Although the transcriptional regulation of gene expression by the clock machinery is well described, the role of the clock in the regulation of post-transcriptional processes, including splicing, remains poorly understood. In the present work, we investigated the putative interplay between the circadian clock and splicing in a cancer context. For this, we applied a computational pipeline to identify oscillating genes and alternatively spliced transcripts in time-course high-throughput data sets from normal cells and tissues, and cancer cell lines. We investigated the temporal phenotype of clock-controlled genes and splicing factors, and evaluated their impact in alternative splice patterns in the Hodgkin Lymphoma cell line HD-MY-Z. Our data points to a connection between clock-controlled genes and splicing factors, which correlates with temporal alternative splicing in several genes in the HD-MY-Z cell line. These include the genes DPYD, SS18, VIPR1 and IRF4, involved in metabolism, cell cycle, apoptosis and proliferation. Our results highlight a role for the clock as a temporal regulator of alternative splicing, which may impact malignancy in this cellular model

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons

Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous—but not individual—depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α–Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability

Intracerebral Hemorrhage and Ischemic Stroke of Different Etiologies Have Distinct Alternatively Spliced mRNA Profiles in the Blood: a Pilot RNA-seq Study.

Whole transcriptome studies have used 3'-biased expression microarrays to study genes regulated in the blood of stroke patients. However, alternatively spliced messenger RNA isoforms have not been investigated for ischemic stroke or intracerebral hemorrhage (ICH) in animals or humans. Alternative splicing is the mechanism whereby different combinations of exons of a single gene produce distinct mRNA and protein isoforms. Here, we used RNA sequencing (RNA-seq) to determine if alternative splicing differs for ICH and cardioembolic, large vessel and lacunar causes of ischemic stroke compared to controls. RNA libraries from 20 whole blood samples were sequenced to 200 M 2 × 100 bp reads using Illumina sequencing-by-synthesis technology. Differential alternative splicing was assessed using one-way analysis of variance (ANOVA), and differential exon usage was calculated. Four hundred twelve genes displayed differential alternative splicing among the groups (false discovery rate, FDR; p < 0.05). They were involved in cellular immune response, cell death, and cell survival pathways. Distinct expression signatures based on usage of 308 exons (292 genes) differentiated the groups (p < 0.0005; fold change >|1.2|). This pilot study demonstrates that alternatively spliced genes from whole blood differ in ICH compared to ischemic stroke and differ between different ischemic stroke etiologies. These results require validation in a separate cohort

FOXP3 interacts with hnRNPF to modulate pre-mRNA alternative splicing

FOXP3 promotes the development and function of regulatory T cells mainly through regulating the transcription of target genes. RNA alternative splicing has been implicated in a wide range of physiological and pathophysiological processes. We report here that FOXP3 associates with heterogeneous nuclear ribonucleoprotein (hnRNP) F through the exon 2-encoded region of FOXP3 and the second quasi-RNA recognition motif (qRRM) of hnRNPF. FOXP3 represses the ability of hnRNPF to bind to its target pre-mRNA and thus modulates RNA alternative splicing. Furthermore, overexpression of mouse hnRNPF in in vitro-differentiated regulatory T cells (Tregs) reduced their suppressive function. Thus, our studies identify a novel mechanism by which FOXP3 regulates mRNA alternative splicing to modulate the function of regulatory T cells

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

RNA-Seq analysis of splicing in Plasmodium falciparum uncovers new splice junctions, alternative splicing and splicing of antisense transcripts.

Over 50% of genes in Plasmodium falciparum, the deadliest human malaria parasite, contain predicted introns, yet experimental characterization of splicing in this organism remains incomplete. We present here a transcriptome-wide characterization of intraerythrocytic splicing events, as captured by RNA-Seq data from four timepoints of a single highly synchronous culture. Gene model-independent analysis of these data in conjunction with publically available RNA-Seq data with HMMSplicer, an in-house developed splice site detection algorithm, revealed a total of 977 new 5' GU-AG 3' and 5 new 5' GC-AG 3' junctions absent from gene models and ESTs (11% increase to the current annotation). In addition, 310 alternative splicing events were detected in 254 (4.5%) genes, most of which truncate open reading frames. Splicing events antisense to gene models were also detected, revealing complex transcriptional arrangements within the parasite's transcriptome. Interestingly, antisense introns overlap sense introns more than would be expected by chance, perhaps indicating a functional relationship between overlapping transcripts or an inherent organizational property of the transcriptome. Independent experimental validation confirmed over 30 new antisense and alternative junctions. Thus, this largest assemblage of new and alternative splicing events to date in Plasmodium falciparum provides a more precise, dynamic view of the parasite's transcriptome