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.     

Regulation of mammalian pre-mRNA splicing

In eukaryotes, most protein-coding genes contain introns which are removed by precursor messenger RNA (pre-mRNA) splicing. Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct proteins. Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression. Mis-regulation of splicing causes a wide range of human diseases. This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing. It also discusses emerging directions in the field of alternative splicing. Supported by the Program of “one Hundred Talented people” of the Chinese Academy of Sciences.     

Stoichiometry of a regulatory splicing complex revealed by single‐molecule analyses

Splicing is regulated by complex interactions of numerous RNA‐binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract‐binding protein (PTB), which regulates tissue‐specific splicing, represses exon 3 of α‐tropomyosin through distant pyrimidine‐rich tracts in the flanking introns. Current models for repression involve either PTB‐mediated looping or the propagation of complexes between tracts. To test these models, we used single‐molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity.     

Identification of a prognostic alternative splicing signature in oral squamous cell carcinoma

Alternative splicing (AS) is critically associated with tumorigenesis and patient's prognosis. Here, we systematically analyzed survival-associated AS signatures in oral squamous cell carcinoma (OSCC) and evaluated their prognostic predictive values. Survival-related AS events were identified by univariate and multivariate Cox regression analyses using OSCC data from the TCGA head neck squamous cell carcinoma data set. The Percent Spliced In calculated by SpliceSeq from 0 to 1 was used to quantify seven types of AS events. A predictive model based on AS events was constructed by least absolute shrinkage and selection operator Cox regression assay and further validated using a training-testing cohort design. Patient survival was estimated using the Kaplan–Meier method and compared with Log-rank test. The receiver operating characteristics curve area under the curves was used to evaluate the predictive abilities of these predictive models. Furthermore, gene–gene interaction networks and the splicing factors (SFs)-AS regulatory network was generated by Cytoscape. A total of 825 survival-related AS events within 719 genes were identified in OSCC samples. The integrative predictive model was better at predicting outcomes of patients as compared to those models built with the individual AS event. The predictive model based on three AS-related genes also effectively predicted patients’ survival. Moreover, seven survival-related SFs were detected in OSCC including RBM4, HNRNPD, and HNRNPC, which have been linked to tumorigenesis. The SF-AS network revealed a significant correlation between survival-related AS genes and these SFs. Our findings revealed a systemic portrait of survival-associated AS events and the splicing network in OSCC, suggesting that AS events might serve as novel prognostic biomarkers and therapeutic targets for OSCC.     

Bioinformatics analysis of alternative splicing

Over the past few years, the analysis of alternative splicing using bioinformatics has emerged as an important new field, and has significantly changed our view of genome function. One exciting front has been the analysis of microarray data to measure alternative splicing genome-wide. Pioneering studies of both human and mouse data have produced algorithms for discerning evidence of alternative splicing and clustering genes and samples by their alternative splicing patterns. Moreover, these data indicate the presence of alternative splice forms in up to 80 per cent of human genes. Comparative genomics studies in both mammals and insects have demonstrated that alternative splicing can in some cases be predicted directly from comparisons of genome sequences, based on heightened sequence conservation and exon length. Such studies have also provided new insights into the connection between alternative splicing and a variety of evolutionary processes such as Alu-based exonisation, exon creation and loss. A number of groups have used a combination of bioinformatics, comparative genomics and experimental validation to identify new motifs for splice regulatory factors, analyse the balance of factors that regulate alternative splicing, and propose a new mechanism for regulation based on the interaction of alternative splicing and nonsense-mediated decay. Bioinformatics studies of the functional impact of alternative splicing have revealed a wide range of regulatory mechanisms, from NAGNAG sites that add a single amino acid; to short peptide segments that can play surprisingly complex roles in switching protein conformation and function (as in the Piccolo C2A domain); to events that entirely remove a specific protein interaction domain or membrane anchoring domain. Common to many bioinformatics studies is a new emphasis on graph representations of alternative splicing structures, which have many advantages for analysis.     

可变剪接调控植物开花的作用机制进展

开花是植物生长发育的关键转折,与种子生产和作物产量密切相关。开花转变受到复杂的基因网络调控,许多开花相关基因通过可变剪接产生多种转录本,调控开花时间。文中从多个角度系统地综述了可变剪接调控植物开花的分子机制,并对将来的研究进行了展望。     

Systematic analysis of survival-associated alternative splicing signatures in clear cell renal cell carcinoma

Alternative splicing (AS) constitutes a major reason for messenger RNA (mRNA) and protein diversity. Increasing studies have shown a link to splicing dysfunction associated with malignant neoplasia. Systematic analysis of AS events in kidney cancer remains poorly reported. Therefore, we generated AS profiles in 533 kidney renal clear cell carcinoma (KIRC) patients in The Cancer Genome Atlas (TCGA) database using RNA-seq data. Then, prognostic models were developed in a primary cohort (N = 351) and validated in a validation cohort (N = 182). In addition, splicing networks were built by integrating bioinformatics analyses. A total of 11 268 and 8083 AS variants were significantly associated with patient overall survival time in the primary and validation KIRC cohorts, respectively, including STAT1, DAZAP1, IDS, NUDT7, and KLHDC4. The AS events in the primary KIRC cohorts served as candidate AS events to screen the independent risk factors associated with survival in the primary cohort and to develop prognostic models. The area under the curve of the receiver-operator characteristic curve for prognostic prediction in the primary and validation KIRC cohorts was 0.84 and 0.82 at 2500 days of overall survival, respectively. In addition, splicing correlation networks revealed key splicing factors (SFs) in KIRC, such as HNRNPH1, HNRNPU, KHDBS1, KHDBS3, SRSF9, RBMX, SFQ, SRP54, HNRNPA0, and SRSF6. In this study, we analyzed the AS landscape in the TCGA KIRC cohort and detected predictors (prognostic) based on AS variants with high performance for risk stratification of the KIRC cohort and revealed key SFs in splicing networks, which could act as underlying mechanisms.     

染色质结构与mRNA可变剪接

mRNA的可变剪接(alternative splicing)是一种由一个mRNA前体(pre-mRNA)通过不同的剪接方式产生多个mRNA变异体(variants)的RNA加工过程。在过去很长一段时间里,人们认为mRNA剪接过程是独立于转录过程的一个转录后RNA加工过程。然而,越来越多的实验证明mRNA剪接在很大程度上是与转录偶联发生的。因此,剪接调控会受到与转录相关因素的调控。本文将对染色质与mRNA剪接调控的相关性和染色质结构调控可变剪接的分子机制进行阐述。     

Regulation of protein diversity by alternative pre-mRNA splicing with specific focus on chondrogenesis

Analysis of the human genome has dramatically demonstrated that the majority of protein diversity is generated by alternative splicing of pre-mRNA. This powerful and versatile mechanism controls the synthesis of functionally different protein isoforms that may be required during specific stages of development from a single gene. Consequently, ubiquitous and/or tissue-specific RNA splicing factors that regulate this splicing mechanism provide the basis for defining phenotypic characteristics of cells during differentiation. In this review, we will introduce the basic mechanisms of pre-mRNA alternative splicing, describe how this process is regulated by specific RNA splicing factors, and relate this to various systems of cell differentiation. Chondrogenesis, a well-defined differentiation pathway necessary for skeletogenesis, will be discussed in detail, with focus on some of the alternatively-spliced proteins known to be expressed during cartilage development. We propose a heuristic view that, ultimately, it is the regulation of these RNA splicing factors that determines the differentiation status of a cell. Studying regulation at the level of pre-mRNA alternative splicing will provide invaluable insights into how many developmental mechanisms are controlled, thus enabling us to manipulate a system to select for a specific differentiation pathway.     

TET‐catalyzed oxidation of intragenic 5‐methylcytosine regulates CTCF‐dependent alternative splicing

Intragenic 5‐methylcytosine and CTCF mediate opposing effects on pre‐mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5‐methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF‐binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF‐mediated alternative splicing through conversion of 5‐methylcytosine to its oxidation derivatives. 5‐hydroxymethylcytosine and 5‐carboxylcytosine are enriched at an intragenic CTCF‐binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5‐methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5‐hydroxymethylcytosine and 5‐methylcytosine at downstream CTCF‐binding sites is a general feature of alternative splicing in naïve and activated CD4⁺ T cells. These findings significantly expand our current concept of the pre‐mRNA “splicing code” to include dynamic intragenic DNA methylation catalyzed by the TET proteins.