表观遗传因素在RNA剪接过程中的作用

RNA剪接是真核生物基因表达过程中的重要环节,增加了蛋白质的多样性和基因表达的可调节性. 日益增多的研究表明,RNA剪接并不是独立的生物过程.RNA Ⅱ型聚合酶(RNA polymerase-Ⅱ, RNA Pol Ⅱ)、核小体定位和组蛋白修饰等因素都与RNA剪接过程密切相关.阐明RNA Pol Ⅱ、核小体定位和组蛋白修饰等因素在RNA剪接过程中的作用,将为剪接位点的准确识别和剪接调控机制的研究提供新思路.本文综述了RNA Pol Ⅱ、核小体定位和组蛋白修饰等因素对RNA剪接的影响以及它们在RNA剪接过程中的调控作用.     

一种新的Srrm1/SRm160可变剪接体经历无义突变介导的mRNA降解

目的:研究基因Srrm1/SRm160的可变剪接。方法:应用RT-PCR研究Srrm1/SRm160的可变剪接,通过蛋白质的翻译抑制和RNA干扰研究剪接异构体是否经历无义突变介导的mRNA降解(NMD)过程。结果:获得Srrm1/SRm160新的可变剪接异构体,该异构体产生提前终止密码子,翻译抑制和RNA干扰证实含有提前终止密码子的剪接体经过NMD而降解。结论:Srrm1/SRm160通过可变剪接和NMD调节自身的表达水平,作为剪接因子进一步调节其他基因的可变剪接。     

乙型肝炎病毒前S基因区缺失突变发生机制的探讨

检测慢性乙型肝炎病毒(HBV)携带者和患者外周血内HBV前S区基因缺失突变的分子结构特点,探讨其发生机理.用聚合酶链反应方法从慢性乙肝患者和携带者血清中扩增出前S区基因片段,克隆、测序,分析缺失发生的结构特点,从而推测这些前S区基因缺失突变的产生机制.从262例慢性乙肝患者和103例无症状HBV携带者体内扩增出前S区片段,共在30例患者和携带者中检测出多种前S区基因缺失突变,主要集中于前S1区的3′端和前S2区的5′端.其中有9例患者和携带者体内存在完全一样的nt3019～nt3201 183bp的缺失突变,该缺失突变符合真核细胞mRNA剪接机制,在此位置上各基因型的序列高度保守.同时有另外两种缺失突变,即nt3019～nt3147 129bp缺失、nt3019～nt3109 91bp缺失也符合该剪接机制.有23种缺失突变部分于重复序列之间,符合逆转录过程中的模板转换机制所导致的缺失.根据前基因组RNA预测出二级结构,仅部分缺失突变在RNA二级结构中对应于局部的结构.此结果表明:HBV在外界因素mRNA的剪接机制和内在因素聚合酶蛋白的功能特点的共同作用下,产生各种突变,不同的机制将导致不同类型的缺失突变.除真核细胞mRNA剪接机制外,逆转录过程中的模板转换是主要机制之一.     

选择性剪接与肿瘤靶向治疗

RNA剪接过程受到多种调节因子作用,以保证前体mRNA剪接的准确性.但是大量研究发现,在人类肿瘤中经常发生选择性剪接的异常或者来自特定癌症基因的剪接调控元件的突变.因此,RN A剪接调节剂作为一类新的癌蛋白和肿瘤抑制因子而逐渐受到关注,并有望通过调节参与致癌基因的RN A而达到治疗肿瘤的效果.改变RN A的异常剪接是治...     

鱼类基因内含子研究进展

内含子是指断裂基因中的非编码区序列,在编码蛋白质前被去除。在高等生物中,内含子的长度远大于外显子,大部分随机突变会发生在内含子中。因此,内含子的存在使高等生物对突变的耐受能力大大增强了。研究表明,内含子可以提高基因表达效率;影响RNA的转录、剪接加工、出核孔以及翻译等过程;启动某些基因的表达;并通过选择性剪接调控基因的表达。内含子功能的研究成果给当前鱼类免疫基因研究开拓了全新的视野。对内含子的分类、剪接、功能以及鱼类内含子研究的新进展进行了综述,并展望了内含子在鱼类免疫基因研究中的应用。     

环形RNA全长组装与定量工具的研究进展

环形RNA是一种广泛存在于真核细胞的内源性RNA,由前体RNA反向剪接而成,不具有5’末端帽子和3’末端poly(A)尾巴,呈封闭环状结构。环形RNA通过miRNA海绵结合等方式参与基因表达调控等许多重要的生物学过程。环形RNA可以通过可变剪接产生不同的环形RNA转录本,因此获取环形RNA转录本内部全长序列信息以及对环形RNA内部可变剪接产物进行精确定量是揭示环形RNA调控功能的前提。生物信息学工具能够高效便捷的处理高通量测序数据,被普遍用来鉴别和分析环形RNA。本文介绍了环形RNA的产生机制以及功能特性,对环形RNA检测、全长序列组装以及定量相关计算工具进行综述。     

非完全互补shRNA对RNA聚合酶II启动子调控的RNAi的影响

RNA干扰（RNAi）是一种转录后基因沉默技术,可有效诱导序列特异性基因沉默．由RNA聚合酶Ⅱ启动子调控表达的小发卡RNA可有效介导RNAi效应,为组织特异性基因沉默提供了一条新的途径．但是,由RNA聚合酶Ⅱ启动子调控表达的小发卡RNA（shRNA）在序列上与靶基因非完全互补对RNAi效应的影响鲜有报道．本文初步探索RNA聚合酶Ⅱ启动子调控表达的shRNA碱基发生突变或缺失对RNAi效应的影响．研究表明,靶向hTERT mRNA的碱基突变shRNA显著降低RNAi效应,而靶向GFP mRNA的碱基缺失shRNA对RNAi效应没有显著影响．本研究为非完全互补shRNA对RNAi效应的进一步深入研究提供了理论与实验依据．     

环状RNA计算预测方法的研究进展

环状RNA(circular RNA,circRNA)是一类具有重要生物作用的内源性RNA,大多在可变剪接过程中通过5'端和3'端反向共价连接形成闭合环状结构.目前,环状RNA的识别策略主要分为两大类:一类方法从高通量测序(RNA-seq)数据中检测反向剪接位点,另一类直接从RNA序列中检测成环特征.由于数据本身和识别...     

NF1微基因及突变体微基因的克隆与鉴定

Ⅰ型神经纤维瘤病(Neurofibromatosis typeⅠ,NF1)是一种常染色体的显性遗传病,其致病性是由NF1基因突变引起。NF1基因m RNA前体受可变剪接的影响,NF1微基因是研究其可变剪接机制的重要工具。本研究从人类外周血中提取基因组DNA,并设计出克隆NF1及突变目的片段的两对引物,利用PCR方法扩增目的片段并导入p EGFP质粒载体,从而构建了分别包含NF1微基因及突变体微基因的载体p EGFP-NF1和p EGFP-NF1m。分别用Bam HⅠ和HindⅢ对NF1微基因及突变体微基因进行双酶切鉴定,均得到预期片段,测序结果正确,表明成功构建了NF1微基因及突变体微基因载体。用转染试剂将NF1微基因和突变体微基因的质粒转染到Hela细胞中,荧光检测说明真核表达载体NF1微基因与突变体微基因成功转染到细胞中。本研究为后续NF1的m RNA前体可变剪接以及调控NF1剪接因子的研究提供了参考。     

Pre-mRNA选择性剪接的调控及选择性剪接数据库

Pre-mRNA选择性剪接是真核生物转录组和蛋白质组多样性的主要来源,也是细胞分化、发育等过程中重要的基因表达调控方式。约95%的人类多外显子基因存在RNA选择性剪接|很多人类基因疾病的发生与RNA剪接错误相关。随着共转录现象的发现,RNA选择性剪接调控机制研究也取得了很大进展。本文分别从序列层面和核小体定位、组蛋白修饰、DNA甲基化及非编码RNA等表观遗传层面,系统地阐述了RNA选择性剪接的调控机制。为便于搜索,本文介绍了近10年来RNA选择性剪接相关的数据库。     

Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZ_EI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.     

The splicing machinery is a genetic modifier of disease severity

Disease severity correlates with the level of correctly spliced RNA transcribed from genes carrying splicing mutations and with the ratio of alternatively spliced isoforms. Hence, a role for splicing regulation as a genetic modifier has been suggested. Here we discuss recent experiments that provide direct evidence that changes in the level of splicing factors modulate the splicing pattern of disease-associated genes. Importantly, modulation of the splicing pattern led to regulation of the protein function and modification of disease severity.     

A DExH/D-box protein coordinates the two steps of splicing in a group I intron

Proteins of the DExH/D family are ATPases that can unwind duplex RNA in vitro. Individual members of this family coordinate many steps in ribonucleoprotein enzyme assembly and catalysis in vivo, but it is largely unknown how the action of these co-factors is specified and precisely timed. As a first step to address this question biochemically, we describe the development of a new protein-dependent group I intron splicing system that requires such an ATPase for coordinating successive steps in splicing. While genetic analysis in yeast has shown that at least five nuclear-encoded proteins are required for splicing of the mitochondrial aI5β group I intron, we show that efficient in vitro splicing of aI5β occurs with only two of these co-factors and, furthermore, they fulfill distinct functions in vitro. The Mrs1p protein stabilizes RNA structure and promotes the first step in splicing. In contrast, a DExH/D protein, Mss116p, acts after the first step and, utilizing ATP hydrolysis, specifically enhances the efficiency of exon ligation. An analysis of Mss116p variants with mutations that impair its RNA-stimulated ATP hydrolysis activity or reduce its ability to unwind duplexes show that the efficiency of ATP hydrolysis is a major determinant in promoting exon ligation. These observations suggest that Mss116p acts in aI5β splicing by catalyzing changes in the structure of the RNA/protein splicing intermediate that promote the second step. More broadly, these observations are consistent with a model in which the “functional-timing” of DExH/D-box protein action can be specified by a specific conformation of its substrate due to the “upstream” activity of other co-factors.     

Identical G+1 to A mutations in three different introns of the type III procollagen gene (COL3A1) produce different patterns of RNA splicing in three variants of Ehlers-Danlos syndrome. IV. An explanation for exon skipping some mutations and not others

Identical G+1 mutations in three different introns of the gene for type III procollagen (COL3A1) that cause aberrant splicing of RNA were found in three probands with life-threatening variants of Ehlers-Danlos syndrome. Because the three mutations were in a gene with multiple and homologous exons, they provided an interesting test for factors that influence aberrant splicing. The G+1 to A mutation in intron 16 caused extensive exon skipping, the G+1 to A mutation in intron 20 caused both use of a cryptic splice site and retention of all the intron sequences, and the G+1 to A mutation in intron 42 caused efficient use of a single cryptic splice site. The different patterns of RNA splicing were not explained by evaluation of potential cryptic splice sites in the introns by either their homology with 5'-splice sites from other genes or by their delta G(0)37 values for binding to U1 RNA. Instead, the results suggested that the patterns of aberrant RNA splicing were primarily determined by the relative rates at which adjacent introns were normally spliced.     

Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences

Information theory-based methods have been shown to be sensitive and specific for predicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the software predicts variants affecting mRNA splicing. Individual information contents (in bits) of reference and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines (U2OS, U251 and A431), which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1-5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.     

Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences

Disease causing aberrations in both tuberous sclerosis predisposing genes, TSC1 and TSC2, comprise nearly every type of alteration with a predominance of small truncating mutations distributed over both genes. We performed an RNA based screening of the entire coding regions of both TSC genes applying the protein truncation test (PTT) and identified a high proportion of unusual splicing abnormalities affecting the TSC2 gene. Two cases exhibited different splice acceptor mutations in intron 9 (IVS9-15G-->A and IVS9-3C-->G) both accompanied by exon 10 skipping and simultaneous usage of a cryptic splice acceptor in exon 10. Another splice acceptor mutation (IVS38-18A-->G) destroyed the putative polypyrimidine structure in intron 38 and resulted in simultaneous intron retention and usage of a downstream cryptic splice acceptor in exon 39. Another patient bore a C-->T transition in intron 8 (IVS8+281C-->T) activating a splice donor site and resulting in the inclusion of a newly recognised exon in the mRNA followed by a premature stop. These splice variants deduced from experimental results are additionally supported by RNA secondary structure analysis based on free energy minimisation. Three of the reported splicing anomalies are due to sequence changes remote from exon/intron boundaries, described for the first time in TSC. These findings highlight the significance of investigating intronic changes and their consequences on the mRNA level as disease causing mutations in TSC.     

Editor's choice: Functional classification of 15 million SNPs detected from diverse chicken populations

Next-generation sequencing has prompted a surge of discovery of millions of genetic variants from vertebrate genomes. Besides applications in genetic association and linkage studies, a fraction of these variants will have functional consequences. This study describes detection and characterization of 15 million SNPs from chicken genome with the goal to predict variants with potential functional implications (pfVars) from both coding and non-coding regions. The study reports: 183K amino acid-altering SNPs of which 48% predicted as evolutionary intolerant, 13K splicing variants, 51K likely to alter RNA secondary structures, 500K within most conserved elements and 3K from non-coding RNAs. Regions of local fixation within commercial broiler and layer lines were investigated as potential selective sweeps using genome-wide SNP data. Relationships with phenotypes, if any, of the pfVars were explored by overlaying the sweep regions with known QTLs. Based on this, the candidate genes and/or causal mutations for a number of important traits are discussed. Although the fixed variants within sweep regions were enriched with non-coding SNPs, some non-synonymous-intolerant mutations reached fixation, suggesting their possible adaptive advantage. The results presented in this study are expected to have important implications for future genomic research to identify candidate causal mutations and in poultry breeding.     

癌症与可变剪接

可变剪接在发育、分化和癌症等过程中发挥着非常重要的作用。近年来,越来越多的研究表明可变剪接与癌症有着密切的关系,许多癌症相关基因受可变剪接调控。由于癌症特异性的剪接变体具有明显的诊断价值,使得对癌症与可变剪接的研究成为热点。简要概述了癌症相关基因的可变剪接、可变剪接变体的鉴定方法、可变剪接与癌症治疗等研究进展。