mRNA选择性剪接的分子机制

真核细胞mRNA前体经过剪接成为成熟的mRNA,而mRNA前体的选择性剪接极大地增加了蛋白质的多样性和基因表达的复杂程度,剪接位点的识别可以以跨越内含子的机制(内含子限定)或跨越外显子的机制(外显子限定)进行。选择性剪接有多种剪接形式：选择不同的剪接位点,选择不同的剪接末端,外显子的不同组合及内含子的剪接与否等。选择性剪接过程受到许多顺式元件和反式因子的调控,并与基本剪接过程紧密联系,剪接体中的一些剪接因子也参与了对选择性剪接的调控。选择性剪接也是1个伴随转录发生的过程,不同的启动子可调控产生不同的剪接产物。mRNA的选择性剪接机制多种多样,已发现RNA编辑和反式剪接也可参与选择性剪接过程。     

基因选择性剪接的生物信息学研究概况

基因选择性剪接现象是真核生物基本而又重要的调控机制。由于基因的选择性剪接在形成生物复杂性和多样性上具有极其重要的作用,同时选择性剪接与许多人类疾病也密切相关。因此,研究基因选择性剪接是一项十分重要的工作。生物信息学作为一门新兴的学科在研究基因选择性剪接上起关键的作用,尤其在研究基因表达调控机制、选择性剪接基因预测以及选择性剪接基因进化上。本文综述了这方面的最新研究进展,为更深入了解真核生物基因的表达调控机理提供依据。     

Alternative promoters in expression of genetic information

Many genes are known to have several promoters. The contribution of alternative promoters to the structural and functional diversity of protein isoforms in eukaryotic cells is considered, including their role in synthesis of identical proteins from different mRNAs, generation of protein isoforms with different and even opposite functions, expression of housekeeping genes, and the variation of the recognition domains of adhesion proteins and receptors. In some cases, alternative promoters allow one gene to produce mRNAs with different open reading frames and, consequently, proteins with no amino acid sequence homology.     

寻找mRNA的选择性剪接

在真核生物的基因中,mRNA选择性剪接现象十分普遍。mRNA选择性剪接导致一个基因多转录本的产生,被认为是高等生物增加蛋白质多样性的主要机制,且已发现与许多人类疾病密切相关。发现这些转录本的选择性剪接位点、新的外显子和外显子组合,乃至获得这些剪接变异体的完整克隆,对于基因功能的深入研究十分必要。简要介绍了几种在mRNA水平探索选择性剪接的方法。     

植物基因选择性剪接:断舍离合,达权知变

选择性剪接是基因转录后的mRNA通过不同的剪接方式产生多样性成熟转录本的过程,是真核生物细胞中一种重要的转录后调控方式。植物基因也常通过这种断舍离合的方式产生一专多能的转录本,达到调节多种生命活动的目的。相较于动物,植物中该领域的研究起步较晚,但近年来也取得了长足的进步。该文综述了植物基因选择性剪接的生物学意义、剪接方式和机制、研究方法以及在生长发育与环境胁迫适应性调节中的作用,并展望了未来的研究方向。     

Analysis of alternative cleavage and polyadenylation in mature and differentiating neurons using RNA-seq data

Background: Most eukaryotic protein-coding genes exhibit alternative cleavage and polyadenylation (APA), resulting in mRNA isoforms with different 3′ untranslated regions (3′ UTRs). Studies have shown that brain cells tend to express long 3′ UTR isoforms using distal cleavage and polyadenylation sites (PASs). Methods: Using our recently developed, comprehensive PAS database PolyA_DB, we developed an efficient method to examine APA, named Significance Analysis of Alternative Polyadenylation using RNA-seq (SAAP-RS). We applied this method to study APA in brain cells and neurogenesis. Results: We found that neurons globally express longer 3′ UTRs than other cell types in brain, and microglia and endothelial cells express substantially shorter 3′ UTRs. We show that the 3′ UTR diversity across brain cells can be corroborated with single cell sequencing data. Further analysis of APA regulation of 3′ UTRs during differentiation of embryonic stem cells into neurons indicates that a large fraction of the APA events regulated in neurogenesis are similarly modulated in myogenesis, but to a much greater extent. Conclusion: Together, our data delineate APA profiles in different brain cells and indicate that APA regulation in neurogenesis is largely an augmented process taking place in other types of cell differentiation.     

SF2/ASF regulates proteomic diversity by affecting the balance between translation initiation mechanisms

Post‐splicing activities have been described for a subset of shuttling serine/arginine‐rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras‐PI 3‐kinase‐Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co‐regulated levels of isoform‐specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5′‐terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap‐independent manner by the action of cis‐acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap‐dependent manner while the other by internal ribosome entry site‐dependent initiation, to show that in vivo over‐expression of SF2/ASF increases the ratio between cap‐dependent and internal ribosome entry site‐dependent translation. Consistently, knocking‐down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor‐2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field. J. Cell. Biochem. 107: 826–833, 2009. © 2009 Wiley‐Liss, Inc.     

Alternative translation start sites and their significance for eukaryotic proteome

Current models of translation initiation and contextual organization of eukaryotic mRNA leader region are reviewed. Hypothesis on frequent usage of several alternative start codons is discussed. Potential contribution of alternative translation start sites to eukaryotic mRNA coding potential is considered.     

Structural and functional diversity generated by alternative mRNA splicing

Alternative mRNA splicing is becoming increasingly recognized as an important mechanism for the generation of structural and functional diversity in proteins. Recent estimations predict that approximately 50% of all eukaryotic proteins can be alternatively spliced. Several lines of evidence suggest that alternative mRNA splicing results in small changes in protein structure and is likely to fine-tune the function and specificity of the affected protein. However, knowledge of how alternative splicing regulates cellular processes on the molecular level is still limited. It is only recently that structures of alternatively spliced proteins have been solved. These studies have shown that alternative splicing affects the structure not only in the vicinity of the splice site but also at long distance.     

真核基因可变剪接研究现状与展望

mRNA前体(pre-mRNA)的可变剪接是控制基因表达和产生蛋白质多样性的重要机制,是功能基因组时代的研究重点之一。生物信息学在识别可变剪接基因及其结构、分析可变剪接的功能和调控方式等方面具有重要作用。除了耗时的实验研究,识别可变剪接基因及其结构主要通过EST、mRNA等转录数据与基因组序列进行比对,获得同一基因的不同结构方式。分析蛋白质产物可对可变剪接的功能进行预测;潜在调控元件的统计分析则可为可变剪接调控机制的研究提供必要的数据。转录数据的时空信息以及比较基因组学对理解可变剪接信息的精确调控将提供重要资料。可变剪接及其调控机制的深入研究将为基因组和蛋白质组之间的对接提供重要的桥梁。     

可变剪切在植物成花转换中的作用

精确调控成花转换,确保植物在适宜环境下开花,对于植物的成功繁殖和物种繁衍至关重要。开花由多种分子机制在转录、转录后和蛋白质水平进行调控。可变剪切(AS)是一种普遍的转录后水平调控过程,可从单个基因产生多个转录本,从而丰富转录组和蛋白质组的多样性。大量研究表明,可变剪切在成花转换过程中发挥重要作用。根据发育和环境条件, AS能够影响mRNA的稳定性和/或蛋白亚型的功能,从而调控开花相关基因的功能转录本和/或功能蛋白水平。揭示成花相关pre-mRNA的AS作用将进一步增进人们对开花相关基因功能以及整个成花转换调控网络的认识。该文归纳了涉及成花转换的AS研究进展,并针对各个调控途径进行总结,以期为进一步研究植物AS和成花转换调控机制提供参考。     

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

It is widely suggested that a eukaryotic mRNA typically contains one translation start site and encodes a single functional protein product. However, according to current points of view on translation initiation mechanisms, eukaryotic ribosomes can recognize several alternative translation start sites and the number of experimentally verified examples of alternative translation is growing rapidly. Also, the frequent occurrence of alternative translation events and their functional significance are supported by the results of computational evaluations. The functional role of alternative translation and its contribution to eukaryotic proteome complexity are discussed.     

Alternative polyadenylation: New insights from global analyses

Recent studies have revealed widespread mRNA alternative polyadenylation (APA) in eukaryotes and its dynamic spatial and temporal regulation. APA not only generates proteomic and functional diversity, but also plays important roles in regulating gene expression. Global deregulation of APA has been demonstrated in a variety of human diseases. Recent exciting advances in the field have been made possible in a large part by high throughput analyses using newly developed experimental tools. Here I review the recent progress in global studies of APA and the insights that have emerged from these and other studies that use more conventional methods.     

Diverse roles of hnRNP L in mammalian mRNA processing: a combined microarray and RNAi analysis

Alternative mRNA splicing patterns are determined by the combinatorial control of regulator proteins and their target RNA sequences. We have recently characterized human hnRNP L as a global regulator of alternative splicing, binding to diverse C/A-rich elements. To systematically identify hnRNP L target genes on a genome-wide level, we have combined splice-sensitive microarray analysis and an RNAi-knockdown approach. As a result, we describe 11 target genes of hnRNP L that were validated by RT-PCR and that represent several new modes of hnRNP L-dependent splicing regulation, involving both activator and repressor functions: first, intron retention; second, inclusion or skipping of cassette-type exons; third, suppression of multiple exons; and fourth, alternative poly(A) site selection. In sum, this approach revealed a surprising diversity of splicing-regulatory processes as well as poly(A) site selection in which hnRNP L is involved.