mRNA前体的选择性剪接

人类基因组草图已基本完成 ,预测人类约有 350 0 0个编码蛋白质的基因 ,只是线虫或果蝇的 2倍[1] 。人类是怎样完成其复杂的生物功能 ?越来越多的证据表明选择性剪接在扩大蛋白质的多样性中发挥重要作用 ,并且有助于解释基因数目与生物复杂程度两者的不一致性。选择性剪接能够从一个基因产生多个转录本 ,从而产生远多于基因数目的蛋白质 ,完成机体的复杂功能及精细调节。1 .ｍＲＮＡ选择性剪接的普遍性目前根据ＥＳＴｓ分析 ,在人类 350 0 0个基因中大约有 40 %的基因具有选择性剪接的形式[1] 。尽管这个数目让人惊讶 ,但这个数目可能比实际…     

Mechano-Regulation of Alternative Splicing

Alternative splicing contributes to the complexity of proteome by producing multiple mRNAs from a single gene. Affymetrix exon arrays and experiments in vivo or in vitro demonstrated that alternative splicing was regulated by mechanical stress. Expression of mechano-growth factor (MGF) which is the splicing isoform of insulin-like growth factor 1(IGF-1) and vascular endothelial growth factor (VEGF) splicing variants such as VEGF₁₂₁, VEGF_165, VEGF₂₀₆, VEGF₁₈₉, VEGF₁₆₅ and VEGF₁₄₅ are regulated by mechanical stress. However, the mechanism of this process is not yet clear. Increasing evidences showed that the possible mechanism is related to Ca²⁺ signal pathway and phosphorylation signal pathway. This review proposes possible mechanisms of mechanical splicing regulation. This will contribute to the biomechanical study of alternative splicing.     

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。     

RNA剪接和剪接调控

ＲＮＡ剪接和剪接调控桂建芳（中国科学院水生生物研究所武汉４３００７２）基因是遗传的功能单位,蛋白是由基因表达的行使生理功能的主体。７０年代以前,人们对基因和蛋白的了解主要是来自于对细菌研究的认识,认为基因、ｍＲＮＡ和蛋白之间是一种线性关系,即基因上的脱氧核苷顺序全部转录成ｍＲＮＡ上的核苷顺序,ｍＲＮＡ上的核苷顺序再全部翻译成蛋白上的氨基酸顺序。     

The Eucaryal tRNA Splicing Endonuclease Recognizes a Tripartite Set of RNA Elements

The tRNA splicing endonuclease cleaves intron-containing tRNA precursors on both sides of the intron. The prevailing belief has been that the enzyme binds only to the mature domain through the invariant bases. We show instead that, for recognition, the endonuclease utilizes distinct sets of structural elements, several of which are within the intron. One subset of recognition elements, localized in the mature domain, is needed for recognition of both cleavage sites, while two other subsets, localized at the exon–intron boundaries, are used for recognition of either one or the other cleavage site. The two cleavage sites are essentially independent: neither is required by the other for cleavage to take place. These results support a two-active-site model for the eucaryal endonuclease.     

Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection

Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3′-half shares high sequence similarity with the TCV 3′ end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3′ end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot ψ₃, which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts.     

基于PCR 技术的人脑基因可变剪接差异性研究

目的:对人脑的基因可变剪接位点差异性进行分析。方法:通过RNA-Seq得到的转录组数据,分析推测出在不同年龄的7对样本的大脑前额叶皮层(prefrontal cortex,PFC)表达的某些基因具有可变剪接位点的差异性;为验证此推测,设计7对引物,提取不同年龄样本的RNA,通过反转录合成cDNA,采用PCR技术扩增目的序列。结果:在已剪接和(或)未剪接产物预期片段处有明显的条带。结论:通过Quantity One软件分析后,确定在不同年龄间存在可变剪接差异。基于PCR技术的基因可变剪接分析对RNA-seq数据集的预测结果进行验证是一种有效新颖的PCR新应用。     

Selective Pressures on RNA Hairpins In Vivo and In Vitro

Comparison of the most stable potential hairpins in the sequences of natural ribozymes with those in the randomized sequences has revealed that the hairpin loop energies are lower than expected by chance. Although these hairpins are not necessarily parts of functional structures, there is a selective pressure to diminish the destabilizing free energies of the hairpin loops. In contrast, no significant bias is observed in the stacking values of the most stable stems. In the ribozymes isolated in vitro the loops of potential hairpins are closer to random values, which can result in less efficient folding rates. Furthermore, the effects of kinetic traps seem to be more significant in the folding pathways of the in vitro isolates due to a potential to form stable stacks incompatible with the functional folds. Similarly to natural ribozyme sequences, the untranslated regions of viral RNAs also form hairpins with relatively low loop free energies. These evolutionary trends suggest ways for efficient engineering of improved RNA constructs on the basis of analysis of in vitro isolates and approaches for the search of regions coding for functional RNA structures in large genome sequences. Received: 12 January 2001 / Accepted: 21 May 2001     

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。     

Catalytic RNA and RNA Splicing

The capacity of Watson-Crick base-pair complementarity to directinformational transactions basic to gene expression has longbeen appreciated. Among RNA molecules, it mediates mRNA-tRNAcodon-anticodon pairing and the 16S rRNA-mRNA Shine-Dalgarnointeraction. More recently, we have come to realize that therole of RNA may transcend that of intermolecular recognition,per se, to include catalysis. Following the tour-de-force studiesof the self-splicing Tetrahymena rRNA precursor, the stage isnow set for the primary role of RNA to be revealed in nuclearpre-RNA splicing, which is catalyzed by a large ribonucleoprotein(RNP) complex in the cell nucleus, called the spliceosome. Theremoval of introns from nuclear pre-messenger RNA (pre-mRNA)shares fundamental properties with certain RNA self-splicingreactions. It therefore seems likely that the major catalyticstrategies in nuclear pre-mRNA splicing are carried out by thesmall nuclear RNAs (snRNAs), which are major constituents ofthe spliceosome.     

Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.     

可变剪接与细胞凋亡调控

细胞凋亡(apoptosis)是多细胞生物的一种基本生命活动,在机体的生长发育、免疫调节及维持内环境稳定等各方面扮演着重要的角色.遗传和生化研究表明,细胞凋亡受到复杂而精细的调控.转录水平、翻译后水平等各种层次的调控,构成了一个复杂的凋亡调控网络.     

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。