选择性多聚腺苷酸化的生物学效应及其调控机制研究进展

真核生物基因的前体mRNA(pre-mRNA)及一些lncRNA在成熟过程中其3'端会发生剪切和多聚腺苷酸化反应(cleavage and polyadenylation, C/P),C/P的发生需要多聚腺苷酸化信号(polyadenylation signal, PAS)的存在。选择性多聚腺苷酸化(alternative cleavage and polyadenylation, APA)是指具有多个PAS的基因,在其mRNA3'端成熟过程中,由于选择不同的PAS,导致产生出多个3'UTR长度和序列组成不同的转录异构体。3'UTR长度和序列的不同会影响mRNA的稳定性、翻译效率、运输和细胞定位等,因此APA是真核生物的一个重要转录后调控方式。近年来,对大量动物、植物及酵母的基因组测序分析发现,APA在真核生物广泛存在,针对APA的生物学效应和调控机制开展了一系列研究。目前已鉴定出许多APA调控的顺式调控元件和反式作用因子。本文重点介绍了APA生物学效应和调控机制的最新研究进展,并探讨了未来APA调控的研究方向。     

细菌RNA聚腺苷酸化修饰及其调控机制与生理作用

聚腺苷酸化 (polyadenylation) 是指在底物RNA的3′-端加上一段聚腺苷酸残基的转录后修饰作用。1971年,第一次发现真核生物mRNA的3′-端存在多聚腺苷酸 (poly (A)) 尾,它保护mRNA免受核酸外切酶攻击,且对于转录终止、mRNA运输及翻译都起到重要作用,学者们一度将该现象认为是真核细胞mRNA的特征之一。时至今日,细菌RNA聚腺苷酸化现象的发现引起了学术界的高度重视,大量的研究结果不仅证明了该种修饰在细菌中普遍存在,而且发现其在细菌RNA的加工、降解及质量监控中扮演重要的角色;然而,与真核生物不同的是,在原核生物中该修饰倾向于使RNA去稳定化,即加速RNA的降解。本文综述了近年来细菌中RNA聚腺苷酸化修饰及其调控机制与生理作用的研究进展。     

水稻OsJMJ718基因可选择性多聚腺苷酸化序列的 克隆及生殖发育期表达模式

可选择性多聚腺苷酸化是真核生物重要的基因调控机制之一, 通过形成不同长度的3'端非翻译区影响信使RNA的稳定性、定位和翻译效率, 从而增加转录本的复杂度。已有研究表明, 拟南芥(Arabidopsis thaliana)中参与DNA去甲基化调控IBM1基因的可选择性多聚腺苷酸化加工受染色质调节因子EDM2调控, 从而影响拟南芥基因组数千基因的CHG甲基化水平, 但该类调控机制是否在其它物种中同样存在仍然未知。以水稻(Oryza sativa)基因组中IBM1同源基因OsJMJ718为研究对象, 利用生物信息学分析和3'RACE实验, 发现IBM1同源基因也存在可选择性多聚腺苷酸化修饰, OsJMJ718基因可能存在9个可选择性多聚腺苷酸化序列。序列比对分析表明, NCBI网站现存日本晴OsJMJ718基因组3'末端序列与9522和明辉63等其它生态型基因组序列组成可能不同。荧光实时定量PCR分析表明, OsJMJ718的9个转录本在水稻生殖发育阶段呈现不同的动态表达模式, 其中TVX5转录本表达量最高。研究获得的OsJMJ718基因可选择性多聚腺苷酸化序列信息及相关的表达模式分析为进一步揭示水稻OsJMJ718基因的可选择性多聚腺苷酸化分子机制和生物学功能奠定了基础。     

聚腺苷酸特异性核糖核酸酶(PARN)的功能多样性

聚腺苷酸尾的降解对于mRNA的质量控制和转录后基因调控十分重要. 在真核生物中,去腺苷酸化是mRNA降解和翻译沉默的首要限速步骤. 3′核糖核酸外切酶--聚腺苷酸特异性核糖核酸酶（poly(A)-specific ribonuclease,PARN）能够高效降解真核生物mRNA的聚腺苷酸尾. PARN不仅在降解mRNA poly(A)尾中发挥关键的作用,还参与DNA损伤、非编码RNA的加工成熟以及肿瘤等疾病过程. PARN是一种多功能酶分子,本文就PARN发现、结构、催化机制和功能多样性进行综述.     

Yeast基因下游二级结构与多聚腺苷作用信号

形成真核生物mRNA 3′末端的多聚腺苷（poly(A)）作用涉及前体mRNA下游的三个元件：效率元件（EE）、定位元件（PE）以及实际的剪切和poly(A)作用位点,实验研究提出了一些EE和PE的碱基序列组成.对180个Yeast基因下游（终止密码子后200个碱基）二级结构进行的详细分析显示,约86%的EE、89%的PE与二级结构中碱基非配对的环(发夹环、膨胀环、内环或多分支环)区或连接单链区有关.这个结果提示,反式因子对EE和PE的识别和作用在一定程度上有赖于EE和PE的二级结构特征.借助mRNA二级结构可以提高对EE和PE位点预测的准确性.     

MicroRNA引起mRNA降解关键核酸酶CAF1和POP2的生物进化分析

MicroRNA(miRNA)参与调控高等真核生物中三分之一以上基因的表达,其中核酸酶CAF1(CCR4-associated factor 1)及其同源基因POP2在miRNA引发的mRNA 3′端多聚腺苷酸(poly(A))的脱腺苷酸化过程中起了关键作用.通过实时定量RT-PCR的方法检测了小鼠各个组织中CAF1和POP2的相对表达情况,发现CAF1和POP2的组织分布特征不同.大多数组织中CAF1的表达水平明显高于POP2,并且组织间差异很大,特别是在大脑、小脑以及睾丸组织中CAF1的表达量很高,而POP2的表达量和变化幅度都较低.蛋白质序列比对发现,CAF1和POP2是一类进化过程中高度保守的核酸外切酶,在酵母、线虫、果蝇和尾索动物代表物种海鞘中都只存在单一基因,而进化到鱼类后产生了两个同源基因——CAF1和POP2,其中CAF1的氨基酸序列保守性较POP2更高,更加接近于原始的单一序列.CAF1和POP2这一对同源基因氨基酸序列的主要差别在蛋白质的C端.鱼类中POP2的C端序列同CAF1的序列较为接近,而在爬行动物之后POP2产生了与CAF1具有明显差异的C端序列,并逐渐趋于稳定.我们的分析结果同已有的功能研究一致,表明可能在miRNA产生后的进化过程中产生了CAF1和POP2两个同源基因,其中CAF1主要担负miRNA调控mRNA脱腺苷酸化的功能,而POP2可能主要参与其它不同的调控作用。     

乙型肝炎病毒表面抗原基因在异源细胞中的表达研究

本研究构建了一系列乙型肝炎病毒表面抗原(HBsAg)基因的表达载体,其中HBsAg基因的启动区被小鼠金属硫蛋白启动子取代;而对HBsAg基因下游非编码区的几种基因元件则进行了不同的删除,并且添加异源基因调控元件。利用Hela细胞瞬时表达系统对各种构建物的表达水平进行分析。实验发现：除了必须取代HBsAg基因原有的启动子外,乙型肝炎病毒(HBv)增强子Ⅰ及其多聚腺苷酸化(poly A)信号对HBsAg在非肝脏细胞中表达也至关重要。但HBsAg的poIy A信号可用异源的poly A信号及猴空泡病毒(sV 40)T抗原基因的剪接信号取代。在HeLa细胞表达系统中,这种取代可进一步提高HBsAg的表达水平。反之,HBV增强子Ⅱ在HeLa细胞中对HBsAg基因的表达无显著作用。实验结果提示：除了已报道的HBV增强子Ⅰ能激活基因本身的启动子外,很可能还存在另外的功能,即对HBsAg转录体起稳定作用。     

严重急性呼吸综合征冠状病毒2膜蛋白对宿主细胞pre-mRNA 3"UTR加工的影响

目的 研究严重急性呼吸综合征冠状病毒2（SARS-CoV-2）膜蛋白对宿主细胞mRNA前体（pre-mRNA）3"非翻译区（UTR）加工的影响。方法 本研究以人肺上皮细胞系A549为模型，利用瞬时转染在细胞内过表达SARS-CoV-2膜蛋白；利用RNA-Seq测序技术及生物信息学分析方法，系统性描绘宿主细胞选择性多聚腺苷酸化（alternative polyadenylation，APA）事件；Metascape数据库对发生显著APA变化的基因进行功能富集分析；RT-qPCR验证靶基因3"UTR长度变化；蛋白质免疫印迹（Western blot）检测目的蛋白表达水平。结果 SARS-CoV-2膜蛋白外源表达后宿主细胞内共813个基因发生显著APA变化。GO和KEGG分析显示，差异APA基因广泛参与有丝分裂细胞周期、调节细胞应激等生物过程，涉及病毒感染和蛋白质加工等。从中进一步筛选出AKT1基因，在IGV软件中显示3"UTR延长；RT-qPCR验证AKT1基因的3"UTR长度变化趋势；Western blot结果显示AKT1蛋白磷酸化水平增加。结论 SARS-CoV-2膜蛋白潜在影响宿主pre-mRNA的3"UTR加工，其中参与多种病毒性生物过程的AKT1基因 3"UTR延长，且其编码的蛋白质功能在细胞内被激活。     

高通量测序技术在可选择性多聚腺苷酸化研究中的应用

可选择性多聚腺苷酸化(alternative polyadenylation,APA)在基因表达调控中起着重要的作用。位于最后一个外显子的APA可以产生具有不同3'UTR长度的mRNA异构体,在细胞中可影响信使RNA(messager RNA,mRNA)的稳定性、翻译效率、转运及亚细胞定位等。随着第二代高通量测序技术的发展,研究人员近年来发展了许多高通量测序文库制备方法,对全基因组APA进行测序和分析。综述了当前研究全基因组水平APA的技术,并且总结了APA与一系列关键生物现象的关系,包括免疫应答、神经反应、胚胎发育和肿瘤发生等。随着APA研究的深入,APA越发成为新的基因转录与翻译调控的研究热点。     

RNA结合蛋白PABPC1的功能及生物学作用

多聚腺苷酸结合蛋白(poly (A) binding protein,PABP)家族通常被认为是mRNA poly (A)尾的一种保护屏障.其中细胞质多聚腺苷酸结合蛋白1 (cytoplasmic poly (A) binding protein-1,PABPC1)在高亲和力作用下能够与mRNA中富含腺苷酸的序列结合,在基因转录后调控中发挥着重要作用.同时PABPC1还参与mRNA的许多代谢通路,包括腺苷酸多聚化/脱腺苷酸化、m RNA转运、m RNA翻译、降解及mircoRNA相关调控.近年来关于PABPC1与生殖细胞的发育、心肌肥大和肿瘤的发生发展的报道屡见不鲜,可见PABPC1与细胞的生长发育有密切联系.本文将主要介绍PABPC1的结构、表达调控、功能及其生物学作用.     

Formation of the 3' end of histone mRNA: getting closer to the end

Nearly all eukaryotic mRNAs end with a poly(A) tail that is added to their 3' end by the ubiquitous cleavage/polyadenylation machinery. The only known exceptions to this rule are metazoan replication-dependent histone mRNAs, which end with a highly conserved stem-loop structure. This distinct 3' end is generated by specialized 3' end processing machinery that cleaves histone pre-mRNAs 4-5 nucleotides downstream of the stem-loop and consists of the U7 small nuclear RNP (snRNP) and number of protein factors. Recently, the U7 snRNP has been shown to contain a unique Sm core that differs from that of the spliceosomal snRNPs, and an essential heat labile processing factor has been identified as symplekin. In addition, cross-linking studies have pinpointed CPSF-73 as the endonuclease, which catalyzes the cleavage reaction. Thus, many of the critical components of the 3' end processing machinery are now identified. Strikingly, this machinery is not as unique as initially thought but contains at least two factors involved in cleavage/polyadenylation, suggesting that the two mechanisms have a common evolutionary origin. The greatest challenge that lies ahead is to determine how all these factors interact with each other to form a catalytically competent processing complex capable of cleaving histone pre-mRNAs.     

Distinct roles of two Yth1p domains in 3'-end cleavage and polyadenylation of yeast pre-mRNAs

Yth1p is the yeast homologue of the 30 kDa subunit of mammalian cleavage and polyadenylation specificity factor (CPSF). The protein is part of the cleavage and polyadenylation factor CPF, which includes cleavage factor II (CF II) and polyadenylation factor I (PF I), and is required for both steps in pre-mRNA 3'-end processing. Yth1p is an RNA-binding protein that was previously shown to be essential for polyadenylation. Here, we demonstrate that Yth1p is also required for the cleavage reaction and that two protein domains have distinct roles in 3'-end processing. The C-terminal part is required in polyadenylation to tether Fip1p and poly(A) polymerase to the rest of CPF. A single point mutation in the highly conserved second zinc finger impairs both cleavage and polyadenylation, and affects the ability of Yth1p to interact with the pre-mRNA and other CPF subunits. Finally, we find that Yth1p binds to CYC1 pre-mRNA in the vicinity of the cleavage site. Our results indicate that Yth1p is important for the integrity of CPF and participates in the recognition of the cleavage site.     

A physical and functional link between splicing factors promotes pre-mRNA 3′ end processing

Polypyrimidine tract-binding protein (PTB) is a splicing regulator that also plays a positive role in pre-mRNA 3′ end processing when bound upstream of the polyadenylation signal (pA signal). Here, we address the mechanism of PTB stimulatory function in mRNA 3′ end formation. We identify PTB as the protein factor whose binding to the human β-globin (HBB) 3′ UTR is abrogated by a 3′ end processing-inactivating mutation. We show that PTB promotes both in vitro 3′ end cleavage and polyadenylation and recruits directly the splicing factor hnRNP H to G-rich sequences associated with several pA signals. Increased binding of hnRNP H results in stimulation of polyadenylation through a direct interaction with poly(A) polymerase. Therefore, our results provide evidence of a concerted regulation of pA signal recognition by splicing factors bound to auxiliary polyadenylation sequence elements.     

A flexible linker region in Fip1 is needed for efficient mRNA polyadenylation

Synthesis of the poly(A) tail of mRNA in Saccharomyces cerevisiae requires recruitment of the polymerase Pap1 to the 3' end of cleaved pre-mRNA. This is made possible by the tethering of Pap1 to the Cleavage/Polyadenylation Factor (CPF) by Fip1. We have recently reported that Fip1 is an unstructured protein in solution, and proposed that it might maintain this conformation as part of CPF, when bound to Pap1. However, the role that this feature of Fip1 plays in 3' end processing has not been investigated. We show here that Fip1 has a flexible linker in the middle of the protein, and that removal or replacement of the linker affects the efficiency of polyadenylation. However, the point of tethering is not crucial, as a fusion protein of Pap1 and Fip1 is fully functional in cells lacking genes encoding the essential individual proteins, and directly tethering Pap1 to RNA increases the rate of poly(A) addition. We also find that the linker region of Fip1 provides a platform for critical interactions with other parts of the processing machinery. Our results indicate that the Fip1 linker, through its flexibility and protein/protein interactions, allows Pap1 to reach the 3' end of the cleaved RNA and efficiently initiate poly(A) addition.     

U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

Insufficient expression of the survival motor neuron (SMN) protein causes spinal muscular atrophy, a neurodegenerative disease characterized by loss of motor neurons. Despite the importance of maintaining adequate SMN levels, little is known about factors that control SMN expression, particularly 3′ end processing of the SMN pre-mRNA. In this study, we identify the U1A protein as a key regulator of SMN expression. U1A, a component of the U1 snRNP, is known to inhibit polyadenylation upon direct binding to mRNA. We show that U1A binds directly and with high affinity and specificity to the SMN 3′-UTR adjacent to the polyadenylation site, independent of the U1 snRNP (U1 small nuclear ribonucleoprotein). Binding of U1A inhibits polyadenylation of the SMN pre-mRNA by specifically inhibiting 3′ cleavage by the cleavage and polyadenylation specificity factor. Expression of U1A in excess of U1 snRNA causes inhibition of SMN polyadenylation and decreases SMN protein levels. This work reveals a new mechanism for regulating SMN levels and provides new insight into the roles of U1A in 3′ processing of mRNAs.