东方蜜蜂微孢子虫基因的可变剪接及可变腺苷酸化解析

本研究旨在基于已获得的第三代纳米孔全长转录组数据对东方蜜蜂微孢子虫Nosema ceranae基因的可变剪接(alternative splicing,AS)和可变多聚腺苷酸化(alternative polyadenylation,APA)进行分析.通过Astalavista软件鉴定东方蜜蜂微孢子虫基因的AS事件类型...     

结合三代测序与二代测序技术揭示蜜蜂球囊菌孢子转录组的复杂性

【目的】蜜蜂球囊菌(Ascosphaera apis)是一种专性侵染蜜蜂幼虫的致死性真菌病原。本研究旨在利用PacBio单分子实时(singlemoleculereal-time,SMRT)测序技术对蜜蜂球囊菌孢子(AaS)中基因的可变剪切(alternative splicing,AS)和可变多聚腺苷酸化(alternative polyadenylation,APA)以及长链非编码RNA (long non-coding RNA,lncRNA)进行鉴定和分析,进而揭示蜜蜂球囊菌孢子中转录组的复杂性。【方法】采用Suppa软件对蜜蜂球囊菌孢子中基因的AS事件进行鉴定。通过RT-PCR对不同类型的AS事件进行验证。采用TAPIS pipeline对蜜蜂球囊菌孢子基因的APA位点进行鉴定。利用MEME软件分析孢子全长转录本的poly(A)剪接位点上游50bp的序列特征并鉴定motif。联用CPC和CNCI软件和比对Swiss-prot数据库的方法预测lncRNA,取三者的交集作为高可信度的lncRNA集合。进一步比较lncRNA和mRNA的转录本长度,外显子数量与长度,内含子长度,GC含...     

基于第三代长读段测序数据解析蜜蜂球囊菌基因的可变剪切与可变腺苷酸化

【目的】蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)是一种专性侵染蜜蜂幼虫的真菌病原,导致的白垩病是严重影响养蜂生产的顽疾,每年给养蜂业造成较大损失。本研究旨在基于已获得的第三代长读段测序数据对球囊菌菌丝(Aam)和孢子(Aas)中基因的可变剪切(alternative splicing,AS)和可变多聚腺苷酸化(alternative polyadenylation,APA)进行深入分析。【方法】利用Astalavista软件鉴定Aam和Aas中基因的AS事件类型。利用IGV浏览器对部分剪切异构体(isoform)的结构进行可视化。通过TAPIS pipeline对Aam和Aas中基因的APA位点进行鉴定。通过MEME软件对Aam和Aas中全长转录本的APA位点上游50bp的序列特征进行分析并对motif进行鉴定。【结果】在Aam中共鉴定到286次AS事件,包括162次RI(Retained intron),87次A3(Alternative 3'splice-site)、32次A5(Alternative 5'splice-site)和5次SE (Skipping exon);在Aas中共鉴定到559次AS事件,包括305次RI、155次A3、85次A5、13次SE和1次MEE (Mutually exclusive exon)。进一步分析发现,现有参考基因组上的多数注释基因结构并不完整;部分注释基因在菌丝和孢子中转录形成的isoform在数量和结构方面均存在差异;部分isoform在参考基因组中没有对应的注释基因。Aam中共鉴定到2748个基因含有1个及以上的APA位点,其中含有1个APA位点的基因数量最多(726,26.42%);Aas中共鉴定到2768个基因含有1个及以上的APA位点,其中含有5个以上APA位点的基因数量最多(1180,42.63%)。部分基因在菌丝和孢子中含有不同的APA位点数。序列特征分析结果显示,球囊菌全长转录本的3'UTR的上下游表现出明显的碱基倾向性,U和A分别富集在3'UTR的上游和下游。此外,在球囊菌全长转录本的APA位点上游鉴定到4个motif,分别是UCUCCU、UCUUCU、CCCACC和CCCCCU。【结论】本研究通过对球囊菌菌丝和孢子中基因的AS和APA进行深入分析,揭示了球囊菌转录组的复杂性,为完善现有的基因组和转录组注释提供了宝贵信息,也为探究AS和APA在球囊菌的基因表达调控中的作用提供了关键基础。     

Wilms’ tumor 1-associating protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences

Wilms’ tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3′-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences.     

Transcriptional landscape of alternative splicing during peripheral nerve injury

Alternative splicing (AS) regulates a variety of biological activities in numerous tissues and organs, including the nervous system. However, the existence and specific roles of AS events during peripheral nerve repair and regeneration remain largely undetermined. In the current study, by mapping splice-crossing sequence reads, we identified AS events and relevant spliced genes in rat sciatic nerve stumps following sciatic nerve crush. AS-related genes at 1, 4, 7, and 14 days post nerve crush were compared with those at 0 day to discover alternatively spliced genes induced by sciatic nerve crush. These injury-induced alternatively spliced genes were then categorized to diseases and biological functions, genetic networks, and canonical signaling pathways. Bioinformatic analysis indicated that these alternatively spliced genes were mainly correlated to immune response, cellular growth, and cellular function maintenance. Our study elucidated AS events following peripheral nerve injury and might help deepen our understanding of the molecular mechanisms underlying peripheral nerve regeneration.     

Identification of an intronic splicing regulatory element involved in auto‐regulation of alternative splicing of SCL33 pre‐mRNA

In Arabidopsis, pre‐mRNAs of serine/arginine‐rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis‐elements and trans‐acting proteins involved in regulating AS. Using a splicing reporter (GFP–intron–GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis‐elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis‐elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP–intron–GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto‐regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis‐element involved in AS of a plant SR gene, and elucidated a mechanism for auto‐regulation of AS of this intron.