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...     

一个肝癌相关长链非编码RNA的克隆及序列分析

最近我们利用新一代测序(next generation sequencing,NGS)技术对肝细胞癌(hepatocellular carcinoma,HCC)患者活检标本及正常对照肝组织样品进行高通量RNA测序(RNA-sequencing,RNA-Seq),在肝癌样品中染色体11q13.1区域检测到几个相邻的RNA-Seq信号峰,而在正常对照组织中没有检测到,且该染色体区域目前尚无已知基因登录,提示这几个RNA-Seq峰可能代表一个或多个未知的新基因.以此为线索,证实这几个RNA-Seq峰来自同一个新基因,并克隆了该基因全长序列,在克隆该基因全长序列时,发现该基因编码的RNA存在多种剪接形式,最长的转录本为3 562 bp.将该基因编码的12条代表性RNA转录本序列递交到美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)的GenBank数据库中,GenBank ID号分别为KC136297~KC136308.该基因编码的RNA没有发现明显的开放阅读框(open reading fragment,ORF),提示该基因可能编码长链非编码RNA(long non-coding RNA,lncRNA).为了探讨该lncRNA基因可能的转录调控机制,我们用生物信息学方法预测了该lncRNA基因潜在启动子区域,发现在其转录起始位点上游-719~-469 bp处有一个潜在的启动子,其中包含7个Sp1、1个STAT5和1个EGR1转录因子结合位点.该lncRNA在肝细胞癌发生发展过程中的作用机制值得进一步深入研究.     

Long noncoding RNA DGCR5 involves in tumorigenesis of esophageal squamous cell carcinoma via SRSF1-mediated alternative splicing of Mcl-1

Long noncoding RNAs (lncRNAs) emerge as essential roles in the regulation of alternative splicing (AS) in various malignancies. Serine- and arginine-rich splicing factor 1 (SRSF1)-mediated AS events are the most important molecular hallmarks in cancer. Nevertheless, the biological mechanism underlying tumorigenesis of lncRNAs correlated with SRSF1 in esophageal squamous cell carcinoma (ESCC) remains elusive. In this study, we found that lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) was upregulated in ESCC clinical samples, which associated with poor prognosis. Through RNA interference and overexpression approaches, we confirmed that DGCR5 contributed to promote ESCC cell proliferation, migration, and invasion while inhibited apoptosis in vitro. Mechanistically, DGCR5 could directly bind with SRSF1 to increase its stability and thus stimulate alternative splicing events. Furthermore, we clarified that SRSF1 regulated the aberrant splicing of myeloid cell leukemia-1 (Mcl-1) and initiated a significant Mcl-1L (antiapoptotic) isoform switch, which contributed to the expression of the full length of Mcl-1. Moreover, the cell-derived xenograft (CDX) model was validated that DGCR5 could facilitate the tumorigenesis of ESCC in vivo. Collectively, our findings identified that the key biological role of lncRNA DGCR5 in alternative splicing regulation and emphasized DGCR5 as a potential biomarker and therapeutic target for ESCC.Subject terms: Tumour biomarkers, Apoptosis     

Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure

The serotonin receptor 2C plays a central role in mood and appetite control. It undergoes pre-mRNA editing as well as alternative splicing. The RNA editing suggests that the pre-mRNA forms a stable secondary structure in vivo. To identify substances that promote alternative exons inclusion, we set up a high-throughput screen and identified pyrvinium pamoate as a drug-promoting exon inclusion without editing. Circular dichroism spectroscopy indicates that pyrvinium pamoate binds directly to the pre-mRNA and changes its structure. SHAPE (selective 2′-hydroxyl acylation analysed by primer extension) assays show that part of the regulated 5′-splice site forms intramolecular base pairs that are removed by this structural change, which likely allows splice site recognition and exon inclusion. Genome-wide analyses show that pyrvinium pamoate regulates >300 alternative exons that form secondary structures enriched in A–U base pairs. Our data demonstrate that alternative splicing of structured pre-mRNAs can be regulated by small molecules that directly bind to the RNA, which is reminiscent to an RNA riboswitch.     

LncRNA882 regulates leukemia inhibitory factor (LIF) by sponging miR-15b in the endometrial epithelium cells of dairy goat

Despite the fact that long noncoding RNAs (lncRNAs) play roles in almost all biological processes, little is known about their biological function in the endometrium during the formation of endometrial receptivity. In this study, a comprehensive analysis of lncRNAs in goat endometrial tissues on Day 5 (prereceptive endometrium, PE) and Day 15 (receptive endometrium, RE) of pregnancy was performed by using RNA-Seq. As a result, 668 differentially expressed lncRNAs (DELs) were found between the PE and RE. Further study showed that lncRNA882, regulated by estrogen (E2) and progestin (P4), could act as competing endogenous RNAs (ceRNAs) for miR-15b, which inhibited the expression of transforming growth factor-b-activated kinase 1 binding protein 3 (TAB3) and then indirectly regulated the level of leukemia inhibitory factor (LIF). This was helpful for the formation of endometrial receptivity in dairy goats. In conclusion, we elucidated the endometrium lncRNA profiles of PE and RE in dairy goats; lncRNA882 acted as a ceRNA for miR-15b and then indirectly regulated the level of LIF in goat endometrial epithelium cells. Thus, this study helped us to better understand the molecular regulation of endometrial receptivity in dairy goats.     

A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression

The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function.     

lncRNA选择性剪接调控的研究进展

长链非编码RNA (lncRNA)选择性剪接是指剪除未成熟lncRNA中的内含子,并将外显子连接起来生成成熟lncRNA的过程.在各类疾病发生和发展过程中,异常的选择性剪接起着重要作用. lncRNA选择性剪接直接参与膀胱癌、结直肠癌、肝癌、神经母细胞瘤等多种肿瘤的发病机制,且与胚胎发育、软骨毛发发育、多系统萎缩症等紧密相关.在此,我们对lncRNA选择性剪接的起因、调控机制以及对疾病影响的研究进展进行综述.此外,本文还介绍了两个与lncRNA选择性剪接相关的数据库(SpliceMap和LNCediting).     

Integrative analysis of competing endogenous RNA networks reveals the functional lncRNAs in heart failure

Heart failure has become one of the top causes of death worldwide. It is increasing evidence that lncRNAs play important roles in the pathology processes of multiple cardiovascular diseases. Additionally, lncRNAs can function as ceRNAs by sponging miRNAs to affect the expression level of mRNAs, implicating in numerous biological processes. However, the functional roles and regulatory mechanisms of lncRNAs in heart failure are still unclear. In our study, we constructed a heart failure‐related lncRNA‐mRNA network by integrating probe re‐annotation pipeline and miRNA‐target interactions. Firstly, some lncRNAs that had the central topological features were found in the heart failure‐related lncRNA‐mRNA network. Then, the lncRNA‐associated functional modules were identified from the network, using bidirectional hierarchical clustering. Some lncRNAs that involved in modules were demonstrated to be enriched in many heart failure‐related pathways. To investigate the role of lncRNA‐associated ceRNA crosstalks in certain disease or physiological status, we further identified the lncRNA‐associated dysregulated ceRNA interactions. And we also performed a random walk algorithm to identify more heart failure‐related lncRNAs. All these lncRNAs were verified to show a strong diagnosis power for heart failure. These results will help us to understand the mechanism of lncRNAs in heart failure and provide novel lncRNAs as candidate diagnostic biomarkers or potential therapeutic targets.     

Investigation of Long Non-coding RNA Expression Profiles in the Substantia Nigra of Parkinson’s Disease

Genetics is considered as an important risk factor in the pathological changes of Parkinson’s disease (PD). Substantia nigra (SN) is thought to be the most vulnerable area in this process. In recent decades, however, few related long non-coding RNAs (lncRNAs) in the SN of PD patients had been identified and the functions of those lncRNAs had been studied even less. In this study, we sought to investigate the lncRNA expression profiles and their potential functions in the SN of PD patients. We screened lncRNA expression profiles in the SN of PD patients using the lncRNA mining approach from the ArrayExpress database, which included GSE20295. The samples were from 11 of PD and 14 of normal tissue samples. We identified 87 lncRNAs that were altered significantly in the SN during the occurrence of PD. Among these lncRNAs, lncRNA AL049437 and lncRNA AK021630 varied most dramatically. AL049437 was up-regulated in the PD samples, while AK021630 was down-regulated. Based on the results, we focused on the potential roles of the two lncRNAs in the pathogenesis of PD by the knockdown of the expression of AL049437 or AK021630 in human neuroblastoma SH-SY5Y cell line. Results indicated that the reduction in AL049437 level increased cell viability, mitochondrial transmembrane potential (Δψm), mitochondrial mass, and tyrosine hydroxylase (TyrH) secretion. By contrast, the knockdown of AK021630 resulted in the opposite effect. Based on these results, we speculated that lncRNA AL049437 likely contributed to the risk of PD, while lncRNA AK021630 likely inhibited the occurrence of PD.