Genome-Wide Analysis of the 5′ and 3′ Ends of Vaccinia Virus Early mRNAs Delineates Regulatory Sequences of Annotated and Anomalous Transcripts
Genome-Wide Analysis of the 5′ and 3′ Ends of Vaccinia Virus Early mRNAs Delineates Regulatory Sequences of Annotated and Anomalous Transcripts
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DOI：
10.1128/jvi.00428-11
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发表时间：
2011-06-01
影响因子：
5.4
通讯作者：
Moss, Bernard
中科院分区：
文献类型：
--
作者：
Yang, Zhilong;Bruno, Daniel P.;Moss, Bernard
痘病毒是一种大型DNA病毒，它编码多亚单位RNA聚合酶、阶段特异性转录因子，以及在受感染动物细胞的细胞质内覆盖和聚腺苷酸化mRNAs的酶。全基因组微阵列和RNA-SEQ技术已被用于分析痘苗病毒(VACV)的转录组，VACV是该家族的原型成员。在这里，我们采用基于标签的方法，结合Solid和Illumina深度测序平台来确定VACV早期mRNA5‘和3’端的精确位置，并绘制可能的转录起始点(TSS)和多聚腺苷酸化位点(PASS)。在104个注释开放阅读框架(ORF)之前发现了单个和聚集的SSs，不包括假基因。在大多数情况下，ORF的上游存在一个15个核苷酸的共识核心基序。然而，该基序也存在于许多其他位置，表明它不足以启动转录。进一步的分析发现，在功能核心基序之后有一个10核苷酸的富含AT的间隔区，这可能有助于DNA的解离。其他推测的TSS出现在可能扩大VACV基因组功能谱系的异常位置。然而，许多异常的TSS缺乏上游核心基序，这增加了它们可能是由真核系统中提出的处理机制引起的。在UUUUUNU终止信号之后，发生了大约40个核苷酸的离散和聚集性传递。然而，大量的PASS之前并没有这个基序，这表明了替代的多腺苷作用机制。在两种类型的PASS的上游都立即发现了富含嘧啶的编码链序列，这意味着VACV 3‘端形成和多聚腺苷化的另一个特征。
Poxviruses are large DNA viruses that encode a multisubunit RNA polymerase, stage-specific transcription factors, and enzymes that cap and polyadenylate mRNAs within the cytoplasm of infected animal cells. Genome-wide microarray and RNA-seq technologies have been used to profile the transcriptome of vaccinia virus (VACV), the prototype member of the family. Here, we adapted tag-based methods in conjunction with SOLiD and Illumina deep sequencing platforms to determine the precise 5' and 3' ends of VACV early mRNAs and map the putative transcription start sites (TSSs) and polyadenylation sites (PASs). Individual and clustered TSSs were found preceding 104 annotated open reading frames (ORFs), excluding pseudogenes. In the majority of cases, a 15-nucleotide consensus core motif was present upstream of the ORF. This motif, however, was also present at numerous other locations, indicating that it was insufficient for transcription initiation. Further analysis revealed a 10-nucleotide AT-rich spacer following functional core motifs that may facilitate DNA unwinding. Additional putative TSSs occurred in anomalous locations that may expand the functional repertoire of the VACV genome. However, many of the anomalous TSSs lacked an upstream core motif, raising the possibility that they arose by a processing mechanism as has been proposed for eukaryotic systems. Discrete and clustered PASs occurred about 40 nucleotides after an UUUUUNU termination signal. However, a large number of PASs were not preceded by this motif, suggesting alternative polyadenylation mechanisms. Pyrimidine-rich coding strand sequences were found immediately upstream of both types of PASs, signifying an additional feature of VACV 3'-end formation and polyadenylation.