Molecular basis for naturally occurring elevated readthrough transcription across the M-F junction of the paramyxovirus SV5.
Molecular basis for naturally occurring elevated readthrough transcription across the M-F junction of the paramyxovirus SV5.
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副粘病毒 SV5 的 M-F 连接处自然发生的通读转录升高的分子基础。
DOI：
10.1006/viro.1998.9266
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Parks,GD
中科院分区：
文献类型：
--
作者：
Rassa,JC;Parks,GD
副粘病毒RNA基因组的转录被认为涉及一种顺序的停止-启动机制，在该机制中，单顺反子mRNAs是通过聚合腺苷酸化和终止3‘上游基因，然后在下游起始点重新启动而产生的。对于一些副粘病毒，跨M-F基因连接处的转录导致合成高水平的双顺反子M-F通读RNA。在副粘病毒SV5感染的细胞中，病毒P、M、SH、HN和L基因的转录本中有15%或更少的转录本被检测到带有3‘端基因的通读产物。相反，∼中40%的SV5FmRNA被检测为双顺反子M-F转录本。对单个SV5基因连接的比较表明，M-F通读转录水平的提高与M基因末端具有最短的引导多聚腺苷基化的U区以及与共识序列不同的基因末端序列相关。我们已经验证了一种假设，即M-F直读转录的升高是由于M基因末端的无效终止信号造成的。建立了一个反向遗传学系统，在该系统中，利用编码SV5M-F或SH-HN基因连接的cDNA源聚合酶组分和双顺反子微基因组，在转基因细胞中重组SV5转录。构建了嵌合的SV5微基因组，以包含来自M-F(∼40%通读)和SH-HN(∼15%通读)连接的10个碱基基因末端序列和U链的交换。对从这些改变的模板合成的RNA进行的Northern杂交分析表明，在M-F基因间隔区的背景下，将M基因末端U链的长度从4个残基增加到6或8个U残基并不会降低M-F的转录读数。相反，包含SH基因末端10个碱基区的嵌合微型基因组导致了非常有效的基因终止和相应的通读转录减少。突变分析表明，位于M基因U链5个碱基3‘端的单个G-A替换足以将M基因末端区域转变为一个有效的多聚腺苷终止信号。这些结果表明，在副粘病毒基因组中，位于U链3‘端的基因末端区域作为转录终止的主要决定因素发挥了作用。讨论了M-F通读转录在副粘病毒生长周期中的可能作用。
Transcription of the paramyxovirus RNA genome is thought to involve a sequential stop-start mechanism whereby monocistronic mRNAs are produced by polyadenylation and termination of a 3′ upstream gene followed by reinitiation at the downstream start site. For a number of paramyxoviruses, transcription across the M-F gene junction results in the synthesis of high levels of a dicistronic M-F readthrough RNA. In cells infected with the paramyxovirus SV5, 15% or less of the transcripts from the viral P, M, SH, HN, and L genes were detected as readthrough products with the 3′ proximal gene. By contrast, ∼40% of the SV5 F mRNA was detected as a dicistronic M-F transcript. A comparison of the individual SV5 gene junctions showed that elevated M-F readthrough transcription correlated with the M gene end having the shortest U tract for directing polyadenylation and a gene end sequence that differs from the consensus sequence. We have tested the hypothesis that elevated M-F readthrough transcription results from an inefficient termination signal at the end of the M gene. A reverse genetics system was established whereby SV5 transcription was reconstituted in transfected cells using cDNA-derived polymerase components and dicistronic minigenomes that encoded either the SV5 M-F or the SH-HN gene junction. Chimeric SV5 minigenomes were constructed to contain exchanges of a 10 base gene end sequence and the U tract from the M-F (∼40% readthrough) and SH-HN (∼15% readthrough) junctions. Northern blot analysis of RNA synthesized from these altered templates showed that, in the context of the M-F intergenic region, increasing the length of the M gene end U tract from four residues to six or eight U residues did not decrease M-F readthrough transcription. In contrast, chimeric minigenomes that contained the 10 base region from the end of the SH gene directed very efficient gene termination and a corresponding decrease in readthrough transcription. Mutational analysis showed that a single G to A substitution located five bases 3′ to the M gene U tract was sufficient to convert the M gene end region to an efficient signal for polyadenylation-termination. These results demonstrate a role for the gene end region located immediately 3′ to the U tract as a major determinant of transcription termination in the paramyxovirus genome. The possible role of M-F readthrough transcription in the paramyxovirus growth cycle is discussed.
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