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首页> 外文期刊>Journal of Molecular Biology >A cis -Acting Element Downstream of the Mouse Mammary Tumor Virus Major Splice Donor Critical for RNA Elongation and Stability
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A cis -Acting Element Downstream of the Mouse Mammary Tumor Virus Major Splice Donor Critical for RNA Elongation and Stability

机译：在小鼠乳腺肿瘤病毒下游的顺式元素主要剪接供体对于RNA伸长和稳定性至关重要

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BackgroundThe mouse mammary tumor virus (MMTV) encodes a functional signal peptide, a cleavage product of envelope and Rem proteins. Signal peptide interacts with a 3′cis-acting RNA element, the Rem-responsive element (RmRE), to facilitate expression of both unspliced genomic (gRNA) and spliced mRNAs. An additional RmRE has been proposed at the 5′ end of the genome, facilitating nuclear export of the unspliced gRNA, whereas the 3′ RmRE could facilitate translation of all other mRNAs, including gRNA. ResultsTo address this hypothesis, a series of mutations were introduced into a 24-nt region found exclusively in the unspliced gRNA. Mutant clones using MMTV or human cytomegalovirus promoters were tested in both transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Nuclear export of the gRNA was affected only in a small mutant subset in stably transfected Jurkat T cells. Quantitative real-time RT-PCR of actinomycin D-treated cells expressing MMTV revealed that multiple mutants were severely compromised for RNA expression and stability. Both genomic and spliced nuclear RNAs were reduced, leading to abrogation of Gag and Env protein expressed from unspliced and spliced mRNAs, respectively. RT-PCRs with multiple primer pairs indicated failure to elongate genomic MMTV transcripts beyond ~500 nt compared to the wild type in a cell line-dependent manner. ConclusionsMMTV contains a novelcis-acting element downstream of the major splice donor critical for facilitating MMTV gRNA elongation and stability. Presence of a mirror repeat within the element may represent important viral/host factor binding site(s) within MMTV gRNA.
机译：背景，小鼠乳腺肿瘤病毒（MMTV）编码功能信号肽，包膜的切割产物和REM蛋白质。信号肽与3'Cis作用RNA元素，REM响应元件（RMRE）相互作用，以促进表达未燃种基因组（GRNA）和剪接MRNA。在基因组的5'末端提出了另外的RMRE，促进了未燃料GRNA的核导出，而3'RMRE可以促进所有其他MRNA的翻译，包括GRNA。结果Sto解决了这个假设，将一系列突变引入专门在未烧蚀的GRNA中发现的24-NT区域。使用MMTV或人巨细胞病毒启动子的突变克隆在短暂和稳定的转染中进行测试，以确定它们对GRNA核导出，稳定性和翻译的影响。只有在稳定转染的Jurkat T细胞中的小突变体亚特区的核导出受到影响。表达MMTV的测量霉素D处理细胞的定量实时RT-PCR显示，对于RNA表达和稳定性，多个突变体严重受损。基因组和剪接核RNA都减少，导致分别出现从未燃烧和剪接的MRNA表达的GAG和ENV蛋白。具有多个引物对的RT-PCR，与细胞系依赖性方式相比，具有多个引物对的未伸长基因组MMTV转录物，其与野生型相比伸长〜500nt。结论MMTV含有下游的新型剪接供体下游，用于促进MMTV GRNA伸长和稳定性。在元素内的镜子重复的存在可以代表MMTV GRNA内的重要病毒/宿主因子结合位点。

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