Functional Analysis of Sequence Motifs Involved in the Polyadenylation of Trichomonas vaginalis mRNAs
Functional Analysis of Sequence Motifs Involved in the Polyadenylation of Trichomonas vaginalis mRNAs
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DOI：
10.1128/ec.05322-11
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发表时间：
2012-06-01
期刊：
影响因子：
--
通讯作者：
Lopez-Villasenor, Imelda
中科院分区：
文献类型：
--
作者：
Fuentes, Vanessa;Barrera, Guadalupe;Lopez-Villasenor, Imelda
真核生物中功能性mRNA的合成涉及前体转录物的加工，包括在3'端添加聚（A）尾。多蛋白复合物识别多聚腺苷酸化信号，通常是后生动物中的六核苷酸AAUAAA，以指导前mRNA的加工。基于对几种cDNA的序列分析，我们以前曾提出，UAAA四核苷酸（可能包括UAA翻译终止密码子）可能是导致人类滴虫病的寄生原虫--阴道毛滴虫中的多聚腺苷酸化信号。本文借助报道基因的瞬时表达系统分析了这一建议。迷走细胞肌动蛋白非编码序列）。当细胞用带有含有UAAA基序的原始3'非翻译区（UTR）序列的质粒转染时，所得的cat mRNA与内源性肌动蛋白mRNA类似地被聚腺苷酸化。UAAA序列中的碱基变化导致报告基因mRNA的多聚腺苷酸化位点发生改变，而UAAA两侧的核苷酸取代则没有。此外，重新定位的UAAA基序重定向的报告mRNA的加工和聚腺苷酸化。此外，定义了用于多聚腺苷酸化的前mRNA切割位点。T.电泳迁移率变动分析显示了具有UAAA基序的迷走蛋白。根据我们的研究结果，我们提供的证据表明，在T。在迷走神经中，UAAA四核苷酸具有与后生动物共有AAUAAA多聚腺苷酸化信号等同的作用。
Synthesis of functional mRNA in eukaryotes involves processing of precursor transcripts, including the addition of a poly(A) tail at the 3' end. A multiprotein complex recognizes a polyadenylation signal, generally the hexanucleotide AAUAAA in metazoans, to direct processing of the pre-mRNA. Based on sequence analysis of several cDNAs, we have previously suggested that the UAAA tetranucleotide (which may include the UAA translation stop codon) could be the polyadenylation signal in Trichomonas vaginalis, a parasitic protozoon that causes human trichomoniasis. This proposal is analyzed here with the aid of a transient-expression system of a reporter gene (cat flanked by T. vaginalis actin noncoding sequences). When cells were transfected with a plasmid bearing the original 3' untranslated region (UTR) sequence containing the UAAA motif, the resulting cat mRNA was polyadenylated similarly to the endogenous actin mRNA. Base changes in the UAAA sequence produced alterations to the polyadenylation site of the reporter mRNAs, while nucleotide substitutions at either side of UAAA did not. Furthermore, relocation of the UAAA motif redirected the processing and polyadenylation of the reporter mRNA. In addition, a pre-mRNA cleavage site for polyadenylation was defined. Interaction of T. vaginalis proteins with the UAAA motif was shown by electrophoretic mobility shift assays. Based on our findings, we provide evidence that in T. vaginalis the UAAA tetranucleotide has a role equivalent to that of the metazoan consensus AAUAAA polyadenylation signal.