mRNA decay proteins are targeted to poly(A)+ RNA and dsRNA-containing cytoplasmic foci that resemble P-bodies in Entamoeba histolytica.
mRNA decay proteins are targeted to poly(A)+ RNA and dsRNA-containing cytoplasmic foci that resemble P-bodies in Entamoeba histolytica.
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DOI：
10.1371/journal.pone.0045966
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
López-Camarillo C
中科院分区：
文献类型：
--
作者：
López-Rosas I;Orozco E;Marchat LA;García-Rivera G;Guillen N;Weber C;Carrillo-Tapia E;Hernández de la Cruz O;Pérez-Plasencia C;López-Camarillo C
在高等真核生物中，mRNA降解和基于RNA的基因沉默发生在被称为加工体（P体）的细胞质病灶中。在原生动物寄生虫中，P体的存在及其在mRNA衰变中的假定作用尚未得到全面解决。P体的鉴定可能提供关于mRNA降解机制如何在低等真核生物中进化的信息。在这里，我们使用免疫荧光和共聚焦显微镜检测，以探讨在人类肠道寄生虫溶组织内阿米巴的mRNA降解蛋白的细胞定位，并发现存在的P-机构的证据。两个mRNA衰变因子，即EhXRN 2核糖核酸外切酶和EhDCP 2脱帽酶，定位在细胞质病灶中的模式类似于P-体组织。考虑到变形虫病灶似乎比高等真核生物中描述的更小，更不圆，我们将其命名为“P体样结构”。这些病灶含有额外的mRNA降解因子，包括参与RNA干扰的EhCAF 1去腺苷酶和EhAGO 2 -2蛋白。生化分析显示EhCAF 1与EhXRN 2共沉淀，但不与EhDCP 2或EhAGO 2 -2共沉淀，因此将去腺苷化与5′-至-3 ′ mRNA降解联系起来。分别用放线菌素D和放线菌酮抑制转录和翻译后，含EhCAF 1的灶的数量显著减少。此外，RNA-FISH分析的结果表明：（i）EhCAF 1与poly（A）+ RNA共定位，（ii）在通过RNA干扰沉默Ehpc 4基因的过程中，EhAGO 2 -2与小干扰RNA共定位在胞质病灶中。我们的观察脱帽，deadenylation和RNA干扰蛋白内的P-体样灶表明，这些结构已被保存后，起源于真核细胞谱系的早期进化。据我们所知，这是第一个研究报告的mRNA衰变蛋白定位在P体样结构在E。溶组织剂我们的研究结果应该打开机会破译mRNA降解和RNA为基础的基因沉默在这个深分支的真核生物的机制。
In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies). In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them “P-body-like structures”. These foci contain additional mRNA degradation factors, including the EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5′-to-3′ mRNA decay. The number of EhCAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i) EhCAF1 colocalized with poly(A)+ RNA and (ii) during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P-body-like structures in E. histolytica. Our findings should open up opportunities for deciphering the mechanisms of mRNA degradation and RNA-based gene silencing in this deep-branching eukaryote.
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影响因子：
3.9
作者：
Kulkarni, Meeta;Ozgur, Sevim;Stoecklin, Georg
通讯作者：
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通讯作者：
Lopez-Camarillo, Cesar
影响因子：
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通讯作者：
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