Cavaiuolo Marina, Kuras Richard, Wollman Francis-André, Choquet Yves, Vallon Olivier
Unité Mixte de Recherche 7141, CNRS/UPMC, Institut de Biologie Physico-Chimique, F-75005 Paris, France.
Nucleic Acids Res. 2017 Oct 13;45(18):10783-10799. doi: 10.1093/nar/gkx668.
In Chlamydomonas reinhardtii, regulation of chloroplast gene expression is mainly post-transcriptional. It requires nucleus-encoded trans-acting protein factors for maturation/stabilization (M factors) or translation (T factors) of specific target mRNAs. We used long- and small-RNA sequencing to generate a detailed map of the transcriptome. Clusters of sRNAs marked the 5' end of all mature mRNAs. Their absence in M-factor mutants reflects the protection of transcript 5' end by the cognate factor. Enzymatic removal of 5'-triphosphates allowed identifying those cosRNA that mark a transcription start site. We detected another class of sRNAs derived from low abundance transcripts, antisense to mRNAs. The formation of antisense sRNAs required the presence of the complementary mRNA and was stimulated when translation was inhibited by chloramphenicol or lincomycin. We propose that they derive from degradation of double-stranded RNAs generated by pairing of antisense and sense transcripts, a process normally hindered by the traveling of the ribosomes. In addition, chloramphenicol treatment, by freezing ribosomes on the mRNA, caused the accumulation of 32-34 nt ribosome-protected fragments. Using this 'in vivo ribosome footprinting', we identified the function and molecular target of two candidate trans-acting factors.
在莱茵衣藻中，叶绿体基因表达的调控主要发生在转录后。它需要细胞核编码的反式作用蛋白因子来促进特定靶标mRNA的成熟/稳定（M因子）或翻译（T因子）。我们使用长链和小RNA测序来生成转录组的详细图谱。小RNA簇标记了所有成熟mRNA的5'端。它们在M因子突变体中的缺失反映了同源因子对转录本5'端的保护作用。通过酶促去除5'-三磷酸可以鉴定出那些标记转录起始位点的共转录本来源的小RNA（cosRNA）。我们检测到另一类源自低丰度转录本的小RNA，它们与mRNA呈反义关系。反义小RNA的形成需要互补mRNA的存在，并且当氯霉素或林可霉素抑制翻译时会受到刺激。我们推测它们源自反义转录本与有义转录本配对产生的双链RNA的降解，这一过程通常会受到核糖体移动的阻碍。此外，氯霉素处理通过使核糖体滞留在mRNA上，导致了32 - 34个核苷酸的核糖体保护片段的积累。利用这种“体内核糖体足迹法”，我们确定了两个候选反式作用因子的功能和分子靶点。
