Impact of RNA binding proteins on translation initiation and elongation.

我们鉴定了-据我们所知-第一个没有解旋酶活性的RNA结合蛋白，促进其靶mrna的翻译。cchc -锌指核酸结合蛋白（CNBP）是哺乳动物胚胎发育所需的真核生物保守的核酸结合蛋白。它含有7个cchc型锌指结构域，被认为可以作为核酸伴侣和转录因子。在这里，我们将所有CNBP亚型鉴定为细胞质信使RNA （mRNA）结合蛋白。利用PAR-CLIP，我们在全基因组范围内以核苷酸水平的分辨率绘制了它在RNA上的结合位点，发现CNBP在人类细胞系中与4178种mrna相互作用，优先在成熟mrna上靠近AUG起始密码子的富含g的基序上相互作用。功能丧失和功能获得分析结合全系统RNA和蛋白质定量显示，CNBP不影响RNA丰度，而是促进其靶标的翻译。这与CNBP的RNA伴侣功能一致，它有助于解析二级结构，从而促进翻译。

We identified the - to our knowledge - first RNA binding protein without helicase activity that promotes translation of its target mRNAs. The CCHC-Zinc Finger, Nucleic Acid Binding Protein (CNBP) is a eukaryote-conserved nucleic-acid binding protein required in mammals for embryonic development. It contains seven CCHC-type zinc-finger domains and was suggested to act as a nucleic acid chaperone, as well as a transcription factor. Here, we identify all CNBP isoforms as cytoplasmic, messenger RNA (mRNA)-binding proteins. Using PAR-CLIP, we mapped its binding sites on RNA at nucleotide-level resolution on a genome-wide scale and found that CNBP interacted with 4178 mRNAs in a human cell line, preferentially at a G-rich motif close to the AUG start codon on mature mRNAs. Loss- and gain-of-function analyses coupled with system-wide RNA and protein quantification revealed that CNBP did not affect RNA abundance, but rather promoted translation of its targets. This is consistent with an RNA chaperone function of CNBP helping to resolve secondary structures, thus promoting translation. The impact of sequence-specific translational regulatory events controlled by RBPs cannot be studied by standard mRNA quantification at a systems-wide scale. Thus, the study of translational regulation affords our group the opportunity to become experts in state-of-the-art methods measuring translation efficiency, including ribosome profiling (Ingolia et al., 2009) and/or approaches to measuring protein abundance, including SILAC and iTRAQ (Ross et al., 2004; Schwanhausser et al., 2009). Combined with the biochemical assays established for the study of CNBP these protocols will allow us to dissect the molecular function of one of the most abundant RBPs in mammalian cells and tissues, YBX1, which we are implicating in translational regulation processes.