Impact of RNA binding proteins on translation initiation and elongation.

据我们所知，我们鉴定了第一个没有解旋酶活性的RNA结合蛋白，该蛋白促进其靶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，并被认为是一种核酸伴侣，以及转录因子。在这里，我们确定所有的CNBP亚型作为细胞质，信使RNA（mRNA）结合蛋白。使用PAR-CLIP，我们在全基因组范围内以核苷酸水平分辨率绘制了其在RNA上的结合位点，发现CNBP与人类细胞系中的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的基序处。结合全系统RNA和蛋白质定量的功能丧失和获得分析显示，CNBP不影响RNA丰度，而是促进其靶点的翻译。这与CNBP的RNA伴侣功能一致，有助于解析二级结构，从而促进翻译。 由RBP控制的序列特异性翻译调控事件的影响不能通过在系统范围内的标准mRNA定量来研究。因此，翻译调控的研究使我们的小组有机会成为测量翻译效率的最先进方法的专家，包括核糖体分析（Ingolia et al.，2009）和/或测量蛋白质丰度的方法，包括SILAC和iTRAQ（Ross等人，2004; Schwanhausser等人，2009年）。结合为CNBP研究建立的生物化学测定，这些方案将使我们能够剖析哺乳动物细胞和组织中最丰富的RBP之一YBX 1的分子功能，我们正在研究其在翻译调控过程中的作用。

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.