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个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。 我们还剖析了高度保守的DEAH盒解旋酶DHX 36/RHAU在体外对DNA和RNA的调控影响，但缺乏对DHX 36靶点和功能的系统范围分析。我们在人类细胞系中绘制了DHX 36与RNA结合的全局图，发现它优先与超过4500种mRNA上的富含G和G4形成序列相互作用。虽然DHX 36敲除（KO）导致靶mRNA丰度的显著增加，但来自这些靶的核糖体占有率和蛋白质输出减少，表明它们被赋予免疫缺陷。考虑到DHX 36靶向，携带G4，优先定位在应激颗粒中，并且DHX 36 KO导致SG形成和蛋白激酶R（PKR/EIF 2AK 2）磷酸化增加，我们推测DHX 36参与rG 4诱导的细胞应激的解决。翻译效率可能受到mRNA稳定性和二级RNA结构的影响。在本文中，作者揭示了DHX 36解旋酶活性的丧失导致非翻译区中具有富含G结构的无活性靶mRNA的积累。 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. We also dissected the regulatory impact of the the highly conserved DEAH-box helicase DHX36/RHAU thought to G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress. Translation efficiency can be affected by mRNA stability and secondary RNA structures. Here the authors reveal that loss of DHX36 helicase activity leads to an accumulation of translationally inactive target mRNAs with G-rich structures in untranslated regions. 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.