Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.     

RNA-protein interactions in vivo: global gets specific

RNA-binding proteins (RBPs) impact every process in the cell; they act as splicing and polyadenylation factors, transport and localization factors, stabilizers and destabilizers, modifiers, and chaperones. RNA-binding capacity can be attributed to numerous protein domains that bind a limited repertoire of short RNA sequences. How is specificity achieved in cells? Here we focus on recent advances in determining the RNA-binding properties of proteins in vivo and compare these to in vitro determinations, highlighting insights into how endogenous RNA molecules are recognized and regulated. We also discuss the crucial contribution of structural determinations for understanding RNA-binding specificity and mechanisms.     

Insights into RNA biology from an atlas of mammalian mRNA-binding proteins

RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.     

mRNPs take shape by CLIPPING and PAIRING

The interaction of RNA-binding proteins (RBPs) with RNA is a crucial aspect of normal cellular metabolism. Yet, the diverse number of RBPs and RNA motifs to which they bind, the wide range of interaction strengths and the fact that RBPs associate in dynamic complexes have made it challenging to determine whether a particular RNA-binding protein binds a particular RNA. Recent work by three different laboratories has led to the development of new tools to query such interactions in the more physiological environs of cultured cells. The use of these methods has led to insights into (1) the networks of RNAs regulated by a particular protein, (2) the identification of new protein partners within messenger ribonucleoprotein particles and (3) the flux of RNA-binding proteins on an mRNA throughout its lifecycle. Here, I examine these new methods and discuss their relative strengths and current limitations.     

Genome-wide identification of protein binding sites on RNAs in mammalian cells

RNA-binding proteins (RBPs) are proteins that bind to the RNA and participate in forming ribonucleoprotein complexes. They have crucial roles in various biological processes such as RNA splicing, editing, transport, maintenance, degradation, intracellular localization and translation. The RBPs bind RNA with different RNA-sequence specificities and affinities, thus, identification of protein binding sites on RNAs (R-PBSs) will deeper our understanding of RNA-protein interactions. Currently, high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is one of the most powerful methods to map RNA-protein binding sites or RNA modification sites. However, this method is only used for identification of single known RBPs and antibodies for RBPs are required. Here we developed a novel method, called capture of protein binding sites on RNAs (RPBS-Cap) to identify genome-wide protein binding sites on RNAs without using antibodies. Double click strategy is used for the RPBS-Cap assay. Proteins and RNAs are UV-crosslinked in vivo first, then the proteins are crosslinked to the magnetic beads. The RNA elements associated with proteins are captured, reverse transcribed and sequenced. Our approach has potential applications for studying genome-wide RNA-protein interactions.     

紫外交联合并串联亲和纯化高效鉴定蛋白复合物组分的RNA结合活性

RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.     

Ribonomic approaches to study the RNA-binding proteome

Gene expression is controlled through a complex interplay among mRNAs, non-coding RNAs and RNA-binding proteins (RBPs), which all assemble along with other RNA-associated factors in dynamic and functional ribonucleoprotein complexes (RNPs). To date, our understanding of RBPs is largely limited to proteins with known or predicted RNA-binding domains. However, various methods have been recently developed to capture an RNA of interest and comprehensively identify its associated RBPs. In this review, we discuss the RNA-affinity purification methods followed by mass spectrometry analysis (AP-MS); RBP screening within protein libraries and computational methods that can be used to study the RNA-binding proteome (RBPome).     

Intrinsic Disorder in Human RNA-Binding Proteins

Although RNA-binding proteins (RBPs) are known to be enriched in intrinsic disorder, no previous analysis focused on RBPs interacting with specific RNA types. We fill this gap with a comprehensive analysis of the putative disorder in RBPs binding to six common RNA types: messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA), ribosomal RNA (rRNA), and internal ribosome RNA (irRNA). We also analyze the amount of putative intrinsic disorder in the RNA-binding domains (RBDs) and non-RNA-binding-domain regions (non-RBD regions). Consistent with previous studies, we show that in comparison with human proteome, RBPs are significantly enriched in disorder. However, closer examination finds significant enrichment in predicted disorder for the mRNA-, rRNA- and snRNA-binding proteins, while the proteins that interact with ncRNA and irRNA are not enriched in disorder, and the tRNA-binding proteins are significantly depleted in disorder. We show a consistent pattern of significant disorder enrichment in the non-RBD regions coupled with low levels of disorder in RBDs, which suggests that disorder is relatively rarely utilized in the RNA-binding regions. Our analysis of the non-RBD regions suggests that disorder harbors posttranslational modification sites and is involved in the putative interactions with DNA. Importantly, we utilize experimental data from DisProt and independent data from Pfam to validate the above observations that rely on the disorder predictions. This study provides new insights into the distribution of disorder across proteins that bind different RNA types and the functional role of disorder in the regions where it is enriched.