Existing high-throughput methods to identify RNA-binding proteins (RBPs) involving capture of polyadenylated RNAs can not recover proteins that interact with non-adenylated RNAs, including lncRNA, pre-mRNA and bacterial RNAs. We present orthogonal organic phase separation (OOPS) which does not require molecular tagging or capture of polyadenylated RNA. We verify OOPS in HEK293, U2OS and MCF10A human cell lines, finding 96% of proteins recovered are bound to RNA. We demonstrate that all long RNAs can be crosslinked to proteins and recover 1838 RBPs, including 926 putative novel RBPs. Importantly, OOPS is approximately 100-fold more efficient than current techniques, enabling analysis of dynamic RNA-protein interactions. We identified 749 proteins with altered RNA binding following release from nocodazole arrest. Finally, OOPS allowed the characterisation of the first RNA-interactome for a bacterium, Escherichia coli. OOPS is an easy to use and flexible technique, compatible with downstream proteomics and RNA sequencing and applicable to any organism.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.

The c‐fyn proto‐oncogene is a member of a family of closely related genes of which c‐src is the prototype. Using peptide antibodies which had been raised against sequences predicted to be specific for the human c‐fyn gene product, the c‐fyn protein was identified. It is a tyrosine kinase with apparent mol. wt of 59 kd that is also phosphorylated and myristylated. Like pp60c‐src and pp62c‐yes, pp59c‐fyn is able to form a stable complex with middle‐T antigen, the transforming protein of polyomavirus. The transformation‐defective middle‐T mutant NG59, which is unable to associate stably with pp60c‐src does not associate with pp59c‐fyn. In contrast to pp60c‐src, complex formation with middle‐T antigen does not lead to a significant increase in the tyrosine kinase activity of pp59c‐fyn. These findings lead us to suggest that middle‐T mediated transformation may be a consequence of the deregulation of several members of the src‐family of protein tyrosine kinases.

Large and small tumor (T)antigens of simian virus 40 were synthesized in vitro with L-cell extracts that had been treated by the method of Palmiter to prevent amino-terminal acetylation of nascent proteins. Partial amino-terminal amino acid sequences of both forms of T-antigen were determined and found to be identical. Methionine residues were located at positions 1 and 14, a lysine residue at position 3, and leucine residues at positions 5, 11, 13, 16, 17, and 19. These amino acid sequence data match perfectly the amino acid sequence predicted from a sequence of nucleotides in the E strand of simian virus 40 DNA which begins near the junction between HindII/III fragments A and C at about 0.65 map units. This strongly suggests that the sequence coding for the amino terminus of both proteins is located at this position. Furthermore, the data are consistent with a model for the synthesis of both forms of T-antigen that predicts that (f) small T-antigen is coded for by a sequence of nucleotides from the 5' end of the early region and (ii) large T-antigen is coded for by nucleotide sequences from two noncontiguous regions of simian virus 40 DNA.

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans‐acting regulatory RNA‐binding proteins (RBPs) are necessary to provide mRNA‐specific translation, and these interact with 5′ and 3′ untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans‐acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans‐acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans‐acting ribosomal proteins in directing the translation of specific mRNAs.This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA–Protein ComplexesTranslation > Translation RegulationTranslation > Translation Mechanisms

Fifty variant forms of simian virus 40 (SV40) large T antigen bearing point, multiple point, deletion, or termination mutations within a region of the protein thought to be involved in DNA binding were tested for their ability to bind to SV40 origin DNA. A number of the mutant large T species including some with point

The ability of polyoma virus to transform cells results primarily from the action of one of the virus-coded early proteins, called middle-T antigen. Middle-T has an associated tyrosine-specific protein kinase activity that can be measured in vitro and results in the phosphorylation of middle-T itself. Almost all mutants so far tested that lack the ability to transform cells, also lack associated kinase activity. Attempts to map within middle-T the tyrosine residue(s) that are phosphorylated in vitro suggest that a likely site of phosphorylation is tyrosine 315 (refs 8-10 and unpublished results). The amino acid sequence preceding Tyr 315 includes a tract of six contiguous glutamic acid residues and bears some homology with that preceding the tyrosine phosphorylated in vivo in pp60v-src, the transforming protein of Rous sarcoma virus, and with a region in the polypeptide hormone, gastrin, preceding a tyrosine that is sulphated. Furthermore, although surprisingly large tracts of middle-T may be removed without affecting its transforming activity, mutants that lack the sequences corresponding to amino acids 311-318 inclusive are transformation defective. Because the likely site of phosphorylation, the homology with pp60v-src and gastrin and the sequence apparently required for transformation all overlap, it has generally been accepted that this region of middle-T may form part of an essential region, possibly an active site on the protein. Here we have used techniques of site-directed and site-specific mutagenesis to probe the sequence requirements in more detail. Contrary to expectation, the results obtained strongly suggest that Tyr 315 and conservation of the surrounding amino acid sequence are not essential for transformation.

A large number of mutations were introduced into the carboxy-terminal domain of pp60c-src. The level of phosphorylation on Tyr-416 and Tyr-527, the transforming activity (as measured by focus formation on NIH 3T3 cells), kinase activity, and the ability of the mutant pp60c-src to associate with the middle-T antigen of polyomavirus were examined. The results indicate that Tyr-527 is a major carboxy-terminal element responsible for regulating pp60c-src in vivo. A good but not perfect correlation exists between lack of phosphorylation at Tyr-527 and increased phosphorylation at Tyr-416, between elevated phosphorylation on Tyr-416 and activated kinase activity, and between activated kinase activity and transforming activity. Phosphorylation of Tyr-527 was insensitive to the mutation of adjacent residues, indicating that the primary sequence only has a minor role in recognition by kinases or phosphatases which regulate it in vivo. Three mutants which have in common a modified Glu-524 residue were phosphorylated on Tyr-416 and Tyr-527 and were weakly transforming. This suggests that other mechanisms besides complete dephosphorylation of Tyr-527 can lead to increased phosphorylation of Tyr-416 and activation of the transforming activity of pp60c-src. Furthermore, the residues between Asp-518 and Pro-525 were required to form a stable complex with middle-T antigen. The proximity of these sequences to Tyr-527 suggests a model in which middle-T activates pp60c-src by binding directly to this region of the molecular and thereby preventing phosphorylation of Tyr-527. Alternatively, middle-T binding may mediate a conformational change in this region, which in turn induces an alteration in the level of phosphorylation at Tyr-527 and Tyr-416.