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.
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