Noncoding translation mitigation.

Translation is pervasive outside of canonical coding regions, occurring in long noncoding RNAs, canonical untranslated regions and introns1-4, especially in ageing4-6, neurodegeneration5,7 and cancer8-10. Notably, the majority of tumour-specific antigens are results of noncoding translation11-13. Although the resulting polypeptides are often nonfunctional, translation of noncoding regions is nonetheless necessary for the birth of new coding sequences14,15. The mechanisms underlying the surveillance of translation in diverse noncoding regions and how escaped polypeptides evolve new functions remain unclear10,16-19. Functional polypeptides derived from annotated noncoding sequences often localize to membranes20,21. Here we integrate massively parallel analyses of more than 10,000 human genomic sequences and millions of random sequences with genome-wide CRISPR screens, accompanied by in-depth genetic and biochemical characterizations. Our results show that the intrinsic nucleotide bias in the noncoding genome and in the genetic code frequently results in polypeptides with a hydrophobic C-terminal tail, which is captured by the ribosome-associated BAG6 membrane protein triage complex for either proteasomal degradation or membrane targeting. By contrast, canonical proteins have evolved to deplete C-terminal hydrophobic residues. Our results reveal a fail-safe mechanism for the surveillance of unwanted translation from diverse noncoding regions and suggest a possible biochemical route for the preferential membrane localization of newly evolved proteins.

Mutation of Methionine to Asparagine but Not Leucine in Parathyroid Hormone Mimics the Loss of Biological Function upon Oxidation.

Human parathyroid hormone (PTH) is an 84-amino acid peptide that contains two methionine (Met) residues located at positions 8 and 18. It has long been recognized that Met residues in PTH are subject to oxidation to become Met sulfoxide, resulting in a decreased biological function of the peptide. However, the mechanism of the lost biological function of PTH oxidation remains elusive. To characterize whether the shift from the hydrophobic nature of the native Met residue to the hydrophilic nature of Met sulfoxide plays a role in the reduction of biological activity upon PTH oxidation, we conducted in silico and in vitro site-directed mutagenesis of Met-8 and Met-18 to the hydrophilic residue asparagine (Asn) or to the hydrophobic residue leucine (Leu) and compared the behavior of these mutated peptides with that of PTH oxidized at Met-8 and/or Met-18. Our results showed that the biological activity of the Asn-8 and Asn-8/Asn-18 mutants was significantly reduced, similar to Met-8 sulfoxide and Met-8/Met-18 sulfoxide analogues, while the functions of Asn-18, Leu-8, Leu-8/Leu-18 mutants, or Met-18 sulfoxide analogues were similar to wild-type PTH. This is rationalized from molecular modeling and immunoprecipitation assay, demonstrating disruption of hydrophobic interactions between Met-8 and Met-18 of PTH and type-1 PTH receptor (PTHR1) upon mutation or oxidation. Thus, these novel findings support the notion that the loss of biological function of PTH upon oxidation of Met-8 is due, at least in part, to the conversion from a hydrophobic to a hydrophilic residue that disrupts direct hydrophobic interaction between PTH and PTHR1.