Genomic innovation of ATD alleviates mistranslation associated with multicellularity in Animalia.

The emergence of multicellularity in Animalia is associated with increase in ROS and expansion of tRNA-isodecoders. tRNA expansion leads to misselection resulting in a critical error of L-Ala mischarged onto tRNAThr, which is proofread by Animalia-specific-tRNA Deacylase (ATD) in vitro. Here we show that in addition to ATD, threonyl-tRNA synthetase (ThrRS) can clear the error in cellular scenario. This two-tier functional redundancy for translation quality control breaks down during oxidative stress, wherein ThrRS is rendered inactive. Therefore, ATD knockout cells display pronounced sensitivity through increased mistranslation of threonine codons leading to cell death. Strikingly, we identify the emergence of ATD along with the error inducing tRNA species starting from Choanoflagellates thus uncovering an important genomic innovation required for multicellularity that occurred in unicellular ancestors of animals. The study further provides a plausible regulatory mechanism wherein the cellular fate of tRNAs can be switched from protein biosynthesis to non-canonical functions.

The first animals evolved around 750 million years ago from single-celled ancestors that were most similar to modern-day organisms called the Choanoflagellates. As animals evolved they developed more complex body plans consisting of multiple cells organized into larger structures known as tissues and organs. Over time cells also evolved increased levels of molecules called reactive oxygen species, which are involved in many essential cell processes but are toxic at high levels. Animal cells also contain more types of molecules known as transfer ribonucleic acids, or tRNAs for short, than Choanoflagellate cells and other single-celled organisms. These molecules deliver building blocks known as amino acids to the machinery that produces new proteins. To ensure the proteins are made correctly, it is important that tRNAs deliver specific amino acids to the protein-building machinery in the right order. Each type of tRNA usually only pairs with a specific type of amino acid, but sometimes the enzymes involved in this process can make mistakes. Therefore, cells contain proofreading enzymes that help remove incorrect amino acids on tRNAs. One such enzyme ­ called ATD ­ is only found in animals. Experiments in test tubes reported that ATD removes an amino acid called alanine from tRNAs that are supposed to carry threonine, but its precise role in living cells remained unclear. To address this question, Kuncha et al. studied proofreading enzymes in human kidney cells. The experiments showed that, in addition to ATD, a second enzyme known as ThrRS was also able to correct alanine substitutions for threonines on tRNAs. However, reactive oxygen species inactivated the proofreading ability of ThrRS, suggesting ATD plays an essential role in correcting errors in cells containing high levels of reactive oxygen species. These findings suggest that as organisms evolved multiple cells and the levels of tRNA and oxidative stress increased, this led to the appearance of a new proofreading enzyme. Further studies found that ATD originated around 900 million years ago, before Choanoflagellates and animals diverged, indicating these enzymes might have helped to shape the evolution of animals. The next step following on from this work will be to understand the role of ATD in the cells of organs that are known to have particularly high levels of reactive oxygen species, such as testis and ovaries.

Combinatorial Labeling Method for Improving Peptide Fragmentation in Mass Spectrometry.

Annotation of peptide sequence from tandem mass spectra constitutes the central step of mass spectrometry-based proteomics. Peptide mass spectra are obtained upon gas-phase fragmentation. Identification of the protein from a set of experimental peptide spectral matches is usually referred as protein inference. Occurrence and intensity of these fragment ions in the MS/MS spectra are dependent on many factors such as amino acid composition, peptide basicity, activation mode, protease, etc. Particularly, chemical derivatizations of peptides were known to alter their fragmentation. In this study, the influence of acetylation, guanidinylation, and their combination on peptide fragmentation was assessed initially on a lipase (LipA) from Bacillus subtilis followed by a bovine six protein mix digest. The dual modification resulted in improved fragment ion occurrence and intensity changes, and this resulted in the equivalent representation of b- and y-type fragment ions in an ion trap MS/MS spectrum. The improved representation has allowed us to accurately annotate the peptide sequences de novo. Dual labeling has significantly reduced the false positive protein identifications in standard bovine six peptide digest. Our study suggests that the combinatorial labeling of peptides is a useful method to validate protein identifications for high confidence protein inference. Graphical Abstract ᅟ.