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Vilardo, E.

Publications and source records attributed to Vilardo, E..

2 recordsLinked to original sources

tRNA expression and modification landscapes, and their dynamics during zebrafish embryo development

tRNA genes exist in multiple copies in the genome of all organisms across the three domains of life. Besides the sequence differences across tRNA copies, extensive post-transcriptional modification adds a further layer to tRNA diversification. Whilst the crucial role of tRNAs as adapter molecules in protein translation is well established, whether all tRNA are actually expressed, and whether the differences across isodecoders play any regulatory role is only recently being uncovered. Here we built upon recent developments in the use of NGS-based methods for RNA modification detection and developed tRAM-seq, an experimental protocol and in silico analysis pipeline to investigate tRNA expression and modification. Using tRAM-seq we analysed the full ensemble of nucleo-cytoplasmic and mitochondrial tRNAs during embryonic development of the model vertebrate zebrafish. We show that the repertoire of tRNAs changes during development, with an apparent major switch in tRNA isodecoder expression and modification profile taking place around the start of gastrulation. Taken together, our findings suggest the existence of a general reprogramming of the expressed tRNA pool, possibly gearing the translational machinery for distinct stages of the delicate and crucial process of embryo development.

molecular biology↗

Cleavage kinetics of human mitochondrial RNase P and contribution of its non-nuclease subunits

RNase P is the endonuclease responsible for the 5-end processing of tRNA precursors (pre-tRNAs). Unlike the single-subunit protein-only RNase P (PRORP) found in plants or protists, human mitochondrial RNase P is a multi-enzyme assembly that in addition to the homologous PRORP subunit comprises a methyltransferase (TRMT10C) and a dehydrogenase (SDR5C1) subunit; these proteins, but not their enzymatic activities, are required for efficient pre-tRNA cleavage. Here we report a detailed kinetic analysis of the cleavage reaction by human PRORP and its interplay with TRMT10C-SDR5C1 using a comprehensive set of mitochondrial pre-tRNAs. Surprisingly, we found that PRORP alone binds pre-tRNAs with nanomolar affinity and can even cleave some of them at reduced efficiency without the other subunits. Thus, the ancient binding mode, involving the tRNA elbow and PRORPs PPR domain, seems retained by human PRORP, and its metallonuclease domain is in principle correctly folded and functional. Our findings support a model according to which the main function of TRMT10C-SDR5C1 is to direct PRORPs nuclease domain to the cleavage site, thereby increasing the rate and accuracy of cleavage. Human PRORPs dependence on the extra tRNA binder appears to have evolved to secure specificity in the cleavage of the structurally degenerating mitochondrial tRNAs.

biochemistry↗