Comparative parallel analysis of RNA ends identifies mRNA substrates of a tRNA splicing endonuclease-initiated mRNA decay pathway

Abstract

Eukaryotes share a conserved messenger RNA (mRNA) decay pathway in which bulk mRNA is degraded by exoribonucleases. In addition, it has become clear that more specialized mRNA decay pathways are initiated by endonucleolytic cleavage at particular sites. The transfer RNA (tRNA) splicing endonuclease (TSEN) has been studied for its ability to remove introns from pre-tRNAs. More recently it has been shown that single amino acid mutations in TSEN cause pontocerebellar hypoplasia. Other recent studies indicate that TSEN has other functions, but the nature of these functions has remained obscure. Here we show that yeast TSEN cleaves a specific subset of mRNAs that encode mitochondrial proteins, and that the cleavage sites are in part determined by their sequence. This provides an explanation for the counterintuitive mitochondrial localization of yeast TSEN. To identify these mRNA target sites, we developed a “comPARE” (comparative parallel analysis of RNA ends) bioinformatic approach that should be easily implemented and widely applicable to the study of endoribonucleases. The similarity of tRNA endonuclease-initiated decay to regulated IRE1-dependent decay of mRNA suggests that mRNA specificity by colocalization may be an important determinant for the degradation of localized mRNAs in a variety of eukaryotic cells.

Recent insights into the structure, function, and regulation of the eukaryotic transfer RNA splicing endonuclease complex

“…The yeast TSEN complex was shown to cleave a stem loop structure within CBP1 mRNA on the mitochondrial outer membrane both in vivo and in vitro. A recent bioinformatic approach termed comPARE (comparative parallel analysis of RNA ends) led to the identification of several additional yeast TSEN mRNA substrates (Hurtig et al, 2021). Interestingly, all these new mRNA substrates encode for mitochondrial proteins, suggesting that the spatial regulation of the TSEN complex influences its substrates.…”

“…To date, no additional substrates have been identified for the human complex but given the difference in cellular localization between yeast and human, the non-tRNA substrates are not likely to be conserved. Adaptation of the comPARE methodology to mammalian cell lines along with 2 0 3 0 -cyclic phosphate and 5 0 -hydroxyl sequencing approaches will hopefully aid in the future determination of non-tRNA substrates of the human complex (Hurtig et al, 2021;Peach et al, 2015;Shigematsu et al, 2019)…”

What connects splicing of transfer RNA precursor molecules with pontocerebellar hypoplasia?

“…As yeast TSEN was demonstrated to cleave certain subtypes of rRNAs and mRNAs, [43,[48][49][50][51] analyses of such cleavage products in patient-derived neuronal cells would offer the possibility to explore yet unknown targets of human TSEN. Putative mRNA targets could be the missing link between TSEN/CLP1 and the mRNA 3′-end processing pathway.…”

“…Several reports associated the yeast TSEN complex with processing of precursor ribosomal RNAs (pre‐rRNAs) and mRNAs encoding mitochondrial proteins. [ 43,48–51 ] Similarly, depletion of the human TSEN2 subunit was shown to impede mRNA 3′‐end processing. [ 30 ] TSEN components and CLP1 can be detected in cytosolic and nuclear fractions of HeLa cells further indicating cellular functions beyond pre‐tRNA splicing.…”

Identification of long non-coding RNAs as substrates for cytoplasmic capping enzyme

“…Future studies will be directed to reveal the role of cap homeostasis in regulating these lncRNAs. For example, analyses using CAGE, teloprime [60] or related techniques coupled with degradome sequencing [61] can validate the sequences obtained via RACE experiments and/or identify new candidate cytoplasmic capping sites. In addition, future studies should be directed to identify specific sequences that might protect these lncRNAs from cellular nucleases and/or target these lncRNAs to the cytoplasmic capping machinery.…”