Nuclear fate of yeast snoRNA is determined by co-transcriptional Rnt1 cleavage

Abstract

Small nucleolar RNA (snoRNA) are conserved and essential non-coding RNA that are transcribed by RNA Polymerase II (Pol II). Two snoRNA classes, formerly distinguished by their structure and ribonucleoprotein composition, act as guide RNA to target RNA such as ribosomal RNA, and thereby introduce specific modifications. We have studied the 5ʹend processing of individually transcribed snoRNA in S. cerevisiae to define their role in snoRNA biogenesis and functionality. Here we show that pre-snoRNA processing by the endonuclease Rnt1 occurs co-transcriptionally with removal of the m7G cap facilitating the formation of box C/D snoRNA. Failure of this process causes aberrant 3ʹend processing and mislocalization of snoRNA to the cytoplasm. Consequently, Rnt1-dependent 5ʹend processing of box C/D snoRNA is critical for snoRNA-dependent methylation of ribosomal RNA. Our results reveal that the 5ʹend processing of box C/D snoRNA defines their distinct pathway of maturation.

DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

“…The utility of the DNAzyme-dependent cleavage in the analysis of rRNA modifications has been shown recently in the context of snoRNAs maturation 13 . The DNAzyme-dependent assay was used to show that lack of 5'-end pre-snoRNA processing affects 2'-O-methylation levels of 25S and 18S rRNA in S. cerevisiae 13 .…”

“…DNAzyme-dependent digestion can be used as a simple and quick method to analyze site-specific RNA 2'-O-methylation 12,13 . DNAzymes cleave RNA if the nucleotide upstream of the cleavage site is not methylated.…”

Records of RNA localization through covalent tagging

“…The presence of tagged snRNAs supports the recent finding that yeast snRNAs are shuttled to the cytoplasm as part of their maturation to prevent inclusion of misprocessed snRNAs in the spliceosome 84 . Tagged snoRNAs may arise through a different form of surveillance, as failure to properly process the guanosine cap of snoRNAs leads to their accumulation into the cytoplasm 86 .…”

“…While snoRNAs are thought to be restricted to the nucleus, failure to properly process their guanosine cap can cause their accumulation in the cytoplasm 86 . Together, the data on ncRNAs suggest tagging captures mature ncRNAs as well as ones undergoing maturation and surveillance.…”

Current approaches for RNA-labelling to identify RNA-binding proteins

“…Although these RBPs have been known to play important cellular roles by interacting with mRNAs (Gerstberger et al 2014;Dixit and Lukeš 2018;Moore and von Lindern 2018), recent work has demonstrated the importance of RBPs and their interactions with various types of noncoding RNA such as microRNAs, small nucleolar RNAs, long-noncoding RNA, piRNA, and viral RNA (Bagni and Lapeyre 1998;Jiang and Coller 2012;Ciafrè and Galardi 2013;Falaleeva et al 2016;Zealy et al 2017;Grzechnik et al 2018;Lei et al 2018;Tedeschi et al 2018;Baou et al 2011;Zhou et al 2018;Qiao et al 2019;Yang et al 2019). The formation of RNP complexes acts to regulate essential cellular functions from cell division to cell death, transcription to post-translational gene regulation, and downstream functions (Clayton and Shapira 2007;Glisovic et al 2008;Kishore et al 2010;Brinegar and Cooper 2016).…”