Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

Abstract

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure2–8. mRNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function – it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding center9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as 100 novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease11–13.

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…Several modified ribonucleosides including 6‐methyladenosine (m 6 A), 5‐methylcytidine (m 5 C), 1‐methyladenosine (m 1 A) and pseudouridine have recently been shown to occur in messenger (m)RNAs and to affect their biogenesis, translation and stability (see e.g. Carlile et al , 2014; Liu & Jia, 2014; Dominissini et al , 2016). Methylated nucleosides can undergo further modification and proteins of the AlkB family of alpha‐ketoglutarate and Fe(II)‐dependent dioxygenases (ALKBH1‐8 and FTO in human cells) can oxidise or even remove modifications in DNA and RNA (Fedeles et al , 2015; Ougland et al , 2015), increasing the dynamics and regulation of RNA modifications and their roles in RNA metabolism.…”

Profiling of m6A RNA modifications identified an age‐associated regulation of AGO2 mRNA stability

“…For example, A‐to‐I editing within a coding region of mRNA may lead to abnormal protein expressions that are not encoded in the genome, and editing within intronic regions could create new splice sites resulting in the inclusion of an undesired sequence in the mature mRNA (Nishikura, 2016). Also, hundreds of pseudouridylated mRNAs were found in mammalian cells and, in response to serum starvation, dynamic changes of pseudouridylation were observed, underscoring its potential as a regulatory element (Carlile et al., 2014). As for RNA methylation, five types of methylation have been identified including N 7 ‐methylguanine (m 7 G at the 5' cap), N 6 ‐methyl adenosine (m 6 A), N 1 ‐methyl adenosine (m 1 A), 5‐methylcytosine (m 5 C), and 2‐ O ‐methylation (2'OME).…”

Properties and reactivity of nucleic acids relevant to epigenomics, transcriptomics, and therapeutics

“…23); [229][230][231] First with single RNAs and reverse transcriptase blockade assays, and more recently in massively parallel format to sequence classes of RNAs or even entire transcriptomes. 224,225,232,233 Many more  sites have been identified than expected and the new challenge is to determine the function of these edited sites. …”