Transcriptome-wide analysis of pseudouridylation of mRNA and non-coding RNAs in Arabidopsis

Abstract

Pseudouridine (Ψ) is widely distributed in mRNA and various non-coding RNAs in yeast and mammals, and the specificity of its distribution has been determined. However, knowledge about Ψs in the RNAs of plants, particularly in mRNA, is lacking. In this study, we performed genome-wide pseudouridine-sequencing in Arabidopsis and for the first time identified hundreds of Ψ sites in mRNA and multiple Ψ sites in non-coding RNAs. Many predicted and novel Ψ sites in rRNA and tRNA were detected. mRNA was extensively pseudouridylated, but with Ψs being under-represented in 3′-untranslated regions and enriched at position 1 of triple codons. The phenylalanine codon UUC was the most frequently pseudouridylated site. Some Ψs present in chloroplast 23S, 16S, and 4.5S rRNAs in wild-type Col-0 were absent in plants with a mutation of SVR1 (Suppressor of variegation 1), a chloroplast pseudouridine synthase gene. Many plastid ribosomal proteins and photosynthesis-related proteins were significantly reduced in svr1 relative to the wild-type, indicating the roles of SVR1 in chloroplast protein biosynthesis in Arabidopsis. Our results provide new insights into the occurrence of pseudouridine in Arabidopsis RNAs and the biological functions of SVR1, and will pave the way for further exploiting the mechanisms underlying Ψ modifications in controlling gene expression and protein biosynthesis in plants.

Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

“…However, this modification is not known to exist in plastids. Pseudouridines (Ψ) were detected in chloroplasts mRNAs [48], but also do not influence reverse transcription. RNA editing, and conversion of cytidines into uridines [49], cannot be distinguished from DMS-caused mutations in reverse transcription.…”

Occurrence and Functions of m6A and Other Covalent Modifications in Plant mRNA

“…Significant enrichment then identifies mRNA intervals likely to contain a modified nucleotide (Dominissini et al, 2012;Meyer et al, 2012;Edelheit et al, 2013;Schwartz et al, 2013;Delatte et al, 2016;Dominissini et al, 2016;Li et al, 2016;Cui et al, 2017). (2) Modified nucleotides are specifically derivatized using unique reactivity with an appropriate compound, and the presence of the derived nucleotide is read either as a mutation signature or as a stop upon reverse transcription (Squires et al, 2012;Carlile et al, 2014;Lovejoy et al, 2014;Schwartz et al, 2014a;Li et al, 2015;David et al, 2017;Enroth et al, 2019;Sun et al, 2019;Zhang et al, 2019b). The cross-linking immunoprecipitation (CLIP) variant miCLIP constitutes an important special case in this category: a modification-specific antibody is UV cross-linked to purified RNA, allowing mutation-dependent mapping of cross-link sites (Ke et al, 2015;Linder et al, 2015) as in other CLIP techniques (Ule et al, 2018).…”

“…In plants, there are now reports on mapping of three distinct covalent nucleotide modifications in the bodies of mRNA: m 6 A (Li et al, 2014c;Wan et al, 2015;Shen et al, 2016;Duan et al, 2017;Anderson et al, 2018;Miao et al, 2019;Parker et al, 2019), 5-methylcytidine (m 5 C; Cui et al, 2017;David et al, 2017;Yang et al, 2019), and pseudouridine (C; Sun et al, 2019). Other types of mRNA modifications, including C-U editing of mitochondrial and plastidial mRNAs (Shikanai, 2006), alternative 59-caps (Kiledjian, 2018;Wang et al, 2019), and untemplated addition of nucleotides to mRNA 39-ends (De Almeida et al, 2018), will not be covered here.…”

“…Cartography of mRNA modifications (i.e. their mapping to specific sites in mRNAs) is very much in its infancy, particularly in plants, in which m 6 A stands out as the so-far best understood mRNA modification, with only limited knowledge available on C and m 5 C. A recent report shows that C exists in hundreds of mRNAs in Arabidopsis (Sun et al, 2019), but the functions of C in mRNA or the identities of pseudouridine synthases responsible for mRNA pseudouridylation remain unknown. Three different studies have mapped hundreds of m 5 C sites in Arabidopsis mRNA, but there is discrepancy in assessments of whether m 5 C is enriched in coding sequences or in 39-untranslated regions (UTRs), perhaps as a consequence of the different tissues used or because of actual inconsistencies between results obtained by the two different m 5 C mapping methods employed (Cui et al, 2017;David et al, 2017).…”

A molecular‐level perspective on the frequency, distribution, and consequences of messenger RNA modifications

“…Several modifications, Ψ, ac 4 C, Cm, and Gm, have abundances approaching those of m 6 A (Arango et al, ; X. Li et al, ; Tardu et al, ; You et al, ). These modifications have been less well studied than m 6 A, but their prevalence and localization in mRNA coding regions suggests that they might also play roles in regulating mRNA function (Arango et al, ; Carlile et al, ; X. Li et al, ; Lovejoy et al, ; Nakamoto, Lovejoy, Cygan, & Boothroyd, ; S. Schwartz et al, ; L. Sun et al, ; You et al, ). Like m 6 A, the positions of some of these modifications appear to be conserved.…”