Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Li, Xiaoyu; Zhu, Ping; Ma, Shiqing; Song, Jinghui; Bai, Jinyi; Sun, Fangfang; Yi, Chengqi

doi:10.1038/nchembio.1836

Cited by 463 publications

(713 citation statements)

References 49 publications

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“…This modification is found at high levels in rRNA in multiple organisms and is highly evolutionarily conserved at multiple sites in rRNA, tRNA, and spliceosomal RNA (Volkin and Cohn 1951;Spenkuch et al 2014;Li et al 2016). Most recently, with the advent of highthroughput sequencing tools, Ψ has also been found in mRNAs (Carlile et al 2014;Lovejoy et al 2014;Schwartz et al 2014;Li et al 2015).…”

Section: Introductionmentioning

confidence: 99%

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the singlecelled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3 ′ ′ ′ ′ ′ -UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.

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Section: Introductionmentioning

confidence: 99%

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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“…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. …”

Section: Pseudouridine Sequencing By Carbodiimide Modificationmentioning

confidence: 99%

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

2016

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Developments in epigenomics, toxicology, and therapeutic nucleic acids all rely on a precise understanding of nucleic acid properties and chemical reactivity. In this review we discuss the properties and chemical reactivity of each nucleobase and attempt to provide some general principles for nucleic acid targeting or engineering. For adenine-thymine and guaninecytosine base pairs, we review recent quantum chemical estimates of their Watson-Crick interaction energy, - stacking energies, as well as the nuclear quantum effects on tautomerism. Reactions that target nucleobases have been crucial in the development of new sequencing technologies and we believe further developments in nucleic acid chemistry will be required to deconstruct the enormously complex transcriptome.

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“…[22][23][24] In human, the reported number of mRNA C sites varies widely from 96 in one study, 23 to 353 in a second, 22 and up to 2084 in a third study. 25 This variation is likely the result of technical differences in experimental design and data analysis. While all three studies relied on the chemical reactivity of C with Ncyclohexyl-N 0 -b-(4-methylmorpholinium) ethylcarbodiimide (CMC), Li et al synthesized a biotinylated CMC derivative for an enrichment step, likely allowing them to identify low abundance sites that the other methods might miss.…”

Section: Mrnamentioning

confidence: 99%

“…In keeping with an emerging theme among mRNA modifications, however, C is dynamic in heat shock, nutrient deprivation, and other stresses. 22,23,25 While 5-methylcytidine (m 5 C) is best known for its central role as an epigenetic mark in DNA, it was also described in early studies of RNA methylation. 3 Mapping of m 5 C in RNA was initially attempted by adapting the bisulfite sequencing technology used for m 5 C in DNA, leading to the identification of approximately 10,000 m 5 C sites in mRNA.…”

Section: Mrnamentioning

confidence: 99%

Publisher's Note

2017

RNA Biology

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RNA modifications have long been known to be central in the proper function of tRNA and rRNA. While chemical modifications in mRNA were discovered decades ago, their function has remained largely mysterious until recently. Using enrichment strategies coupled to next generation sequencing, multiple modifications have now been mapped on a transcriptome-wide scale in a variety of contexts. We now know that RNA modifications influence cell biology by many different mechanisms -by influencing RNA structure, by tuning interactions within the ribosome, and by recruiting specific binding proteins that intersect with other signaling pathways. They are also dynamic, changing in distribution or level in response to stresses such as heat shock and nutrient deprivation. Here, we provide an overview of recent themes that have emerged from the substantial progress that has been made in our understanding of chemical modifications across many major RNA classes in eukaryotes.

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Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Cited by 463 publications

References 49 publications

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

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

Publisher's Note

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