ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

Cozen, Aaron E.; Quartley, Erin; Holmes, Andrew; Hrabeta-Robinson, Eva; Phizicky, Eric M.; Lowe, Todd M.

doi:10.1038/nmeth.3508

Cited by 374 publications

(434 citation statements)

References 42 publications

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“…We note that PSI-seq is optimized to detect Ψ's in RNAs that do not have other RT stops due to secondary structure or other modifications, whereas tRNAs are heavily modified and contain extensive secondary structure. A number of the CMCT-independent stops we observed were found in the region between positions 35 and 45 of the tRNAs, which are known to contain RT-blocking modifications (data not shown; Cozen et al 2015). Because the majority of Ψ's in tRNAs are typically located 5 ′ of positions 35-45, these blocks significantly lowered our power to detect tRNA Ψ's.…”

Section: Psi-seq Identifies Predicted and Tgpus1-dependent ψ'S In Toxmentioning

confidence: 88%

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: Psi-seq Identifies Predicted and Tgpus1-dependent ψ'S In Toxmentioning

confidence: 88%

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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“…Our laboratory and the Lowe/Phizicky laboratories independently developed tRNAseq methods that first removed many base methylations using AlkB-derived enzymes before cDNA synthesis (Cozen et al 2015;Zheng et al 2015). We also used a thermophilic reverse transcriptase (TGIRT) that could more efficiently read through base methylations than the commonly used superscript RTs.…”

Section: Identification Of Trna Methylation Sitesmentioning

confidence: 99%

“…Recently, our group and Lowe/Phizicky laboratories published Illumina sequencing methods for tRNA: DM-tRNAseq (demethylase tRNA sequencing) and ARM-seq (AlkBfacilitated RNA methylation sequencing) (Cozen et al 2015;Zheng et al 2015). The principle of both methods is to use the E. coli AlkB demethylase and its mutant as central components to remove m 1 A, N3-methyl-cytosine (m 3 C), and N1-methyl-guanosine (m 1 G) modifications at the Watson-Crick face in tRNA prior to cDNA synthesis.…”

Section: Introductionmentioning

confidence: 99%

tRNA base methylation identification and quantification via high-throughput sequencing

Clark¹,

Evans²,

Dominissini

et al. 2016

RNA

167

224

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Eukaryotic transfer RNAs contain on average 14 modifications. Investigations of their biological functions require the determination of the modification sites and the dynamic variations of the modification fraction. Base methylation represents a major class of tRNA modification. Although many approaches have been used to identify tRNA base methylations, including sequencing, they are generally qualitative and do not report the information on the modification fraction. Dynamic mRNA modifications have been shown to play important biological roles; yet, the extent of tRNA modification fractions has not been reported systemically. Here we take advantage of a recently developed high-throughput sequencing method (DM-tRNA-seq) to identify and quantify tRNA base methylations located at the Watson-Crick face in HEK293T cells at single base resolution. We apply information derived from both base mutations and positional stops from sequencing using a combination of demethylase treatment and cDNA synthesis by a thermophilic reverse transcriptase to compile a quantitative "Modification Index" (MI) for six base methylations in human tRNA and rRNA. MI combines the metrics for mutational and stop components from alignment of sequencing data without demethylase treatment, and the modifications are validated in the sequencing data upon demethylase treatment. We identify many new methylation sites in both human nuclear and mitochondrial-encoded tRNAs not present in the RNA modification databases. The potentially quantitative nature of the MI values obtained from sequencing is validated by primer extension of several tRNAs. Our approach should be widely applicable to identify tRNA methylation sites, analyze comparative fractional modifications, and evaluate the modification dynamics between different samples.

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“…This fact limits its use, considering the high tRNA sequence homology. Lowe and co-workers developed the AlkB-facilitated RNA methylation sequencing (ARM-seq), which demethylates m1A, m3C, and m1G, commonly found in tRNA (Cozen et al 2015). Besides, comparative methylation analysis using ARM-seq provides a transcriptome-scale map of these modifications.…”

Section: The State Of the Trna Population In The Cellmentioning

confidence: 99%

Protein folding and tRNA biology

2017

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Polypeptides can fold into tertiary structures while they are synthesized by the ribosome. In addition to the amino acid sequence, protein folding is determined by several factors within the cell. Among others, the folding pathway of a nascent polypeptide can be affected by transient interactions with other proteins, ligands, or the ribosome, as well as by the translocation through membrane pores. Particularly, the translation machinery and the population of tRNA under different physiological or adaptive responses can dramatically affect protein folding. This review summarizes the scientific evidence describing the role of translation kinetics and tRNA populations on protein folding and addresses current efforts to better understand tRNA biology. It is organized into three main parts, which are focused on: (i) protein folding in the cellular context; (ii) tRNA biology and the complexity of the tRNA population; and (iii) available methods and technical challenges in the characterization of tRNA pools. In this manner, this work illustrates the ways by which functional properties of proteins may be modulated by cellular tRNA populations.

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ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

Cited by 374 publications

References 42 publications

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

tRNA base methylation identification and quantification via high-throughput sequencing

Protein folding and tRNA biology

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