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Promoted Read-through and Mutation Against Pseudouridine-CMC By An Evolved Reverse Transcriptase

ABSTRACT: Promoted Read-through and Mutation Against Pseudouridine-CMC By An Evolved Reverse Transcriptase

PROVIDER: PRJNA1121393 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29325551.fastq.gz	Fastqsanger.gz
	SRR29325552.fastq.gz	Fastqsanger.gz
	SRR29325553.fastq.gz	Fastqsanger.gz
	SRR29325554.fastq.gz	Fastqsanger.gz
	SRR29325555.fastq.gz	Fastqsanger.gz

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Promoted Read-through and Mutation Against Pseudouridine-CMC By An Evolved Reverse Transcriptase

Project description:Pseudouridine (Y) is an abundant chemical modification that plays critical roles in cellular RNA metabolism and functions, and in RNA therapeutic applications. Transcriptome-wide mapping technologies present major advantages in discovering the functional contexts and crucial effector proteins of Y. Current mapping strategies for Ys are limited in identifying Ys at base-resolution within consecutive uridine sequence contexts where Y occurs frequently. Here we report “Mut-Y-seq” that utilities a recently evolved reverse transcriptase (“RT-1306”) coupled with the selective labeling of Y by the CMC treatment to identify Ys in mRNAs and non-coding RNAs from human cells. We showed that RT-1306 showed significantly promoted read-through and mutation signatures against the CMC-Y adduct; the mutation signatures generated by RT-1306 can discern the position of Y in consecutive uridine sequence contexts. We quantitatively characterized the Y-identification efficiencies by Mut-Y-seq under variable chemical treatment conditions by the receiver operating characteristic curve analysis and revealed the presence of potential false positive discoveries generated by side-reactions on unmodified uridines. Finally, the intersection of sites identified by orthogonal chemical treatments (i.e. CMC and bisulfite) produced a high-confidence list of Y sites in mRNAs and non-ribosomal long non-coding RNAs from HEK-293T cells. This report presents datasets for biological functional studies of endogenous Y sites, and sheds light on the combination of the reverse transcriptase engineering and selective chemical reactions for further expanding the toolkit for studying RNA chemical modifications.

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Promoted Read-through and Mutation Against Pseudouridine-CMC by an Evolved Reverse Transcriptase.

Project description:Pseudouridine (Ψ) is an abundant RNA chemical modification that can play critical roles in the biological functions of RNA, and RNA-therapeutic applications. Current Ψ detection methods are limited in identifying Ψs at base-resolution in U-rich sequence contexts, where Ψ occurs frequently. The N-cyclohexyl N'-(2-morpholinoethyl)carbodiimide (CMC) can selectively label Ψ in RNA by forming the CMC-Ψ adduct. Here we report that an evolved reverse transcriptase ("RT-1306") shows promoted read-through and mutation against the CMC-Ψ. The mutation signature can resolve the occurrence of Ψs within UU-containing sequences. We developed "Mut-Ψ-seq" utilizing CMC and RT-1306 for transcriptome-wide mapping of Ψ at base-resolution. The mutation signatures robustly identify reported Ψs in human rRNAs via the ROC analysis, and elongated CMC reaction duration increases the detection sensitivity of Ψ. We report a high-confidence list of Ψ sites in polyA-enriched RNAs from HEK-293T cells identified by orthogonal chemical treatments (CMC and bisulfite). The mutation signatures resolve the position of Ψ in UU-containing sequences, revealing diverse occurrence of Ψs in such sequences. This work provides new methods and datasets for biological research of Ψ, and demonstrates the potential of combining the reverse transcriptase engineering and selective chemical labeling to expand the toolkit for RNA chemical modifications studies.

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