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Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

ABSTRACT: Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

PROVIDER: PRJNA1010332 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27276100_1.fastq.gz	Fastqsanger.gz
	SRR27276100_2.fastq.gz	Fastqsanger.gz
	SRR27276101_1.fastq.gz	Fastqsanger.gz
	SRR27276101_2.fastq.gz	Fastqsanger.gz
	SRR27276102_1.fastq.gz	Fastqsanger.gz

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Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Project description:Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of Ψ at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a Ψ-to-C transition. Compared to previous methods, BACS allowed the precise identification of Ψ positions, especially in densely modified Ψ regions and consecutive uridine sequences. BACS successfully detected all known Ψ sites in human rRNA and spliceosomal snRNAs and generated the first quantitative Ψ map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant Ψ114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of Ψ in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of Ψ in future studies.

2024-07-31 | GSE241849 | GEO

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome.

Project description:Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. Here, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS), which enables Ψ-to-C transitions, for quantitative profiling of Ψ at single-base resolution. BACS allowed the precise identification of Ψ positions, especially in densely modified Ψ regions and consecutive uridine sequences. BACS detected all known Ψ sites in human rRNA and spliceosomal small nuclear RNAs and generated the quantitative Ψ map of human small nucleolar RNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine editing sites and N1-methyladenosine. Depletion of pseudouridine synthases TRUB1, PUS7 and PUS1 elucidated their targets and sequence motifs. We further identified a highly abundant Ψ114 site in Epstein-Barr virus-encoded small RNA EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of Ψ in their viral transcripts or genomes, shedding light on differences in pseudouridylation across virus families.

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BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution

Project description:Pseudouridine (Ψ) is an abundant RNA modification that is present in and impacts the functions of diverse non-coding RNA species, including rRNA, tRNA, snRNA, etc. Pseudouridine also exists in mammalian mRNA and likely exhibits functional roles; however, functional investigations of pseudouridine in mammalian mRNA have been hampered by the lack of a quantitative method that detects Ψ at base precision. We have recently developed Bisulfite-Induced Deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA Ψ distribution transcriptome-wide at single-base resolution. Here, we describe an optimized BID-seq protocol to generate highly reproducible results, displaying nearly zero background deletions at unmodified uridines after bisulfite treatment and uncovering 8,407 Ψ sites starting from as little as 10 ng mouse embryonic stem cell (mESC) polyA+ RNA, with the steps that are fast in both library preparation and data analysis. This optimized BID-seq workflow takes 5 days to complete and includes four main sections: RNA preparation, library construction, next-generation sequencing (NGS), and data analysis. The library construction can be completed by researchers who have basic knowledge and skills in molecular biology and genetics. In addition to the experimental protocol, we provide BID-pipe (https://github.com/y9c/pseudoU-BIDseq), a user-friendly data analysis pipeline for BID-seq, requiring only basic bioinformatic and computational skills to uncover Ψ signatures from NGS data.

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BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution

Project description:BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution

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Transcriptome-wide Profiling of Multiple RNA Modifications Simultaneously at Single-base Resolution

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