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Isoform characterization of m6A in single cells identifies its role in RNA surveillance [Direct RNA-seq for UPF1 knockdown]

ABSTRACT: Isoform characterization of m6A in single cells identifies its role in RNA surveillance [Direct RNA-seq for UPF1 knockdown]

PROVIDER: PRJNA1260438 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR33472400_1.fastq.gz	Fastqsanger.gz
	SRR33472401_1.fastq.gz	Fastqsanger.gz
	SRR33472402_1.fastq.gz	Fastqsanger.gz
	SRR33472403_1.fastq.gz	Fastqsanger.gz

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Isoform characterization of m6A in single cells identifies its role in RNA surveillance [Direct RNA-seq for UPF1 knockdown]

Project description:The distribution of m6A across various RNA isoforms and its heterogeneity within single cells are still not well understood. Here, we develop m6A-isoSC-seq, which employs both Oxford Nanopore long-read and Illumina short-read sequencing on the same 10x Genomics single-cell cDNA library with APOBEC1-YTH induced C-to-U mutations near m6A sites. Through m6A-isoSC-seq on a pooled sample of three cell line origins, we unveil a profound degree of m6A heterogeneity at both the isoform and single-cell levels. Through comparisons across single cells, we identify widespread specific m6A methylation on certain RNA isoforms, usually those mis-processed RNA isoforms. Compared to the coding isoforms of the same genes, the expression of highly methylated misprocessed RNA isoforms is more sensitive to METTL3 depletion. These mis-processed RNAs tend to have excessive m6A sites in coding regions, which are targets of CDS-m6A decay (CMD). This study offers undocumented insights into the role of m6A on RNA surveillance.

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Isoform characterization of m6A in single cells identifies its role in RNA surveillance [Direct RNA-seq for SMG1i treated]

Project description:Isoform characterization of m6A in single cells identifies its role in RNA surveillance [Direct RNA-seq for SMG1i treated]

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Project description:Metazoan histone mRNAs are a unique class of mRNAs that lack the poly(A) tail present in all other eukaryotic transcripts. Instead, they end in a conserved stem-loop (SL) structure, necessitating a decay mechanism that is distinct from deadenylation-initiated degradation. Here, combining cryoEM with chemical crosslinking mass spectrometry (CXMS) and functional approaches, we elucidate molecular mechanisms of initiation of histone mRNA decay. At the end of S-phase, the RNA helicase UPF1, the exoribonuclease 3’hExo and stem-loop binding protein SLBP all contribute to histone mRNA degradation, although how they are mechanistically coupled remained unknown. The cryoEM structure of an UPF1:SL RNA complex, presented here, shows that binding of UPF1 partially melts the RNA stem in the absence of ATP, harnessing the free energy derived from RNA-binding to unwind RNA. This melting event primes the SL-RNA for decay by 3’hExo. Using biochemical and cellular analyses, we demonstrate that SLBP directly engages the UPF1 helicase core to attenuate its unwinding activity and prevent premature degradation. Activation of UPF1 at a later stage promotes SL-RNA decay. We provide direct evidence that UPF1, SLBP and 3’hExo form a degradosome-like assembly that functionally couples SL unwinding and degradation, highlighting a dynamic and intricate network of UPF1-centric interactions that orchestrates timely histone mRNA decay.

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Isoform characterization of m6A in single cells identifies its role in RNA surveillance [Direct RNA-seq for SMG1i treated]

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Isoform characterization of m6A in single cells identifies its role in RNA surveillance [m6A-isoSC-seq (Illumina)]

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