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Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells

ABSTRACT: Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells

PROVIDER: PRJNA826336 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18740938_1.fastq.gz	Fastqsanger.gz
	SRR18740938_2.fastq.gz	Fastqsanger.gz
	SRR18740939_1.fastq.gz	Fastqsanger.gz
	SRR18740939_2.fastq.gz	Fastqsanger.gz
	SRR18740940_1.fastq.gz	Fastqsanger.gz

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Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells

Project description:N6-methyladenosine (m6A) plays critical roles in gene expression control by recruiting the cytoplasmic reader proteins YTHDF1,2 and 3. Recently, the function of YTHDF proteins and whether they bind to shared or distinct sets of methylated mRNAs in cells has been the subject of debate. Here, we developed TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. Applying TRIBE-STAMP to the YTHDF proteins revealed that most target mRNAs are shared among the three YTHDF proteins. Surprisingly, we also found that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime. Furthermore, we show that YTHDF proteins bind sequentially to target methylated mRNAs, with YTHDF1 binding before YTHDF2 or YTHDF3. Our data reveal shared molecular interactions among the YTHDF proteins and support a model in which YTHDF1 and YTHDF3 are unlikely to promote rapid mRNA decay.

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Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells.

Project description:RNA-binding proteins (RBPs) regulate nearly every aspect of mRNA processing and are important regulators of gene expression in cells. However, current methods for transcriptome-wide identification of RBP targets are limited, since they examine only a single RBP at a time and do not provide information on the individual RNA molecules that are bound by a given RBP. Here, we overcome these limitations by developing TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. We applied TRIBE-STAMP to the cytoplasmic m6A reader proteins YTHDF1, YTHDF2, and YTHDF3 and discovered that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime, providing new insights into the function of YTHDF proteins in cells. TRIBE-STAMP is a highly versatile approach that enables single-molecule analysis of the targets of RBP pairs simultaneously in the same cells.

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