Dataset Information

RNA-interactome capture in human embryonic stem cells (hESCs)

ABSTRACT: Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carried out a proteome-wide screen and identified 810 proteins that directly bind RNA in hESCs. We determined the RBP catalog by using RNA-interactome capture (RIC), a method based on UV light-mediated cross-linking (CL) of RNAs to proteins in living cells, followed by oligo(dT) purification of poly(A)-RNA-protein complexes and mass spectrometry analysis of captured proteins. As control, we applied a similar strategy to non-cross-linked (non-CL) samples. To uncover the identity of the eluted proteins, we performed in-solution tryptic digestion of CL and non-CL eluates and analyzed their contents by a high-resolution mass spectrometer (Q-Exactive Plus). We then performed differential proteome analysis between CL and non-CL eluates, resulting in a set of 810 high-confidence protein groups, defined as the hESC RNA-interactome. RIC was carried out in four independent biological replicates. This data accompanies the manuscript: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells".

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Stem Cell

DISEASE(S): Disease Free

SUBMITTER: Shlomi Dvir

LAB HEAD: Yael Mandel-Gutfreund

PROVIDER: PXD025194 | Pride | 2023-05-05

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	HUMAN_refProteome_uniprot_canonical_UP000005640_70639_seq_release_140516.fasta	Fasta
	QEP2_YMA_CL_Eluate_1_1_180816_EasySpray.raw	Raw
	QEP2_YMA_CL_Eluate_1_1_Exclusion_300816_EasySpray.raw	Raw
	QEP2_YMA_CL_Eluate_2_1_180816_EasySpray.raw	Raw
	QEP2_YMA_CL_Eluate_2_1_Exclusion_300816_EasySpray.raw	Raw

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Publications

Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells.

Dvir Shlomi S Argoetti Amir A Lesnik Chen C Roytblat Mark M Shriki Kohava K Amit Michal M Hashimshony Tamar T Mandel-Gutfreund Yael Y

Cell reports 20210601 9

Embryonic stem cell (ESC) self-renewal and cell fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during early stem cell diff ...[more]

PMID: 34077720

Similar Datasets

Project description:Here, we performed RNA-interactome capture (RIC) on nuclear fractions from human embryonic stem cells (hESCs). The poly(A)+ RNA-bound proteome was determined by UV light-mediated cross-linking (CL) of RNAs to proteins in living cells, followed by nuclei isolation, oligo(dT) purification of poly(A)-RNA-protein complexes, and mass spectrometry analysis of captured proteins. As a control, we applied a similar strategy to non-cross-linked (non-CL) samples. RIC was performed in four independent biological replicates. This data accompanies the manuscript: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells". Abstract: "Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that directly bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during exit from pluripotency and early lineage specification. Moreover, we show that nearly 200 RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly bound by this factor. Intriguingly, over 20 percent of the proteins detected in our study are putative DNA- and RNA-binding proteins (DRBPs), among them key transcription factors (TFs). Using fluorescently labeled RNA and seCLIP (single-end enhanced crosslinking and immunoprecipitation) experiments, we discover that the pluripotency-associated STAT3 and OCT4 TFs interact with RNA in hESCs and confirm the direct binding of STAT3 to the conserved NORAD long-noncoding RNA. Taken together, our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated, reinforcing the importance of post-transcriptional regulation in stem cell biology".