Reevaluation by the CRISPR/Cas9 knockout approach revealed that multiple pluripotency-associated lncRNAs are dispensable for pluripotency maintenance while Snora73a/b is essential for pluripotency exit

Jul 12, 2024Science China. Life sciences

Removing several stem cell-related long RNAs does not affect stem cell maintenance, but Snora73a/b is needed for stem cells to start changing

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Circadian Biology on OpenScience ↗PubMed ↗DOI ↗

Abstract

Disruption of 8 lncRNAs previously thought to promote pluripotency in mouse embryonic stem cells showed no impact on pluripotency maintenance or cell proliferation.

All 8 lncRNAs were found to be dispensable for maintaining pluripotency and proliferation in mouse embryonic stem cells when individually or collectively disrupted.
Single-cell transcriptomic analysis revealed minimal effects on pluripotency gene expression and cell identity following lncRNA knockout.
Small hairpin RNAs used for lncRNA knockdown were found to downregulate pluripotency genes, suggesting off-target effects contributed to observed pluripotency defects.
Knockout and knockdown of the linc1343 lncRNA led to high expression of pluripotency genes and failure to form cystic structures during embryoid body differentiation.
Reintroducing RNA products from the linc1343 locus showed that two snoRNAs could rescue defects in pluripotency silencing during embryoid body differentiation.

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Many long noncoding RNAs (lncRNAs) have been identified through siRNA-based screening as essential regulators of embryonic stem cell (ESC) pluripotency. However, the biological and molecular functions of most lncRNAs remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 lncRNAs previously reported to promote pluripotency in mouse ESCs. Unexpectedly, all of these lncRNAs were dispensable for pluripotency maintenance and proliferation in mouse ESCs when disrupted individually or in combination. Single-cell transcriptomic analysis also showed that the knockout of these lncRNAs has a minimal impact on pluripotency gene expression and cell identity. We further showed that several small hairpin RNAs (shRNAs) previously used to knock down lncRNAs caused the downregulation of pluripotency genes in the corresponding lncRNA-knockout ESCs, indicating that off-target effects likely responsible for the pluripotency defects caused by these shRNAs. Interestingly, linc1343-knockout and linc1343-knockdown ESCs failed to form cystic structures and exhibited high expression of pluripotency genes during embryoid body (EB) differentiation. By reintroducing RNA products generated from the linc1343 locus, we found that two snoRNAs, Snora73a and Snora73b, but not lncRNAs, could rescue pluripotency silencing defects during EB differentiation of linc1343 knockout ESCs. Our results suggest that the 8 previously annotated pluripotency-regulating lncRNAs have no overt functions in conventional ESC culture; however, we identified snoRNA products derived from an annotated lncRNA locus as essential regulators for silencing pluripotency genes.

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Reevaluation by the CRISPR/Cas9 knockout approach revealed that multiple pluripotency-associated lncRNAs are dispensable for pluripotency maintenance while Snora73a/b is essential for pluripotency exit

Abstract

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