Abstract
The DiGeorge syndrome critical region gene 8 (Dgcr8) knockout strategy has been widely used to study the function of canonical microRNAs (miRNAs) in vitro and in vivo. However, primary miRNA (pri-miRNA) transcripts are accumulated in Dgcr8 knockout cells due to interrupted processing. Whether abnormally accumulated pri-miRNAs have any function is unknown. Here, using clustered regularly interspaced short palindromic repeats system/CRISPR-associated protein 9 (CRISPR/Cas9), we successfully knocked out the primary microRNA-290~295 (pri-miR-290~295) cluster, the most highly expressed miRNA cluster in mouse embryonic stem cells (ESCs), in Dgcr8 knockout background. We found that the major defects associated with Dgcr8 knockout in mouse ESCs, including higher expression of epithelial-to-mesenchymal transition (EMT) markers, slower proliferation, G1 accumulation, and defects in silencing self-renewal, were not affected by the deletion of pri-miR-290~290 cluster. Interestingly, the transcription of neighboring gene nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 12(Nlrp12) was upregulated upon the deletion of the pri-miR-290~295 cluster. Together, our results suggested that the major defects in Dgcr8 knockout ESCs were not due to the accumulation of pri-miR-290~295, and the deletion of miRNA genes could affect the transcription of neighboring DNA elements.
Highlights
The essential roles of microRNAs in modulating gene expression and various cellular processes have been extensively reported [1]
RNA-Seq analysis of the wild type and DiGeorge syndrome critical region gene 8 (Dgcr8) knockout embryonic stem cells (ESCs) showed that pri-miR-290~295 transcripts were significantly accumulated in Dgcr8 knockout ESCs (Figure 1A,B, Table S1)
Accumulated reads from miR-290~295 cluster accounted for ~70% of total accumulated reads from all mapped miRNA loci, indicating that pri-miR-290~295 was the major contributor to the pool of accumulated pri-miRNAs in Dgcr8 knockout ESCs
Summary
The essential roles of microRNAs (miRNAs) in modulating gene expression and various cellular processes have been extensively reported [1]. The potential role of pri-miR-290~295 in regulating the cell cycle, pluripotency maintenance, and self-renewal was investigated by comparing Dgcr8/ pri-miR-290~295 double knockout to Dgcr knockout ESCs. We showed that the deletion of primary transcripts of the miR-290~295 cluster had no overt effects for these processes. We showed that the deletion of primary transcripts of the miR-290~295 cluster had no overt effects for these processes In both wild type and Dgcr knockout ESCs, the deletion of pri-miR-290~295 locus led to the upregulation in the transcription of neighboring gene Nlrp. The accumulation of pri-miR-290~295 transcripts was not responsible for major defects observed in Dgcr knockout ESCs. the deletion of miRNA genes might impact the transcription of neighboring genes through mechanisms independent of miRNA production and targeting
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