Canonical MicroRNA Activity Facilitates but May Be Dispensable for Transcription Factor-Mediated Reprogramming

摘要

Highlights ? Reprogramming may be initiated and maintained solely by transcription factors ? miRNA activity as a whole facilitates reprogramming ? Canonical miRNAs may be dispensable for reprogramming Summary MicroRNAs (miRNAs) are important regulators of reprogramming of somatic cells into induced pluripotent stem cells (iPSCs); however, it is unclear whether miRNAs are required for reprogramming and whether miRNA activity as a whole facilitates reprogramming. Here we report on successful reprogramming of mouse fibroblasts and neural stem cells (NSCs) lacking Dgcr8 , a factor required for the biogenesis of canonical miRNAs, by Yamanaka factors, albeit at decreased efficiencies. Though iPSCs derived from Dgcr8 -deficient mouse fibroblasts or NSCs were able to self-renew and expressed pluripotency-associated markers, they exhibited poor differentiation potential into mature somatic tissues, similar to Dgcr8 ?/? embryonic stem cells. The differentiation defects could be rescued with expression of DGCR8 cDNA. Our data demonstrate that while miRNA activity as a whole facilitates reprogramming, canonical miRNA may be dispensable in the derivation of iPSCs. prs.rt("abs_end"); Introduction MicroRNAs (miRNAs) are short, endogenous, non-coding RNAs that repress gene expression post-transcriptionally by destabilizing and/or repressing translation of target mRNAs. In the canonical biogenesis pathway, primary microRNA transcripts (pri-miRNAs) are processed in the nucleus by the microprocessor complex, which consists of the RNase III enzyme DROSHA and the double-stranded RNA-binding protein DGCR8, to generate ～70-nt precursor miRNAs (pre-miRNAs). The pre-miRNAs are then exported to the cytoplasm by EXPORTIN-5 and further processed by another RNase III enzyme, DICER, to generate ～22-nt mature miRNAs ( Figure?S1 ) ( Kim et?al., 2009 ). More than 400 miRNAs have been identified in the human ( Landgraf et?al., 2007 ), and up to 60% of all human genes may be regulated by miRNAs ( Friedman et?al., 2009 ). Given the potentially vast regulatory influence of miRNAs on gene expression and the critical roles of these molecules in embryo development ( Bartel, 2009 and Sun and Lai, 2013 ), it is not surprising that miRNAs have emerged as important regulators in reprogramming somatic cells into induced pluripotent stem cells (iPSCs). Together with the Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC) ( Takahashi and Yamanaka, 2006 ), co-expression of the miRNA cluster 302/367 or 106a/363; members of the miR-302, miR-294, or miR-181 family; or miR-93 and miR-106b greatly enhance iPSC derivation efficiency ( Judson et?al., 2013 , Li et?al., 2011 , Liao et?al., 2011 , Lin et?al., 2011 and Subramanyam et?al., 2011 ). Furthermore, expression of the miR-302/367 cluster or miR-200c, miR-302, and miR-369 without the Yamanaka factors is sufficient to reprogram human and mouse fibroblasts ( Anokye-Danso et?al., 2011 and Miyoshi et?al., 2011 ). How these miRNAs promote reprogramming is only partially understood. Several mechanisms have been proposed, such as acceleration of mesenchymal to epithelial transition and antagonism of the activities of let-7 family miRNAs, MBD2, NR2F2, and/or other reprogramming suppressors ( Hu et?al., 2013 , Judson et?al., 2013 , Lee et?al., 2013 , Liao et?al., 2011 and Melton et?al., 2010 ). In addition to the miRNAs that promote reprogramming, several miRNAs that inhibit reprogramming, such as the let-7 family members, have been reported ( Melton et?al., 2010 and Unternaehrer et?al., 2014 ). Therefore, it remains unclear whether miRNA activity as a whole facilitates reprogramming and whether miRNAs are required to convert somatic cells into iPSCs. Previous attempts to reprogram Dicer null mouse embryonic fibroblasts (MEFs) were unsuccessful ( Kim et?al., 2012 ); however, this observation cannot rule out a requirement of miRNAs in reprogramming because DICER is also critical for the biogenesis of several other small RNAs, such as endogenous small hairpin RNAs (shRNAs), mirtrons, and endogenous small interfering RNAs (siRNAs) ( Figure?S1 ) ( Babiarz et?al., 2008 ). In this study, we addressed the question of whether miRNAs are required for generating iPSC by reprogramming mouse cells that lack Dgcr8 , a factor required specifically for the biogenesis of canonical miRNAs ( Figure?S1 ), including all miRNAs implicated in reprogramming ( Babiarz et?al., 2008 , Judson et?al., 2013 and Wang et?al., 2007 ). We report that Dgcr8 -deficient fibroblasts and NSCs can be reprogrammed by the Yamanaka factors, albeit at decreased efficiencies. These results demonstrate that while canonical miRNAs as a whole facilitate reprogramming, they may be dispensable for the derivation of iPSCs. Results Reprogramming of Dgcr8 Δ/Δ MEFs and Tail Tip Fibroblasts To assess the requirement of miRNAs in iPSC derivation, we first tested whether Dgcr8 -deficient MEFs and tail tip fibroblasts (TTFs) could be reprogrammed by Yama