Abstract
Post‐transcriptional repression of gene expression by miRNAs occurs through transcript destabilization or translation inhibition. mRNA decay is known to account for most miRNA‐dependent repression. However, because transcript decay occurs co‐translationally, whether target translation is a requirement for miRNA‐dependent transcript destabilization remains unknown. To decouple these two molecular processes, we used cytosolic long noncoding RNAs (lncRNAs) as models for endogenous transcripts that are not translated. We show that, despite interacting with the miRNA‐loaded RNA‐induced silencing complex, the steady‐state abundance and decay rates of these transcripts are minimally affected by miRNA loss. To further validate the apparent requirement of translation for miRNA‐dependent decay, we fused two lncRNA candidates to the 3’‐end of a protein‐coding gene reporter and found this results in their miRNA‐dependent destabilization. Further analysis revealed that the few natural lncRNAs whose levels are regulated by miRNAs in mESCs tend to associate with translating ribosomes, and possibly represent misannotated micropeptides, further substantiating the necessity of target translation for miRNA‐dependent transcript decay. In summary, our analyses suggest that translation is required for miRNA‐dependent transcript destabilization, and demonstrate that the levels of coding and noncoding transcripts are differently affected by miRNAs.
Highlights
Post-transcriptional regulation of gene expression by microRNAs is widespread in eukaryotes and impacts diverse biological processes in health and disease (Bartel, 2004; Bartel, 2009)
Since post-transcriptional regulation by miRNAs occurs in the cytoplasm (Bartel, 2018), and does not directly impact the levels of nuclear long noncoding RNAs (lncRNAs), we first classified lncRNAs based on their subcellular localization
We took advantage of publicly available Halo-enhanced Ago2 pull-down (HEAP) data in mESCs (Li et al, 2020) to assess whether cytosolic lncRNAs are associated with miRNAs loaded into RISC (miRISC)
Summary
Post-transcriptional regulation of gene expression by microRNAs (miRNAs) is widespread in eukaryotes and impacts diverse biological processes in health and disease (Bartel, 2004; Bartel, 2009). Most mature miRNAs are the product of a relatively complex biogenesis process. Primary miRNA transcripts that generally depend on RNA Polymerase II for transcription are initially processed by the nuclear enzyme DROSHA and its cofactor DGCR8 into a premature hairpin RNA ~60 nucleotides in length (pre-miRNA transcript; Lee et al, 2003). Pre-miRNAs are exported into the cytoplasm, where they undergo a second round of processing by DICER, resulting in a ~22 nucleotide long double-stranded RNA duplex (Hutvagner et al, 2001). Loss-of-function mutations in any of the miRNA-processing factors result in complete depletion of most miRNA species (Kim et al, 2016). Argonaute proteins (AGO) bind mature miRNAs and guide target recognition of the RNA-inducing silencing complex (RISC). Target recognition relies primarily on complementarity between the miRNA seed region (position 2–8 of the mature miRNA) and miRNA recognition elements (MREs) in the target (Bartel, 2018)
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