Mouse let-7 miRNA populations exhibit RNA editing that is constrained in the 5′-seed/ cleavage/anchor regions and stabilize predicted mmu-let-7a:mRNA duplexes

MicroRNAs, a species of small noncoding RNA, induce post-transcriptional silencing of genes through mRNA decay and/or translational repression in the RNA induced silencing complex (RISC) (Ambros 2001; Bartel and Chen 2004; Wienholds and Plasterk 2005; Carthew 2006). However, more recent reports indicate that miRNAs can also activate or enhance translation (Vasudevan et al. 2007; Place et al. 2008). The active mature miRNA is processed in two steps from a primary miRNA (pri-miR) that is transcribed through Pol II or Pol III promoters. The pri-miR is processed into a precursor miRNA (pre-miR) by the RNaseIII-domain containing RNASEN (also known as DROSHA) enzyme (in the nucleus) and subsequently the pre-miR is processed into a ∼22-nucleotide (nt) dsRNA by another RNaseIII-domain containing enzyme, DICER1, once in the cytoplasm (Ambros 2001; Bartel and Chen 2004; Wienholds and Plasterk 2005; Carthew 2006). The ∼22-nt dsRNAs that are generated by RNASEN (DROSHA)/DICER1 enzymatic action contain 5′-phosphates and 3′ 2-nt overhangs. One of the strands (mature miRNA or guide RNA) is selectively loaded onto the RISC and the other strand (passenger RNA or miR*) is typically degraded. The guide RNA binds mRNA targets within the RISC to establish a miRNA:mRNA duplex. The transcripts that are regulated by a specific miRNA are determined by the binding of its 5′-seed (nucleotides 2–8) and anchor (nucleotides 13–16) with target sequences in the 3′ untranslated region (UTR) of cognate mRNAs (Rajewsky 2006; Grimson et al. 2007). In general, perfect or near-perfect complementarity yields stable duplexes that lead to mRNA decay (Hornstein et al. 2005). In contrast, imperfect miR:mRNA binding represses translation, without destroying the target mRNA. A number of recent reports reveal that ADAR-mediated A-to-I RNA editing affects the regulation of pri- and pre-miRNA processing (Bass 2006; Yang et al. 2006; Habig et al. 2007; Kawahara et al. 2007; Ohman 2007) and redirects the target repertoire by modifying the 5′-seed (Blow et al. 2006; Liang and Landweber 2007). Recently, high levels of terminal U-insertion and A-insertion were reported in mature miRNAs from a large cohort of 26 cell types (Landgraf et al. 2007). Internal mismatches were reported at a lower frequency. The relatively small number of sequences (∼1300 clones/tissue library) and of mismatches per miRNA per tissue observed in their study made it difficult to distinguish between PCR/sequencing error and other sources of these discrepancies, such as post-transcriptional modification. Recently developed “next-generation sequencing” (NGS) technologies, including Illumina sequencing (formerly known as Solexa sequencing) and 454 Life Sciences (Roche) pyrosequencing, afford an unprecedented opportunity to uncover novel aspects of miRNA biogenesis, processing, and function. To study the extent of the murine developmental miRNA-transcriptome, we applied Illumina and 454-GS20 amplicon sequencing strategies (Lu et al. 2005; Margulies et al. 2005; Rajagopalan et al. 2006; Ruby et al. 2006; Morin et al. 2008) to characterize small RNAs (<30 nt) expressed in mouse tissues. Ovary (Illumina), mouse embryonic pancreas E14.5 (454), and the insulin-secreting βTC-3 cell line (454) were sequenced (Efrat et al. 1988; Lynn et al. 2007). We then carried out a systematic study of nucleotide changes in mature miRNAs within a cell type through intraspecies comparison. From this work we have discovered RNA editing of internal nucleotides that are constrained in the 5′-seed and cleavage/anchor region nucleotides of predicted miRNA:mRNA duplexes associated with the mmu-let-7 family. The modifications also increase the stability of down-regulated mmu-let-7a:mRNA targets. Our results suggest a dynamic relationship between miRNAs and target genes that may play an important role in establishing the target repertoire and regulating mRNA decay versus translational repression for a given miRNA in a given cell.