Analysis of miR-376 RNA cluster members in the mouse inner ear.

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that regulate diverse developmental and physiological processes (Kapranov et al. 2007). This group of RNAs can negatively regulate protein expression at the post-transcriptional level by translational inhibition and/or mRNA degradation, mostly through base pairing with the 3′-UTR of their target mRNAs (Ambros 2003). Bioinformatic data indicate that a single miRNA could potentially bind to hundreds of mRNA targets, thus controlling various cellular functions (Bartel 2004). MicroRNAs genes (known as Mir genes) appear as single genes or in cluster of several Mir genes that are cotranscribed as a single transcription unit. MiRNAs are transcribed as parts of longer molecules called pri-miRNAs, which are processed in the nucleus into hairpin RNAs of 70–100 nucleotides (nt), called pre-miRNAs, by the double-stranded RNA (dsRNA)-specific ribonuclease Drosha (Lee et al. 2004). These hairpins are then transported to the cytoplasm, where they act as substrates for a second double-strand specific ribonuclease called Dicer and miRNA duplex of 20–22nt is obtained (Bernstein et al. 2003; Lee et al. 2004, Kim 2005). Studies have shown that pri-miRNAs of certain miRNA genes are subject to RNA editing that converts adenosine to inosine (A→I) (Luciano et al. 2004; Blow et al. 2006; Yang et al. 2006). This A-to-I RNA editing is catalysed by members of the ADAR (adenosine deaminases acting on RNA) gene family (Bass 2002; Nishikura 2006). Editing of pri-miRNAs could alter their processing including suppression of the Dicer or inhibition of the Drosha cleavage step. For instance, editing of pri-miR-151 by ADAR1 interferes with Dicer processing (Kawahara et al. 2007a), whereas that of pri-miR-142 by ADAR1 and ADAR2 suppresses Drosha processing and consequently affects the expression of the mature miRNA levels (Yang et al. 2006). Furthermore, editing of pri-miRNAs could also lead to the expression of edited mature miRNAs that are likely to repress genes different from those targeted by unedited miRNAs (Kawahara et al. 2007b). The miR-376 cluster of miRNA genes is located on human chromosome 14 and syntenic regions of the distal end of mouse chromosome 12 (Seitz et al. 2004). Expression of miR-376 RNAs has been reported in the developing embryos, and adult tissues, including the cochlea (Poy et al. 2004; Seitz et al. 2004; Friedman et al. 2009a; Wang et al. 2010). The four human miR-376 RNAs (miR-376a1, miR-376a2, miR-376b, miR-376c previously known as miR-368) and three mouse miR-376a-c RNAs share a high degree of sequence identity (Kawahara et al. 2007b; miRBase 18; genome.ucsc.edu/). It has also been shown that the miR-376 cluster members are transcribed into one long primary RNA transcript that can be subject to extensive and simultaneous editing by adenosine deaminase at one or two specific sites (+4 and +44) in select human and mouse tissues and subregions of the brain tissue (Kawahara et al. 2007b) during the processing of pri-miRNAs to mature miRNA (Luciano et al. 2004; Blow et al. 2006; Yang et al. 2006). The +4 site is edited by ADAR2, whereas the +44 site is selectively edited by ADAR1. Finding of the edited forms of mature miR-376 RNAs in certain tissues, such as brain, implicated that, unlike the case of pri-miR-142 and pri-miR-151, editing of pri-miR-376 RNAs at the two sites does not interfere with the Drosha and Dicer cleavage steps. A→I editing of miR-376 cluster transcripts is tissue specific, leading to the silencing of a different set of genes and tissue-specific regulation of certain gene products. Some miR-376 cluster members (pri-miR-376a2, pri-miR-376b and pri-miR-376c) are nearly 100% edited at the +44 site in the human cortex and medulla, whereas no editing was detected at the +4 site of human pri-miR-376a1 in liver and at the +44 site of mouse pri-miR-376a in all tissues (Kawahara et al. 2007b). Missense mutations in phosphoribosylpyrophosphate synthetase 1 (PRPS1) are associated with a spectrum of non-syndromic to syndromic hearing loss (de Brouwer et al. 2010; Liu et al. 2010). Both of the two editing sites in pri-miR-376 RNAs (+4 and + 44) are located within the functionally critical 5′ proximal ‘seed’ sequences of miRNA-376-5p and −3p strands, suggesting that the edited mature miR-376 RNAs may target genes different from those targeted by the unedited miR-376 isoform RNAs. The transcript of PRPS1 contains multiple binding sites for the edited version of miR-376a-5p within its 3′UTR that controls its transcription levels. This editing of miR-376a-5p is tissue specific. In the brain cortex, heart and kidney, it is edited, whereas in the liver almost no editing occurs. Because only the edited miR-376a-5p silences the expression of PRPS1, the activity of this target gene is repressed in a tissue-specific manner (Kawahara et al. 2007b). However, it is still unknown whether the pri-miRNAs editing of miR-376 cluster members occurs in the inner ear. In this study, we examined the pri-miRNAs editing of miR-376 cluster members in the mouse inner ear. We used quantitative real-time PCR (q-RT-PCR) and in situ hybridization to examine the expression pattern of miR-376a, b in the developing and adult mouse inner ear.