Biogenesis of miRNAs

Th e biogenesis of miRNAs is diverse following several diff erent pathways, refl ecting control of the processes by diff erent mechanisms and evolutionary fl exibility. Th e pathways are generally divided into canonical and alternative. Th e canonical pathway refers to RNA polymerase II (RNAP II) transcripts processed by two RNase III1 enzymes, Drosha and Dicer, generating the majority of miRNAs. Th e alternative pathways generate non-canonical miRNAs usually bypassing one of the RNase III processing steps. Th ese pathways came into light following analyses of data from deep sequencing and high-throughput sequencing technologies, revealing miRNAs that only partially meet the classical criteria (Fig. 1a). Canonical miRNA precursors are transcribed by RNAP II into long primary transcripts (pri-miRNAs) of several thousand nucleotides in length that acquire 5¢ cap and 3¢ poly(A) tails at their ends (Lee et al. 2004). RNAP III can also transcribe sequences to produce miRNAs as is the case for the human chromosome 19 miRNA cluster (C19MC) (Bohmert et al. 1998). Oft en, the primary transcripts are organized as polycistronic primary transcripts consisting a miRNA cluster (Peters and Meister 2007). Th ese clusters are highly conserved implying important regulatory and functional roles (Elbashir et al. 2001b). Either in clusters or alone, the resulting transcripts are capped, spliced and polyadenylated (Cai et al. 2004; Lee et al. 2004). Subsequently, pri-miRNAs are cleaved in the nucleus by Drosha, an RNase III endonuclease, in complex with the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region gene 8), also known as Pasha (Partner of Drosha) in D. melanogaster and Pash-1 in C. elegans, forming the microprocessor complex. DGCR8 interacts directly with the pri-miRNA guiding Drosha to cleave the 5¢ and 3¢ arms of the pri-miRNA hairpin producing a pre-miRNA with a 5¢ phosphate, a 3¢ hydroxyl and a 2-nt overhang at its 3¢ end that serves as a recognition site for the second processing step (Denli et al. 2004; Gregory et al. 2004; Han et al. 2004; Landthaler et al. 2004; Miyoshi et al. 2010; Yang and Lai 2011). An unexpected origin of miRNAs was revealed when it was reported that introns might process to functional miRNAs, also revealing an alternative

pathway for their biogenesis. Th ese pre-miRNAs/introns, called mirtrons, can bypass Drosha cleavage given that the intron resulting from mRNA splicing has the appropriate size to form a hairpin resembling a precursor miRNA (pre-miRNA) (Okamura et al. 2007; Ruby et al. 2007). Th us far, 11 human mirtrons have been predicted (Berezikov et al. 2007). However, research on the biogenesis of mirtrons revealed a subset of predicted mirtrons that are splicing-independent, thus termed “simtrons” (splicing-independent mirtron-like miRNAs) (Havens et al. 2012). Simtrons do not follow the canonical miRNA pathway either, but they are Drosha-dependent. Either originated from the canonical or the mirtron pathway, the resulting pre-miRNA consists of ~ 60-75 nucleotides with a hairpin shape and a two-nucleotide overhang at its 3¢ end (Lee et al. 2002; Bartel 2004; Cai et al. 2004; Han et al. 2004; Singh et al. 2008). Another alternative biogenesis pathway and an exception to the Drosha-processing revealed upon studies on non-coding RNAs contained in vault particles known as vault RNAs (vRNAs or vtRNAs)2. It was shown that vRNAs generate small vault RNAs bypassing Drosha processing, while Dicer is involved in svRNA cleavage (Persson et al. 2009)3. When a pre-miRNA is correctly processed (i.e bearing a short stem loop and a 3¢ overhang), it can be exported to the cytoplasm through successful binding to Exportin 5 in complex with Ran-GTP (Yi et al. 2003; Lund et al. 2004). Further processing in the cytoplasm by Dicer, a RNase III endonuclease, removes the loop of the pre-miRNA leaving another 3¢ overhang to the other end (reviewed in (Kim 2005a). Th e previous processes result to a mature miRNA of 19-24 nucleotide duplex with two nucleotides protruding as overhangs at each 3¢ end. Th erefore, in an appropriate loop sequence in the nucleus, Drosha generates one end of pre-mature miRNA (a two-nt 3¢ overhang), while in the cytoplasm Dicer measures ~22 nucleotides from the pre-existing end (generated by Drosha) producing the other end of the miRNA through appropriate cleavage of the loop.