miRNAs are produced in distinct steps in nuclear and cytoplasmic compartments. In the canonical miRNA biogenesis pathway (, left panel), primary miRNA transcripts (pri-miRNAs) are generated by RNA polymerase II and then cleaved into ~70-nucleotide precursor-miRNAs (pre-miRNAs) in the nucleus by the microprocessor complex, comprised of a RNAse III enzyme called Drosha and a dsRNA binding protein DiGeorge syndrome Critical Region Gene 8 (DGCR8)5, 6
. Pre-miRNAs are subsequently trafficked by Exportin 5 to the cytoplasm, where they are further cleaved by another RNAse III enzyme called Dicer at a site 22nt away from the Drosha cleavage site into mature miRNA duplexes 5, 7
. Dicer is in a complex that contains the dsRNA binding protein (Transactivating Region Binding Protein) TRBP, PACT (Protein Activator of PRK), and Loquious (Loqs). This complex further separates the two RNA strands of the miRNA/miRNA* duplex, incorporates one RNA strand (miRNA) into Ago and releases the other strand (miRNA*), which is typically subject to degradation 8–10
. The miRNA-containing Ago is then assembled into miRNA-induced Silencing Complex (miRISC), which recognizes the target mRNA via incomplete sequence complementarity between the miRNA and mRNA targets. Nucleotides 2–8 of miRNAs are critical for the target recognition through perfect complementarity with the target, and are thus called the “seed motif” 11, 12
. A single miRNA can recognize multiple targets, regulating up to several hundred different species of mRNAs. In addition, multiple miRNAs can act simultaneously on one mRNA target. Such combinatorial modes of action allow several hundreds of miRNAs to modulate the expression of thousands of mRNA species at the posttranslational levels. Such regulation is conceivably achieved via decreased translational efficiency and/or reduced mRNA stability 13
. Recently, Bartel and colleagues found that decreased mRNA levels constitutes most (~80%) of the reduced protein production14
Three different pathways of miRNA biogenesis: the canonical pathway (left panel), the microprocessor –independent pathway (middle panel), and the Dicer-independent pathways (right panel). For details, see text.
In addition to the canonical pathway, there are microprocessor-independent and Dicer-independent pathways (, middle panel). In the microprocessor-independent pathway (, middle panel), the cropping function by Drosha is replaced by three different mechanisms: (1) by spliceosome for mirtron pathway; (2) by Dicer for snoRNA-, tRNA-, and shRNA-derived precursor; and (3) by tRNase Z for tRNA-derived pathway 15–20
. Recently, Dicer-independent miRNAs were discovered in zebrafish and mammals 21, 22
. In this pathway (, right panel), the RNA cleavage activity of Ago2 mediates the maturation of pre-miR-451. miR-451 is unique in that its 5′-end was defined by Drosha cut, yet its 3′-end is flexible and extends over the loop region of the hairpin with a length range of 20–30 nt. Moreover, 1–5 non-templated uridine residues were also found at the 3′-end of longer reads, implying that the Ago2 cleavage products undergo uridylation and subsequent trimming by a nuclease. These alternative pathways illustrate the ability of cells to exploit a wide variety of mechanisms to generate miRNAs and to execute gene regulation.