Features of canonical miRNA and mirtron biogenesis. Canonical miRNAs are transcribed as long primary transcripts whose hairpin structures are cleaved by the nuclear Drosha RNAse III enzyme to release pre-miRNAs. Mirtrons are short hairpin introns that are spliced and then debranched, yielding pre-miRNA mimics. Both types of hairpins are exported from the nucleus by Exportin-5, then cleaved by the cytoplasmic RNAse III enzyme Dicer; this yields a duplex of ~21–24 nt RNAs. One RNA product, termed the mature miRNA, is preferentially loaded into an Argonaute (AGO) protein and guides it to complementary transcripts for regulation. The other duplex strand, termed the miRNA* species, is favored for degradation and accumulates to a lower level than the miRNA. The schematic was adapted from a published model [54].

Changes in miRNA sequence can diversify miRNA activity. (A) Typical families of miRNAs can be classified according to shared 7mer motifs located at positions 2–8, also known as the miRNA “seed” (green box). Shown are the three members of the Drosophila miR-9 family; note that their sequences are identical through position 8, but strongly diverge beginning with position 9. The members of this family are inferred to have at least some common targets because of their shared seed, although their target properties are likely somewhat distinct because of their divergent 3′ ends. (B) An atypical family of Drosophila miRNAs which differ in a seed position (red box). These miRNAs are inferred to have mostly non-overlapping target sets. (C) Example of a miRNA that is edited in human and mouse, miR-376-5p. Of several edited positions identified, the A-I conversion at position 4 is significant as it is inferred to redirect its targeting capacity.

Changes in Drosha or Dicer processing can diversify miRNA activity. (A) mir-2a-1 and mir-2a-2 share 27 consecutive nucleotides along their miRNA-producing arms; therefore many sequences can be mapped to either genomic locus (# loci=2). However, their unique miRNA* sequences (# loci=1) allow their respective miRNA strands to be deconvolved based on miRNA/miRNA* duplexes with ~2-nt 3′ overhangs (B). This reveals the 5′ ends of miR-2a-1 and miR-2a-2 to be shifted by 2 nt with respect to each other (C). (D) An example of a rare miRNA locus that appears to be subject to alternative Dicer processing, yielding equal numbers of the distinct miRNAs miR-210.1 and miR-210.2. Note that the mir-2 and mir-210 cloning data depict the most abundant isoforms recovered from large-scale sequencing data [28]; less abundant reads mapped to these loci are not shown.

Changes in spatial or temporal expression can diversify miRNA activity. (A) miR-13b-1 and miR-13b-2 are identical miRNAs produced from loci on different chromosomes. Their non-redundant activity is evidenced by the distinct expression of their primary transcripts in the central nervous system (B) and the gut and musculature (C). (D) The let-7 sisters comprise related miRNAs with distinct temporal expression. (E) The levels of miR-48/miR-84/ miR-241 peak during the transition from L2 to L3, while the level of let-7 peaks during the transition from L4 to the adult. (F) Genetic hierarchy of the control of L2-L3 transition by miR-48/miR-84/miR-241 and control of the L4-adult transition by let-7. Note that let-7 is a unique regulator of lin-41 at this developmental stage since it requires 3′ compensatory pairing that is specific to let-7; mir-48/miR-84/miR-241 may repress hbl-1 at both stages.

Acquisition of miRNA* functionality can diversify miRNA activity. While bulk miRNA* species are preferentially degraded, a substantial fraction of miRNA* species are actively sorted into AGO complexes and are used to repress endogenous targets. The schematic was adapted from a published model [66].

Antisense miRNA transcription and processing can diversify miRNA activity. The mir-iab-4 hairpin in the Drosophila Bithorax Complex (BX-C) is transcribed on its antisense strand as the mir-iab-8 hairpin. Thus, four different small RNAs are produced from a single hairpin locus in embryos. The 5p miRNAs of mir-iab-4 and mir-iab-8 directly regulate other Hox genes; stronger regulatory interactions are depicted with darker lines. The schematic was adapted from a published model [86].

Typical genomes encode many miRNA-like hairpins but only a limited set of genuine miRNA hairpins that are specifically processed by the canonical miRNA or mirtron machinery (Figure 1). The large surplus of genomic hairpins that occur frequently throughout the genome may serve as a breeding ground of nascent miRNAs that may accidentally enter the miRNA processing pathway at some low frequency. If they provide a useful function to the organism, these may eventually be stabilized as genuine miRNA genes.