Models for the biogenesis of canonical miRNAs, mirtrons, and hpRNAs in Drosophila. All three originate in the nucleus as RNA polymerase II transcripts containing inverted repeats, and are presumably capped and polyadenylated species. Canonical miRNAs are first cleaved by the Pasha/Drosha complex to release the pre-miRNA hairpin. Mirtrons are short intronic hairpins that are spliced and debranched to yield pre-miRNA mimics. Both types of pre-miRNAs are exported to the cytoplasm via Exportin-5, where they are cleaved by the Dicer-1/Loqs complex. The usual fate of the resulting small RNA duplex is for one strand to be preferentially loaded into AGO1 as the mature miRNA, while its partner miRNA* strand is preferentially degraded. However, a fraction of miRNA species are transferred into AGO2 and methylated at their 3' ends; a fraction of miRNA* strands are also transferred into AGO proteins. Hairpin RNA processing is not dependent on Exportin-5, and so the relevant factor is not known (?); possibly it transits the conventional mRNA export pathway. In the cytoplasm, Dicer-2 and Loqs are required for hpRNA maturation, but it is not known if these function as a discrete complex or not (?). The typical fate of the resulting small RNA duplexes is their preferential loading into AGO2 and 3' methylation as mature siRNAs. However, some hpRNA products associate with AGO1. A Dicer-2/R2D2 complex loads exogenously-derived siRNAs into AGO2, but it is not known if this is true for hpRNA-derived siRNAs (?).

Examples of canonical microRNA, mirtron, and hairpin RNA precursors from Drosophila. (A) A canonical miRNA precursor includes a lower duplex stem that is bound by Pasha/DGCR8 and cleaved by the Drosha RNAse III enzyme. The resultant pre-miRNA hairpin is cleaved on the terminal loop side by the Dicer RNAse III enzyme (specifically Dicer-1 in Drosophila) to yield a miRNA/miRNA* duplex. One duplex strand (in this case the bantam species, shaded in green) is preferentially loaded into an Argonaute protein, resulting in its preferred stability and higher cloning frequency. The numbers of cloned bantam/bantam* reads are from Ruby et al.58 (B) A mirtron is a short hairpin intron; shown is an example from the CG6695 gene. The hairpin ends coincide with the spliced intron, and thus the left RNA begins with a splice donor (here, GUGGGU) and the right RNA ends with a splice acceptor (here, CAG). Both canonical miRNAs and mirtrons are processed on their loop sides by Dicer-1. However, while mature miRNAs collectively derive from both left and right hairpin arms, the mature miRNA of Drosophila mirtrons almost always derives from the right arm (shaded in green). Reads are from Ruby et al.26 (C) A hairpin RNA is an inverted repeat transcript whose duplex region is usually much larger than that of canonical miRNAs; in this case, it is ~400 bp. hp-CG18854, like some other hpRNA loci, contains a large unstructured loops and is also spliced. Thus, the duplex regions are separated by substantial intervals of genomic sequence. hpRNAs generate small RNA duplexes with 3' overhangs, as is the case for canonical miRNAs and mirtrons. However, hpRNAs alone generate multiple cloned duplexes that are phased, likely because they are processed by Dicer-2. The dominant species of a given duplex can derive from either the left or right arm (as shaded in green); reads are from Okamura et al.18