Alternative miRNA biogenesis pathways and the interpretation of core miRNA pathway mutants

Abstract

Since the establishment of a canonical animal microRNA biogenesis pathway driven by the RNase III enzymes Drosha and Dicer, an unexpected variety of alternative mechanisms that generate functional microRNAs have emerged. We review here the many Drosha-independent and Dicer-independent microRNA biogenesis strategies characterized over the past few years. Beyond reflecting the flexibility of small RNA machineries, the existence of noncanonical pathways has consequences for interpreting mutants in the core microRNA machinery. Such mutants are commonly used to assess the consequences of "total" microRNA loss, and indeed, they exhibit many overall phenotypic similarities. Nevertheless, ongoing studies reveal a growing number of settings in which alternative microRNA pathways contribute to distinct phenotypes among core microRNA biogenesis mutants.

Figure 1. Canonical and major alternative miRNA biogenesis pathways in animals

(A) Canonical animal miRNAs are generated through consecutive cleavages of hairpin precursors by two RNase III enzymes. In the nucleus, the single strand-double strand junction of the pri-miRNA hairpin is recognized by DGCR8, which positions the catalytic site of the RNase III enzyme Drosha. This cleavage generates a ~55-70 pre-miRNA hairpin that is exported to the cytoplasm, where it is cleaved towards the terminal loop end by the RNase III enzyme Dicer. The miRNA/miRNA* duplexes are loaded into miRNA-class Argonaute effectors (in mammals, Ago1-4). One of the duplex strands is preferentially retained in Ago to form the functional RNA-induced silencing complex. (B) Many Drosha/DGCR8-independent pathways can generate pre-miRNA-like hairpins that serve as Dicer substrates. Mirtrons are short intronic hairpins that are excised by splicing and linearized by lariat debranching; tailed mirtrons require further resection by nucleases, e.g. 3′-5′ resection of 3′ tailed mirtrons by the RNA exosome. RNA pol III-transcribed MHV68 tRNA-shRNA fusions are processed into pre-miRNA-like hairpins with defined 5′ and 3′ ends as a result of RNaseZ cleavage and pol III termination, respectively. Endo-shRNAs without lower stems for Drosha/DGCR8 processing may derive from pol III transcription or cleavage by as yet unknown endo- or exonucleases. These non-canonical miRNAs, like canonical miRNAs, are incorporated to Ago1-4. Endogenous substrates with extended dsRNA character, including hpRNAs, transposable elements (TEs), antisense pseudogenes and natural antisense transcripts (NATs), are directly cleaved by Dicer to generate siRNAs. These may potentially sort to all of the mammalian Agos, but presumably only those that load Ago2 can fulfill target slicing. (C) Pri-mir-451 is processed by Drosha/DGCR8, and the resulting ~18-bp pre-mir-451 is directly incorporated to Ago2. The Slicer activity of Ago2 cleaves the 3′ arm of pre-mir-451, giving rise to ac-pre-mir-451, which is further resected by an as yet unknown mechanism to generate mature miR-451.

Figure 2. Summary of tRNA and snoRNA-derived small RNAs

During typical tRNA maturation, the 5′ leader and 3′ trailer of the pre-tRNA are removed by RNase P and RNase Z cleavages, respectively, followed by 3′ CCA addition. Subfragments of tRNAs are frequently observed, many of which reflect routine tRNA turnover and are not regulatory in nature. However, a number of specific pathways have been observed that extend the regulatory range of tRNA loci. In the case of tRNA-Ile/mir-1983, an alternative fold of the pre-tRNA adopts an extensive hairpin that permits cleavage by Dicer. Some mature tRNA cloverleafs may also serve as Dicer substrates and/or generate subfragments that load Ago proteins; this is most prominent in Tetrahymena where the Piwi protein Twi12 carries 3′ tRNA fragments exclusively. Some RNaseZ-generated tRNA 3′ trailers associate preferentially with Ago3/4 and regulatory activity of these trailers was reported, although this is not necessarily mediated by Ago complexes. Under stress conditions, mature tRNA are cut into halves, which may associate with unknown complexes to exert regulatory roles. A number of box C/D and box H/ACA snoRNAs can also give rise to Ago-associated, miRNA-like species in a Drosha/DGCR8-independent, Dicer-dependent manner.

Figure 3. Direct cleavage of RNA substrates by RNase III enzymes

RNase III enzymes may have regulatory roles that are independent of miRNA production. (A) mRNAs bearing short hairpin structures in untranslated or coding regions can be recognized and cleaved by Drosha/DGCR8, resulting in mRNA destabilization. The best-characterized example of this is cleavage of the dgcr8 mRNA by Drosha. (B) The pol III-transcribed long double-stranded Alu transcripts are toxic to cells unless they are cleaved by Dicer to short, inert fragments. In addition, direct dicing of viral replication intermediates may contribute to antiviral defense in Drosophila.