How does Dicer-1 know what a pre-miRNA should look like?

Both small interfering RNAs (siRNAs) and microRNAs (miRNAs) are produced via the “dicing” action of Dicer enzymes. Insects have two Dicer proteins with specialized functions: Dicer-1 that processes precursor miRNAs (pre-miRNAs) and Dicer-2 that dices long double-stranded RNAs (dsRNAs). Here, HFSP Career Development Award holders Yukihide Tomari and Hervé Seitz and their colleagues show the unique mechanism by which fly Dicer-1 specifically recognizes the pre-miRNA structure.

HFSP Career Development Award holders Yukihide Tomari and Hervé Seitz and colleagues
authored on Tue, 06 December 2011

Small RNAs, such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) play no small role in gene regulation. Both siRNAs and miRNAs are born double-stranded from their precursor RNAs via the “dicing” action of Dicer enzymes. While mammals and worms have a single Dicer, insects have two Dicer proteins with specialized functions: Dicer-1 that liberates miRNA/miRNA* duplexes from precursor miRNAs (pre-miRNAs) and Dicer-2 that dices long double-stranded RNAs (dsRNAs) into siRNA duplexes. How do Dicer-1 and Dicer-2 recognize their cognate substrate RNAs exclusively? It was recently shown that Dicer-2 itself is a promiscuous enzyme that can dice both pre-miRNAs and long dsRNAs, but its partner protein R2D2 and inorganic phosphate render Dicer-2 into an enzyme specific for long dsRNAs (Cenik et al., 2011). However, the mechanism by which Dicer-1 specifically recognizes pre-miRNAs was unknown.

Pre-miRNAs have characteristic features, including a 2-nt 3´ overhang, a ~25-nt long double-stranded stem region which often contains internal mismatches, and a terminal single-stranded loop. To understand the substrate recognition mechanism by fly Dicer-1, HFSP Career Development Award holder Yukihide Tomari and colleagues took a classical biochemical approach; they purified recombinant Dicer-1 and assessed its dicing activity towards a systematic series of pre-miRNA structural variants. Altogether their data suggest that the N-terminal helicase domain of Dicer-1 binds to the terminal loop and checks the loop size. Dicer-1 also measures the distance from the 3´ overhang to the terminal single-stranded loop via the PAZ domain and the helicase domain, respectively (Figure). Dicer-1 then cuts the pre-miRNA at a fixed distance (~22 nt) from the 3´ overhang by its RNase III domains (Figure). This unique mechanism allows fly Dicer-1 to strictly inspect the authenticity of pre-miRNA structures. In contrast, recombinant human Dicer could not discriminate the pre-miRNA structural variants, agreeing with its dual functionality in processing both pre-miRNAs and long dsRNAs.

Given that insect Dicer-1 has the strict substrate specificity, could pre-miRNAs in insects conserve more restricted structures than those in other organisms? To this end, HFSP Career Development Award holder Hervé Seitz took a bioinfomatic approach. His data indicate that the loop size and the overhang-to-loop distance are highly similar across species, suggesting that insect Dicer-1 proteins have evolved to exclusively recognize the pre-miRNA structure, rather than that miRNA genes have evolved to adapt their structures to Dicer’s substrate specificity. Together, this collaborative, interdisciplinary work highlights the unique mechanism by which insect Dicer-1 specifically recognizes the pre-miRNA structure, and its biological meaning.

Reference

Recognition of the pre-miRNA structure by Drosophila Dicer-1. Tsutsumi A, Kawamata T, Izumi N, Seitz H, *Tomari Y.  Nat Struct Mol Biol. 2011 Sep 18;18(10):1153-8.

Other reference

Phosphate and R2D2 restrict the substrate specificity of Dicer-2, an ATP-driven ribonuclease.  Cenik ES, Fukunaga R, Lu G, Dutcher R, Wang Y, Tanaka Hall TM, Zamore PD.  Mol Cell. 2011 Apr 22;42(2):172-84.

Pubmed link