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. 2012 Oct 26;151(3):533-46.
doi: 10.1016/j.cell.2012.09.027. Epub 2012 Oct 11.

Dicer partner proteins tune the length of mature miRNAs in flies and mammals

Ryuya Fukunaga  1 , Bo W Han, Jui-Hung Hung, Jia Xu, Zhiping Weng, Phillip D Zamore
Affiliations

Dicer partner proteins tune the length of mature miRNAs in flies and mammals

Ryuya Fukunaga et al. Cell. .

Erratum in

Abstract

Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. Alternative splicing of the loquacious (loqs) mRNA generates three distinct Dicer partner proteins. To understand the function of each, we constructed flies expressing Loqs-PA, Loqs-PB, or Loqs-PD. Loqs-PD promotes both endo- and exo-siRNA production by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: a specific subset of miRNAs requires Loqs-PB. Surprisingly, Loqs-PB tunes where Dicer-1 cleaves pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The longer form of miR-307a represses glycerol kinase and taranis mRNA expression. The mammalian Dicer-partner TRBP, a Loqs-PB homolog, similarly tunes where Dicer cleaves pre-miR-132. Thus, Dicer-binding partner proteins change the choice of cleavage site by Dicer, producing miRNAs with target specificities different from those made by Dicer alone or Dicer bound to alternative protein partners.

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Figures

Figure 1
Figure 1. Loqs-PB is Necessary and Sufficient to Maintain Female Germline Stem Cells
(A) Drosophila and human Dicer partners. (B) Drosophila germarium architecture. TF, terminal filament. CC, cap cells. SS, spectrosomes. Green, fusomes. Somatic cells are gray, germline are pink. Ovaries from 10-day-old females were stained with anti-Hu li tai shao (Hts) antibody (green) to detect the spectrosome and fusome, anti-Vasa antibody (red) to identify germ cells, and DAPI (blue) to highlight DNA. White arrowheads mark GSCs. (C) The number of GSCs per germarium (mean ± SD) was measured for each genotype in 10–15 randomly selected germaria from pooled ovaries from >30 flies. Eight z axis images, spanning 20 μm, were acquired for each germarium. See also Figure S1 and Table S1.
Figure 2
Figure 2. Loqs-PB Helps Dcr-1 Produce a Subset of miRNAs, Whereas Loqs-PD Helps Dcr-2 Produce Endo-siRNAs and Exo-siRNAs
(A) Silencing of white mRNA by a long hairpin RNA trigger corresponding to white exon 3 (GMR-wIR) was measured by using eye pigment from 3- to 4-day-old female fly eyes. Data are mean ± SD for five biological replicates. Figure S2A presents data from male heads. (B–D) Sequencing of small RNAs from female heads expressing the wIR RNAi trigger and from ovaries. (B) Normalized number of reads (ppm) for esi-1.1, esi-1.2, and esi-2.1, white siRNAs, miRNA (red) and miRNA* (blue) from fly heads and ovaries. Only miRNAs and miRNA* strands whose abundance was > 100 ppm in at least one library were analyzed. Figure S2C presents additional head data. (C) Abundance and 5′ position of wIR-derived siRNAs in heads. Antisense siRNAs are shown in red, sense in blue. (D) Change in cis-NAT and transposon-derived endo-siRNA abundance in heads. See also Figures S2, S3 and S4 and Tables S2, S3, and S4.
Figure 3
Figure 3. Loqs-PB Changes the Size Distribution of Small RNAs Cleaved from Pre-miR-307a In Vivo
The sequence, length, and abundance of ovary small RNAs from control and mutant females bearing Loqs-expressing transgenes was measured by deep sequencing. (A) Mean length of all miRNA isomirs in the transgenic, mutant flies compared to control (w1118). Only miRNAs whose total isomir abundance was more than 100 ppm in w1118 were analyzed. (B) Normalized abundance of each isomir of miR-307a* (blue) and miR-307a (red) for each genotype. Here and below, we considered only isomirs whose sequence variation reflected a change solely in the site Dicer cleaved. (C) Abundance of miR-9b isomirs. (D) Abundance of miR-316 isomirs. See also Figure S5.
Figure 4
Figure 4. Purified Loqs-PB, but Not Loqs-PA, Tunes the Length of Small RNAs from Pre-miR-307a
(A) Pre-miR-307a and pre-let-7 (100 nM) were incubated with recombinant Dcr-1 or Dcr-2 (10 nM) supplemented with Loqs-PA or Loqs-PB (10 nM) for 2 hr. The products were resolved by denaturing polyacrylamide electrophoresis and detected by northern hybridization. (B) Quantification of three independent replicates of the experiment in (A) and Figures S6H and S6I. Chimeric pre-miRNA comprised the pre-miR-307a stem and the pre-let-7 loop or the pre-let-7 stem and the pre-miR-307a loop. (C) Pre-miR-307 (30 nM) was incubated with purified, recombinant Dcr-1 alone (8 nM) or supplemented with Loqs-PA or Loqs-PB (8 nM). For sites of the 32P-radiolabel see Figure S6A. (D) The stoichiometry of Loqs-PB in complex with Dcr-1 was determined by titration. Products were detected by quantitative northern hybridization. The inflection point in the curves, 0.85 molecules of Loqs-PB per molecule of Dcr-1, suggests the two proteins form a 1:1 complex. Data are reported as mean ± SD for three independent trials. See also Figures S6 and S7.
Figure 5
Figure 5. miR-307a21-mer and miR-307a23-mer Have Distinct Targets
(A) Pairing of miR-307a21-mer (purple) and miR-307a23-mer (green) to luciferase reporter target sites (black). (B) Silencing of Renilla luciferase reporters bearing 3′ UTR target sites for miR-307a21-mer or miR-307a23-mer. Reporters had six sites with mismatches at positions 9–11, or four or eight sites matching the seed of miR-307a21-mer or miR-307a23-mer. Renilla luciferase expression relative to a firefly luciferase internal control was determined at different concentrations of transfected miRNA/miRNA*. Data were normalized to a transfection control without miR-307a/miR-307*. (C) The reporter data for Renilla mRNA bearing eight or no target sites matching the seeds of miR-307a21-mer or miR-307a23-mer was analyzed to determine the extent of repression at the highest concentration of transfected miR-307/miR-307* duplex. (D) Data for Renilla mRNA bearing the 3′ UTR of Gk or tara mRNA. miRNAs were transfected at 20 nM. Data are mean ± SD for three independent trials.
Figure 6
Figure 6. miR-307a23-mer, but Not miR-307a21-mer, Represses Gk and tara
(A) Relative abundance of Gk and tara mRNA in ovaries of control and mutant flies with Loqs-expressing transgenes, determined by quantitative RT-PCR (mean ± SD for three biological replicates) and mRNA-Seq. (B) Proposed functions of Dicer partner proteins in flies.
Figure 7
Figure 7. Mammalian TRBP Tunes Where Dicer Cleaves Pre-miR-132
(A) Length distribution of miR-132 and miR-132* isomirs in MEFs measured by sequencing. (B) Pre-miR-132 (100 nM), bearing a single 32P-radiolabel (asterisk), was incubated for 2 hr with purified, recombinant human Dicer (10 nM) or Dicer supplemented with PACT or TRBP (10 nM). Data are mean ± SD for four independent trials. See also Table S5.
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