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. 2016 Jul 8;44(12):5924-35.
doi: 10.1093/nar/gkw276. Epub 2016 Apr 19.

Poly(A)-binding proteins are required for microRNA-mediated silencing and to promote target deadenylation in C. elegans

Affiliations

Poly(A)-binding proteins are required for microRNA-mediated silencing and to promote target deadenylation in C. elegans

Mathieu N Flamand et al. Nucleic Acids Res. .

Abstract

Cytoplasmic poly(A)-binding proteins (PABPs) link mRNA 3' termini to translation initiation factors, but they also play key roles in mRNA regulation and decay. Reports from mice, zebrafish and Drosophila further involved PABPs in microRNA (miRNA)-mediated silencing, but through seemingly distinct mechanisms. Here, we implicate the two Caenorhabditis elegans PABPs (PAB-1 and PAB-2) in miRNA-mediated silencing, and elucidate their mechanisms of action using concerted genetics, protein interaction analyses, and cell-free assays. We find that C. elegans PABPs are required for miRNA-mediated silencing in embryonic and larval developmental stages, where they act through a multi-faceted mechanism. Depletion of PAB-1 and PAB-2 results in loss of both poly(A)-dependent and -independent translational silencing. PABPs accelerate miRNA-mediated deadenylation, but this contribution can be modulated by 3'UTR sequences. While greater distances with the poly(A) tail exacerbate dependency on PABP for deadenylation, more potent miRNA-binding sites partially suppress this effect. Our results refine the roles of PABPs in miRNA-mediated silencing and support a model wherein they enable miRNA-binding sites by looping the 3'UTR poly(A) tail to the bound miRISC and deadenylase.

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Figures

Figure 1.
Figure 1.
PAB-1 and PAB-2 interact with miRISC in Caenorhabditis elegans embryos. (A) ALG-1/2 western blot analysis of 2′-O-Me pull-downs. 5′ Biotinylated 2′-O-Me oligos containing miR-35, CeBantam or both sites were used to pull down the miRISC WT strain (N2) embryonic extract. ALG-1 and ALG-2 were detected using near-infrared fluorescent western blot (LiCOR). (B) Proteins identified in MuDPIT analyses of a dual site (CeBantam + miR-35) 2′-O-Me pull-down. Only proteins which were detected in two independent purifications and were absent from pull-downs with mutated binding sites are included here. For each protein, the number of peptides (#pep) found in each experiment and the coverage (%cov) of these peptides for the full-length protein are indicated. (C) Venn diagram comparing detected interactions with the dual site (CeBantam + miR-35) pull-down to single-site RISC pull-down, and AIN-2-GFP IP. (D) A GST pull-down using either GST or GST-PAIP2 was performed either on WT (N2) or mutant pab-2(0) (ok1851) embryonic extract in the presence of 0.1 ng/μl RNAse A. The bound proteins were analyzed on SDS-PAGE and detected by western blot.*: non-specific band.
Figure 2.
Figure 2.
pab-1 and pab-2 genetically cooperate with let-7 and lsy-6 miRNAs. (A) pab-1 and pab-2 genetically interact with the let-7 miRNA. let-7(n2853) animals (L1) were fed with bacterially-expressed dsRNA against the indicated gene, or L4 animals injected with dsRNA (B) and F1 animals were scored for bursting vulva phenotype at the permissive temperature (16°C). Results shown are representative of at least two experimental and biological replicates. (C) pab-2 genetically interacts with lsy-6. pab-2(0)(ok1851) were crossed with OH3646 (otIs114(Plim-6::GFP, rol-6(d));lsy-6(ot150). Animals were scored for expression of GFP in the ASEL.
Figure 3.
Figure 3.
Biochemical depletion of PAB1 and PAB-2 delays deadenylation. (A) PAB-1 and PAB-2 levels were assessed by western blot in GST- or GST-PAIP2-treated extracts. A fraction of the extract was analyzed by SDS-PAGE followed by western blotting. (B) ×ばつ-miR-35-p(A)86 was subjected to an in vitro deadenylation assay in the presence of a miR-35 or miR-1 2′-O-Me inhibitor in GST- or GST-PAIP2-treated extracts. RNA was extracted and analyzed by UREA-PAGE. (C) ×ばつ-miR-52-p(A)86 was subjected to an in vitro deadenylation assay in GST- or GST-PAIP2-treated extracts. RNA was extracted and analyzed by UREA-PAGE. (D) Human PABC1 restores deadenylation in the Caenorhabditis elegans embryonic extract. PAB-1/2-depleted extract was supplemented with 115 nM of human PABC1 or GST proteins and a radiolabeled ×ばつ-miR-35-p(A)86 was subjected to deadenylation and analyzed by UREA-PAGE. T1/2 is the half deadenylation time (min).
Figure 4.
Figure 4.
Depletion or impairment of PAB-1 and PAB-2 prevents miRNA-mediated silencing in vitro. The ×ばつ-miR-35-p(A)86 reporter was subjected to a translational repression assay in GST- or GST-PAIP2-treated extracts (A) or in presence of 2.5μM soluble GST or GST-PAIP2 (B). Translational activity was monitored through measurement of RL activity in the presence of miR-35 2′-O-Me inhibitor or a non-cognate miR-1 2′-O-Me inhibitor. The ×ばつ-miR-52-p(A)86 reporter was subjected to a translational repression assay in GST- or GST-PAIP2-treated extracts (C) or in presence of 5 μM soluble GST or GST-PAIP2 (D). Translational activity was monitored through measurement of RL activity in the presence of miR-52 2′-O-Me inhibitor or a non-cognate miR-58 2′-O-Me inhibitor.
Figure 5.
Figure 5.
PAB-1 and PAB-2 are required for miRNA-mediated silencing independently of the poly(A) tail. (A) Design for the RNA used in the experiment. A capped transcript containing a Renilla luciferase ORF and six miR-35 binding sites in its 3′UTR, but lacking a poly(A) tail. (B) The reporter was incubated in an embryonic translation extract in the presence of 2′-O-Me inhibitor (miR-35) or non-specific inhibitor (miR-1). Translation activity was monitored through measurement of RL activity. Repression activity of the ×ばつ-miR-35-p(A)0 transcript was assayed in (C) GST- or (D) GST-PAIP2-treated extracts. Translation counts were monitored over time in the presence of miR-35 or miR-1 2′-O-Me inhibitors. (E) ×ばつ-miR-35-p(A)0 was incubated in GST- or GST-PAIP2-treated extracts. RNA was extracted and analyzed by UREA-PAGE. *: significance in a one-sided Welch t-test.
Figure 6.
Figure 6.
3′UTRs modulate PABP contribution in miRNA-mediated deadenylation. (A) Design of the RNAs used in the experiment. Capped transcripts encode the Renilla luciferase ORF and either three or six miR-35-binding sites in its 3′UTR, followed by a linker of 32 nt (L32) or 262 nt (L262). (B) ×ばつ-miR-35-p(A)86 L32 and L262 were subjected to an in vitro deadenylation assay in GST- or GST-PAIP2-treated extract. (C) ×ばつ-miR-35-p(A)86 L32 and L262 were subjected to an in vitro deadenylation assay in GST- or GST-PAIP2-treated extract. (D) The 3′UTR-poly(A) loop model of PABP function in miRNA-mediated deadenylation. PABPs enables miRNA-binding sites by facilitating access to the poly(A) tail to miRISC and to the associated deadenylase. Longer 3′UTRs exacerbate, and shorter 3′UTRs suppress PABP dependency. T1/2 correspond to the half deadenylation time (min).

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