HPat a Decapping Activator Interacting with the miRNA Effector Complex

<div><p>Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study we performed split-affinity purifications in <i>Drosophila</i> cells and provide evidence for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors we demonstrate the importance of NOT1 for this interaction. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation.</p></div

Co-purification of HPat (A) or AGO1 (B) with GW182 in NOT1 knockdown cells.

<p><b>A, B:</b> Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD) or NOT1 (NOT1 KD). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–5 and 14–18, in B: lanes 1–6 and 15–21. IPs in A lanes 6 and 19, in B lanes 7 and 22), anti-c-myc (Input in A: lanes 7–12 and 20–23. IPs in A lanes 13 and 24) or anti-AGO1 antibody (Input in B: lanes 8–13 and 23–29. IPs in B lanes 14 and 30). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. <b>C, D:</b> The amount of Myc-HPat/HA-GW182 (<b>C</b>) or AGO1/HA-GW182 (<b>D</b>) in immunoprecipitates (IP) from lysates of control and NOT1 knockdown cells. The IP was normalized (Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071860#pone.0071860.s004" target="_blank">Figure S4</a>) and the value of the control IP set to 1. <b>E:</b> Analysis of <i>NOT1</i> mRNA levels in knockdown cells compared to control cells treated with dsYFP RNA. The levels of <i>NOT1</i> mRNA in total RNA of input samples were analyzed by RT-qPCR and normalized to <i>rp49</i> mRNA levels. The values of dsYFP treated cells were set to 1. <b>F:</b> Upregulation of endogenous miRNA targets in knockdown cells. Total RNA of input samples were analyzed by RT-qPCR for changes of <i>CG5123</i> mRNA levels in NOT1 knockdown cells. mRNA levels were normalized to <i>rp49</i> mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s <i>t</i> test and significance values are as follows: ns, not significant; *, p<0.005; **, p<0.001.</p

The interaction of HPat and GW182 protein.

<p>Immunoprecipitation analysis of <i>Drosophila</i> S2 cell lysates using anti-HPat (<b>A</b>) or anti-GW182 (<b>B, C</b>) antibodies or preimmune sera. Input (lane 1) and immunoprecipitates (lanes 2, 3) were separated on SDS-PAGE and analyzed by Western blot analysis using anti-HPat, anti-GW182 or anti-AGO1 antibody. In A) and B) 1.5% of the input (total clarified cell lysate) and 40% of the immunoprecipitate were separated on a SDS-PAGE, while in C) 2.5% of the input and only 10% of the immunoprecipitate were separated. The asterix indicates cross-reactivity of the secondary antibody with the immunoglobulin heavy chain of the antibody used for immunoprecipitation.</p

Co-purification of HPat with GW182 in EDC4 and Dcp1 (A), or XRN1 (B) knockdown cells.

<p><b>A, B:</b> Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD), EDC4 and Dcp1 (EDC4/Dcp1 KD, <b>A</b>) or XRN1 (XRN1 KD, <b>B</b>). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–3 and 11–14, in B: lanes 1–5 and 14–18. IPs in A: lanes 4 and 16, in B: lanes 6 and 19) or anti-c-myc antibody (Input in A: lanes 5–9 and 17–21, in B: lanes 7–12 and 20–25. IPs in A: lanes 10 and 22, in B: lanes 13 and 26). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. <b>C:</b> The amount of Myc-HPat/HA-GW182 in immunoprecipitates (IP) from lysates of control, EDC4 and Dcp1, or XRN1 knockdown cells. The IP was normalized (Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071860#pone.0071860.s005" target="_blank">Figure S5</a>) and the value of the control IP set to 1. <b>D:</b> Analysis of <i>EDC4, Dcp1,</i> and <i>XRN1</i> mRNA levels. The levels of <i>EDC4, Dcp1,</i> and <i>XRN1</i> mRNA in total RNA of input samples were analyzed by RT-qPCR and normalized to <i>rp49</i> mRNA levels. The values of dsYFP treated cells were set to 1. <b>E:</b> Upregulation of endogenous miRNA targets in knockdown cells. <i>CG6770</i> mRNA levels in total RNA of EDC4/Dcp1, XRN1, and YFP knockdown cells were analyzed by RT-qPCR. mRNA levels were normalized to <i>rp49</i> mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s <i>t</i> test and significance values are as follows: ns, not significant; *, p<0.02; **, p<0.001; ***, p<0.0001.</p