Role of Rck-Pat1b binding in assembly of processing-bodies

Abstract

The DEAD box RNA helicase Rck and the scaffold protein Pat1b participate in controlling gene expression at the post-transcriptional level by suppressing mRNA translation and promoting mRNA decapping. In addition, both proteins are required for the assembly of processing (P)-bodies, cytoplasmic foci that contain stalled mRNAs and numerous components of the mRNA decay machinery. The C-terminal RecA-like domain of Rck interacts with the N-terminal acidic domain of Pat1b. Here, we identified point mutations in human Rck and Pat1b that prevent the two proteins from binding to each other. By analyzing interaction-deficient mutants in combination with knockdown and rescue strategies in human HeLa cells, we found that Pat1b assembles P-bodies and suppresses expression of tethered mRNAs in the absence of Rck binding. In contrast, Rck requires the Pat1b-binding site in order to promote P-body assembly and associate with the decapping enzyme Dcp2 as well as Ago2 and TNRC6A, two core components of the RNA-induced silencing complex. Our data indicate that P-body assembly occurs in a step-wise manner, where Rck participates in the initial suppression of mRNA translation, whereas Pat1b in a second step triggers P-body assembly and promotes mRNA decapping.

Keywords: PatL1/Pat1b; RNA helicase; RNA-induced silencing complex; Rck/p54; decapping; mRNA degradation; processing body; translation suppression.

Figure 1. Identification of the Rck-binding motif in Pat1b. (A) Alignment of the Pat1/Pat1b N-terminal region from different species: Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Dr, Danio rerio; Xl, Xenopus laevis; Hs, Homo sapiens. Acidic amino acids are depicted in blue, conserved residues are shadowed in yellow and mutated residues are highlighted in red. The Pat1b domains are schematically represented below the alignment: A, acidic domain; N, N-terminal domain; H, homology domain and C, C-terminal domain. (B) HEK293 cells were transiently transfected with HA-tagged Rck together with either YFP alone, YFP-Pat1b or YFP-Pat1b-4A. After 1 d, cytoplasmic lysates (input) were prepared for immunoprecipitation (IP) with GFP-binder. The YFP- and HA-tagged proteins were detected by western blotting. (C) HEK293 cells were transiently transfected with YFP alone, YFP-Pat1b, YFP-Pat1b-4A or the Pat1b fragments YFP-Pat1b-A, -N, -AN or -HC. After IP with GFP-binder, the YFP-tagged proteins as well as endogenous Rck, Ago2 and TNRC6A were detected by western blotting.

Figure 2. Pat1b functions are not affected by lack of Rck binding. (A) Pat1b fused to PP7 coat protein (cp) was tethered to a FLB-PP7bs reporter mRNA that encodes firefly luciferase (FL) linked to β-globin and contains six copies of the PP7-binding site (bs) in its 3′UTR. HeLa cells were transiently transfected with FLB or FLB-PP7bs reporter genes together with a Renilla luciferase (RL) control reporter and either HA-PP7cp, HA-PP7cp-Pat1b or HA-PP7cp-Pat1b-4A. Cytoplasmic lysates were prepared after 24 h to measure FL/RL activity (upper panel). FLB mRNA levels were determined from the same lysates by northern blot analysis, normalized to RPS7 mRNA and quantified (lower panel). Average values ± SD from three repeat experiments were plotted in the graph. (B) Localization of YFP-Pat1b was determined in transiently transfected HeLa cells by immunofluorescence microscopy. P-bodies were counterstained in red using an antibody that crossreacts with Hedls in the cytoplasm. Images were acquired by spinning disc confocal microscopy at 100x magnification, maximum projections of z-stacks are depicted. Size bar, 10 µm. (C) Localization of YFP-Pat1b-4A was determined as in (B). (D) Localization of YFP-Pat1b-dA lacking the 84 amino acid long N-terminal acidic A-domain was determined as in (B).

Figure 3. Rescue of P-body assembly by Pat1b. (A) HeLa cells were transfected either with control siRNA D0 or with si-Pat1b T3, at 50 nM final concentration, twice over a period of 4 d. Total cell lyates were prepared for detection of endogenous Pat1b and Rck by western blotting. (B–E) HeLa cells were transfected with siRNA D0 or T3 twice over 4 d. Plasmids encoding (B and C) YFP, (D) YFP-Pat1b-T3r or (E) YFP-Pat1b-4A-T3r were included in the second transfection. T3r designates silent point mutations that render the Pat1b mRNA resistant to the T3 siRNA. Cells were processed for immunoflourescence microscopy as in Figure 2B, P-bodies were counterstained in red using an antibody that crossreacts with Hedls in the cytoplasm. Images were acquired by spinning-disc confocal microscopy at 60x magnification; maximum projections of z-stacks are depicted. Size bar, 20 µm.

Figure 4. Identification of the Pat1b-binding domain in Rck. (A) Schematic representation of human Rck with its helicase motifs in the N-terminal (green) and C-terminal RecA-like domain (beige). (B) HEK293 cells were transiently transfected with HA-Pat1b together with YFP, YFP-tagged full-length Rck (1–483) or Rck fragments 1–300 and 301–483. After 1 d, cytoplasmic lysates (input) were prepared for immunoprecipitation (IP) with HA antibody. The YFP- and HA-tagged proteins were detected by western blotting.

Figure 5. Identification of the Pat1b-binding site in Rck. (A) Based on the crystal structure of the C-terminal RecA-like domain of human Rck by Tritschler et al. (2009, see ref. 32), surface-exposed residues were highlighted according to their negative (blue) or positive charge (yellow). Patches of positively charged amino acids were mutated to alanine in the m1-5 mutants, as indicated. (B) HEK293 cells were transiently transfected with YFP or YFP-Pat1b together with HA-tagged Rck, either wt or mutants m1-5. After 1 d, cytoplasmic lysates (input) were prepared for IP with GFP-binder. The YFP- and HA-tagged proteins were detected by western blotting. (C) HEK293 cells were transiently transfected with YFP, YFP-tagged wt Rck or the corresponding mutants m1-m5. After 1 d, cytoplasmic lysates (input) were prepared for IP with GFP-binder. The YFP-tagged proteins were detected by western blotting along with endogenous Pat1b. (D) The same IP was performed followed by detection of endogenous Dcp2 and Edc3 by western blot analysis. (E) HEK293 cells were transiently transfected with YFP, YFP-Rck or YFP-Rck-m4. After 1 d, cytoplasmic lysates (input) were prepared for IP with GFP-binder. The YFP-tagged proteins were detected by western blotting along with endogenous Ago2 and TNRC6A.

Figure 6. Rck requires Pat1b-binding for P-body localization. (A) Localization of YFP-Rck (green) was determined in transiently transfected HeLa cells by immunofluorescence microscopy as described for Figure 2B. P-bodies were counterstained in red using an antibody that crossreacts with Hedls in the cytoplasm. Images were acquired by spinning disc confocal microscopy at 60x magnification, maximum projections of z-stacks are depicted. Merged pictures are shown in the panel on the right side. The circles delineate cells expressing YFP-Rck; size bar, 10 µm. (B) Localization of YFP-Rck-m4 was determined as in (A). (C) Quantification shows the percentage of transfected cells in which YFP-Rck or YFP-Rck-m4 is localized in P-bodies. Average percentages ± SD from n = three repeat experiments are depicted, > 100 cells were assessed in each experiment.

Figure 7. Rescue of P-bodies by Rck requires Pat1b-binding. (A) Rck was knocked down by transfection of siRNA R1 twice over a period of 4 d in HeLa cells. Western blot analysis shows expression of Rck along with 14-3-3 as loading control. (B) After transfection of control siRNA D0, YFP was expressed in HeLa cells and P-bodies were visualized by immunoflourescence microscopy as in Figure 2B. (C) After knockdown of Rck by siRNA R1, YFP was expressed in HeLa cells and P-bodies were visualized as in (B). (D) Quantification shows the percentage of transfected containing P-bodies. Average percentages ± SD from n = three repeat experiments are depicted, > 100 cells were assessed in each experiment. (E and F) After knockdown of Rck by siRNA R1, (E) YFP-Rck-R1r or (F) YFP-Rck-m4-R1r was expressed in HeLa cells and P-bodies were visualized as in (B) using an antibody that crossreacts with Hedls in the cytoplasm. R1r designates silent point mutations that render the Rck mRNA resistant to the R1 siRNA.

Figure 8. Model of step-wise P-body assembly by Rck and Pat1b, based on previously published data and this study. mRNAs are bound by Rck as an early step in translation suppression. Rck then associates through a positively charged patch on helix 11 of its C-terminal RecA-like domain with the conserved DxxND(D/E)TFG motif in the acidic, N-terminal domain of Pat1b. In turn, Pat1b triggers assembly of P-bodies through its aggregation-prone N-domain, and enhances mRNA decapping though its N- and C-domains.