A conserved PUF-Ago-eEF1A complex attenuates translation elongation

Abstract

PUF (Pumilio/FBF) RNA-binding proteins and Argonaute (Ago) miRNA-binding proteins regulate mRNAs post-transcriptionally, each acting through similar, yet distinct, mechanisms. Here, we report that PUF and Ago proteins can also function together in a complex with a core translation elongation factor, eEF1A, to repress translation elongation. Both nematode (Caenorhabditis elegans) and mammalian PUF-Ago-eEF1A complexes were identified, using coimmunoprecipitation and recombinant protein assays. Nematode CSR-1 (Ago) promoted repression of FBF (PUF) target mRNAs in in vivo assays, and the FBF-1-CSR-1 heterodimer inhibited EFT-3 (eEF1A) GTPase activity in vitro. Mammalian PUM2-Ago-eEF1A inhibited translation of nonadenylated and polyadenylated reporter mRNAs in vitro. This repression occurred after translation initiation and led to ribosome accumulation within the open reading frame, roughly at the site where the nascent polypeptide emerged from the ribosomal exit tunnel. Together, these data suggest that a conserved PUF-Ago-eEF1A complex attenuates translation elongation.

Figure 1

FBF-1 binds CSR-1 to repress target mRNA. (a) FBF-1 co-IPs with CSR-1. Transgenic GFP::FBF-1 (FBF-1) was immunoprecipitated from adult hermaphrodites with or without RNase. Bound proteins (Pellets) were probed on Western blots for indicated proteins. CSR-1 and EFT-3 were both detected in FBF-1 IPs, whereas DRH-3 and actin were not. Two CSR-1 isoforms exist in vivo^,, and both co-IP with FBF-1. CSR-1 did not co-IP with GFP::Tubulin (Tub). (b) CSR-1 is expressed in the mitotic zone. Top, diagram of csr-1 locus. The q791 deletion removes key domains resulting in a frameshift. Arrows, alternative promoter elements. Bottom: CSR-1 antibody stains the wild-type mitotic zone cytoplasm (*, distal end of the gonad; dashed line, mitotic zone/transition zone boundary), but not in a csr-1 mutant. (c, d) Adult germlines expressing GFP::H2B (green) under control of wild-type (WT) or FBE-lacking (ΔFBE) gld-1 3′ UTR; nuclei seen with DAPI (blue). Marked include: *, distal end; open triangle, distal-most GFP-positive cell; closed triangle, distal-most bright GFP; dashed line, mitotic zone/transition zone boundary. (c) Control RNAi, empty vector. GFP::H2B normally repressed in distal region (top panels), but expands into distal germ cells without FBEs (lower panels). (d) csr-1(RNAi). GFP::H2B expanded more distally than control in panel c when reporter harbors an FBE. (e) GFP::H2B extent after RNAi against genes indicated, scoring only sterile animals to ensure effective RNAi. Blue columns, most distal germ cell row with GFP::H2B-positive cell; red columns, mitotic zone/transition zone boundary. (f) Percent cells in mitotic zone with bright GFP::H2B.

Figure 2

FBF-1/CSR-1/EFT-3 ternary complex formation reduces EFT-3 GTPase activity. (a) GST-FBF-1 and His₆-CSR-1(MID-PIWI) were co-expressed in the pETDuet system. Affinity selection was performed with either glutathione to select GST-FBF-1 or Ni₂₊ to select His₆-MID-PIWI respectively. GST-FBF-1 and His₆-MID-PIWI associate. His₆-MID-PIWI degradation products are indicated (*). (b) As in panel a, but GST-FBF-1 was co-expressed with CSR-1 PAZ (His₆-PAZ). FBF-1 and His₆-PAZ do not associate. (c, d) CSR-1 refers to His₆-CSR-1(MID-PIWI); FBF-1 refers to GST-FBF-1. (c) EFT-3 binds the FBF-1/CSR-1 complex. Recombinant EFT-3 was added to wild-type FBF-1, FBF-1(F344R), FBF-1/CSR-1 or FBF-1(F344R)/CSR-1 prepared from bacteria (Inputs, 25% loaded). Glutathione was used to select GST-FBF-1 (wild-type and mutant) after incubation (Pellets, 50% loaded). EFT-3 (arrowhead) specifically associated with wild-type FBF-1/CSR-1, but not with FBF-1 alone or with mutant FBF-1(F344R)/CSR-1. We note that the FBF-1/CSR-1/EFT-3 complex forms with apparent 1:1:1 stoichiometry. (d) FBF-1/CSR-1 specifically inhibits EFT-3 GTPase activity. [³²P]-γ-GTP was incubated with EFT-3, catalytically-dead EFT-3(H95L), EFT-3 and FBF-1, EFT-3/FBF-1/CSR-1 or EFT-3 and FBF-1(F344R)/CSR-1. Liberated P_i was scintillation counted after phase extraction.

Figure 3

PUM2/Ago/eEF1A complex inhibits protein production. (a) Human PUM2 promotes eEF1A association with Ago. Western blots using Flag antibody to detect 3xFlag-PUM2 and Flag-HA-AGOs1-3 (top) oreEF1A antibody (bottom). PUM2 co-expression dramatically enhanced eEF1A co-IP. (b) RNA binding by PUM2 variants. Recombinant PUM2, PUM2(F868R), PUM2(T752E) and PUM2(H852A) at increasing concentrations (triangle) were assayed for binding the hunchback PBE sequence. (c) Complex formation with PUM2 variants. AGO co-precipitates with all PUM2 mutants except PUM2(T752E). eEF1A co-precipitates with wild-type and PUM2(H852A) but not with PUM2(F868R) or PUM2(T752E). Equivalent amounts of recombinant PUM2 proteins were added to reticulocyte lysate (Input) in the presence of RNase. (d) PUM2 variant summary. (e) PBE-dependent translation repression of nonadenylated reporter mRNA. Recombinant GST or GST-tagged PUM2 variants, nonadenylated firefly luciferase mRNA with 3xPBEs in its 3′ UTR and control nonadenylated Renilla luciferase reporter were added to reticulocyte lysate. To monitor PBE-dependent translation, firefly luciferase production was normalized to Renilla luciferase production. Only wild-type PUM2 inhibited translation of nonadenylated firefly luciferase mRNA. Note that protein output was not corrected for mRNA level since all reactions contained same initial quantity of reporter mRNAs, and wild-type PUM2 reactions had more final mRNA than others. (f) As in panel e, but with polyadenylated mRNAs.

Figure 4

Kinetics of PUM2 translational repression. (a,b) PUM2 proteins were incubated with reporter mRNAs in reticulocyte lysate for 5 minutes to ensure ribosome loading; at time = 0, excess mRNA cap analog was added to inhibit de novo 40S ribosomal subunit loading. Luciferase production was monitored every 30-seconds. (a) Renilla luciferase production. Left, kinetics of Renilla luciferase production were equivalent for PUM2 and PUM2(RBD); right, enlargement of early time points. (b) Firefly luciferase production. Left, firefly luciferase production was delayed with PUM2 compared to PUM2(RBD) and dramatically inhibited midway through the reaction. The PUM2 curve was similar to that of PUM2(RBD) from the ~5 to 8 min time points (albeit with a 30 second delay), suggesting that a fully-functional repressive complex forms slowly. Right, enlargement of data points when firefly luciferase is first produced. (c) To estimate translational elongation rate, we compared the time required to first produce either firefly or Renilla luciferase (firefly luciferase is 319 residues longer than Renilla luciferase). In reactions with PUM2(RBD), the first firefly luciferase protein was detected 2.5 minutes after the first Renilla luciferase (4.5 minutes for firefly vs. 2 minutes for Renilla). From this difference, we infer an estimated translational elongation rate (eTER) of 2.12 a.a./sec (319 residues/150 second). In reactions with wild-type PUM2, the same calculation yields an eTER of 1.77 a.a/sec. This modest decrease was seen at a time well before PUM2 becomes fully repressive (8 minutes after cap analog addition). Therefore, PUM2 may affect translation elongation using multiple mechanisms.

Figure 5

Human PUM2 attenuates translation elongation. (a) Ribosomal footprinting protocol. In vitro translation reactions were performed with radiolabeled firefly luciferase mRNA harboring 3xPBEs, reactions quenched with cycloheximide and treated with RNase. RNA from monoribosome fractions was collected and hybridized to a spotted array with oligonucleotides complementary to firefly mRNA. Signals from monoribosome-bound RNA fragments were normalized with input RNA fragments. (b) PUM2 affects ribosome position. PUM2, PUM2(ABD), PUM2(EBD) or PUM2(RBD) were added to reticulocyte lysate, and ribosomal footprinting was performed as diagrammed in panel a. At the 5′ end of the ORF, ribosomal density is equivalent for all samples, a level that continues across the ORF when PUM2(ABD, EBD and RBD) mutants are added. With wild-type PUM2, ribosomal density peaks at ~100–140 nts and decreases to a level lower than that seen with PUM2 mutants. Therefore translation is attenuated during elongation. Note: samples with wild-type PUM2 had more input RNA signal due to stabilized mRNA (Supplementary Fig. 6b), which dampens the signal observed for ribosomal footprints once normalized. (c) As in panel b, but footprinting was performed with a mutant firefly luciferase mRNA deleted for ~500 nts after the start codon (red). As above, wild-type PUM2 caused ribosomes to accumulate over the first ~40–140 nts. Ribosomal density again dropped after this site of accumulation to a level lower than that seen with the PUM2 mutants, which all had equivalent ribosomal density across the ORF. (d) Model for how the PUF/Ago/eEF1A complex attenuates translation elongation. See text for explanation.