UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant

Abstract

Messenger RNA decay mediated by the c-fos major protein coding-region determinant of instability (mCRD) is a useful system for studying translationally coupled mRNA turnover. Among the five mCRD-associated proteins identified previously, UNR was found to be an mCRD-binding protein and also a PABP-interacting protein. Interaction between UNR and PABP is necessary for the full destabilization function of the mCRD. By testing different classes of mammalian poly(A) nucleases, we identified CCR4 as a poly(A) nuclease involved in the mCRD-mediated rapid deadenylation in vivo and also associated with UNR. Blocking either translation initiation or elongation greatly impeded poly(A) shortening and mRNA decay mediated by the mCRD, demonstrating that the deadenylation step is coupled to ongoing translation of the message. These findings suggest a model in which the mCRD/UNR complex serves as a "landing/assembly" platform for formation of a deadenylation/decay mRNA-protein complex on an mCRD-containing transcript. The complex is dormant prior to translation. Accelerated deadenylation and decay of the transcript follows ribosome transit through the mCRD. This study provides new insights into a mechanism by which interplay between mRNA turnover and translation determines the lifespan of an mCRD-containing mRNA in the cytoplasm.

Figure 1.

Identification of UNR as the c-fos mCRD-binding protein in mCRD-associated protein complex. (A, left) The GST fusion proteins tested were shown by 10% SDS-PAGE and Coomassie blue staining. (Right) The ability of each mCRD-associated protein to bind c-fos mCRD was tested by gel electrophoretic mobility shift assay (GEMSA). (B) Specificity of binding of GST-UNR or GST-PABP to c-fos mCRD was demonstrated by competition GEMSA. (C) Binding ability of GST-UNR or GST-PABP to poly(A) RNA was tested by GEMSA. (D) Competition GEMSA showing that mCRD RNA cannot compete with poly(A) RNA for PABP binding. (E) Competition GEMSA showing that poly(A)₂₅ RNA outcompetes mCRD RNA for PABP binding.

Figure 2.

UNR-binding motifs in the c-fos mCRD are involved in mRNA destabilization. (A) Mapping of UNR-binding motifs in the 87-nt minimal functional element of c-fos mCRD. (Top) Sequences of the 87-nt mCRD and its derivatives. Purine residues are shown in gray, and the three purine stretches (PuS I, PuS II, and PuS III) are indicated. (Bottom) Different portions of mCRD RNA were [³²P]-labeled and tested for their ability to bind GST-UNR (0.5 pmole) by GEMSA. The results are summarized on the right of the top panel (+ indicates detection of band-shifted complexes; - indicates band-shifted complex is not readily detectable). (B, top) Physical map of β-globin mRNA carrying the 87-nt mCRD (BBBspc + cd87). Solid lines indicate 5′ and 3′ UTRs from β-globin mRNA. (Rectangle) Protein coding region from β-globin. (Radial box) The 87-nt c-fos mCRD. A 314-nt protein coding sequence from rat GAPDH (gray box-marked spacer) is referred to as “spc” in the names for hybrid mRNAs. (Bottom) In vivo functional tests of individual or combination of UNR-binding motifs in mRNA decay by Northern blot analysis. BBB mRNAs were expressed constitutively and served as an internal control. The times given at the top correspond to hours after serum stimulation. Poly(A)^- RNA was prepared in vitro by treating RNA sample from early time point with oligo(dT) and RNase H. The amount of tested RNAs on Northern blots was quantitated by phosphorimaging, normalized to the amount of BBB RNA, and plotted semilogarithmically.

Figure 3.

Knocking down UNR expression slows down the deadenylation and decay of c-fos mCRD-containing mRNA. (A) Western blots of whole-cell lysate showing knock-down of UNR protein expression by the specific (UNR) siRNA and not by the nonspecific (NS) siRNA. (B) Northern blots showing decay and deadenylation of BBBspc + cd87 mRNA isolated from NIH 3T3 cells transfected with either UNR-specific or nonspecific siRNA. BBB mRNA was expressed constitutively and served as an internal standard. Poly(A) lengths were compared by calculating from the difference in electrophoretic mobility between each message and cognate Poly(A)^- RNA. Deadenylation curves were plotted as described previously (Shyu et al. 1991).

Figure 4.

Identification and characterization of the PABP-binding activity of UNR. (A) GST pull-down assay examining the interactions between UNR and other mCRD-associated proteins. Each of the in vitro translated [³⁵S]methionine-labeled proteins with (+) or without (-) RNase A treatment was incubated with immobilized GST-UNR on glutathione column. The bound protein complexes were eluted and analyzed by SDS-PAGE (10%) followed by Coomassie blue staining for GST-UNR (lower right) and then autoradiography for labeled proteins (top right). (Left) Coomassie blue-stained SDS-PAGE gel showing 20% of the in vitro translated proteins used for pull-down assay. (B) MBP-UNR but not MBP alone interacts with GST-PABP. Equal moles of GST-PABP and MBP-UNR fusion proteins were mixed together and applied to glutathione-column. Bound proteins were eluted from the column and analyzed by SDS-PAGE (7.5%) followed by Coomassie blue staining. (C) UNR can interact with PABP associated with poly(A). In vitro translated [³⁵S]methionine-labeled UNR was incubated with GST-PABP immobilized glutathione-Sepharose that had been preincubated with (+) or without (-) poly(A) RNA as described in Materials and Methods Bound protein complexes were eluted and analyzed by SDS-PAGE (10%) followed by Coomassie blue staining for GST-PABP (lower right) and then autoradiography (top right) for labeled UNR. (Left) Twenty percent of the in vitro translated proteins used for pull-down assay. (D) Co-IP of UNR and PABP. COS7 cells were transfected with vector alone or the vector expressing HA-tagged UNR. Cytoplasmic lysates were prepared and incubated with anti-HA agarose in the absence (-) or presence (+) of RNase A. Following precipitation, bound proteins were analyzed by Western blotting using various antibodies as indicated. The lysate panel shows 6% of the input.

Figure 5.

Identification and functional analysis of UNR-binding sites in PABP. (A) Schematic diagram of GST-PABP and its derivatives. +++, ++; and + indicate relative levels of labeled proteins pulled down, and - denotes not detectable. (B) GST pull-down assay mapping UNR-binding sites in PABP. GST-PABP or its derivatives (Imataka et al. 1998; Khaleghpour et al. 2001) was immobilized on glutathione-Sepharose beads. [³⁵S]-methionine-labeled UNR or Paip1 was incubated with GST-tagged PABP, or its derivative proteins immobilized glutathione-Sepharose as indicated. The bound protein complexes were eluted and analyzed by SDS-PAGE (11%) followed by Coomassie blue staining (top) for GST-fusion proteins and then autoradiography (bottom) for labeled proteins. Twenty percent of the in vitro translated proteins used for pull-down assay were shown on the bottom right. The results of UNR binding to PABP and its derivatives are summarized in A. (C) Co-IP experiments showing that overexpression of PABP(166-289) interferes with the interaction between UNR and endogenous PABP. HA-UNR and GST-PABP (166-289) are expressed individually or in combination as indicated (+ indicates present in lysate; - indicates absent in lysate). (D) Western blot analysis showing the expression of GST-tagged PABP (166-289) in the cytoplasm of NIH 3T3 cells. The blot was probed with an antibody against the GST-tag and a control antibody against α-tubulin. (E) Northern blot analysis showing deadenylation and decay of BBBspc + cd87 mRNA in the absence (vector) or presence of ectopically expressed PABP (166-289). BBB mRNA was served as an internal standard. Poly(A)^- RNA preparation was carried out as described in the legend for Figure 2. (F) Comparison of deadenylation (top) and decay (bottom) kinetics of BBBspc + cd87 mRNA in the absence (open circle) or presence (filled circle) of ectopically expressed PABP (166-289). Data quantitation and plotting were as described in the legend for Figure 3.

Figure 6.

Identification of a poly(A) nuclease involved in mCRD-directed mRNA turnover. (A) Northern blots showing mRNA deadenylation and decay of the BBBspc + cd87 mRNA in absence (vector) or presence of ectopically expressed poly(A) nucleases, CCR4, CCR4-mt, PAN2, PAN2-mt, PARN, and PARN-mt. Corresponding deadenylation rate of the BBBspc + cd87 mRNA in each case was plotted. Plates of cells for the same time-course experiment were mixed and reseeded 10 h after transfection, which allows endogenous GAPDH mRNA to be served as a control for sample handling and loading. (B) Western blot analysis of cytoplasmic lysates showing that all ectopically expressed poly(A) nucleases can be readily detected by anti-HA (for CCR4 and PAN2) or anti-V5 tag (for PARN) antibody. (C) Immunoprecipitation experiment showing co-IP of UNR and CCR4.

Figure 7.

Ribosome transit is required to trigger accelerated deadenylation for mCRD-mediated mRNA turnover. (A, left) Physical maps and Northern blots showing deadenylation and decay of BFB and hp-BFB hybrid mRNAs. Solid lines indicate 5′ and 3′ UTRs from β-globin mRNA. (Rectangles) Protein-coding region from c-fos mRNA. (Radial box) c-fos mCRD. (hp) Hairpin structure. Northern blots showing deadenylation and decay of BFB and hp-BFB mRNA transcribed from the c-fos promoter in serum-induced NIH3T3 cells. (Right) Semi-log plot showing the deadenylation kinetics of BFB, hp-BFB, and BBB mRNA. α-globin/GAPDH (control) mRNAs were expressed constitutively and served as an internal standard. The times given at the top correspond to minutes or hours after serum stimulation. Deadenylation curves were plotted as described previously (Shyu et al. 1991). (B) Polysome profiles showing that the hairpin inserted in the 5′ UTR of BFB mRNA efficiently and specifically blocked the ongoing translation of the message. (Left) Evaluation of positions and distributions of the 40S, 60S, 80S, and polysome by running RNA samples from each fraction on a 1% nondenaturing agarose gel. (Right) Distribution of BFB, hp-BFB, and c-fos mRNAs as identified by Northern blotting. (C) Physical maps and Northern blots showing deadenylation and decay of BBBspc, cBBBspc + cd87, and cBBBspc + cd87(PTC) mRNAs. Note that cBBBspc + cd87 and cBBBspc + cd87(PTC) mRNAs are expressed from intronless cDNA constructs. The schematic drawings are as described in the legend for Figure 2. (PTC) Premature termination codon. (D) Semi-log plot showing deadenylation kinetics of the three transcripts: cBBBspc + cd87(PTC; open circles), BBBspc (open triangles), and cBBBspc + cd87 (filled circles).