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ELAV/Hu proteins inhibit p27 translation via an IRES element in the p27 5'UTR - PubMed

  • ️Tue Jan 01 2002

ELAV/Hu proteins inhibit p27 translation via an IRES element in the p27 5'UTR

Michael Kullmann et al. Genes Dev. 2002.

Abstract

p27Kip1 restrains cell proliferation by binding to and inhibiting cyclin-dependent kinases. To investigate the mechanisms of p27 translational regulation, we isolated a complete p27 cDNA and identified an internal ribosomal entry site (IRES) located in its 5'UTR. The IRES allows for efficient p27 translation under conditions where cap-dependent translation is reduced. Searching for possible regulators of IRES activity we have identified the neuronal ELAV protein HuD as a specific binding factor of the p27 5'UTR. Increased expression of HuD or the ubiquitously expressed HuR protein specifically inhibits p27 translation and p27 IRES activity. Consistent with an inhibitory role of Hu proteins in p27 translation, siRNA mediated knockdown of HuR induced endogenous p27 protein levels as well as IRES-mediated reporter translation and leads to cell cycle arrest in G1.

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Figures

Figure 1
Figure 1

The p27 5′UTR. (A) Structure of the largest p27 5′UTR. The largest human p27 5′UTR is aligned to the 5′ flanking region of the mouse p27 genomic sequence (accession nos. U77915 and U10440). The upstream ORF (uORF) is marked with a line. (B) p27 mRNA contains an extended 5′UTR that is produced independently from cell cycle position. RNAse protection of 14 μg RNA isolated from proliferating (lane 4) or arrested HeLa cells (lane 5, lovastatin; lane 6, thymidin). Total RNA or 14 μg yeast tRNA hybridized to a 681-nucleotide antisense fragment containing the p27 5′UTR from nucleotides −3 to −575. Protected RNAs were detected by autoradiography. The major protection products are indicated by a clamp; additional protection products are indicated by arrows. (Lane 1) RNA marker; fragment length in nucleotides is indicated. (Lanes 2,3) Yeast tRNA (14 μg) with (lane 3) or without (lane 2) RNase treatment. (Lanes 4–6) RNA from proliferating (lane 4), lovastatin-arrested (lane 5), or thymidine-arrested (lane 6) HeLa cells hybridized and treated with RNase.

Figure 2
Figure 2

The p27 mRNA contains an internal ribosomal entry site (IRES). (A) Bicistronic reporter plasmids. The p27 5′UTR fragments indicated (Δc from −461 to −3; Δd from −417 to −3; and Δe from −356 to −3) were inserted in the intercistronic region of pRF and phpRF. The open boxes represent the SV40 promoter; the light gray and the dark gray boxes represent the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) coding sequence, respectively. phpRF contains a palindromic sequence upstream of Rluc that allows the formation of a stable hairpin in the mRNA known to reduce cap-dependent translation. (B) Relative Renilla luciferase and firefly luciferase activities. Adherent HeLa cells were transfected with bicistronic plasmids and pSV-β-Gal as a control. Both firefly (top) and Renilla (bottom) luciferase activities were normalized to β-galactosidase and expressed relative to the values obtained from pRF, which was set to 1. The ratio between the relative firefly and Renilla luciferase activities for the constructs is indicated below the diagram. (Right) A random sequence of the GAPDH mRNA fails to possess IRES activity. (C) Northern blot analysis of bicistronic mRNA expression. Total RNA (7 μg) was hybridized with a specific probe for the Renilla luciferase coding region, stripped, and rehybridized with a probe for the firefly luciferase coding region. Positions of 18S and 28S ribosomal RNAs are indicated. The generation of an additional firefly mRNA from a plasmid containing the largest 5′UTR (pRF-5′UTR and phpRF-5′UTR) may reflect promotor activity or RNA cleavage.

Figure 3
Figure 3

The IRES domain of the p27 5′UTR confers resistance to translational repression by PI3-kinase inhibitor LY294002. (A) Protein synthesis rates of HeLa cells after addition of PI3-kinase inhibitor LY294002. Cells treated with LY294002 or solvent (ethanol) for 0 min, 10 min, 2 h, 5 h, or 24 h were labeled with (35S)-methionine for an additional 30 min. The incorporation of radioactivity into proteins in LY294002 treated cells was plotted as a percentage of the incorporation in solvent-treated cells. (B) Analysis of PI3-kinase inhibition on the translation of monocistronic mRNAs with p27 5′UTR leaders. Transfected HeLa cells were incubated for 24 h with LY294002 or solvent. Luciferase acivities were normalized to luciferase mRNA levels determined in Northern blots. (C,D) Endogenous p27 protein and mRNA levels are unaltered in LY294002-treated cells. HeLa cells were incubated with LY294002 (LY) or the solvent (C). p27 mRNA levels (top) were analyzed by Northern blot of total RNA with probes specific for p27 mRNA and GAPDH mRNA (bottom). The 18S small ribosomal RNA serves as an additional loading control. (D) Protein levels of p27 (top), p21 (middle), or α-tubulin (bottom) were detected by immunoblotting using monoclonal antibodies. (E) Synthesis rates of p27 are unaltered in the presence of LY294002. After 23-h incubation with LY294002 or solvent (control), adherent HeLa cells were pulse-labeled with (35S) methionine and (35S) cysteine for 1 h. Extracts adjusted for equal amounts of protein were boiled and p27 was immunoprecipitated. p27 synthesis was quantified after SDS-PAGE using a PhosphorImager. (Left) The average of six experiments is represented. (Right) Incorporation of radioactivity in total protein was determined by TCA-precipitation of protein extracts of identical protein concentrations from solvent (C) or LY294002 (LY)-treated cells and liquid scintillation counting. Three experiments are represented. (F) Flow cytometry analysis of cell cycle distribution of adherent HeLa cells after 24 h treatment with LY294002 or solvent.

Figure 4
Figure 4

HuD and HuR proteins bind to the p27 5′UTR. (A) Identification of HuD as a factor binding to the p27 5′UTR. A protein array of 37,200 colonies expressing human cDNAs was hybridized with a radioactive-labeled p27 5′UTR. Each colony was spotted twice onto a nylon membrane in a defined orientation. The autoradiography shows an area of the filter that contains the HuD cDNA-expressing cells (left). Specificity of HuD binding was demonstrated by stripping and second hybridization with a 32P-labeled GAPDH mRNA. (B) Recombinant HuD protein was UV cross-linked to either the p27 5′UTR or the GAPDH mRNA. As a control, the RNA binding YB-1 protein was analyzed. Protein–RNA complexes were resolved by SDS-PAGE and the dried gels exposed to a film. While YB-1 binds to the p27 5′UTR and to the GAPDH mRNA, HuD specifically binds to the p27 5′ leader. (C) HuR protein binds to the p27 5′UTR. FLAG-tagged HuR was overexpressed in HeLa cells and protein extracts were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was first processed for Northwestern analysis using a radioactive-labeled fragment of the p27 5′UTR and thereafter for immunodetection of HuR-proteins with an HuR-specific antibody. FLAG-tagged fusion and endogenous HuR proteins are indicated by arrows. The additional band in the Northwestern analysis between the endogenous and FLAG-tagged HuR represents a yet uncharacterized nonspecific RNA-binding protein present in HeLa cell extracts. (D) A U-rich element known to bind HuR lacks significant IRES activity. 293T cells were transfected with plasmids expressing bicistronic mRNA that contains fragments of the p27 5′UTR in the intercistronic region, as indicated above the bar graph. Firefly and Renilla luciferase activities were determined, and the ratio between firefly and Renilla luciferase was calculated. The ratio for the empty vector pRF was set to 1.

Figure 5
Figure 5

HuD and HuR repress p27 translation. 293T cells were cotransfected with luciferase reporter plasmids, expression vectors for HuD or HuR, and pSV-β-Gal. Twenty-four hours after transfection the relative luciferase activity and β-galactosidase activities were determined. (A) Cells expressing firefly luciferase with or without the p27 5′UTR (pGL3 or pGL5′UTR) or the IRES domain of the p27 5′UTR (pGL-IRES) were cotransfected with HuD, HuR, or empty-vector DNA. Normalized luciferase translation rates are expressed relative to that of pGL3 (set to 1). (B) FLAG-tagged HuR and HuD were expressed in 293T cells. Expression level of both proteins were compared by immunoblotting with an anti FLAG antibody. (C) HuR and HuD inhibit the p27-IRES activity. pRF, pRF-IRES, and phpRF-IRES were cotransfected with expression vectors for HuD or HuR. IRES activities were determined as ratios of firefly to Renilla luciferase activities relative to the empty vector. Representative results of at least three independent experiments are shown in A and C.

Figure 6
Figure 6

(A) Reduced HuR correlates with induced p27 in density-arrested fibroblasts. Human diploid fibroblasts were grown into contact inhibition, and HuR and p27 levels were compared by immunoblotting to those of proliferating fibroblasts. (B–D) Knockdown of HuR expression by RNA interference induces p27 protein. HeLa cells were transfected with siRNAs targeting HuR mRNA (siHuR). (B) Western blot analysis of HuR RNAi in HeLa cells. Twenty-five micrograms of total cell extract from either control-transfected (siRNA) or siHuR-transfected cells were isolated 24 h after transfection, and protein extracts were separated by SDS-PAGE and analyzed for p27, HuR, or α-tubulin. (C) Northern analysis of HuR RNAi in HeLa cells. Ten micrograms of total RNA were separated and immobilized on a nylon membrane. The filter was sequentially hybridized with 32P-labeled probes specific for p27, HuR, and GAPDH mRNAs. Relative hybridization signals for p27 and GAPDH were determined using a PhosphorImager, and the calculated relative ratio of these mRNAs is given below the blot. (D) Down-regulation of HuR expression induces p27-IRES activity. HeLa cells were transfected with the bicistronic constructs pRF, pRF-IRES, and phpRF-IRES, together with siHuR and pSV-β-Gal. Cells were harvested 24 h after transfection and extracts analyzed for Renilla and firefly luciferase expression. As a reference a scrambled siRNA was cotransfected (control). IRES activities were determined as ratios of firefly to Renilla luciferase activities relative to the empty vector. Three sets of independent experiments are shown. Samples of one experiment are shown in the same gray tone (e.g., experiment 1: scramble 1 and HuR 1).

Figure 6
Figure 6

(A) Reduced HuR correlates with induced p27 in density-arrested fibroblasts. Human diploid fibroblasts were grown into contact inhibition, and HuR and p27 levels were compared by immunoblotting to those of proliferating fibroblasts. (B–D) Knockdown of HuR expression by RNA interference induces p27 protein. HeLa cells were transfected with siRNAs targeting HuR mRNA (siHuR). (B) Western blot analysis of HuR RNAi in HeLa cells. Twenty-five micrograms of total cell extract from either control-transfected (siRNA) or siHuR-transfected cells were isolated 24 h after transfection, and protein extracts were separated by SDS-PAGE and analyzed for p27, HuR, or α-tubulin. (C) Northern analysis of HuR RNAi in HeLa cells. Ten micrograms of total RNA were separated and immobilized on a nylon membrane. The filter was sequentially hybridized with 32P-labeled probes specific for p27, HuR, and GAPDH mRNAs. Relative hybridization signals for p27 and GAPDH were determined using a PhosphorImager, and the calculated relative ratio of these mRNAs is given below the blot. (D) Down-regulation of HuR expression induces p27-IRES activity. HeLa cells were transfected with the bicistronic constructs pRF, pRF-IRES, and phpRF-IRES, together with siHuR and pSV-β-Gal. Cells were harvested 24 h after transfection and extracts analyzed for Renilla and firefly luciferase expression. As a reference a scrambled siRNA was cotransfected (control). IRES activities were determined as ratios of firefly to Renilla luciferase activities relative to the empty vector. Three sets of independent experiments are shown. Samples of one experiment are shown in the same gray tone (e.g., experiment 1: scramble 1 and HuR 1).

Figure 7
Figure 7

siRNA-mediated knockdown of HuR induces cell cycle arrest in G1. (A) Growth curve of HeLa cells after HuR knockdown. Cells were transfected with siRNA oligonucleotides on day 0 and day 1, and cell numbers were determined. (B) Cell cycle distribution of HeLa cells transfected with siRNA directed against HuR. Cell cycle phase distribution using BrdU and propidium iodide was determined by flow cytometry.

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