Department of Life Science, Pohang University of Science and Technology, San31, Hyoja-Dong, Pohang, Kyungbuk 790-784, Korea1
Author for correspondence: Sung Key Jang.Fax +82 562 279 2199. e-mail sungkey{at}postech.ac.kr
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Abstract |
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Introduction |
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The IRES-dependent translation requires most of the initiation factors used in cap-dependent translation (Pestova et al., 1996a , b
; Scheper et al., 1992
). However, it is generally believed that some RNA-binding proteins that specifically interact with IRES elements are also required for IRES-dependent translation. One such protein is a cytoplasmic RNA-binding protein of 57 kDa known as polypyrimidine tract-binding protein (PTB) or heterogeneous nuclear ribonucleoprotein (hnRNP) I. It has been suggested that PTB regulates pre-mRNA splicing in the nucleus (Gil et al., 1991
; Patton et al., 1991
) and enhances IRES-dependent translation of encephalomyocarditis virus (EMCV) mRNA in the cytoplasm (Borovjagin et al., 1994
; Jang & Wimmer, 1990
; Witherell et al., 1995
).
Another cellular protein, human La autoantigen (also known as p52 and SS-B) has also been shown to stimulate IRES-dependent translation of poliovirus mRNA and hepatitis C virus (HCV) mRNA. Originally, La protein was identified as an autoantigen recognized by sera from patients with systemic lupus erythematosus and Sjögrens syndrome (Tan, 1989 ). The La protein belongs to a group of RNA-binding proteins containing the RNA recognition motif (Kenan et al., 1991
). La protein is involved in regulation of initiation and termination of transcription by RNA polymerase III (Gottlieb & Steitz, 1989a
, b
; Maraia et al., 1994
; Maraia, 1996
). In addition, La protein is associated with polymerase II RNA transcripts such as U1 RNA (Madore et al., 1984
) as well as viral RNAs including EpsteinBarr virus-encoded RNAs (Toczyski & Steitz, 1991
; Lee & Deng, 1992
), adenovirus VA RNAs (Francoeur & Mathews, 1982
), vesicular stomatitis virus leader RNAs (Kurilla & Keene, 1983
), the 5' NTR of picornaviruses (Meerovitch et al., 1993
; Svitkin et al., 1994a
), and the HIV TAR sequence (Chang et al., 1994
). In the case of poliovirus and HCV, addition of purified La to rabbit reticulocyte lysates (RRL) stimulates virus IRES-dependent translation (Meerovitch et al., 1993
; Svitkin et al., 1994a
). The binding of La protein to the HIV TAR sequence alleviates the translational repression exerted by the TAR sequence on a downstream reporter gene (Svitkin et al., 1994b
). All these observations support the conclusion that the La antigen has a role in the translational regulation of some mRNAs.
In this report, we confirm the translational enhancing effect of PTB on certain EMCV mRNAs by depleting the translation mixture of PTB and then re-adding purified PTB to it. Interestingly, surplus PTB reduces translation driven by EMCV IRES. The inhibitory effect of surplus PTB can be alleviated by the addition of purified La protein. PTB outcompetes La protein in the binding to the same site(s) on the EMCV IRES. Possible roles of PTB and La protein in EMCV IRES-dependent translation are discussed.
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Methods |
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In vitro transcription.
Plasmid DNAs were purified by the polyethylene glycol precipitation method (Sambrook et al., 1989 ) and then linearized with appropriate restriction enzymes. The linearized DNAs were extracted with phenolchloroform and ethanol-precipitated. Transcription was performed by incubation with T7 RNA polymerase (Boehringer Mannheim) for 90 min at 37 °C as described by the manufacturer. To yield capped mRNA, 1 mM m7GpppG (Pharmacia Biotech) was included in the transcription reaction mixture. The concentration of the RNA transcripts was determined by UV spectrophotometry. BamHI-digested pSK(+)/CAT
-ECAT DNA was used to produce the dicistronic mRNA (CAT
-EMCV IRES-CAT). 32P-Labelled EMCV IRES (nt 260488) or a full-length EMCV IRES probe was produced by in vitro transcription of pBS-ECAT digested with HindIII or BalI in the presence of [
-32P]UTP (NEN).
Depletion of endogenous PTB from RRL and HeLa lysate.
Partial depletion of endogenous PTB from the micrococcal nuclease-treated RRL (Promega) was performed as described by Niepmann (1996) . RRL (1 ml) was adjusted with 250 mM potassium acetate and incubated with 75 µl poly(U)Sepharose (Pharmacia Biotech) for 30 min at 4 °C with gentle agitation. The resin was then removed by centrifugation. This step was repeated twice. Cytoplasmic S-10 extracts of HeLa S3 cells were prepared as described by Oh et al. (1998)
. The endogenous PTB from HeLa S3 extracts was removed by poly(U)Sepharose as a step in the depletion of the RRL.
In vitro translation.
In vitro translation in the RRL was performed in 20 µl reaction mixtures including control or depleted RRL plus mRNA at a final concentration of 6 nM. Translation reactions of EMCV mRNAs in the HeLa cytoplasmic extracts were performed in 12·5 µl translation mixture containing 40 nM mRNA as described by Rose et al. (1978) . Translation reactions were carried out for 1 h at 30 °C in the presence of [35S]methionine (NEN). Translation products were analysed by 15% SDSPAGE. The intensity of the autoradiographic images was enhanced by fluorography using salicylic acid. The gel was dried and exposed to Kodak XAR-5 or Agfa Curix RP1 for 1218 h. Efficiency of the translation was measured with a densitometer (Bioimage 50S Series; B. I. System) or phosphorimager.
Protein purification.
Human La cDNA from HeLa total mRNA was cloned in pGEX-KG using the RTPCR method. La protein was produced in E. coli cells and the cell pastes were harvested and resuspended in lysis buffer [20 mM sodium phosphate (pH 7·2), 10 mM EDTA, 10 mM EGTA, 0·5 mM PMSF, 1 mM DTT, 300 mM NaCl]. After lysis, pre-swollen glutathione Sepharose beads (Pharmacia Biotech) were incubated with the cell extracts for 2 h at 4 °C. After centrifugation, the pelleted beads were washed three times with lysis buffer. To cleave the La protein from GSTLa, the resuspended pellet beads were incubated with thrombin in cleavage buffer [50 mM TrisHCl (pH 8·0), 150 mM NaCl, 2·5 mM CaCl2, 0·1% DTT] for 3 h at room temperature. Glutathione Sepharose beads were removed by centrifugation, and then the supernatant containing the cleavage product of GSTLa was loaded onto a glutathione Sepharose 4B column equilibrated with lysis buffer. The flowthrough from the glutathione Sepharose 4B column containing most of the La was applied directly to a poly(U)Sepharose column (Pharmacia Biotech). Bound La was eluted with a linear gradient of 0·12·0 M NaCl. Fractions were analysed on a gel and those containing La were pooled and dialysed against LD buffer [16·2 mM HEPESKOH (pH 7·5), 50 mM KCl, 0·5 mM DTT, 0·1 mM EDTA, 10% glycerol]. The purification steps for recombinant PTB have been described elsewhere (Oh et al., 1998 ).
UV cross-linking of labelled RNAs and proteins.
UV cross-linking reactions were performed essentially as described by Meerovitch et al. (1989) with slight modifications. RNA probes (2x105 to 4x105 c.p.m.) purified with push columns (Stratagene) were incubated at 30 °C for 30 min with 75 µg RRL or 40 µg HeLa extract in 30 µl reaction mixture. After RNA binding, the reaction mixtures were irradiated with UV light on ice for 30 min using a UV-Stratalinker (Stratagene). Unbound RNA was removed by digestion with 20 µg RNase A, 200 U RNase T1 and 1 U RNase V1 (cobra venom nuclease; Pharmacia Biotech) at 37 °C for 20 min. The RNAprotein complexes were analysed by 12% or 15% SDSPAGE followed by autoradiography.
Western blot analysis.
HeLa extracts or RRL were resolved by 12% SDSPAGE and then transferred to a nitrocellulose paper (Amersham). The membrane was blocked overnight with 5% skimmed milk in TBS buffer [20 mM TrisHCl (pH 7·5), 150 mM NaCl, 0·5% Tween 20] and then incubated with anti-La MAb (3B9) for 2 h. The antibody was kindly provided by M. Bachmann, Institut für Physiologische Chemie, Johannes-Gutenberg Universität, Germany. Horseradish peroxidase-conjugated anti-mouse IgG was used as secondary antibody. To visualize bands, the membrane was developed with the ECL method following the suppliers instructions (Amersham).
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Results |
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La protein alleviates the inhibitory activity of excess PTB
It has been suggested that La protein binds to poliovirus and EMCV IRES and plays an important role in poliovirus-dependent translation (Meerovitch et al., 1993 ; Svitkin et al., 1994a
; Witherell & Wimmer, 1994
). We investigated the effect of La protein on translation driven by EMCV IRES in dicistronic configuration [Fig. 1a
, upper panel (CAT
-ECAT)]. The identity of purified La was confirmed by Western blot analysis using a MAb against human La (3B9; data not shown). We also confirmed that La protein bound strongly to HCV 5' NTR, enhancing internal initiation via HCV IRES (data not shown; Ali & Siddiqui, 1997
). The addition of 1 µg purified recombinant La to the poly(U)-depleted RRL alleviated the inhibitory effect of PTB on EMCV IRES-dependent translation (Fig. 1b
, compare lanes 6 and 10). BSA, a negative control protein, did not alleviate this inhibitory effect (Fig. 1b
, lane 15). La protein by itself did not enhance the translation driven by EMCV IRES in poly(U)-depleted RRL (Fig. 2a
) or in the presence of 4 ng PTB (Fig. 2b
). Translational activation by La protein in the presence of excess PTB (250 ng) was dosage-dependent (Fig. 2c
, compare lanes 37). The more specific depletion of endogenous PTB in the RRL was performed by using biotinylated RNA corresponding to EMCV IRES (nt 260488) which strongly binds to PTB; the same interplay between PTB and La protein was also observed (data not shown).
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Discussion |
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PTB may enhance EMCV IRES-dependent translation by facilitating the RNA to assume a proper conformation of the IRES. In this case, PTB may function as a scaffolding protein holding RNA segments in a certain conformation. Several biochemical properties support this hypothesis. Firstly, deletion mutants of PTB that bound to the EMCV IRES with wild-type affinity and specificity did not enhance EMCV translation (Kaminski et al., 1995 ). Secondly, PTB binds to several sites on the EMCV IRES (Jang & Wimmer, 1990
; Kolupaeva et al., 1996
; Witherell et al., 1993
, 1995
; Witherell & Wimmer, 1994
). Lastly, PTB can form oligomers in solution and it contains several RNA-binding domains (Oh et al., 1998
; Perez et al., 1997
).
Based on the biochemical properties of PTB and La protein, we can speculate how excess PTB may inhibit translation of EMCV IRES-dependent translation and how La protein could alleviate the inhibitory effect. Surplus PTB could be deleterious to IRES function, since non-specific binding of the excess PTB to the IRES element could inhibit IRES function by blocking the binding of other essential cellular factor(s). One of the possible essential factors is La protein, which was shown to play an important role in poliovirus IRES-dependent translation (Svitkin et al., 1994a ). Purified La protein bound well to the EMCV IRES (Fig. 7
, lane 6), and PTB inhibited La protein binding to the EMCV IRES (Fig. 7
, lanes 2 and 3). In such a scenario, inhibition and restoration of translation by PTB and La protein could be explained as follows. Residual La protein in the poly(U)-depleted RRL may not be enough to compete with the excess PTB for RNA-binding. Addition of excess La protein to the translation mixture lets translation resume because of adequate competition for RNA binding with the surplus PTB. In this case, La protein plays an active role in EMCV IRES-dependent translation. Alternatively, La protein may play a passive role in EMCV IRES-dependent translation by preventing abnormal binding of PTB to the EMCV IRES with its RNA helicase activity (Bachmann et al., 1990
; Huhn et al., 1997
; Xiao et al., 1994
). Prevention of abnormal binding of PTB to the EMCV IRES may thus result in the alleviation of the inhibitory effect of PTB.
Rather large quantities of purified La protein were required for the restoration of the EMCV IRES-dependent translation (Fig. 2c). This may have been due to the specific activity of the La protein expressed in E. coli. The purified protein may not have possessed full activity possibly because of a lack of modification or for other reasons. Alternatively, the limiting factor removed in the poly(U)-depleted RRL may not be the La protein at all, but a factor related to the activity of La protein. This putative factor may enhance La protein activity by facilitating RNA binding of La protein to a specific site. The detailed activational mechanism of PTB and La protein in translation remains to be elucidated.
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Acknowledgments |
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References |
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Received 29 April 1999;
accepted 2 August 1999.