Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Husargatan 3, Box 582, 751 23 Uppsala, Sweden1
Author for correspondence: Stefan Schwartz. Fax +46 18 509 876. e-mail stefan.schwartz{at}imim.uu.se
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Abstract |
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Introduction |
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Most eukaryotic cellular mRNAs contain a Cap structure at the 5' end and a poly(A) tail at the 3' end. These structures have multiple functions and affect splicing, transport, translation and stability of mRNAs. For example, the Cap and the poly(A) tail act synergistically to promote translation of cellular mRNAs (Sachs, 1997 ; Wickens et al., 1997
). The HCV RNAs lack a poly(A) tail and Cap structure, but the 5' UTR contains an internal ribosome entry site (IRES) that is required for initiation of translation of HCV mRNAs (Lai, 1998
; Lemon & Honda, 1997
; Wang & Siddiqui, 1995
). In addition, it appears that the HCV 3' UTR stimulates translation initiation at the HCV IRES (Ito et al., 1998
).
The Cap and the poly(A) tail interact with cellular proteins that protect the mRNAs from exonucleolytic degradation (Sachs, 1997 ; Wickens et al., 1997
). Decapping and deadenylation precede mRNA degradation (Ross, 1995
). Thus, the presence of the Cap and the poly(A) tail on the mRNAs results in protection of the mRNAs from RNases and prevents untimely degradation of the cellular mRNAs. For example the poly(A) binding protein binds to the poly(A) tail and inhibits premature mRNA degradation. Interestingly, the poly(A) binding protein stabilizes mRNAs in the absence of a poly(A) tail, if tethered to the mRNA (Coller et al., 1998
), demonstrating that it is the poly(A) binding protein and not the poly(A) tail itself that protects the mRNA from degradation. Since HCV mRNAs lack Cap and a poly(A) tail, they may be sensitive to degradation and therefore may interact with cellular proteins that prevent premature degradation. We used a recently described in vitro RNA degradation assay that reproduces regulated mRNA stability (Ford et al., 1999
; Ford & Wilusz, 1999
) to study the half-life of in vitro-synthesized HCV RNAs representing the 3' ends of HCV RNAs of positive and negative polarities.
Our results show that the 3' end of positive polarity HCV RNA is sensitive to cytosolic, cellular RNases and that the cellular La protein binds to the HCV 3' UTR and inhibits premature degradation of the viral mRNA.
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Methods |
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Purification of recombinant proteins.
His-hnRNP C1 and His-La proteins were prepared by using HiTrap chelating columns according to the manufacturers instructions (Pharmacia Biotech) with minor changes. Briefly, a 400 ml culture of bacterial cells transformed with the appropriate plasmid was induced with IPTG (0·1 mM) for 2 h at 37 °C. The cells were pelleted in a Beckman centrifuge at 6000 g. The pellets were resuspended in ice-cold PBS and lysed by 30 s bursts of sonication followed by incubation in 1% Triton X-100 on ice. Cell debris was removed by centrifugation and the supernatants were filtered through a 0·45 µm filter and loaded onto HiTrap chelating columns. Bound proteins were eluted with EDTAphosphate buffer pH 7·4 (1 mM Na2HPO4, 1 mM NaH2PO4, 50 mM NaCl) with the following different concentrations of EDTA: 20, 40, 60 and 100 mM, respectively. The protein concentrations were determined by comparison with a serial dilution of BSA on Coomassie-stained SDSpolyacrylamide gels.
Preparation of cell extracts.
Cytoplasmic extract was prepared by lysis of HeLa cells in a Dounce homogenizer in buffer A (20 mM Tris pH 7·8, 5 mM MgCl2, 0·1 mM EDTA, 0·1 mM DTT, 5% glycerol and 2 mg/ml Aprotinin) followed by centrifugation at 10000 g for 10 min (Dignam et al., 1983 ). The supernatant was collected and centrifuged for 90 min at 100000 g. The supernatant from this step was designated S100 and the pellet was resuspended in buffer A containing 0·65 M KCl and centrifuged through 30% sucrose. Resuspension of the pellet, consisting of the majority of intact cell organelles, in buffer A containing 0·65 M KCl releases proteins associated with the cell organelles and microsomes. The supernatant from this centrifugation step was designated P100. S100 contains the soluble portion of the cell, the cytosol. The protein concentration was determined by the Bradford method.
Western blotting.
This was done as described previously (Tan & Schwartz, 1995 ) except that an anti-La monoclonal antibody, La4B6 (ICN Pharmaceuticals), was used as primary antibody at a dilution of 1:1000. Horseradish peroxidase-conjugated anti-mouse Ig antibody (Amersham) was used as secondary antibody at a dilution of 1:10000. The samples were boiled prior to loading on 12% SDSpolyacryalamide gels. Bands were visualized by using the ECL detection system (Amersham).
RNA degradation assay.
An in vitro RNA degradation assay that had been shown previously to reproduce intracellular RNA degradation was used (Ford et al., 1999 ; Ford & Wilusz, 1999
). One fmol of radiolabelled HCV RNA was incubated in a total volume of 20 µl containing 2% polyvinyl alcohol, 0·8 mM ATP, 17·5 mM creatine phosphate and 1·4 mg/ml cellular extract at 30 °C. Reactions were stopped by addition of stop buffer (400 mM NaCl, 25 mM TrisHCl pH 7·6 and 0·1% SDS) and the samples were subjected to phenolchloroform extraction followed by ethanol precipitation. The BSA that was added in some experiments was a commercially available product that had been acetylated to inactivate nucleases (Life Technologies). The samples were loaded on denaturing 6% ureapolyacryalamide gels and RNA levels were monitored by autoradiography. All experiments were performed several times and in triplicate. In all experiments shown here, the standard error was less than 20%.
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Results |
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Discussion |
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In yeast (Saccharomyces cerevisiae) cells, 5'3' and 3'5' exonuclease pathways appear to be distinct (Jacobs et al., 1998 ; Mitchell et al., 1997
). However, both pathways start with a shortening of the poly(A) tail. This step is followed either by removal of the Cap and 5'3' exonuclease degradation or complete removal of the poly(A) tail and 3'5' exonuclease degradation. In addition to these two pathways, a poly(A) tail-independent endonucleolytic pathway has been described. Less is known about mRNA degradation in mammalian cells. The premature degradation of mammalian mRNAs containing mRNA instability elements appears to start with deadenylation, followed by degradation of the mRNA body (Ross, 1995
). Many mRNAs lacking poly(A) tail also lack Cap, suggesting that the 5'3' exonuclease degradation pathway described for yeast cells is also operational in mammalian cells. It has also been shown that RNA instability elements in the 3' UTR of cellular mRNAs may be attacked by endonucleases (Binder et al., 1994
). The HCV 3' UTR may be the target of both exo- and endonucleases since it lacks a poly(A) tail and contains a U-rich region, a hallmark of many unstable cellular mRNAs (Ross, 1995
). Interestingly, a number of mammalian RNases found in the RNaseA super family show a clear preference for poly(U) as a substrate over the other homoribopolymers (Sorrentino & Libonati, 1997
). The RNase4 family strongly prefers poly(U) over other RNA substrates (Hofsteenge et al., 1998
). For this reason, one may speculate that the poly(U) tract, which is conserved among various HCV sequences, is particularly sensitive to RNases and must be protected from rapid degradation.
The presence of both exo- and endonucleases in cell extracts has been described (Brewer, 1999 ; Ford et al., 1999
). We speculate that the RNases degrading the HCV 3'(+) RNA may be similar to those described by Ford et al. (1999)
which target deadenylated RNAs with AU-rich RNA instability elements for rapid degradation. Indeed, the HCV 3'(+) RNA contains a U-rich region which is not present in the HCV 3'(-) RNA and which may be the target for the cellular RNases as discussed above. In contrast, RNases that have been described previously to be located in the P100 fraction of mammalian cell cytoplasm extracts (Brewer & Ross, 1988
; Wennborg et al., 1995
) apparently do not target the HCV RNAs for degradation. Binding of the La protein to the U-rich region may protect the HCV RNA from premature degradation by both exo- and endonucleases in the infected cell.
There are also cellular mRNAs that lack poly(A) tails. Histone mRNAs constitute a rare example of an unpolyadenylated cellular mRNA. They contain a stemloop structure at the 3' end that interacts with cellular factors. Production of histone protein is restricted to the S-phase in the cell cycle and ceases at the end of the S-phase, partly as a result of rapid degradation of histone mRNAs. Specific degradation of histone mRNAs in vitro has been reproduced and was shown to be dependent on cellular factors in an S130 extract (McLaren et al., 1997 ). This effect was augmented by the addition of histone protein. Interestingly, these investigators showed that addition of La protein to their in vitro histone degradation assay significantly stabilized the histone mRNA (McLaren et al., 1997
). Similarly to reslts presented here, these investigators demonstrated that the presence of the La protein stabilized an unpolyadenylated RNA in an in vitro RNA degradation assay.
The La protein binds to RNA polymerase III-synthesized RNAs such as tRNAs, 5S rRNAs, U6 snRNA and the cytoplasmic Y RNAs. The target for the La protein on these RNAs is an oligouridylate sequence at their 3' ends. Interestingly it has been proposed that the La protein stabilizes the Y RNAs and the U6 snRNA. Small cytoplasmic Y RNAs are normally found in Ro ribonucleoprotein particles (RNPs) together with Ro and La proteins. These RNAs are specifically and rapidly degraded in apoptotic cells (Rutjes et al., 1999 ). In conjunction with degradation, the La protein is released from the complex, suggesting that the La protein may protect the Y RNAs from premature degradation (Rutjes et al., 1999
). In addition, studies on the role of the yeast La homologous protein 1 (Lhp1p) and its effects on RNA polymerase III transcripts in yeast have shown that La stabilizes newly synthesized U6 snRNAs and acts as a chaperone during assembly of the RNA into U6 snRNPs (Pannone et al., 1998
).
The La protein has also been shown to interact with unspliced RNAs produced by hepatitis B virus (HBV) (Heise et al., 1999 ), a DNA virus that, similar to HCV, replicates in the liver of the infected individual. It was shown that La binds to a stemloop structure located in a sequence on the HBV RNAs that had been shown previously to regulate the levels of HBV RNAs. Treatment of cells expressing HBV RNAs with inflammatory cytokines such as interferon-
and tumour necrosis factor-
suppresses HBV gene expression by reducing the half-lives of the HBV RNAs. Interestingly, it appeared that cytokine-induced signal transduction pathways regulate the stability of the HBV RNAs by affecting the binding of the La protein to a 91 nucleotide RNA element on the HBV RNAs (Heise et al., 1999
). The authors speculated that the La protein affects the HBV RNA half-life, constitutively and in response to cytokines (Heise et al., 1999
). Here we have shown that the HCV 3' UTR is rapidly degraded in vitro and that binding of the La protein to the HCV 3' UTR stabilizes the HCV RNA. It would be interesting to investigate if inflammatory cytokines affect the HCV RNA half-lives in hepatocytes and if the effects on the HCV RNAs are mediated by the La protein.
In summary, the La protein has been shown to stabilize unpolyadenylated cellular RNAs, i.e. histone mRNAs, Y RNAs and U6 snRNAs (McLaren et al., 1997 ; Pannone et al., 1998
; Rutjes et al., 1999
) and here we show that La protects HCV RNAs from premature degradation by interacting with sequences in the HCV 3' UTR.
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Acknowledgments |
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References |
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Received 19 June 2000;
accepted 2 October 2000.