Portal de Periódicos da CAPES

La Protein Is Associated with Terminal Oligopyrimidine mRNAs in Actively Translating Polysomes

2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês

10.1074/jbc.m300722200

1083-351X

Beatrice Cardinali, Claudia Carissimi, Paolo Gravina, Paola Pierandrei‐Amaldi,

RNA Research and Splicing

La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5′ end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40–60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells. La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5′ end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40–60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells. Regulation of translation can be achieved by modulation of the activity of general translation factors or by specific interactions of regulatory proteins with control sequences located in the 5′- or 3′-untranslated regions (UTR) 1The abbreviations used are: UTR, untranslated region; hnRNP, heterogeneous nuclear ribonucleoprotein; TOP, terminal oligopyrimidine; rp, ribosomal proteins; CNBP, cellular nucleic acid-binding protein; mRNP, messenger ribonucleoprotein; DTT, dithiothreitol; RT, reverse transcription; eEF1α, eukaryotic elongation factor 1α. of mRNAs. The interaction of specific proteins with typical structural elements located in different mRNAs characterizes classes of functionally related mRNAs that are subjected to a concerted control as suggested in the recently proposed hypothesis of the posttranscriptional operons (1Keene J.D. Tenenbaum S.A. Mol. Cell. 2002; 9: 1161-1167Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). A classical example is the interaction of the iron-responsive element-binding protein with the iron-responsive element of different mRNAs for proteins involved in iron metabolism (2Rouault T.A. Harford J.B. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 655-670Google Scholar). Similarly, in erythroid cells, two different members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, hnRNP E1 and hnRNP E2, participate in controlling the stabilization of human α-globin mRNA (3Weiss I.M. Liebhaber S.A. Mol. Cell Biol. 1995; 15: 2457-2465Crossref PubMed Google Scholar, 4Kiledjian M. Wang X. Liebhaber S.A. EMBO J. 1995; 14: 4357-4364Crossref PubMed Scopus (219) Google Scholar) and the translational silencing of lipoxigenase mRNA (5Ostareck D.H. Ostareck-Lederer A. Wilm M. Thiele B.J. Mann M. Hentze M.W. Cell. 1997; 89: 597-606Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). This indicates that one protein in different complexes can participate in different but functionally related post-transcriptional control pathways in the same cell and at the same stage of differentiation. Another case of concerted regulation of many mRNAs is represented by the interaction of La protein with the class of terminal oligopyrimidine (TOP) mRNAs, which code for functionally related proteins and are coordinately controlled at the translational level (6Cardinali B. Di Cristina M. Pierandrei-Amaldi P. Nucleic Acids Res. 1993; 21: 2301-2308Crossref PubMed Scopus (41) Google Scholar, 7Pellizzoni L. Lotti F. Rutjes S.A. Pierandrei-Amaldi P. J. Mol. Biol. 1998; 281: 593-608Crossref PubMed Scopus (67) Google Scholar, 8Pierandrei-Amaldi P. Campioni N. Beccari E. Bozzoni I. Amaldi F. Cell. 1982; 30: 163-171Abstract Full Text PDF PubMed Scopus (91) Google Scholar). La is an evolutionary conserved and abundant RNA-binding protein (9van Venrooij W.J. Slobbe R.L. Pruijn G.J. Mol. Biol. Rep. 1993; 18: 113-119Crossref PubMed Scopus (57) Google Scholar, 10Wolin S.L. Cedervall T. Annu. Rev. Biochem. 2002; 71: 375-403Crossref PubMed Scopus (340) Google Scholar) present mostly in the nucleus where it is found associated with newly synthesized RNA polymerase III transcripts and is implicated in the termination and initiation of transcription by this polymerase (11Stefano J.E. Cell. 1984; 36: 145-154Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 12Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 841-850Crossref PubMed Scopus (174) Google Scholar, 13Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 851-861Crossref PubMed Scopus (303) Google Scholar, 14Maraia R.J. Kenan D.J. Keene J.D. Mol. Cell Biol. 1994; 14: 2147-2158Crossref PubMed Scopus (135) Google Scholar, 15Fan H. Sakulich A.L. Goodier J.L. Zhang X. Qin J. Maraia R.J. Cell. 1997; 88: 707-715Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), in tRNA processing (16Yoo C.J. Wolin S.L. Cell. 1997; 89: 393-402Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 17Lin-Marq N. Clarkson S.G. EMBO J. 1998; 17: 2033-2041Crossref PubMed Scopus (44) Google Scholar) and in transport and nuclear retention of some polymerase III transcripts (18Boelens W.C. Palacios I. Mattaj I.W. RNA. 1995; 1: 273-283PubMed Google Scholar, 19Simons F.H. Broers F.J. Van Venrooij W.J. Pruijn G.J. Exp. Cell Res. 1996; 224: 224-236Crossref PubMed Scopus (52) Google Scholar, 20Grimm C. Lund E. Dahlberg J.E. EMBO J. 1997; 16: 793-806Crossref PubMed Scopus (57) Google Scholar). However, despite the mainly nuclear localization, cytoplasmic functions have been ascribed to La. Several studies have documented that La promotes the translation of certain viral RNA by binding to the 5′-UTR (21Meerovitch K. Svitkin Y.V. Lee H.S. Lejbkowicz F. Kenan D.J. Chan E.K. Agol V.I. Keene J.D. Sonenberg N. J. Virol. 1993; 67: 3798-3807Crossref PubMed Google Scholar, 22Svitkin Y.V. Pause A. Sonenberg N. J. Virol. 1994; 68: 7001-7007Crossref PubMed Google Scholar, 23Chang Y.N. Kenan D.J. Keene J.D. Gatignol A. Jeang K.T. J. Virol. 1994; 68: 7008-7020Crossref PubMed Google Scholar, 24Ali N. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2249-2254Crossref PubMed Scopus (248) Google Scholar) and cellular mRNAs, similar to the X-linked inhibitor of apoptosis protein mRNA and the human immunoglobulin heavy chain-binding protein mRNA in vivo and in vitro (25Holcik M. Korneluk R.G. Mol. Cell Biol. 2000; 20: 4648-4657Crossref PubMed Scopus (197) Google Scholar, 26Kim Y.K. Back S.H. Rho J. Lee S.H. Jang S.K. Nucleic Acids Res. 2001; 29: 5009-5016Crossref PubMed Scopus (82) Google Scholar). The common feature characterizing all of these La binding RNAs is the presence of a stretch of pyrimidine with which La interacts. As mentioned above, La interacts with TOP mRNAs in vertebrates. This class of mRNAs, which includes mRNAs for ribosomal proteins (rp-mRNAs) and for other factors involved in the production and function of the translational apparatus, is characterized by a similar 5′-UTR that starts with a TOP sequence (reviewed in Refs. 27Meyuhas O. Avni D. Shama S. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 363-388Google Scholar and 28Meyuhas O. Hornstein E. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 671-693Google Scholar) and is coordinately regulated at the translational level in a specific growth-dependent manner. This occurs through a modulation of the distribution of TOP mRNAs between translating polysomes and non-translating mRNPs that results in the increased or decreased coordinate synthesis of the corresponding proteins (8Pierandrei-Amaldi P. Campioni N. Beccari E. Bozzoni I. Amaldi F. Cell. 1982; 30: 163-171Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 29Geyer P.K. Meyuhas O. Perry R.P. Johnson L.F. Mol. Cell Biol. 1982; 2: 685-693Crossref PubMed Scopus (93) Google Scholar, 30Loreni F. Amaldi F. Eur. J. Biochem. 1992; 205: 1027-1032Crossref PubMed Scopus (48) Google Scholar). It has been found in amphibian and mammalian somatic cells that the 5′-UTR of TOP mRNAs is responsible for the control (31Mariottini P. Amaldi F. Mol. Cell Biol. 1990; 10: 816-822Crossref PubMed Scopus (72) Google Scholar) and that the TOP sequence is the cis element of the regulation, also by cooperating with a downstream region (32Levy S. Avni D. Hariharan N. Perry R.P. Meyuhas O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3319-3323Crossref PubMed Scopus (286) Google Scholar). Furthermore, reciprocal transfection experiments demonstrated that the mechanisms underlying the control in mammalian and amphibian cells are conserved (33Avni D. Shama S. Loreni F. Meyuhas O. Mol. Cell Biol. 1994; 14: 3822-3833Crossref PubMed Scopus (135) Google Scholar). The conservation of the TOP sequence in these mRNAs and their coordinate translation in vivo suggested that some proteins might have a role in the regulation by binding the typical sequences. An extensive in vitro binding analysis was carried out using normal and mutated forms of the 5′-UTR and of the TOP sequence (6Cardinali B. Di Cristina M. Pierandrei-Amaldi P. Nucleic Acids Res. 1993; 21: 2301-2308Crossref PubMed Scopus (41) Google Scholar, 7Pellizzoni L. Lotti F. Rutjes S.A. Pierandrei-Amaldi P. J. Mol. Biol. 1998; 281: 593-608Crossref PubMed Scopus (67) Google Scholar, 34Pellizzoni L. Cardinali B. Lin-Marq N. Mercanti D. Pierandrei-Amaldi P. J. Mol. Biol. 1996; 259: 904-915Crossref PubMed Scopus (67) Google Scholar, 35Pellizzoni L. Lotti F. Maras B. Pierandrei-Amaldi P. J. Mol. Biol. 1997; 267: 264-275Crossref PubMed Scopus (93) Google Scholar), some of which are known to disrupt the control in vivo (32Levy S. Avni D. Hariharan N. Perry R.P. Meyuhas O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3319-3323Crossref PubMed Scopus (286) Google Scholar). The outcome of these studies lead to the identification of two proteins: La and the cellular nucleic acid-binding protein (CNBP). These studies, which utilized either extracts or recombinant proteins, determined that La interacts with the TOP sequence both in Xenopus laevis and in mammalian cells while CNBP binds to the downstream region of the 5′-UTR (6Cardinali B. Di Cristina M. Pierandrei-Amaldi P. Nucleic Acids Res. 1993; 21: 2301-2308Crossref PubMed Scopus (41) Google Scholar, 7Pellizzoni L. Lotti F. Rutjes S.A. Pierandrei-Amaldi P. J. Mol. Biol. 1998; 281: 593-608Crossref PubMed Scopus (67) Google Scholar, 34Pellizzoni L. Cardinali B. Lin-Marq N. Mercanti D. Pierandrei-Amaldi P. J. Mol. Biol. 1996; 259: 904-915Crossref PubMed Scopus (67) Google Scholar, 35Pellizzoni L. Lotti F. Maras B. Pierandrei-Amaldi P. J. Mol. Biol. 1997; 267: 264-275Crossref PubMed Scopus (93) Google Scholar, 36Kaspar R.L. Kakegawa T. Cranston H. Morris D.R. White M.W. J. Biol. Chem. 1992; 267: 508-514Abstract Full Text PDF PubMed Google Scholar, 37Zhu J. Hayakawa A. Kakegawa T. Kaspar R.L. Biochim. Biophys. Acta. 2001; 1521: 19-29Crossref PubMed Scopus (36) Google Scholar). Further studies indicated that the 5′- UTR can assume alternative conformations, probably stabilized by the two proteins, compatible with different translational activities (7Pellizzoni L. Lotti F. Rutjes S.A. Pierandrei-Amaldi P. J. Mol. Biol. 1998; 281: 593-608Crossref PubMed Scopus (67) Google Scholar). The role of La in the translational control of TOP mRNAs has been assayed in vivo by functional studies in Xenopus cell lines stably transfected with constructs expressing normal and mutated La. It has been found that this protein has a stimulatory effect on the translation of three endogenous TOP mRNAs, rp-L4 and rp-S1 mRNAs and translation factor eEF1α mRNA, as measured by their association with polysome (38Crosio C. Boyl P.P. Loreni F. Pierandrei-Amaldi P. Amaldi F. Nucleic Acids Res. 2000; 28: 2927-2934Crossref PubMed Scopus (78) Google Scholar). Consistent results were obtained in the same study when La-expressing plasmids were co-transfected in human cells together with a plasmid expressing a reporter mRNA carrying a normal or a mutated TOP sequence (38Crosio C. Boyl P.P. Loreni F. Pierandrei-Amaldi P. Amaldi F. Nucleic Acids Res. 2000; 28: 2927-2934Crossref PubMed Scopus (78) Google Scholar). Finally, a recent report in which a TOP/La and CNBP-based translation control system was used in mammalian cells (39Schlatter S. Senn C. Fussenegger M. Biotechnol. Bioeng. 2003; 83: 210-225Crossref PubMed Scopus (23) Google Scholar) supports these results. Taking into account the above mentioned evidence that La binds the regulatory region of TOP mRNAs in vitro and activates their translation in vivo, we wanted to check whether La was associated with the endogenous mRNAs engaged with polysomes, the cellular compartment devoted to translation. Moreover, we determined the amount of La protein in the cytoplasm to establish whether it is present in this compartment to an extent consistent with binding and translational control of TOP mRNAs. We show that La is present in mRNP complexes engaged in translation and is bound to polysomes via mRNA. Furthermore, we have confirmed an association of La protein with TOP mRNAs in vivo, in agreement with the previous in vitro binding data and with the observed positive role in their translation. Biological Materials—X. laevis adults were purchased from Nasco (Fort Atkinson, WI). Artificially fertilized eggs were obtained from hormone-stimulated X. laevis females according to a previously reported procedure (40Rusconi S. Schaffner W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5051-5055Crossref PubMed Scopus (110) Google Scholar). Jelly coat was removed after fertilization for 5 min with 0.2 m Tris, pH 8.8, and 3 mm DTT, extensively washed with 0.1× Barth solution (41Gurdon J.B. The Control of Gene Expression in Animal Development. Oxford University Press, Oxford1974Google Scholar) and incubated in the same solution containing streptomycin (250 units/ml) and penicillin (250 μg/ml) at 22 °C. Preparation of Nuclear and Cytoplasmic Samples—Groups of oocytes, dissected from a piece of ovary, were defolliculated by hand and kept in 0.2% bovine serum albumin in the Barth solution. Nuclei were dissected by forceps in Barth, 10% glycerol, and 1 oocyte in 1 ml of solution, which was changed with every dissection, and accumulated in ice in 100 μl of TKM homogenization buffer: 10 mm Tris-HCl, pH 7, 0.15 m KCl, 4 mm MgCl2, plus 0.05% Triton X-100, 0.5 mm DTT, and 100 μg/ml leupeptin (Sigma), which prevents La cleavage (34Pellizzoni L. Cardinali B. Lin-Marq N. Mercanti D. Pierandrei-Amaldi P. J. Mol. Biol. 1996; 259: 904-915Crossref PubMed Scopus (67) Google Scholar). Cytoplasms were accumulated in Eppendorf tubes in ice. Oocyte nuclei were homogenized by a pipette tip, and then an appropriate volume of 5× electrophoresis sample buffer was added. Whole oocytes and cytoplasms were homogenized in glass/Teflon homogenizers in the same buffer, centrifuged at 300 × g to remove yolk platelets, and brought to the concentration of electrophoresis sample buffer as above. Stage 35 embryos were washed in 0.1 Barth solution and homogenized in the same solution as the oocytes, and then the sample was prepared for SDS-PAGE as above. Polysome Preparation and Analysis—Stage 35 embryos (30 individuals) were washed several times in sterile 0.1× Barth solution and homogenized in 1 ml of TKM buffer containing 0.05% Triton X-100, 0.5 mm DTT, 50 units/ml RNase inhibitor (Amersham Biosciences), and 100 μg/ml leupeptin. Until homogenization, the temperature was kept at 22 °C to avoid abrupt redistribution of mRNAs between polysomes and mRNPs because of temperature changes and then all of the procedure was carried out in ice. The homogenate was centrifuged at 500 × g for 5 min at 4 °C in a Sorvall SS34 rotor. The supernatant was collected and centrifuged at 10000 × g for 10 min. The supernatant (S10) was collected, and the pellet (P10) was resuspended in 1 ml of homogenization buffer plus RNase inhibitor. Corresponding amounts of S10 and P10 were loaded on 15–50% sucrose gradients in TNaM buffer (30 mm Tris-HCl, pH 7.4, 100 mm NaCl, and 5 mm MgCl2) and centrifuged for 2 h in a Beckman SW41 rotor at 37,000 rpm at 4 °C to separate polysomes from mRNP particles. Sodium was substituted for potassium in the gradient buffer solution to avoid SDS precipitation during RNA extraction. Gradients were automatically collected, monitoring the optical density at 260 nm, and the fractions were kept in ice. An aliquot of each fraction was precipitated with 1 volume of isopropylic alcohol in the presence of carrier RNA at 3 μg/ml. In scaled up experiments, 1 ml of the P10 fraction was loaded for further purification on sucrose steps in TNaM (1 ml of 19% sucrose on top of 1 ml of 27% sucrose) and centrifuged in polycarbonate tubes in a 65 Ti Beckman rotor at 100,000 × g for1hto pellet polysomes and clean them of contaminating S10 fraction. Sucrose steps were carefully removed in the cold, each pellet (P100) was kept in ice and resuspended in 200 μl of TNaM, 0.5 mm DTT, 50 units/ml RNase inhibitor, and 100 μg/ml leupeptin, and centrifuged at 300 × g for 2 min to remove debris and used for further analysis. All of the manipulations for preparing and analyzing polysomes were carried out in the cold room. EDTA, Micrococcal Nuclease, and High Salt Treatment—For EDTA experiments, six P100 pellets (30 embryos each) were pooled and divided in two parts. One was used as control, the other was made as 30 mm EDTA, pH 7.4, and both were incubated for 15 min in ice. The samples then were loaded on 15–30% sucrose gradients in TNaM buffer, the first containing 10 mm MgCl2 and the second containing 10 mm EDTA instead of magnesium. Gradients were centrifuged at 37,000 rpm for 4.5 h at 4 °C to pellet polysomes and separate ribosomal subunits. Gradient fractions of 1 ml were collected while the optical density profile at 260 nm was monitored. The pellet was resuspended in 1 ml of gradient buffer, and then one-tenth of pellet and one-tenth of gradient fractions were precipitated with 1 volume of isopropylic alcohol in the presence of carrier RNA at 3 μg/ml to be processed for RNA analysis. The remaining part of each fraction was precipitated for protein analysis with 6 volumes of ethanol/acetone/methanol (2:1:1) at –20° in the presence of 10 μg/ml bovine serum albumin. For nuclease digestion experiments, six P100 pellets were dissolved as indicated above, brought to a final volume of 2 ml with the same buffer, and divided into two parts. One was used as control, and the other was treated with micrococcal nuclease (Amersham Biosciences) at 0.02 units/μl for 20 min in ice in the presence of 1 mm CaCl2. The reaction then was stopped by the addition of EGTA at 3 mm final concentration. Samples were loaded on top of 1 ml of 19% sucrose step in TNaM and centrifuged at 170,000 × g for 1 h at 4 °C. Supernatants and steps were collected, and the pellets were dissolved in 1 ml of TNaM buffer. One-tenth of each fraction was precipitated for RNA analysis, and the remainder was precipitated for protein analysis as described above. Of the protein samples, one-tenth was used for Coomassie Blue staining and the rest was used for Western blot. For high salt treatment, six P100 pellets were dissolved as above for treatment with 0.5 m KCl. Control and treated samples were incubated for 30 min in ice with gentle stirring, and then they were centrifuged and processed as in the micrococcal nuclease experiment with the exception that the supernatants were dialyzed before precipitation for 1 h versus 100 volumes of TNaM plus 0.5 mm DTT and 10 μg/ml leupeptin. Immunoprecipitation and RT-PCR Analysis—Polysome pellets (P100) from stage 35 Xenopus embryos were dissolved in TNN buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.1% Nonidet P-40) containing 650 units/ml RNase inhibitor, 100 μg/ml tRNA, and 200 μg/ml leupeptin and incubated with 30 mm EDTA for 5 min in ice to dissociate ribosomal subunits. Following EDTA treatment, the sample was diluted to 20 mm EDTA and incubated four times for 20 min each at 4 °C rotating with 20 μl of protein A-Sepharose (Amersham Biosciences) to preclear the extract. After preclearing, the sample was divided into four parts. Two were kept as samples before precipitation, and the other two were incubated, respectively, with 3 μg of preimmune IgGs or with affinity-purified anti-La antibodies previously bound to protein A-Sepharose for 1 h at 4 °C. The beads were then washed 5 times with TNN-300 buffer (50 mm Tris-HCl, pH 7.4, 300 mm NaCl, and 0.1% Nonidet P-40). The RNAs coimmunoprecipitated with the antibody-antigen complex were eluted by incubating the beads with proteinase K buffer at 55 °C for 30 min (42Sambrook J. Russell D.W. Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar), extracted with phenol-chloroform, and precipitated with 0.3 m sodium acetate, pH 5.2, and 2.5 volumes of cold ethanol in the presence of 2 μg/ml glycogen (Roche Applied Science) as carrier. Similarly, the RNA from the two non-immunoprecipitated samples was extracted. RNAs were then analyzed by RT-PCR using primers specific for the following mRNAs: rp-L4, (sense) 5′-AGTGAGCAAACTTGCTGGTC-3′, (antisense) 5′-ACATACGTCCACCACGACAC-3′ (43Loreni F. Ruberti I. Bozzoni I. Pierandrei-Amaldi P. Amaldi F. EMBO J. 1985; 4: 3483-3488Crossref PubMed Scopus (65) Google Scholar); rp-S1, (sense) 5′-TGGTTTCCCTGAAGGAAGTG-3′, (antisense) 5′-ATGATGAAACGGAGGACACC-3′ (44Pellizzoni L. Crosio C. Pierandrei-Amaldi P. Gene (Amst.). 1995; 154: 145-151Crossref PubMed Scopus (12) Google Scholar); eEF1-α, (sense) 5′-ATGCACCATGAAGCCCTTAC-3′, (antisense) 5′-GGCATATCCAGCACCAATCT-3′ (45Krieg P.A. Varnum S.M. Wormington W.M. Melton D.A. Dev. Biol. 1989; 133: 93-100Crossref PubMed Scopus (342) Google Scholar); β-actin, (sense) 5′-GGACTTTGAGCAGGAGATGG-3′, (antisense) 5′-CAAGGAAAGATGGCTGGAAG-3′ (GenBank™ accession number AF079161); and CNBP, (sense) 5′-TTGTGATCTGCAGGAGGATG-3′, (antisense) 5′-TTCTGTTCATCAGCGTGCTC-3′ (47De Dominicis A. Lotti F. Pierandrei-Amaldi P. Cardinali B. Gene (Amst.). 2000; 241: 35-43Crossref PubMed Scopus (21) Google Scholar). The amplified products were analyzed by Southern blot hybridization using as probes the following specific oligonucleotides corresponding to an internal region of each amplified product labeled at the 5′ end with [γ-32P]dATP: rp-L4, 5′-CCAAACAAGTGCTGAATCGTGGGGA-3′; rp-S1, 5′-GTCTGGCTGTGAGGAGAGCTTGCT-3′; eEF1-α, 5′-AGATTGACCCACCAATGGAAGCTGG-3′; β-actin, 5′-GAGCTATGAGCTGCCTGACGGACAA-3′; and CNBP, 5′-ATTGCCAAGGACTGTAAGGAGCCCAG-3′. Filters were hybridized at 37 °C in 50% formamide overnight and washed at 42 °C in 2× SSC and 0.5% SDS for 1 h and then exposed to 3MM films for autoradiography. Densitometric analysis of the films was carried out by ImageQuant 1.1 software. To identify the linear range of amplification, the RNA from each sample was subjected to a semiquantitative RT-PCR for 20, 25, 30, and 35 cycles using primers specific for the different TOP mRNAs. Following Southern hybridization, the signals obtained for each RNA were quantified. In all of the cases, the 25-cycle reaction proved to be within the linear range of amplification and this condition was used for the experiments. RNA Preparation and Analysis—Precipitates from gradient fractions were pooled as indicated and subjected to RNA extraction with the proteinase K/SDS/phenol-chloroform-isoamylic alcohol procedure and precipitated with absolute ethanol. The precipitates were resuspended, loaded on 0.8% denaturing agarose gel, and analyzed by Northern blot with probes obtained by the random primer method in the presence of 50 μCi of [α-32P]dATP. Hybridization and washing conditions were performed according to standard procedures. Filters were exposed to 3MM films for autoradiography. Protein Analysis—Protein precipitated from gradient fractions were analyzed on 12% acrylamide gels, blotted on nitrocellulose paper (Schleicher & Schuell), and then analyzed by Western blot with rabbit antisera versus Xenopus recombinant La (34Pellizzoni L. Cardinali B. Lin-Marq N. Mercanti D. Pierandrei-Amaldi P. J. Mol. Biol. 1996; 259: 904-915Crossref PubMed Scopus (67) Google Scholar), Xenopus CNBP fragment 159–178 (47De Dominicis A. Lotti F. Pierandrei-Amaldi P. Cardinali B. Gene (Amst.). 2000; 241: 35-43Crossref PubMed Scopus (21) Google Scholar), and Xenopus nucleoplasmin monoclonal antibodies (48Wedlich D. Dreyer C. Cell Tissue Res. 1988; 254: 295-300Crossref PubMed Scopus (8) Google Scholar) according to Pellizzoni et al. (34Pellizzoni L. Cardinali B. Lin-Marq N. Mercanti D. Pierandrei-Amaldi P. J. Mol. Biol. 1996; 259: 904-915Crossref PubMed Scopus (67) Google Scholar). Western blots were revealed with the ECL kit (Pierce). When necessary, quantitation of band intensity was determined with the ImageQuant program. Evaluation of Cytoplasmic La in Xenopus Cells—Since we were interested in a cytoplasmic function of La, we wanted first to have an idea of the amount of La in the cytoplasm and determine whether this was sufficient for binding TOP mRNAs. Experimental determination of La in the cytoplasm has been impaired by the diffusion of La from the nuclei and nuclear breakage during conventional extract preparations from cells (10Wolin S.L. Cedervall T. Annu. Rev. Biochem. 2002; 71: 375-403Crossref PubMed Scopus (340) Google Scholar). For this reason here we took advantage of the oocyte system, which allows a clean separation of the two-cell compartments. Single defolliculated stage V-VI oocytes were manually enucleated under the microscope and immediately collected in separate dishes. Nuclei, cytoplasms, and whole oocyte extracts were prepared as indicated under "Experimental Procedures" and analyzed by Western blot. The nucleus/cytoplasm separation was checked by reacting the same filter with La antibodies and with antibodies against the abundant nucleoplasmin as a nuclear marker (48Wedlich D. Dreyer C. Cell Tissue Res. 1988; 254: 295-300Crossref PubMed Scopus (8) Google Scholar) and CNBP as a cytoplasmic marker (49Warden C.H. Krisans S.K. Purcell-Huynh D. Leete L.M. Daluiski A. Diep A. Taylor B.A. Lusis A.J. Genomics. 1994; 24: 14-19Crossref PubMed Scopus (42) Google Scholar) as shown in Fig. 1A. To quantitate the relative amount of La in the compartments, we loaded on the gel the cytoplasmic extract equivalent to 1 oocyte and the nuclear extract equivalent to one-tenth and one-twentieth of one oocyte. Fig. 1B shows, as an example, the results obtained in one of the three experiments performed. The amount of La protein found in a total oocyte extract corresponds to that present in the equivalent amount of nuclear plus cytoplasmic extract (Fig. 1B, lanes 1, 3, and 4), confirming that most of the protein is localized in the nucleus. Quantitation analysis of band intensity of La from nuclear and cytoplasmic extracts of this (Fig. 1B, lanes 2, 3, and 4) and other similar experiments indicated that cytoplasmic La is approximately 2–4% of nuclear La. This value is compatible with data obtained by immunoprecipitation of oocyte nuclear and cytoplasmic extracts (19Simons F.H. Broers F.J. Van Venrooij W.J. Pruijn G.J. Exp. Cell Res. 1996; 224: 224-236Crossref PubMed Scopus (52) Google Scholar). Although very appropriate to determine the relative amount of La in the cell cytoplasm, the oocyte is not as convenient for our polysome studies as the somatic cells of stage 35 embryos where TOP mRNAs translation is significantly more active than in oocy