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• W2088156779 abstract "Developmental stages of the trypanosome life cycle differ in their morphology, biology, and biochemical properties. Consequently, several proteins have to be tightly regulated in their expression to allow trypanosomes to adapt rapidly to sudden environmental changes, a process that might be of central importance for parasite survival. However, in contrast to higher eukaryotic cells, trypanosomes do not seem to regulate gene expression through regulation of transcription initiation. These parasites make use of post-transcriptional regulatory mechanisms and modification of mRNA half-life is a relevant one. Trans-acting factors binding to cis-elements that affect mRNA stability of mature transcripts have not been identified in these cells. In this work, a novel U-rich RNA-binding protein (TcUBP-1) from Trypanosoma cruzi, the agent of Chagas disease, was identified. Its structure includes an RNA recognition motif, a nuclear export signal, and auxiliary domains with glycine- and glutamine-rich regions. TcUBP-1 recognizes the 44-nucleotide AU-rich RNA instability element located in the 3′-untranslated region of mucin SMUG mRNAs (Di Noia, J. M., D'Orso, I., Sanchez, D. O., and Frasch, A. C. (2000)J. Biol. Chem. 275, 10218–10227) as well as GU-rich sequences. Over-expression of TcUBP-1 in trypanosomes decreases the half-life of SMUG mucin mRNAs in vivo but does not affect the stability of other parasite mRNAs. Because TcUBP-1 is developmentally regulated, it might have a relevant role in regulating protein expression during trypanosome differentiation, allowing a correct expression pattern of U-rich-containing mRNAs. Developmental stages of the trypanosome life cycle differ in their morphology, biology, and biochemical properties. Consequently, several proteins have to be tightly regulated in their expression to allow trypanosomes to adapt rapidly to sudden environmental changes, a process that might be of central importance for parasite survival. However, in contrast to higher eukaryotic cells, trypanosomes do not seem to regulate gene expression through regulation of transcription initiation. These parasites make use of post-transcriptional regulatory mechanisms and modification of mRNA half-life is a relevant one. Trans-acting factors binding to cis-elements that affect mRNA stability of mature transcripts have not been identified in these cells. In this work, a novel U-rich RNA-binding protein (TcUBP-1) from Trypanosoma cruzi, the agent of Chagas disease, was identified. Its structure includes an RNA recognition motif, a nuclear export signal, and auxiliary domains with glycine- and glutamine-rich regions. TcUBP-1 recognizes the 44-nucleotide AU-rich RNA instability element located in the 3′-untranslated region of mucin SMUG mRNAs (Di Noia, J. M., D'Orso, I., Sanchez, D. O., and Frasch, A. C. (2000)J. Biol. Chem. 275, 10218–10227) as well as GU-rich sequences. Over-expression of TcUBP-1 in trypanosomes decreases the half-life of SMUG mucin mRNAs in vivo but does not affect the stability of other parasite mRNAs. Because TcUBP-1 is developmentally regulated, it might have a relevant role in regulating protein expression during trypanosome differentiation, allowing a correct expression pattern of U-rich-containing mRNAs. AU-rich element Trypanosoma cruzi U-rich RNA-binding protein electrophoresis mobility shift assay 3′-untranslated region small mucin gene family nuclear export signal RNA recognition motif embryonic lethal abnormal vision ribonucleoprotein human RNP glutathione S-transferase expressed sequence tag nucleotide base pair(s) pRIBOTEX vector mitogen-activated protein kinase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Messenger RNA degradation/stabilization is one of the mechanisms that control gene expression in higher eukaryotic cells (1Beelman C.A. Parker R. Cell. 1995; 81: 179-183Abstract Full Text PDF PubMed Scopus (571) Google Scholar). One well characterized cis-element that regulates mRNA stability is an AU-rich element (ARE)1found in the 3′UTR of short-lived mRNAs (2Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3123) Google Scholar, 3Caput D. Beutler B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1216) Google Scholar) such as those of proto-oncogenes and cytokines (interleukin-1, -2, -3, and -10) (4Chen C.Y. Del Gatto-Konczak F. Wu Z. Karin M. Science. 1998; 280: 1945-1949Crossref PubMed Scopus (329) Google Scholar, 5Stoecklin G. Hahn S. Moroni C. J. Biol. Chem. 1994; 269: 28591-28597Abstract Full Text PDF PubMed Google Scholar, 6Kishore R. Tebo J.M. Kolosov M. Hamilton T.A. J. Immunol. 1999; 162: 2457-2461PubMed Google Scholar). Each ARE represents a combination of functionally and structurally distinct sequence motifs such as the AUUUA pentamer, the UUAUUUA(U/A)(U/A) nonamer, and stretches of uridines and/or U-rich domains that range in size from 50 to 150 bp. It was shown that ARE-directed mRNA decay is linked to cell transformation, cell growth and differentiation, cell adhesion, and immune response regulation (7Chen C.A. Shyu A. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1679) Google Scholar). An effect of the ARE on translation efficiency was also described both positively (8Kontoyiannis D. Pasparakis M. Pizarro T.T. Cominelli F. Kollias G. Immunity. 1999; 10: 387-398Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar) and negatively (9Sokolowski M. Zhao C. Tan W. Schwartz S. Oncogene. 1997; 15: 2303-2319Crossref PubMed Scopus (51) Google Scholar). RNA-binding proteins that exhibit affinity for, and interact with, ARE sequences have been identified in higher eukaryotes. Many of these factors contain highly conserved RNA binding domains that place them within RNA recognition motif (RRM) superfamily (10Kim Y.J. Baker B.S. Mol. Cell. Biol. 1993; 13: 174-183Crossref PubMed Scopus (54) Google Scholar). Two kinds of trans-acting factors were described that have opposite functions. The ELAV protein HuR increases stability of ARE-containing mRNAs when over-expressed in transfected fibroblasts (11Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (746) Google Scholar). Conversely, AUF-1/hnRNP D (12Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar) mediates a destabilizing activity of the mRNAs containing ARE sequences within their 3′UTR (13Loflin P. Chen C.Y. Shyu A.B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (263) Google Scholar). It was proposed that ELAV proteins may bind the ARE-containing mRNAs in the nucleus and transport them to the cytoplasm, where they either find access to the translational apparatus or are released for rapid degradation (14Gao F.B. Keene J.D. J. Cell Sci. 1996; 109: 579-589Crossref PubMed Google Scholar). Other hnRNPs, such as hnRNP A1 and C (15Hamilton B.J. Nagy E. Malter J.S. Arrick B.A. Rigby W.F. J. Biol. Chem. 1993; 268: 8881-8887Abstract Full Text PDF PubMed Google Scholar), hnRNP A0 (16Myer V.E. Steitz J.A. RNA. 1995; 1: 171-182PubMed Google Scholar), and some RNA-binding proteins displaying catalytic activities such as glyceraldehyde-3-phosphate-dehydrogenase (17Nagy E. Rigby W.F. J. Biol. Chem. 1995; 270: 2755-2763Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar), AUH, an enoyl-CoA hydratase (18Nakagawa J. Waldner H. Meyer-Monard S. Hofsteenge J. Jeno P. Moroni C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2051-2055Crossref PubMed Scopus (125) Google Scholar), were also shown to be AU-rich RNA-binding proteins. Trypanosomes, protozoan parasites from the order Kinetoplastida, cause widespread diseases in humans, domestic animals, and wildlife that include infections of medical and veterinary importance. Studies of trypanosomes have revealed a number of unusual mechanisms occurring during transcription and RNA maturation. Among them are the RNA editing in mitochondrial transcripts (19Stuart K. Verh. K. Acad. Geneeskd. Belg. 1998; 60: 63-74PubMed Google Scholar), polycistronic transcription, andtrans-splicing. Polycistronic transcripts are processed to mature mRNAs through trans-splicing on the 5′-end and poly(A) addition on the 3′-end (20Matthews K.R. Tschudi C. Ullu E. Genes Dev. 1994; 8: 491-501Crossref PubMed Scopus (221) Google Scholar). This scenario makes it difficult to consider that trypanosomes regulate protein expression through regulation of transcription initiation, because unrelated mRNAs might be present in the same polycistron (21Teixeira S.M. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 1995; 270: 22586-22594Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In addition, RNA polymerase II promoters have been difficult to detect in trypanosomes. Two putative promoter regions were described as transcriptionally void regions upstream from the highly transcribed actin andHsp70 genes (22Ben Amar M.F. Jefferies D. Pays A. Bakalara N. Kendall G. Pays E. Nucleic Acids Res. 1991; 19: 5857-5862Crossref PubMed Scopus (31) Google Scholar, 23Lee M.G. Mol. Cell. Biol. 1996; 16: 1220-1230Crossref PubMed Scopus (47) Google Scholar). However, one example of RNA polymerase II promoter was recently identified in the spliced leader genes of the parasite Leptomonas seymouri (24Gilinger G. Bellofatto V. Nucleic Acids Res. 2001; 29: 1556-1564Crossref PubMed Scopus (98) Google Scholar). Thus, trypanosomes are interesting organisms in the analysis of post-transcriptional regulatory mechanisms affecting mRNA expression patterns (25Roditi I. Furger A. Ruepp S. Schurch N. Butikofer P. Mol Biochem. Parasitol. 1998; 91: 117-130Crossref PubMed Scopus (89) Google Scholar). Cis-regulatory elements altering the half-life of mature mRNAs have been identified, most of them located on the 3′-untranslated regions (3′UTR) (26Hotz H.R. Hartmann C. Huober K. Hug M. Clayton C. Nucleic Acids Res. 1997; 25: 3017-3026Crossref PubMed Scopus (118) Google Scholar, 27Coughlin B.C. Teixeira S.M. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 2000; 275: 12051-12060Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). One example is the 16-mer loop localized in the 3′UTR of procyclic mRNAs ofTrypanosoma brucei that confers mRNA stability and improves translation efficiency (28Furger A. Schurch N. Kurath U. Roditi I. Mol. Cell. Biol. 1997; 17: 4372-4380Crossref PubMed Scopus (135) Google Scholar). Also, different 3′UTRs and intergenic regions from trypanosome mRNAs were shown to influenceluciferase expression by changing the steady-state level and/or the translation efficiency of a luc mRNA reporter (29Nozaki T. Cross G.A. Mol. Biochem. Parasitol. 1995; 75: 55-67Crossref PubMed Scopus (64) Google Scholar). Trypanosoma cruzi transcripts, such as the surface proteins FL-160 (30Weston D. La Flamme A.C. Van Voorhis W.C. Mol. Biochem. Parasitol. 1999; 102: 53-66Crossref PubMed Scopus (39) Google Scholar), amastin (27Coughlin B.C. Teixeira S.M. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 2000; 275: 12051-12060Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), gp72, and gp85 (29Nozaki T. Cross G.A. Mol. Biochem. Parasitol. 1995; 75: 55-67Crossref PubMed Scopus (64) Google Scholar), also contain regulatory sequences that modulate mRNA stability. Although these results suggest the possible importance of cis-acting elements in mRNA stability or instability, thetrans-acting factors involved in these processes have not yet been identified in trypanosomes. T. cruzi is the protozoan parasite agent of Chagas disease. The parasite has two hosts, an insect vector and mammals (including man). The parasite is covered by different highlyO-glycosylated mucins that are regulated developmentally. The stage of the parasite present in the insect vector (epimastigote) expresses members of a small mucin family named TcSMUG whose core proteins are encoded in about 70 different genes (31Di Noia J.M. D'Orso I. Sanchez D.O. Frasch A.C. J. Biol. Chem. 2000; 275: 10218-10227Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The forms of the parasite present in the bloodstream of the mammalian host have larger mucins (60–200 kDa, compare with 35–50 kDa in epimastigotes) encoded by about 500 different genes (32Di Noia J.M. D'Orso I. Aslund L. Sanchez D.O. Frasch A.C. J. Biol. Chem. 1998; 273: 10843-10850Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 33Pollevick G.D. Di Noia J.M. Salto M.L. Lima C. Leguizamon M.S. de Lederkremer R.M. Frasch A.C. J. Biol. Chem. 2000; 275: 27671-27680Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Developmentally regulated expression of these mucins in the different parasite stages is relevant because they might accomplish different functions in relation to parasite survival (34Frasch A.C. Parasitol. Today. 2000; 16: 282-286Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). In previous work, we have identified an ARE on the 3′UTR of the mRNAs encoding the core protein ofT. cruzi small mucins (TcSMUG), which affect mRNA stability. Transfection experiments, using a cat reporter gene flanked by mucin intergenic regions bearing or not ARE sequences within its 3′UTR, showed that this cis-element destabilizes mucin mRNAs in a stage-specific manner (31Di Noia J.M. D'Orso I. Sanchez D.O. Frasch A.C. J. Biol. Chem. 2000; 275: 10218-10227Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). These results suggest that different trans-acting factors might bind mucin transcripts in vivo and selectively regulate the stability in a different manner throughout parasite development (35D'Orso I. Frasch A.C.C. J. Biol. Chem. 2001; 276: 15783-15793Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In this work, we have identified a gene encoding a developmentally regulated U-rich RNA-binding protein named TcUBP-1. It specifically recognizes the 44-nucleotide SMUG mucin AU-rich RNA instability element previously described by us (31Di Noia J.M. D'Orso I. Sanchez D.O. Frasch A.C. J. Biol. Chem. 2000; 275: 10218-10227Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), as well as GU-rich sequences. Transfection experiments allowed us to demonstrate that TcUBP-1 is able to selectively destabilize SMUG mucin transcripts in vivobut not mRNAs lacking AU-rich elements within their 3′UTR. These results suggest that TcUBP-1 is involved in the regulation of mRNA expression mediated by U-rich elements. T. cruziCL-Brener cloned stock (36Zingales B. Pereira M.E. Oliveira R.P. Almeida K.A. Umezawa E.S. Souto R.P. Vargas N. Cano M.I. da Silveira J.F. Nehme N.S. Morel C.M. Brener Z. Macedo A. Acta Trop. 1997; 68: 159-173Crossref PubMed Scopus (72) Google Scholar) was used. Different forms of the parasites were obtained as described previously (37Gonzalez Cappa S.M. Bijovsky A.T. Freilij H. Muller L. Katzin A.M. Medicina. 1981; 41: 119-120PubMed Google Scholar). Epimastigote cultures were taken in logarithmic growth phase at a cell density of 3 × 107/ml and treated with actinomycin D (Sigma) at a final concentration of 10 μg/ml, which is known to inhibit transcription in trypanosomatids (28Furger A. Schurch N. Kurath U. Roditi I. Mol. Cell. Biol. 1997; 17: 4372-4380Crossref PubMed Scopus (135) Google Scholar, 38Charest H. Zhang W.W. Matlashewski G. J. Biol. Chem. 1996; 271: 17081-17090Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). TcUBP-1 open reading frame (GenBankTM accession number AF372519) was cloned in the BamHI and EcoRI restriction sites of pRIBOTEX vector (39Martinez-Calvillo S. Lopez I. Hernandez R. Gene. 1997; 199: 71-76Crossref PubMed Scopus (64) Google Scholar), kindly provided by Dr. R. Hernandez (Universidad Nacional de Mexico). Transfections were carried out as described previously (31Di Noia J.M. D'Orso I. Sanchez D.O. Frasch A.C. J. Biol. Chem. 2000; 275: 10218-10227Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). RNA was purified using TRIzol reagent following the manufacturer's instructions (Life Technologies, Inc.) Northern blots were carried out as described (40Fourney R.M. Miyakoshi J. Day III, R.S. Paterson M. Focus. 1978; 10: 5-9Google Scholar). The SMUG and 24SαrRNA DNA probes were obtained as described previously (31Di Noia J.M. D'Orso I. Sanchez D.O. Frasch A.C. J. Biol. Chem. 2000; 275: 10218-10227Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Ductin DNA probe was obtained from a clone with high homology to mammalian ductin. 2R. Verdún and D. O. Sánchez, unpublished data. Hsp70 probe was obtained from the EST clone number TENG-0042 (GenBankTM accession number AI034982). Genomic DNA of T. cruziCL-Brener clone was digested with the indicated restriction enzymes (New England Biolabs). The DNA fragments were separated by electrophoresis in 0.8% agarose gel, transferred by capillarity (41Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 9.31-9.57Google Scholar) to Zeta Probe Nylon-N membranes (Bio-Rad), and UV cross-linked using a UVP CL-1000 cross-linker. Filters were hybridized with the probes described below (RRM and AUX) using hybridization solution containing 0.5 m NaH2PO4, 7% SDS, 1 mm EDTA, and 1% bovine serum albumin, at 58 °C for low stringency conditions and 65 °C for high stringency conditions. The RRM probe was made from clone TcUBP-1 by polymerase chain reaction using oligonucleotides NH2-L (5′-CGGGATCCATGAGCCAAATTGTTGTTTC-3′) and RNP-II (5′-CGGAATGTAGTCACCTGGAG-3′). Probe AUX was made using oligonucleotides NES-sec (5′-AACAAGCGGTTGAAGGTAGCG-3′) and COOH-L (5′-CGGAATTCTTACCTTCGAACAGGACGGGC-3′) and was labeled with [α-32P]dCTP (PerkinElmer Life Sciences). Reverse transcription was performed on total RNA from the epimastigote stage of the parasite using the Superscript II kit following the manufacturer' instructions (Life Technologies, Inc.). Primer oligo(dT)18(5′-GCGACTCCGCGGCCGCG(T)18-3′) was used for cDNA synthesis. The first polymerase chain reaction was performed using the oligonucleotides ME (5′-TACAGTTTCTGTACTATATTG-3′) and RNP-I (5′-AAACTTCACAATCCATAGGCC-3′). These products were cloned in pGEMT-Easy vector (Promega) and sequenced. All plasmids for in vitrotranscription were constructed as follows. Complementary DNA oligonucleotides, corresponding to the sense and antisense strands of each RNA, were annealed and cloned into the EcoRI andHindIII sites of the vector pBS(−) (Stratagene). All of the RNA probes had the same flanking region, GGATC, in the 5′-end and one A at the 3′-end. Transcription of sense sequences was performed with 1 μg of HindIII-digested plasmids using T7 RNA-polymerase (Promega) in the presence of [α-32P]UTP (800 Ci/mmol, PerkinElmer Life Sciences) and 500 μm ATP, CTP, and GTP. All transcripts were purified on an 8 murea-12% polyacrylamide gel. For total extract preparation, parasites were resuspended in lysis buffer (0.75% CHAPS detergent, 1 mm MgCl2, 1 mm EGTA, 5 mm β-mercapthoethanol, 10 mm Tris-HCl, pH 7.6, and 10% glycerol) and supplemented with protease inhibitors phenylmethylsulfonyl fluoride, 1 mm, andtrans-epoxysuccinyl-l-leucylamido(4)guanidino), 50 μm (Sigma). After 30 min on ice, the extract was centrifuged at 10,000 × g and the supernatant was stored at −70 °C. TcUBP-1 cDNA was cloned into the BamHI and EcoRI restriction endonuclease sites of the pGEX-2T vector (Amersham Pharmacia Biotech) generating a glutathione S-transferase (GST) fusion and transformed in Escherichia coli strain DH5αF′Iq. Cultures were induced with 0.2 mm isopropyl β-d-thiogalactopyranoside for 3 h at 37 °C. Recombinant proteins were purified using GST-agarose columns (Sigma). Binding reactions were performed with different amounts of TcUBP-1 protein, 10,000 cpm of RNA probe, 10 mm Tris-HCl (pH 7.6), 5% glycerol, 100 mm KCl, 5 mm MgCl2, 1 μg/ml bovine serum albumin, and 50 ng/μl tRNA (Sigma) in a 20-μl final volume reaction mixture and incubated for 10 min at 37 °C. When indicated, heparin was added at a concentration of 1 μg/ml. Each reaction was loaded directly onto a 7% polyacrylamide gel (acrylamide/bisacrylamide, 38:2) 0.5× TBE (Tris borate-EDTA), to perform an electrophoretic mobility shift assay (EMSA). For competition assays, TcUBP-1 recombinant protein was incubated simultaneously with the indicated amounts of unlabeled and labeled RNAs. All homoribopolymers were from Sigma. For the determination of the Kd between TcUBP-1 and different RNAs, increasing amounts of protein were incubated with the labeled RNA and resolved as described above. Purified GST-TcUBP-1 was injected into rabbits with Freund's adjuvant two times at 3-week intervals. For Western blot analysis, protein samples fractionated on 10% SDS-polyacrylamide gels were transferred to Hybond C nitrocellulose membranes (Amersham Pharmacia Biotech), probed with anti GST-TcUBP-1 antibodies, and developed using horseradish peroxidase-conjugated anti-rabbit antibodies and the Supersignal® West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer's instructions. Signals were quantitated using the 1D Image Analysis Software (Kodak Digital Science). The presence of functional AU-rich elements in mucin mRNAs from trypanosomes (31Di Noia J.M. D'Orso I. Sanchez D.O. Frasch A.C. J. Biol. Chem. 2000; 275: 10218-10227Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 35D'Orso I. Frasch A.C.C. J. Biol. Chem. 2001; 276: 15783-15793Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) suggests the existence oftrans-acting factors binding to these elements. During a search for new T. cruzi genes (42Aguero F. Verdun R.E. Frasch A.C. Sanchez D.O. Genome Res. 2000; 10: 1996-2005Crossref PubMed Scopus (46) Google Scholar), we identified ESTs and Genome Sequence Surveys having homologies with eukaryotic RNA-binding proteins. One of these cDNA clones has a deduced amino acid sequence with identities with Sex lethal (SXL) (43Merendino L. Guth S. Bilbao D. Martinez C. Valcarcel J. Nature. 1999; 402: 838-841Crossref PubMed Scopus (221) Google Scholar), Rbp9 (44Kim Y.J. Baker B.S. J. Neurosci. 1993; 13: 1045-1056Crossref PubMed Google Scholar), and members of the ELAV family (45Good P.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4557-4561Crossref PubMed Scopus (273) Google Scholar) (Figs.1 and 2). The putative protein encoded by this T. cruzi cDNA clone, for which the complete sequence was obtained by reverse transcription-polymerase chain reaction, was named TcUBP-1 for T. cruzi U-rich RNA-binding protein. This 1017-base pair clone had a 638-base pair coding region.Figure 2The RRM and a putative nuclear export signal of TcUBP-1 resemble those of higher eukaryotes RNA-binding proteins. A, amino acid alignment of the amino-terminal region and the RRM of TcUBP-1 with higher eukaryote U-rich RNA-binding proteins: ELAV member HuC, D. melanogaster SXL, and Rbp9. The identities are boxed, and the gaps are introduced for best alignment. The arrow indicates the separation of the amino-terminal region and the RRM motif. The RNP-1 and -2 motifs are indicated above each comparison.B, comparison among the NES of TcUBP-1, mPKIα, HIV-1 Rev, MAPK, and cyclin B1. The position of three of four conserved Leu residues is indicated in bold letters and aligned vertically.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TcUBP-1 presents a short Gln-rich amino-terminal composed of 34 amino acids followed by one RNA recognition motif having the canonical RNP-2 and RNP-1 motifs (46Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1729) Google Scholar). The RRM motif from the T. cruziprotein (Fig. 1, A and B) highly resembles those present in higher eukaryote RNA-binding proteins, such as Rbp9, HuC, and SXL among others (Fig.2 A). The similarity between them ranges between 40–55%, and the RNP-1 and -2 motifs are the most conserved regions. This degree of conservation might be because of the tertiary structure of the RRM motif required for RNA recognition (47Handa N. Nureki O. Kurimoto K. Kim I. Sakamoto H. Shimura Y. Muto Y. Yokoyama S. Nature. 1999; 398: 579-585Crossref PubMed Scopus (317) Google Scholar). After the RRM motif, a putative NES, or LEU-rich nuclear export signal (48Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1741) Google Scholar), is present (Fig. 1). This region has three of the four LEU expected to be conserved (49Dreyfuss G. Hentze M. Lamond A.I. Cell. 1996; 85: 963-972Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) as compared with the functional NES sequences present in MAPK, mPKIα, HIV1-Rev, and cyclin B1 proteins (50Izaurralde E. Mattaj I.W. Cell. 1995; 81: 153-159Abstract Full Text PDF PubMed Scopus (209) Google Scholar) (Fig. 2 B). Toward the carboxyl terminus, two auxiliary domains are present. First is a short Gly-rich region that resembles the repeats in hnRNP A1; an ARE-binding protein in which the intracellular localization determinant for targeting it to the nucleus has been identified in the Gly-rich domain of the protein (51Siomi H. Dreyfuss G. J. Cell Biol. 1995; 129: 551-560Crossref PubMed Scopus (444) Google Scholar). Second, a glutamine-rich carboxyl-terminal region is present. Both Gly- and Gln-rich auxiliary domains, like the ones present in TcUBP-1, were described as being involved in protein-protein interactions in other eukaryotic cells (52Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). A Southern blot of T. cruzi genomic DNA using the RRM region as a probe (Fig.3 A) revealed a number of bands under low stringency, whereas only a few bands were observed under high stringency conditions (Fig. 3 B, panels I andII, respectively). A probe for the auxiliary region gave similar results, although fewer bands were observed under low stringency conditions (data not shown). These results indicated that TcUPB-1 might be part of a gene family whose members have greater homologies in the RRM motif than in the auxiliary domains. Two clones related in sequence and structure to TcUBP-1, containing the same RRM motif and different auxiliary domains, were recently identified in our laboratory. 3I. D'Orso and A. C. C. Frasch, unpublished data. Searches in the Genome Sequence Survey and EST T. cruzi and T. brucei data bases were conducted using the RRM sequence. Four new sequences were identified (GenBankTM accession numbers AZ050633, AQ638822, AQ637856, and AQ654405). These clones share about 35–50% identity in the RRM motif, and the RNP-1 and RNP-2 motifs are the most conserved region of the proteins. Additionally, a search in the Sanger Center Leishmania majorand T. brucei (www.sanger.ac.uk/Projects/) data bases was performed. Two clones highly similar to TcUBP-1 were identified (GenBankTM accession numbers AQ846565 and 0.15543) (see Fig. 3 C). The similarity index between them is about 70%, the RRM motif and the putative NES sequence being the most conserved regions of the proteins. These results suggest that highly similar sequences of TcUBP-1 are conserved in different protozoan parasite species. We have shown that a 44-nt AU-richcis-element localized on the 3′UTR of the TcSMUG mucin family was important in conferring mRNA instability in a stage-specific manner (31Di Noia J.M. D'Orso I. Sanchez D.O. Frasch A.C. J. Biol. Chem. 2000; 275: 10218-10227Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). To determine whether TcUBP-1 recognizes this RNA sequence (named SMUG-L-AU), an in vitro binding reaction was performed (Fig.4 A). The in vitrotranscribed SMUG-L-AU RNA was incubated with GST, GST-TcUBP-1, or without protein, and the reactions were resolved in a native EMSA. TcUBP-1 bound to the SMUG-L-AU RNA and the binding was not affected by the addition of heparin, an unspecific competitor. Control experiments using GST protein were negative (Fig. 4 A). The ribonucleoprotein complex formed by TcUBP-1 and the SMUG-L-AU RNA template was efficiently competed with poly(U) but not with poly(A) or poly(C) RNA homopolymers. Poly(G) somewhat reduced the binding of TcUBP-1 to the RNA probe but much less efficiently than poly(U) (Fig.4 B). The binding of TcUBP-1 to the SMUG-L-AU probe seemed to be to a single RNA site, because only one shifted band was obtained within a large range of TcUBP-1 protein amount (Fig. 4 C). We also searched for the minimal sequence requirement for TcUBP-1 to bind the SMUG-L-AU RNA. Three RNAs from SMUG-L-AU were used to performin vitro binding and EMSA (Fig. 4 D). The binding to the 31-nt SMUG-L-AU-1 RNA was reduced as compared with the complete SMUG-L-AU RNA, showing that the two AUUUUA motifs in the boundaries of this RNA are important for an efficient binding of TcUBP-1. The binding to the UUAUUUAUU nonamer (SMUG-L-AU-2 RNA) was detectable only after over-exposing the film. This interaction was slightly improved when the nonamer was present within a short U-rich context, as happens with the SMUG-L-AU-3 RNA, which has two additional uridines outside the nonamer RNA oligonucleotide (Fig. 4 D). Thus, the entire 44-nt SMUG-L-AU RNA seems to be required for an efficient binding, since deletion of some U-rich stretches within this sequence greatly affects TcUBP-1 recognition. Because a number of T. cruzi transcripts have AU-rich sequences within their 3′UTR, we tested whether TcUBP-1 s" @default.
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• W2088156779 date "2001-09-01" @default.
• W2088156779 modified "2023-10-16" @default.
• W2088156779 title "TcUBP-1, a Developmentally Regulated U-rich RNA-binding Protein Involved in Selective mRNA Destabilization in Trypanosomes" @default.
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• W2088156779 doi "https://doi.org/10.1074/jbc.m102120200" @default.
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