Interaction of U2AF65 RS region with pre-mRNA of branch point and promotion base pairing with U2 snRNA

Science

Volumn 273, Issue 5282, 1996, Pages 1706-1709

  Valcárcel, Juan ^a,b   Gaur, Rajesh K ^a   Singh, Ravinder ^a   Green, Michael R ^a  

^a UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL   (United States)

^b EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

RIBONUCLEOPROTEIN; ARGININE; CROSS LINKING REAGENT; MESSENGER RNA; NUCLEAR PROTEIN; RNA PRECURSOR; SERINE; SMALL NUCLEAR RNA; SPLICING FACTOR U2AF;

AMINO ACID SUBSTITUTION; ARTICLE; BASE PAIRING; CROSS LINKING; MOLECULAR INTERACTION; NONHUMAN; PRIORITY JOURNAL; PROTEIN DNA INTERACTION; PROTEIN DOMAIN; PROTEIN STRUCTURE; RNA METABOLISM; SPLICEOSOME; AMINO ACID SEQUENCE; BIOLOGICAL MODEL; CHEMISTRY; CONFORMATION; DNA BASE COMPOSITION; METABOLISM; MOLECULAR GENETICS; NUCLEOTIDE SEQUENCE; RNA SPLICING;

AMINO ACID SEQUENCE; ARGININE; BASE COMPOSITION; BASE SEQUENCE; CROSS-LINKING REAGENTS; MODELS, GENETIC; MOLECULAR SEQUENCE DATA; NUCLEAR PROTEINS; NUCLEIC ACID CONFORMATION; RIBONUCLEOPROTEINS; RNA PRECURSORS; RNA SPLICING; RNA, MESSENGER; RNA, SMALL NUCLEAR; SERINE; SPLICEOSOMES;

EID: 0030595187     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5282.1706     Document Type: Article

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37. Buffer D: 20 mM Hepes (pH 8.0), 0.2 mM EDTA, 20% glycerol, 0.05% NP-40, and 1 mM dithiothreitol.
38. m-MINX (pre-mRNA positions 150 to 160 substituted by UAUGAUAGUGA). The corresponding pre-mRNAs were generated by in vitro transcription of Bam HI-digested templates.
39. Plasmids pT 7U2 [A. M. Kleinschmidt et al., Nucleic Acids Res. 17, 4817 (1989)], pT 7U2ΔBPRS (deletion of U2 snRNA positions 37 to 42), and pHU1A [J. R. Patton, R. J. Patterson, T. Pederson, Mol. Cell. Biol. 7, 4030 (1987)] were used to transcribe U2 snRNA (positions 1 to 71), ΔBPRS, and U1 snRNA, respectively.
40. Plasmids pT 7U2 [A. M. Kleinschmidt et al., Nucleic Acids Res. 17, 4817 (1989)], pT 7U2ΔBPRS (deletion of U2 snRNA positions 37 to 42), and pHU1A [J. R. Patton, R. J. Patterson, T. Pederson, Mol. Cell. Biol. 7, 4030 (1987)] were used to transcribe U2 snRNA (positions 1 to 71), ΔBPRS, and U1 snRNA, respectively.
41. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val: W, Trp; and Y, Tyr.
42. 35 cDNA [M. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 89, 8769 (1992)]. RS (SF2): positions 887 to 1039 of ASF/SF2 cDNA [H. Ge et al., Cell 66, 373 (1991); A. R. Krainer et al., ibid., p. 383].
43. 35 cDNA [M. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 89, 8769 (1992)]. RS (SF2): positions 887 to 1039 of ASF/SF2 cDNA [H. Ge et al., Cell 66, 373 (1991); A. R. Krainer et al., ibid., p. 383].
44. 35 cDNA [M. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 89, 8769 (1992)]. RS (SF2): positions 887 to 1039 of ASF/SF2 cDNA [H. Ge et al., Cell 66, 373 (1991); A. R. Krainer et al., ibid., p. 383].
45. 65 cDNA fragment encoding amino acids 94 to 475, flanked by Bam HI and Eco RI, into the same sites in pGEX-2T [D. B. Smith and K. S. Johnson, Gene 67,31 (1988)]. Digestion of this construct with Bam HI was used to obtain all the RS mutant derivatives.
46. Inserts were obtained by annealing complementary oligodeoxyribonucleotides corresponding to sequences encoding the RS repeats or mutant derivatives.
47. 65 fusion with a TEV protease cleavage site between the two polypeptides. The fusion protein was expressed in bacteria, purified, and cleaved with TEV protease as described by Parks et al., (ibid.).
48. 65 fusion with a TEV protease cleavage site between the two polypeptides. The fusion protein was expressed in bacteria, purified, and cleaved with TEV protease as described by Parks et al., (ibid.).
49. M. Mirfakhrai and A. M. Weiner, Nucleic Acids Res. 21, 3591 (1993).
50. 65 promoted interactions between U2 snRNA and a variety of pre-mRNAs from Drosophila to human genes.
51. Primer extension mapping of psoralen cross-links has been shown to give rise to more than one primer extension stop from a single cross-link [G. Ericson and P. Wollenzien, Anal. Biochem. 174, 215 (1988); J. Teare and P. Wollenzien, Nucleic Acids Res. 18, 855 (1990)].
52. Primer extension mapping of psoralen cross-links has been shown to give rise to more than one primer extension stop from a single cross-link [G. Ericson and P. Wollenzien, Anal. Biochem. 174, 215 (1988); J. Teare and P. Wollenzien, Nucleic Acids Res. 18, 855 (1990)].
53. We thank M. Jacobson and T. Pederson for the U1 snRNA clone, L. Chiang and J. Kielty for technical support, T. O'Toole for secretarial assistance, and F. Gebauer, K. Neugebauer, and members of the Green lab for advice and comments on the manuscript. Supported by European Molecular Biology Organization and Spanish Ministerio de Educatión y Ciencia fellowships to J.V., Leukemia Society of America postdoctoral and special fellowships to R.S., and a grant from the National Institutes of Health to M.R.G.