![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Research Papers
Division of Biology, California Institute of Technology, Pasadena 91125.
Abstract
U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the T7U6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. C. McGrail and R. T. O'Keefe The U1, U2 and U5 snRNAs crosslink to the 5' exon during yeast pre-mRNA splicing Nucleic Acids Res., February 11, 2008; 36(3): 814 - 825. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. J. Brenner and C. Guthrie Assembly of Snu114 into U5 snRNP requires Prp8 and a functional GTPase domain RNA, May 1, 2006; 12(5): 862 - 871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. A. TURNER, C. M. NORMAN, M. J. CHURCHER, and A. J. NEWMAN Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome RNA, March 1, 2006; 12(3): 375 - 386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. RYAN, C. H. KIM, J. B. MURRAY, C. J. ADAMS, P. G. STOCKLEY, and J. ABELSON New tertiary constraints between the RNA components of active yeast spliceosomes: A photo-crosslinking study RNA, August 1, 2004; 10(8): 1251 - 1265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. VALADKHAN and J. L. MANLEY Characterization of the catalytic activity of U2 and U6 snRNAs RNA, July 1, 2003; 9(7): 892 - 904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hartmuth, H. Urlaub, H.-P. Vornlocher, C. L. Will, M. Gentzel, M. Wilm, and R. Luhrmann Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method PNAS, December 24, 2002; 99(26): 16719 - 16724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Gottschalk, C. Bartels, G. Neubauer, R. Lührmann, and P. Fabrizio A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly Mol. Cell. Biol., May 1, 2001; 21(9): 3037 - 3046. [Abstract] [Full Text]
|
|
|
|
|
|
S. Massenet, Y. Motorin, D. L. J. Lafontaine, E. C. Hurt, H. Grosjean, and C. Branlant Pseudouridine Mapping in the Saccharomyces cerevisiae Spliceosomal U Small Nuclear RNAs (snRNAs) Reveals that Pseudouridine Synthase Pus1p Exhibits a Dual Substrate Specificity for U2 snRNA and tRNA Mol. Cell. Biol., March 1, 1999; 19(3): 2142 - 2154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-R. Liu, B. Sargueil, and C. W. J. Smith Detection of a Novel ATP-Dependent Cross-Linked Protein at the 5' Splice Site-U1 Small Nuclear RNA Duplex by Methylene Blue-Mediated Photo-Cross-Linking Mol. Cell. Biol., December 1, 1998; 18(12): 6910 - 6920. [Abstract] [Full Text]
|
|
|
|
|
|
B. Laggerbauer, T. Achsel, and R. Luhrmann The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro PNAS, April 14, 1998; 95(8): 4188 - 4192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Ruby Dynamics of the U1 Small Nuclear Ribonucleoprotein during Yeast Spliceosome Assembly J. Biol. Chem., July 11, 1997; 272(28): 17333 - 17341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. O'Day, G. Dalbadie-McFarland, and J. Abelson The Saccharomyces cerevisiae Prp5 Protein Has RNA-dependent ATPase Activity with Specificity for U2 Small Nuclear RNA J. Biol. Chem., December 27, 1996; 271(52): 33261 - 33267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Wiest, C. L. O'Day, and J. Abelson In Vitro Studies of the Prp9·Prp11·Prp21 Complex Indicate a Pathway for U2 Small Nuclear Ribonucleoprotein Activation J. Biol. Chem., December 27, 1996; 271(52): 33268 - 33276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D J Field and J D Friesen Functionally redundant interactions between U2 and U6 spliceosomal snRNAs. Genes & Dev., February 15, 1996; 10(4): 489 - 501. [Abstract] [PDF]
|
|
|
|
 |
|
 P Fabrizio, S Esser, B Kastner, and R Luhrmann Isolation of S. cerevisiae snRNPs: comparison of U1 and U4/U6.U5 to their human counterparts Science, April 8, 1994; 264(5156): 261 - 265. [Abstract] [PDF]
|
|
|
|
|
|
E Lund and J. Dahlberg Cyclic 2',3'-phosphates and nontemplated nucleotides at the 3' end of spliceosomal U6 small nuclear RNA's Science, January 17, 1992; 255(5042): 327 - 330. [Abstract] [PDF]
|
|
|
|
|
|
T Tani and Y Ohshima mRNA-type introns in U6 small nuclear RNA genes: implications for the catalysis in pre-mRNA splicing. Genes & Dev., June 1, 1991; 5(6): 1022 - 1031. [Abstract] [PDF]
|
|
|
|
|
|
T P Hausner, L M Giglio, and A M Weiner Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes & Dev., December 1, 1990; 4(12a): 2146 - 2156. [Abstract] [PDF]
|
|
|
|
|
|
P Fabrizio and J Abelson Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing Science, October 19, 1990; 250(4979): 404 - 409. [Abstract] [PDF]
|
|
|
|
|
|
R Bordonne, J Banroques, J Abelson, and C Guthrie Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo. Genes & Dev., July 1, 1990; 4(7): 1185 - 1196. [Abstract] [PDF]
|
|
|
|
|
|
D S McPheeters, P Fabrizio, and J Abelson In vitro reconstitution of functional yeast U2 snRNPs. Genes & Dev., December 1, 1989; 3(12b): 2124 - 2136. [Abstract] [PDF]
|
|
