Abstract

Eukaryotic genes contain introns which interrupt the continuity of the genetic information. Introns are removed from an RNA transcript of the gene by RNA splicing. This project continues an investigation of the mechanism of splicing of two kinds of RNA splicing in yeast: tRNA and mRNA. tRNA precursors are spliced in a two-step reaction. An endonuclease removes the intron and a ligase, requiring ATP, joins the exons together. The endonuclease will be purified to homogeneity. The mechanism by which it recognizes and cleaves the precursor will be studied. The ligase has already been purified to homogeneity. Further studies will explore its domain structure and the mechanism of the ligase reaction. Studies on a synthetic tRNA-Phe precursor will elucidate the role of secondary and tertiary structure of the mature domain in substrate recognition. mRNA precursors are spliced on a large particle called the spliceosome. Yeast mutants have been isolated called rna2-11 which define components of the spliceosome. The RNA gene products will be purified using an in vitro complementation assay. These products, once purified, can be used to define and characterize other RNA and protein components of the spliceosome. The spliceosome will be purified and characterized by electron microscopy and biochemical fractionation. The pathway by which it is assembled and disassembled will be determined. A possible role of hnRNPs in mRNA splicing will also be investigated.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM032637-13
Application #
2176674
Study Section
Special Emphasis Panel (NSS)
Project Start
1983-03-01
Project End
1996-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
13
Fiscal Year
1995
Total Cost
Indirect Cost

  Institution

Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
078731668
City
Pasadena
State
CA
Country
United States
Zip Code
91125

  Publications

Ryan, Daniel E; Kim, Chang Hee; Murray, James B et al. (2004) New tertiary constraints between the RNA components of active yeast spliceosomes: a photo-crosslinking study. RNA 10:1251-65
Perriman, Rhonda; Barta, Imre; Voeltz, Gia K et al. (2003) ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA. Proc Natl Acad Sci U S A 100:13857-62
Ryan, Daniel E; Abelson, John (2002) The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly. RNA 8:997-1010
Ryan, Daniel E; Stevens, Scott W; Abelson, John (2002) The 5' and 3' domains of yeast U6 snRNA: Lsm proteins facilitate binding of Prp24 protein to the U6 telestem region. RNA 8:1011-33
Story, R M; Li, H; Abelson, J N (2001) Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii. Proc Natl Acad Sci U S A 98:1465-70
Wagner, J D; Jankowsky, E; Company, M et al. (1998) The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes. EMBO J 17:2926-37
Arenas, J E; Abelson, J N (1997) Prp43: An RNA helicase-like factor involved in spliceosome disassembly. Proc Natl Acad Sci U S A 94:11798-802
Kim, C H; Ryan, D E; Marciniec, T et al. (1997) Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro. EMBO J 16:2119-29
Kim, C H; Abelson, J (1996) Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site. RNA 2:995-1010
O'Day, C L; Dalbadie-McFarland, G; Abelson, J (1996) The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA. J Biol Chem 271:33261-7

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