Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5

Barham K. Abu Dayyeh, Tiffani K. Quan, Marygrace Castro, Stephanie W. Ruby

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Pre-mRNA binding to the yeast U2 small nuclear ribonucleoprotein (snRNP) during prespliceosome formation requires ATP hydrolysis, the highly conserved UACUAAC box of the branch point region of the pre-mRNA, and several factors. Here we analyzed the binding of a radiolabeled 2′-O-methyl oligonucleotide complementary to U2 small nuclear RNA to study interactions between the UACUAAC box, U2 snRNP, and Prp5p, a DEAD box protein necessary for prespliceosome formation. Binding of the 2′-O-methyl oligonucleotide to the U2 snRNP in yeast cell extract was assayed by gel electrophoresis. Binding was rapid, enhanced by ATP, and dependent on the integrity and conformation of the U2 snRNP. It was also stimulated by Prp5p that was found to associate physically with U2 snRNP. In vitro heat inactivation of the temperature-sensitive prp5-1 mutant extract decreased oligonucleotide binding to U2 and the ATP enhancement of binding by 3-fold. Furthermore, the temperature-sensitive prp5-1 mutation maps to the ATP-binding motif I within the helicase-like domain. Thus the catalytic activity of Prp5p likely promotes a conformational change in the U2 snRNP.

Original languageEnglish (US)
Pages (from-to)20221-20233
Number of pages13
JournalJournal of Biological Chemistry
Volume277
Issue number23
DOIs
StatePublished - Jun 7 2002
Externally publishedYes

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U2 Small Nuclear Ribonucleoproteins
Adenosine Triphosphate
Oligonucleotides
Proteins
RNA Precursors
Yeast
Yeasts
Temperature
Cell Extracts
Electrophoresis
Conformations
Hydrolysis
Catalyst activity
Hot Temperature
Gels
Cells
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5. / Abu Dayyeh, Barham K.; Quan, Tiffani K.; Castro, Marygrace; Ruby, Stephanie W.

In: Journal of Biological Chemistry, Vol. 277, No. 23, 07.06.2002, p. 20221-20233.

Research output: Contribution to journalArticle

Abu Dayyeh, Barham K. ; Quan, Tiffani K. ; Castro, Marygrace ; Ruby, Stephanie W. / Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 23. pp. 20221-20233.
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