Probing interactions between the U2 small nuclear ribonucleoprotein and the DEAD-box protein, Prp5.
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.
Abu Dayyeh
Barham K Abu
BK
Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Cancer Research and Treatment Center, Albuquerque, New Mexico 87131, USA.
Quan
Tiffani K
TK
Castro
Marygrace
M
Ruby
Stephanie W
SW
D001483:Base Sequence; D002384:Catalysis; D053487:DEAD-box RNA Helicases; D005656:Fungal Proteins / Q000378:metabolism*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D020365:RNA Helicases / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D015335:Molecular Probes; D012333:RNA, Messenger; D017413:Ribonucleoprotein, U2 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C064447:PRP5 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases