The 5' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly.
AUTOR(ES)
Khellil, S
RESUMO
Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=333725Documentos Relacionados
- Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly.
- Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly.
- Interplay between U2 snRNP and 3' splice factor(s) for branch point selection on human beta-globin pre-mRNA.
- Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes.
- Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution.