Premium
In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein
Author(s) -
Rain JeanChristophe,
Legrain Pierre
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.7.1759
Subject(s) - biology , rna splicing , yeast , genetics , sequence (biology) , conserved sequence , nucleotide , nucleic acid sequence , saccharomyces cerevisiae , peptide sequence , gene , rna
Pre‐mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision. We previously developed a pre‐mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se . Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5′ splice site lead to pre‐mRNA export, whereas 3′ splice site mutations do not. A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre‐mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre‐mRNA. In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially. Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.