Abstract:
Most human protein coding genes contain coding (exon) and noncoding (intron)
sequences. In the process of precursor-mRNA splicing, introns are removed and exons
spliced together from the precursor-mRNA transcripts of such genes. Precursor-mRNA
splicing is catalyzed by the spliceosome - a dynamic assembly composed of five sub-
complexes or U snRNPs. To catalyze splicing, the U snRNPs assemble onto a
precursor-mRNA in an ordered pathway to form the E, A, B, B* and C intermediates.
Difficulties in capturing these intermediates has hindered their characterization and
structural investigation, particularly of the B complex which progresses to a catalytically
active state or B* complex following displacement of the U1 snRNP from its pre-mRNA
binding site. We hypothesize that through a site-specific engineered crosslink between
a thiol-modified precursor-mRNA and a mutagenized subunit of U1 snRNP, dissociation
of U1 snRNP can be prevented and a pre-catalytic B complex reversibly trapped for
mechanistic and structural studies. We report on the design, expression, purification,
and characterization of mutant variants of a U1 snRNP subunit to be used to engineer a
disulfide crosslink and their crosslinking to a thiol-modified oligonucleotide.
