|prevalence of splicing mutations|| account for about 15% of genetic diseases|
-beta-thalassaemias: mutation of conserved G at beginning of intron leads to use of several cryptic sites
-OR mutation within the intron leads to new 5' splice site
-makes mutant globins
|snRNPS|| -snRNA complexes with Sm proteins via the Sm binding site. |
-discovered bc Sm proteins are common antigens in lupus.
-short conserved sequences in snRNA that interact with pre-mRNA
-U^ is the most highly conserved snRNA.
|Evidence that snRNPS are important in splicing|| if you added labeled pre-mRNA and allow snrps to bind, then add rnase, you get a pre-mrna fragment. (bind and chew experiments)|
-genetic suppression experiments: mutate the 5' splice site and then engineer compensatory snRNA base changes.
-shows U1, U2, U4, U6 are essential
|RNase H experiments|| make a DNA-RNA duplex usig the snRNA|
-add RNase H, which can only work on a duplex hydrid.
-add splicing substrate. lack of activity indicates that snrp is essential.
|Spliceosome|| -contains 50-100 proteins|
-splicing factors bind the poly-pyrimidine tract upstream of 3'splice site
-U1/U2 base pairing to 5' splice/branch sites
-conformational rearrangements powered by ATP