Ds. Mcpheeters et al., Interaction of the yeast DExH-box RNA helicase Prp22p with the 3 ' splice site during the second step of nuclear pre-mRNA splicing, NUCL ACID R, 28(6), 2000, pp. 1313-1321
Using site-specific incorporation of the photochemical cross-linking reagen
t 4-thiouridine, we demonstrate the previously unknown association of two p
roteins with yeast 3' splice sites. One of these is an unidentified similar
to 122 kDa protein that cross-links to 3' splice sites during formation of
the prespliceosome. The other factor is the DExH-box RNA helicase, Prp22p.
With substrates functional in the second step of splicing, only very weak
cross-linking of Prp22p to intron sequences at the 3' splice site is observ
ed. In contrast, substrates blocked at the second step exhibit strong cross
-linking of Prp22 to intron sequences at the 3' splice site, but not to adj
acent exon sequences. In vitro reconstitution experiments also show that th
e association of Prp22p with intron sequences at the 3' splice site is depe
ndent on Prp16p and does not persist when release of mature mRNA from the s
pliceosome is blocked. Taken together, these results suggest that the 3' sp
lice site of yeast introns is contacted much earlier than previously envisi
oned by a protein of similar to 120 kDa, and that a transient association o
f Prp22p with the 3' splice site occurs between the first and second cataly
tic steps.