The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 214

to over 200 m M depending on the RNA species tested. 106-108 Although the
fragment showed a very low affinity for binding RNAs, it did show a pref-
erence for binding duplex RNAs containing four-way junctions. 108 In acti-
vated human spliceosomes, U2 and U6 snRNAs are predicted to form such a
structure. 53,69 What made this RNase H-like domain particularly interesting
was that the amino acids corresponding to its active site are positioned adja-
cent to residues in Prp8 that were previously shown to crosslink to the 5 0
splice site in precatalytic spliceosomes. 109 However, the efficiency of the
formation of this crosslink was reduced as the precatalytic spliceosomes
progressed to become catalytically active spliceosomal complexes. 109 The
observed decrease in crosslinking efficiency could be interpreted to indicate
that this interaction is disrupted in activated spliceosomes. Alternatively, the
interaction may persist, but the formation of the crosslink itself is abolished
due to a minor change in the environment of the crosslinked residues. If this
degenerate RNase H-like domain is indeed positioned in proximity to the 5 0
splice site in catalytically active spliceosomes, it is possible that it may par-
ticipate in stabilizing the active conformation of the snRNAs, positioning
the substrates in the active site, and/or coordinating catalytic metal ions.
While the domain by itself cannot bind RNA or metal ions, it is possible
that in the presence of other active site elements it may form part of the sub-
strate or metal ion-binding pockets. Despite these intriguing possibilities, the
role of Prp8 in spliceosomal catalysis remains uncertain.
10. THE ROLE OF SPLICEOSOMAL PROTEINS
IN REMODELING THE CATALYTIC CORE
At least eight DExD/H-box proteins are associated with spliceosomes at
various steps of the spliceosomal cycle, and several showweak helicase activity
in vitro . 110,111 The spliceosomal helicases are thought to play central roles in
remodeling RNA-RNA and/or RNA-protein interactions, and in several
cases their remodeling activity seems to be tied to the spliceosomal cycle
and quality control mechanisms. 58,96 One of the most interesting remodeling
helicases in the spliceosome is the U5-associated DExD/H-box protein Brr2.
Similar to Prp8, Brr2 is a large protein ( 2100 amino acids in yeast and
human) and is closely associated with the snRNAs. Current data indicate that
Brr2 is responsible for disrupting the interaction of U6 with its base-pairing
partners in both precatalytic and post-catalytic spliceosomes. 85,112 The protein
contains two helicase-like domains followed by a Sec63 domain of unknown
function. The ATPase and helicase activities of Brr2, at least in vitro ,are