Biology Reference
In-Depth Information
DEAxD/H box helicases are governing structural rearrangements to shape the active complex. However,
many of these ATPases function in a generic way hydrolyzing also other RNA species. Thus, they
have to be specifically activated for catalyzing the correct structural rearrangements, not least to ensure
fidelity of the splicing reaction. It was proposed that additional splicing factors interact with DExD/H
box helicases to direct their activity to specific substrates during the assembly process.
SF3b125 was detected only in low amounts in the SF3b subcomplex, but not associated with the
17S U2 snRNP, and hPrp5 was present in the 17S U2 snRNP, but not in the SF3b subcomplex [Will
et al.
, 2002]. The protein hPrp5 exhibits an ATP independent function, which stabilizes U2 snRNP to
the BP [Perriman
et al.
, 2003], and was proposed to function as bridge between the U1 snRNP and
the U2 snRNP at the time of A-complex formation [Xu
et al.
, 2004]. Accompanying the U2 snRNP
rearrangements, catalyzed by hPrp5 under ATP hydrolization, the contacts of the protein SF3a60 to the
U2 snRNA are significantly reduced upon association of U2 with the BP in the A-complex [Dybkov
et
al.
, 2006]. Another DExD/H box helicase, which is required for U2 snRNP / BP interaction, is UAP56,
whose recruitment also depends on the protein U2AF65 [Fleckner
et al.
, 1997]. The reactions of the
individual assembly stages are described and formalized in Supplementary Table S1.
B-complex
(
active spliceosome
)
In this stage, U4, U5, and U6 snRNPs enter the assembly pathway as tri-snRNP complex. This
subcomplex is formed in a separate way, where U4 and U5 snRNP assemble, similar to U1 snRNP, from
a family of seven RNA binding proteins, termed Sm proteins [Will and L uhrmann, 2001; Liu
et al.
,
2006], whereas the U6 snRNA is bound by a different set of proteins termed Sm-like proteins [Beggs,
2005]. Subsequently, the U4 snRNP and the U6 snRNP form a duplex via base pairing of their snRNAs,
resulting in a structural conformation, which is stabilized by several additional proteins, for example
hSnu13, Prp3, Prp4, CypH and Prp31 [Liu
et al.
, 2006]. The U5 snRNP contains several proteins,
important for structural rearrangements prior to the first catalytic step of splicing, most important the
DExD/H box helicases Brr2 and Prp28 and the two proteins Snu114 and Prp8 [Laggerbauer
et al.
,
1998; Liu
et al.
, 2006]. An interaction between Prp6 and Prp31 was proposed to serve as bridging
step between the U4/U6 snRNP complex and the U5 snRNP, preparing the formation of the U4/U6
U5 tri-snRNP complex [Schaffert
et al.
, 2004]. The additional proteins Snu66, Sad1 and 27K, help to
stabilize the intermediate tri-snRNP complex. The latter is recruited to the spliceosome which, however,
is still catalytically inactive. An intermediate state, designated pre-catalytic B0 complex, was shown
to accomodate a highly flexible tri-snRNP structure, possibly for integrating other components, for
example, the protein Prp19 [Boehringer
et al.
, 2004].
Prior to the conformational rearrangement required for spliceosome activation, U1 snRNP is dissoci-
ating from the 5'ss enabling U6 snRNP to contact the donor splice site. This step involves the ATPase,
Prp28, which was found to counteract the stabilizing effect of the U1 component U1C with the U1
snRNA [Chen
et al.
, 2001]. Prp5 could leave the spliceosome at this stage as it was demonstrated to
function mainly before or during A-complex assembly [Will
et al.
, 2002]. The U5 snRNP components
Brr2, Prp8 and Snu114 are critically for unwinding the U4/U6 snRNA stemloop. This step resembles a
G-protein activating mechanism, where Snu114 enters a GTP dependent state, and subsequently activates
the helicase Brr2 [Turner
et al.
, 2004; Small
et al.
, 2006]. This conformational change also involves
Prp19, a protein of the Nineteenth complex (NTC) [Makarova
et al.
, 2004]. Brr2 is further involved in
interactions with Prp16 and U1-70K [van Nues and Beggs, 2001; Liu
et al.
, 2006]. After unwinding,
the U4/U6 snRNA duplex, U2 and U6 snRNP establish several interactions with the premRNA via their
snRNAs, whereas a binding motif within the U6 snRNA directly contacts the 5'ss. Subsequent release of
