Biology Reference
In-Depth Information
of proteins present in the nucleus, which are recycled for repeated rounds of spliceosome assembly. The
mature spliceosome catalyzes the splicing reaction in eukyaryotic messenger RNAs.
E-complex
(
commitment complex
)
The recognition of the 5' splice site (5'ss) is initiated by the early interaction of the U1 specific protein
U1C with the 5'ss sequence [Du and Rosbash, 2002]. Additionally, interactions via phosphorylated RS
domains of the protein U1-70K with splicing enhancing factors, which bind to nearby located specific
enhancer motifs, can direct the U1 snRNP to a specific 5'ss [Cao and Garcia-Blanco, 1998]. It was
demonstrated that 5'ss with low complementarity to the canonical eukaryotic 5'ss are still selected by
U1 snRNP due to interactions between U1 snRNP proteins and splice enhancing proteins, such as
ASF/SF2 or TIA1 [Cao and Garcia-Blanco, 1998; F orch
et al.
, 2002]. This is in agreement with the
earlier observation that donor site selection by U1 snRNP can be maintained by the SR protein, SC35,
in case of an impaired donor recognition motif in the U1 snRNA sequence [Tarn and Steitz, 1994]. In
contrast, a weak 5'ss might also be selected by variants of the U1 snRNA [Kyriakopoulou
et al.
, 2006].
This led to the interesting hypothesis that 5'ss selection within the E-complex might be possible even
without the binding of U1 snRNP. Experiments indicate that the presence of higher concentrations of
SR proteins enable such a spliceosomal pathway [Crispino
et al.
, 1996]. At the 3' end of the intron,
E-complex assembly involves U2 auxiliary factor, U2AF, which is a heterodimer of a 65 and 35 kDa
subunit. Together with SF1 (in mammals designated branch point binding protein, mBBP), these factors
recognize via their RNA recognition motifs (RRM) the polypyrimidine tract, 3'ss and branch point,
respectively. This step was shown to occur in a coordinated way involving cooperativity between SF1
and U2AF65 [Berglund
et al.
, 1998], which is mediated by interactions between the RS domains of these
proteins [Shen and Green, 2004]. Additional interactions reported during E-complex assembly include
the bridging of U1 snRNP at the 5'ss with factors bound to the branch point and 3'ss, for example, by
action of the SR protein FBP11 [Abovich and Rosbash, 1997]. It was also shown that at this stage,
the U2 snRNP can be in close proximity to the U1 snRNP, suggesting the dependency of E-complex
formation on the presence of factors required for the next assembly stage [Das
et al.
, 2000, D onmez
et
al.
, 2007]. A summary of models of E-complex assembly is depicted in Fig. 2.
A-complex
(
pre-spliceosome
)
The A-complex contains the stably integrated U2 snRNP, which is assembled out of two heteromeric
subcomplexes SF3a and SF3b via an intermediate 17S complex. Prior to their contacts to the U2 snRNA,
SF3a and SF3b are formed by several interactions between U2 core components (
e.g.
, SF3b155 with
SF3b14 or SF3a120 with SF3a60) [Dybkov
et al.
, 2006]. In this stage, U2 snRNP interacts with the
branch point site via base pairing between U2 snRNA and the pre-mRNA under consumption of ATP
[D onmez
et al.
, 2007]. Additionally, the transition from the E-complex to the A-complex is supported by
the SF3b protein, SF3b155, which binds at both sides of the branch point and interacts simultaneously
with the U2AF65 subunit and the SF3b14 protein [Gozani
et al.
, 1998; Spadaccini
et al.
, 2006]. The ATP
requirement during A-complex formation has been reasoned by two other U2 snRNP proteins, SF3b125
and hPrp5, which are members of the DExD/H family and may function either as helicases or RNPases
[Xu
et al.
, 2004]. Such auxiliary enzymes could be recruited from proximal cajal bodies or speckles
[Will
et al.
, 2002].
These helicase like proteins have been shown to function as generic ATPases that bind and hydrolyze
NTP to unwind double-stranded RNAs (dsRNAs) [Staley and Guthrie, 1998]. In contrast, the ADP-
bound form can modulate the annealing of complementary RNA strands. In context of the spliceosome
