Biology Reference
In-Depth Information
Prp8, which is the most conserved spliceosomal protein, is thought to
play such a role in the spliceosomal active site. Prp8 is arguably the most
interesting of the spliceosomal proteins, as it is unusually conserved with
61% identity between yeast and human.
86
However, it has few clearly dis-
tinguishable functional motifs in its
2300 amino acid length, and the func-
tional domains that have been discerned so far are degenerate and likely
perform functions unrelated to the cellular role of the original founding
motif.
86
On the other hand, Prp8 clearly plays a very critical role in the
spliceosomal active site. It has been crosslinked to the 5
0
and 3
0
splice sites
and the branch site of premessenger RNAs, in addition to U5 and U6
snRNAs, which indicates that it is present in the spliceosomal catalytic core
and directly contacts the critical players in the splicing reaction.
86,90
Further,
it has been shown that Prp8 interacts with several key spliceosomal proteins
including Snu114 and Brr2 (see below).
86,91,92
Mutations in Prp8 are associated with a wide spectrum of spliceosomal
defects including alterations in the ability of spliceosomes to reject sub-
optimal splice sites and suppression of defects caused by mutations in other
spliceosomal factors such as Prp28, Brr2, U4, and U6 snRNAs.
86,93,94
A subset of Prp8 mutants selectively modulates the efficiency of either
the first or the second step of splicing, especially in suboptimal sub-
strates.
58,86,95-97
These observations are best explained by a hypothesis stat-
ing that Prp8 is involved in stabilization of alternative, mutually exclusive
active site conformations that are either poised for catalysis of the first or
the second step of splicing, and that certain Prp8 mutants might lead to selec-
tive hyperstabilization of one of these states.
58,96,98
While significant evi-
dence suggests that Prp8 is likely involved in positioning of the substrates
and stabilization of the active site, no direct evidence currently exists that
suggests or refutes its additional involvement in metal ion coordination or
direct participation in spliceosomal catalysis.
This uncertainty stems from the fact that very little is known about the
way Prp8 performs its function. An innovative transposon-mediated
screening assay indicated that large regions of Prp8 are highly sensitive
to insertion of transposons and likely function as a single structural unit,
although it was possible to insert transposons or even divide Prp8 into
two fragments at a number of other positions without the loss of viabil-
ity.
90,92
Apart from a degenerate nuclear localization signal close to its
N
terminus and a degenerate RRM (RNA recognition motif ) motif in
the middle of the protein, only two other functional domains have been
identified in this
2300 amino acid-long protein.
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