The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 213

A degenerate variant of the MPN/Jab1 domain that is associated with
deubiquitinating enzymes is found near the C terminus of Prp8. 86 Analysis
of the high-resolution structure of a fragment of Prp8 containing this
domain has shown that the metal ion-binding site of the isopeptidase center
is impaired, and thus it likely does not function as a deubiquitinating
enzyme. 99,100 In vitro , a fragment of Prp8 containing this domain could
directly bind to ubiquitin with an affinity that was comparable to other
known ubiquitin-binding proteins. 101 Several known Prp8 mutations that
disrupted splicing also disrupted in vitro ubiquitin binding, thus raising the
possibility of a functional role for ubiquitination in splicing regulation.
Importantly, proteomic analyses have indicated that several spliceosomal
factors, including Sad1, Snu114, Rse1, and Prp8 itself, are ubiquitinated
in vivo , and furthermore the spliceosomal core protein Prp19 exhibits E3
ubiquitin ligase activity in vitro . 101-105 Further, ubiquitin plays a role in
the formation and maintenance of tri-snRNP, possibly by modulating
Prp8-mediated regulation of the function of Brr2. 102 Alternatively, it is also
possible that the MPN/Jab1 domain functions as a protein-protein interac-
tion platform. A number of mutations in Prp8 which fall in this domain
result in the hereditary blindness retinitis pigmentosa, 86 and these mutations
weakened the interaction of Prp8 with Brr2 and Snu114. 99
The high-resolution structure of another fragment of Prp8 has been
determined that encompasses a highly conserved region (69% amino acid
identity between yeast and human) located close to its C-terminal domain.
Analysis of the structure revealed the presence of a b -hairpin finger resem-
bling those found in ribosomal proteins and a degenerate RNase H-like
domain. 106-108 While the overall geometry of the RNase H-like motif at
the level of secondary and tertiary structure was well conserved, the primary
sequence showed a much lower level of conservation and only one of the
three catalytic residues is present at the active site. The conserved residue,
an aspartate, is involved in coordinating two catalytic metal ions in the active
site of canonical RNase H domains. In one study, mutation of this residue in
Prp8 in yeast resulted in no detectable growth defect, which could indicate
redundancy or lack of a critical function. 108 In another study, its effect could
not be examined since the mutation induced misfolding of the protein frag-
ment. 107 No metal ion binding was observed by the region corresponding to
the active site of the RNase H domain when the crystals were grown in as
much as 200 mM MgCl 2 , indicating that the degenerate active site lacks
metal ion-binding ability. Further, the fragment of Prp8 containing this
domain showed a very weak RNA-binding ability, with a K d of 20 m M