Molecular Interactions of Prp8p
Prp8 protein is a highly conserved, pre-mRNA splicing factor. It is a component of spliceosomal U5 snRNPs and is at the catalytic centre of the spliceosome complex in which the splicing reaction occurs. The spliceosome is a highly dynamic RNA-protein complex and Prp8p plays a key role in regulating interactions and conformational changes in the catalytic centre. We have studied the interactions of Prp8p with other splicing factors in Saccharomyces cerevisiae. Prp8p is an exceptionally large protein (280 kDa in yeast). We have investigated the domain structure of Prp8p and its regions of interaction with other U5 snRNP proteins.
Boon et al, 2006; Figure 1.
For example the RNA helicase, Brr2p, was shown to bind to both the N-terminal region and the C-terminal region of Prp8p both in vivo and in vitro.
van Nues & Beggs, 2001; Figure 1.
Review of Prp8p:
Mutations of several highly conserved residues in the C-terminus of human PRP8 correlate with a severe form of blindness, autosomal dominant retinitis pigmentosa (RP). Significantly, seven mutations that cause RP in humans, and which affect residues that are identical in the yeast and human proteins, destabilise the C-terminal Prp8p-Brr2p interaction. We obtained evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks Brr2p, and which depends on a nuclear localisation signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP apparently occurs within the nucleus. We found that the introduction of RP mutations in yeast Prp8p results in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore proposed a novel assembly pathway for U5 snRNP complexes, which is disrupted by mutations that cause RP in humans.
Boon et al., 2007; Figure 2.
In view of the high level of conservation of the U5 snRNP, it seems likely that this novel pathway may be conserved in metazoa and that it has significance for the regulation of U5 snRNP function.
Aar2p and the U5 snRNP
The cytoplasmic precursor U5 snRNP that lacks Brr2p contains, instead, Aar2p, which is absent in mature U5 snRNPs.
Boon et al., 2007; Figure 2.
We showed that Aar2p and Brr2p bind to overlapping regions in the C-terminal of Prp8p, suggesting a potential competition for binding. Subsequently, we showed that Aar2p is phosphorylated in vivo and that a phospho-mimetic mutation in Aar2p leads to disruption of the Aar2p-Prp8p complex in favour of the Brr2p-Prp8p complex. We proposed that Aar2p acts as a U5 snRNP assembly factor that regulates the incorporation of Prp8p, possibly to safeguard against non-specific RNA binding to Prp8p and/or premature activation of Brr2p helicase activity.
Ongoing structure-function studies in collaboration with structural biologist Markus Wahl (Freie University, Berlin) are revealing more details about this regulatory pathway.
Cwc21p
Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. We showed that Cwc21p binds directly to two key U5 snRNP proteins, Prp8p and Snu114p. Using a combination of proteomic techniques we showed that the N-terminus of Prp8p contains an intra-molecular fold that is a Snu114p and Cwc21p interacting domain (SCwid; Figure 3). Cwc21p also binds directly to the C–terminus of Snu114p. Genetic and functional interactions between Cwc21p and other splicing factors suggest that Cwc21p functions at the catalytic centre of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrated that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.
Functions of Brr2p RNA helicase
Eight RNA helicases are thought to regulate conformational changes during the cycle of spliceosome assembly, catalysis and disassembly.
Cordin et al., 2012; Cordin & Beggs, 2012
However, little is known about the helicases mechanism of action and their regulation. Amongst these, Brr2p has an unusual architecture, with two helicase modules. Only the amino-terminal helicase module is catalytically active, and the role of the carboxy-terminal module is unclear.
Using in vivo UV-crosslinking and RNA-Seq we obtained evidence for a specific mechanism for Brr2p unwinding the U4/U6 duplexed RNAs in assembling spliceosomes. In addition, we identified a novel function for Brr2p, driving conformational rearrangements within the spliceosome between the two steps of splicing, helping to reposition the substrate RNA in the catalytic centre for the second splicing reaction.
Hahn et al., 2012; Figure 4.
Combining genetic and biochemical approaches, we also showed that the C-terminus of Brr2p interacts with other spliceosomal RNA helicases, including Prp2p and Prp16p, modulating their ATPase activity. We propose that the second helicase module of Brr2p evolved as a regulatory domain, controlling the activities of multiple splicing helicases at the catalytic centre of the highly dynamic spliceosome complex (Hahn et al., in preparation; Figure 4).