Co-workers:

The mechanism and machinery of RNA splicing are highly conserved throughout eukaryotes, and the budding yeast Saccharomyces cerevisiae makes an excellent model system, permitting the  application of genetic approaches in parallel with molecular studies. In addition to investigating the functions and molecular interactions of yeast splicing factors, we are interested in links between RNA splicing and other metabolic processes such as transcription.  Our approaches include: biochemical analyses of splicing in vitro, quantitative RT-PCR, chromatin immunoprecipitation, in vivo RNA-protein cross-linking and molecular genetics.

Eight RNA helicases are thought to regulate conformational changes during the cycle of spliceosome assembly, catalysis and disassembly. However, little is known about how the  helicases themselves are regulated. 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. Combining genetic and biochemical approaches, we showed that the C-terminus of Brr2p interacts with other spliceosomal RNA helicases, including Prp2p and Prp16p, altering their ATPase activity. These interactions depend on both the conformational state of Brr2p and on the catalytic states of its helicase partners. They are also affected by the presence of RNA. We propose that the second helicase module of Brr2p evolved as a pivotal regulatory domain, controlling the activities of multiple splicing helicases at the catalytic centre of the highly dynamic spliceosome complex (Figure 1; Olivier Cordon and Daniela Hahn).

We previously found evidence for a cytoplasmic precursor  U5 snRNP that lacks Brr2p and,

instead, contains Aar2p, which is absent in mature U5 snRNPs. The association of Brr2p with the

U5 snRNP apparently occurs within the nucleus. In humans, certain mutations in PRP8 cause the

retinitis pigmentosa form of blindness. We found that introducing these mutations into 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 that is disrupted by rp mutations (Boon et al., 2007). More recently, 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. Thes data show that Aar2p acts as a U5 snRNP assembly factor that regulates the incorporation of Brr2p,  possibly to safeguard against non-specific RNA binding to Prp8p and/or premature activation of  Brr2p activity (Figure 2; Vanessa Cristão).

Selected publications:

 Alexander, R., Innocente, S., Barrass, J.D. and Beggs, J.D. (2010) Splicing causes RNA polymerase to pause in yeast. Mol Cell 40,582-593.

Alexander, R., Barrass, J.D., Dichtl, B., Kos, M., Obtulowicz, T., Robert, M-C., Koper, M., Karkusiewicz, I., Mariconti, L., Tollervey, D., Dichtl, B., Kufel, J., Bertrand, E. and Beggs, J.D. (2010) RiboSys, a high resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3’ end processing in Saccharomyces cerevisiae. RNA 16, 2570–2580.

Kudla, G., Granneman, S., Hahn, D., Beggs, J.D. and Tollervey, D. (2011) Mapping in vivo RNA-RNA interactions by crosslinking, ligation and sequencing of hybrids. PNAS, doi 10.1073/PNAS.1017386108.