The Rader Lab, based at the University of Northern BC in Prince George, 800 km north of Vancouver, investigates the biological function and mechanism of pre-mRNA splicing. Most of our work originally focused on assembly of the U4/U6 di-snRNP in the yeast S. cerevisiae (Rader 2002, Stanek 2003) and the role of the U4 snRNA in spliceosome assembly (Hayduk 2012). Recently, we have shifted our attention to the red alga Cyanidioschyzon merolae.
C. merolae is an extremophile, tolerating pH as low as 0.5 and temperatures up to 60 C. Intriguingly to us, it has only 27 annotated introns in its genome, corresponding to an intron density of ~0.5% (compared to > 90% in humans). As the ancestral alga is known to have been intron-rich, this raises the question of why an otherwise genetically austere microbe would retain 27 introns and all of the associated machinery to splice them out.
To address this question, we are using transcriptomics and proteomics to study changes in splicing in response to varying environmental conditions. We are also deleting introns from the genome to assess their importance to growth under normal and stressed conditions. Through a combination of knockouts, splicing reporters, and biochemistry, we aim to reveal what determines splicing efficiency in this organism, and specifically whether it is primarily determined through targeted, regulatory pathways or by a mass action mechanism.
We are also investigating the composition and function of the C. merolae spliceosome, the splicing machine that carries out the chemical steps of splicing. Surprisingly, we discovered that C. merolae lacks the U1 snRNP that is normally responsible for the first step of intron recognition (Stark 2015). We subsequently showed that, in contrast to all other eukaryotes where this has been studied, there is only a single Lsm complex, and that it has roles in both splicing in the nucleus as well as in cytoplasmic mRNA decapping and decay (Reimer 2017). This work also demonstrated that the decapping and decay protein Pat1 is not only nuclear, but is tightly and abundantly associated with splicing particles (Reimer 2017).
To better characterize the composition and mechanism of the C. merolae spliceosome, we are using a combination of biochemistry and structural biology to investigate whether the snRNPs are pre-associated, how they recognize the 5’ splice site in the absence of U1, and whether the spliceosome is functionally coupled to the Pat1 degradation machinery.
We welcome applications from enthusiastic and well-prepared students and postdocs to join our dynamic team in Prince George.
17. Garside, E.L, Whelan, T.A., Stark, M.R., Rader, S.D., Fast, N.M., and A.M. MacMillan. Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle that Correlates with Splicing Complexity across Diverse Taxa, Journal of Molecular Biology 2019, 431:2543-2553.
16. Reimer, K.A., Stark, M.R., Aguilar, L.-C., Stark, S.R., Burke, R.D., Moore, J., Falhman, R.P., Yip, C.K., Kuroiwa, H., Oeffinger, M., and S.D. Rader. The sole LSm complex in Cyanidioschyzon merolae associates with pre-mRNA splicing and mRNA degradation factors, RNA 2017, 23:952-967.
15. Black, C.S., Garside, E.L., MacMillan, A.M., and S.D. Rader. Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae. Protein Science, 2016, 25(4):911-916.
14. Hudson, A.J., Stark, M.R., Fast, N.M., Russell, A.G., and S.D. Rader. Splicing diversity revealed by reduced spliceosomes in C. merolae and other organisms. RNA Biology, 2015 DOI: 10.1080/15476286.2015.1094602.
13. Stark, M.R., Dunn, E.A., Dunn, W.S.C., Grisdale, C., Daniele, A., Halstead, M., Fast, N., and S.D. Rader. A dramatically reduced spliceosome in Cyanidioschyzon merolae. Proceedings of the National Academy of Sciences, 2015, 112:E1191-E1200.
12. Hayduk, A.J., Stark, M.R., and S.D. Rader. In vitro reconstitution of yeast splicing with U4 snRNA reveals multiple roles for the 3’ stem-loop. RNA, 2012, 18:1075-1090.
11. Dunn, E.A., and S.D. Rader. The Secondary Structure of U6 snRNA: Implications for Spliceosome Assembly. Biochemical Society Transactions, 2010, 38(4):1099-1104.
10. Kim, S., Gorrell, A., Rader, S.D., and C.H. Lee. Endoribonuclease activity of human APE1 revealed by a real-time fluorometric assay. Analytical Biochemistry, 2009, 398:69-75.
9. Aukema, K.G., Chohan, K.K., Plourde, G.L., Reimer, K.B., and S.D. Rader. Small molecule inhibitors of yeast pre-mRNA splicing. ACS Chemical Biology, 2009, 4(9):759-768.
8. Thompson, M.D., Aukema, K.G., O’Bryan, D.M., Rader, S.D., and B.W. Murray. Plasmid sonication improves sequencing efficiency and quality in the Beckman-Coulter CEQTM8000. BioTechniques, 2008, 45(3):327-329.
7. Stark, M.R., Pleiss, J., Deras, M., Scaringe, S., and S.D. Rader. An RNA ligase-mediated method for the creation of large, synthetic RNAs. RNA, 2006, 12:2014-2019.
6. Stanek, D., Rader, S.D., Klingauf, M., and K. M. Neugebauer. Targeting of U4/U6 snRNP Assembly Factor SART3/p110 to Cajal Bodies. Journal of Cell Biology, 2003, 160: 505-516.
5. Rader, S.D., and C. Guthrie. An Lsm-Interaction Motif in Prp24 Required for Efficient U4/U6 Formation. RNA, 2002, 8: 1378-1392.
4. Sauter, N.K., Mau, T., Rader, S.D., and D.A. Agard. Structure of the Complex Between Alpha-Lytic Protease and Its Pro Region. Nature Structural Biology, 1998, 5(11): 945-50.
3. Rader, S.D., and D.A. Agard. Conformational Substates in Enzyme Mechanism: The 120 K Structure of Alpha-Lytic Protease at 1.5 Å Resolution. Protein Science, 1997, 6(7): 1375-1386.
2. Sohl, J.L., Shiau, A.K., Rader, S.D., Wilk, B.J., and D.A. Agard. Inhibition of Alpha-Lytic Protease by Pro Region C-terminal Steric Occlusion of the Active Site. Biochemistry, 1997, 36(13): 3894-3902.
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