Research Summary

Eukaryotic genes are interrupted by insertions of noncoding sequences, or introns. The removal of these introns by splicing during gene expression is essential. Failure to splice precisely can have catastrophic consequences, including disease. Splicing is catalyzed by the spliceosome, a molecular machine composed of protein and RNA parts. The RNA parts play key roles in substrate binding and catalysis. Rearrangements of these RNAs couple spliceosome activation to substrate binding and deactivation to product release. Our long-term goal is to understand the inner workings of this extraordinary machine. To pursue this goal, we employ budding yeast, which allows for a combined approach of genetics and biochemistry. Specifically, our goals are

(i) to elucidate the mechanisms that enhance fidelity in splicing,

(ii) to determine the functions of the spliceosome’s RNA parts and

(iii) to elaborate the mechanisms for turning the spliceosome on and off.

Although fidelity in transcription and translation has been well-characterized, our understanding of fidelity in splicing is poor. By a novel biochemical assay, we discovered a role for a member of the ubiquitous DEAD-box ATPase family, which includes factors that translocate along RNA. Our studies validate a general mechanism for establishing fidelity in RNA-dependent processes. The spliceosome’s RNA parts are highly conserved but many of their functions remain enigmatic. Through a molecular genetic analysis of two mutually exclusive RNA structures, we found evidence that the spliceosome toggles between two states that modulate interactions with the substrate during catalysis. The mechanisms that turn the spliceosome on and off remain a mystery. We discovered that the spliceosome is controlled by an elegant mechanism requiring a GTPase and ubiquitylation. Our data suggest that spliceosome dynamics are controlled by a signal transduction mechanism that senses the identity and status of the substrate.

Selected Publications

Zhao C, Bellur DL, Lu S, Zhao F, Grassi MA, Bowne SJ, Sullivan LS, Daiger SP, Chen LJ, Pang CP, Zhao K, Staley JP, and Larsson C. (2009) Autosomal dominant retinitis pigmentosa caused by a mutation in SNRNP200, a gene required for unwinding of U4/U6 snRNAs. J Hum Genet. 2009 Oct 28. [Epub ahead of print] (PubMed)

Mefford, MA, Staley, JP. (2009) U2/U6 helix I promotes both catalytic steps of pre-mRNA splicing and rearranges in between these steps. RNA, 15:1386-1397. (PubMed)

Bellare, P., Small, E.C., Huang, X., Wohlschlegel, J.A., Staley, J.P. and Sontheimer, E.J. (2008) A Role for Ubiquitin in the Spliceosome Assembly Pathway. Nature Structural and Molecular Biology, 15:444-451. (PubMed)

Hilliker A.K., Mefford M.A., and Staley J.P. (2007) U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing. Genes Dev., 21(7):821-34. (PubMed)

Mayas, R. M. & Staley, J. P. (2006) DEAD on. Nature Structural and Molecular Biology, 13:954-955. (PubMed)

Small, E.C., Leggett, S.R., Winans, A.A. & Staley, J.P. (2006) The EF-G-like GTPase Snu114 Regulates Spliceosome Dynamics Mediated by Brr2p, a DExD/H-box ATPase. Molecular Cell, 23:389-399. (PubMed)

Mayas, R.M., Maita, H. & Staley, J. P. (2006) Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nature Structural and Molecular Biology, 13:482-490. (PubMed)

Leeds, N. B., Small, E. C., Hiley S. L., Hughes, T. R. & Staley, J. P. (2006) The Splicing Factor Prp43p, a DEAH box ATPase, Functions in Ribosome Biogenesis. Molecular and Cellular Biology, 26:513-522. (PubMed)

Hilliker, A. K. and Staley, J. P. (2004) Multiple functions for the invariant AGC triad of U6 snRNA. RNA, 10:921-928. (PubMed)

Staley, J. P. (2002) Hanging on to the branch. Nature Structural Biology, 9:5-7. (PubMed)

Staley, J. P. and Guthrie, C. (1999) An RNA switch at the 5 splice site requires ATP and the DEAD box protein Prp28p. Molecular Cell, 3:55-64. (PubMed)

Staley, J. P. and Guthrie, C. (1998) Mechanical devices of the spliceosome: motors, clocks, springs and things. Cell, 92:315-326. (PubMed)