Nuclear pre-mRNA splicing is ubiquitous in all eukaryotes and is required to excise introns before mRNA is translated by the ribosome. Further, splicing enables a critical layer in the regulation of gene expression as well as a flexibility through alternative splicing that affords complexity in higher organisms, but splicing is also susceptible to mutations and underlies at least 15% of human diseases. Splicing is mediated by the spliceosome, a dynamic machine composed of both protein and noncoding RNA parts. Our lab investigates the role of these RNA components in substrate binding and catalysis, the mechanisms for proofreading splice site choice, the regulation of splicing, and the role for ATPase motors in dynamics and fidelity. Using budding yeast as a model system, we pursue these areas through a wide range of approaches, from single molecule microscopy and chemical biology to biochemistry, genetics, and genomics. To learn more about our research projects, summarized below, read our publications and visit our lab website, by clicking the link on the sidebar.
Over three decades ago, the small nuclear RNA (snRNA) components of the spliceosome were proposed to mediate catalysis within the spliceosome. With the Piccirilli lab, we have provided definitive evidence that the spliceosome utilizes RNA to catalyze splicing. This work has raised new questions regarding the mechanism of catalysis.
The integrity of biological processes requires high fidelity. We have discovered that the splicing substrate is proofread at the two chemical steps of splicing by parallel mechanisms involving rejection and then discard by ATPase motors. Despite these advances, fundamental questions remain.
The spliceosome must dynamically assemble anew on every substrate and disassemble once splicing is complete. We have revealed snRNA rearrangements that underlie these dynamics and defined ATPase motors that drive these dynamics. Still, many mysteries remain to be solved.
Fica SM†, Mefford MA†, Piccirilli JA & Staley JP. Evidence for formation of a catalytic triplex in the spliceosome. Nature Structural and Molecular Biology, 21:464-71. †Co-first authors. (NSMB)
Wlodaver A & Staley JP. The DExD/H-box ATPase Prp2 destabilizes and proofreads the catalytic RNA core of the spliceosome, published online. (RNA)
Fica SM†, Tuttle N†, Novak N, Li N-L, Lu J, Koodathingal P, Dai Q, Staley JP*, and Piccirilli JA*. (2013) RNA catalyzes nuclear pre-mRNA splicing, Nature. 2013 Nov 14;503(7475):229-34. †Co-first authors; *Co-corresponding authors; See Nature website for accompanying News & Views, "Metal ghosts in the splicing machine". (Nature)
Koodathingal P & Staley JP. (2013) Splicing fidelity: DExD/H-box ATPases as molecular clocks. RNA Biology, 10:1073-1079. (PubMed)
Kannan R, Hartnett S, Voelker RB, Berglund JA, Staley JP & Baumann P. (2013) Intronic sequence elements impede exon ligation and trigger a discard pathway that yields functional telomerase RNA in fission yeast. Genes Dev. Mar 15;27(6):627-38. (PubMed)
Semlow DR, Staley JP. (2012) Staying on message: ensuring fidelity in pre-mRNA splicing. Trends Biochem Sci. 37(7):263-73. (PubMed)
Koodathingal P, Novak T, Piccirilli JA and Staley JP. (2010) The DEAH box ATPases Prp16 and Prp43 cooperate to proofread 5' splice site cleavage during pre-mRNA splicing. Molecular Cell, 39:385-395. (PubMed)
Mayas RM, Maita H, Semlow DR & Staley JP (2010). The spliceosome discards intermediates via the DEAH box ATPase Prp43p. PNAS, 107:10020-10025. (PubMed)
Zhao C, Bellur DL, Lu S, Zhao F, Grassi MA, Bowne SJ, Sullivan LS, Daiger SP, Chen LJ, Pang CP, Zhao K, Staley JP & Larsson C. (2009) Autosomal dominant retinitis pigmentosa caused by a mutation in SNRNP200, a gene required for unwinding of U4/U6 snRNAs. Am. J. Hum. Genet, 85:617-627. (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 EC, Huang X, Wohlschlegel JA, Staley JP & Sontheimer EJ. (2008) A Role for Ubiquitin in the Spliceosome Assembly Pathway. Nature Structural and Molecular Biology, 15:444-451. (PubMed)
Hilliker AK, Mefford MA and Staley JP. (2007) U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing. Genes Dev., 21(7):821-34. (PubMed)
Small EC, Leggett SR, Winans AA & Staley JP. (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 RM, Maita H & Staley JP. (2006) Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nature Structural and Molecular Biology, 13:482-490. (PubMed)
Leeds NB, Small EC, Hiley SL, Hughes TR & Staley, JP. (2006) The Splicing Factor Prp43p, a DEAH box ATPase, Functions in Ribosome Biogenesis. Molecular and Cellular Biology, 26:513-522. (PubMed)