Research Projects

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Proofreading and Splicing Fidelity

The integrity of biological processes requires high fidelity and specificity, the discrimination of optimal substrates from suboptimal substrates. This requirement is challenging for many biological systems, however, because suboptimal substrates closely resemble optimal substrates. Consequently, differences in initial binding of a substrate to an enzyme are insufficient to distinguish substrates, and these biological systems have evolved proofreading mechanisms to sort   substrates   after   an   initial   binding   step.  In   gene  expression,  our   understanding  of proofreading mechanisms  in  splicing   is   poor,  in comparison  with  our   understanding of  proofreading mechanisms in transcription and translation, but  recent  progress  has  revealed  fundamental themes (Semlow & Staley, 2012, TiBS; Koodathingal & Staley, 2013, RNA Biology).

To elucidate proofreading mechanisms in splicing, we have focused on mechanisms that operate at  the catalytic stage. We have discovered that the two chemical steps of splicing, branching and exon ligation, are proofread by parallel mechanisms in which a member of the DEAH box subfamily of ATPases kinetically competes with the chemical step, permitting splicing of optimal substrates but rejecting suboptimal substrates (Mayas et al., 2006, Nat Struct Mol Biol; Koodathingal et al., 2010, Mol Cell). This rejection step is reversible, necessitating a subsequent step to discard the substrate from the spliceosome.

We have discovered that discard is mediated at both chemical steps by a single DEAH box ATPase that also functions to release the excised intron when splicing is complete, thereby defining this ATPase as a general splicing terminator (Mayas et al., 2010, PNAS; Koodathingal et al., 2010, Mol Cell). In collaboration with the Baumann lab, at the Stower’s Institute, we have further found that one of these proofreading pathways also functions in the biogenesis of telomerase RNA in fission yeast (Kannan et al., 2013, G&D). Despite these advances, intriguing and fundamental questions remain regarding the mechanisms of fidelity.