Spliceosome Mechanism Dissected at the Single Molecule Level

Summary

Principal Investigator: Nils G Walter
Abstract: DESCRIPTION (provided by applicant): Spliceosome mechanism dissected at the single molecule level ABSTRACT: The spliceosome is a multi-megadalton RNA-protein complex that catalyzes in all eukaryotes the removal of introns and the ligation of exons during splicing of pre mRNAs. In humans, 94% of all pre-mRNAs undergo alternative splicing, which allows for the dynamic expression of various protein isoforms from a single gene through cell- and tissue-specific networks of regulated splicing events. It is estimated that up to 50% of all mutations leading to human disease act through disrupting the splicing code. Due to the availability of unique genetic and biochemical manipulation tools, the budding yeast Saccharomyces cerevisiae has long provided a central model system for dissecting the mechanism of eukaryotic pre-mRNA splicing. Despite 25 years of study, however, there is still little known about the compositional and conformational rearrangements, timing, and coordination associated with yeast spliceosome function. To address this challenge, we recently have developed single molecule fluorescence resonance energy transfer (smFRET) assays that have begun to dissect pre-mRNA conformational changes during splicing. In particular, we have identified a small, efficiently spliced yeast pre-mRNA, in which donor and acceptor fluorophores could be placed in the exons adjacent to the 5'and 3'splice sites, and have used it to show that the spliceosome operates close to thermal equilibrium. Here, we propose to follow up on this advance and begin to dissect the mechanism of splicing at the single molecule level. In Specific Aim 1, we will test the hypothesis that specific sets of conformational fluctuations lead to splicing, by adding to our tool set: (i) shuttered illumination combined with advanced hidden Markov modeling and in situ hybridization to faithfully track the conformational dynamics of single pre- mRNA substrate molecules over the entire time course of splicing;(ii) depletion-complementation approaches to introduce functionally active, fluorophore labeled small nuclear RNA (snRNA) and protein components of the spliceosome for coincidence analysis (CIA);(iii) covalent, small-tag fluorophore labeling approaches to non-invasively mark functional protein factors of the spliceosome;and (iv) an optimized affinity purification technique to isolate specific spliceosomal complexes with fluorophore labeled components for focused probing. In Specific Aim 2, we will follow up on our observation that our intron exhibits significant secondary structure, placing its flanking exons much closer than expected from their linear sequence distance. We will test the hypothesis that this secondary structure has a functional impact by introducing a systematic set of mutations that first impair, and then restore the predicted secondary structure, and by testing each mutant for splicing. In Specific Aim 3, we will dissect the mechanistic role of DExD/H-box helicase Prp2 in preparing the activated Bact. spliceosome for the first step of splicing by rearranging it into the B* complex with exposed pre- mRNA branch point. Taken together, these advances will pave the way for, over the funding period, extensive mechanistic studies of yeast splicing and for studying alternative splicing in humans in the longer term.
Funding Period: 2012-02-01 - 2015-11-30
more information: NIH RePORT

Top Publications

  1. pmc Metal ions: supporting actors in the playbook of small ribozymes
    Alexander E Johnson-Buck
    Department of Chemistry, University of Michigan, 930 N University, Ann Arbor, MI 48109 1055, USA
    Met Ions Life Sci 9:175-96. 2011
  2. pmc Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting
    Sethuramasundaram Pitchiaya
    Single Molecule Analysis in Real Time SMART Center, University of Michigan, Ann Arbor, MI 48109 1055, USA
    Methods 63:188-99. 2013
  3. pmc Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing
    Ramya Krishnan
    Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA
    Nat Struct Mol Biol 20:1450-7. 2013

Research Grants

  1. Pre-mRNA Splicing Mechanisms
    Melissa J Moore; Fiscal Year: 2013

Detail Information

Publications4

  1. pmc Metal ions: supporting actors in the playbook of small ribozymes
    Alexander E Johnson-Buck
    Department of Chemistry, University of Michigan, 930 N University, Ann Arbor, MI 48109 1055, USA
    Met Ions Life Sci 9:175-96. 2011
    ....
  2. pmc Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting
    Sethuramasundaram Pitchiaya
    Single Molecule Analysis in Real Time SMART Center, University of Michigan, Ann Arbor, MI 48109 1055, USA
    Methods 63:188-99. 2013
    ....
  3. pmc Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing
    Ramya Krishnan
    Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA
    Nat Struct Mol Biol 20:1450-7. 2013
    ..The spliceosome thus functions as a biased Brownian ratchet machine where a helicase unlocks thermal fluctuations subsequently rectified by a cofactor 'pawl', a principle possibly widespread among the many helicase-driven RNPs. ..

Research Grants30

  1. Pre-mRNA Splicing Mechanisms
    Melissa J Moore; Fiscal Year: 2013
    ..Only by gaining a better understanding the cellular machinery mediating this process will we ultimately be able to treat such splicing-related diseases. ) ..