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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Santa Cruz Electronic Theses and Dissertations (2012)

Pre-mRNA splicing is an essential step in eukaryotic gene expression. Splicing is catalyzed by the spliceosome, a large and highly dynamic macromolecular machine in the cell. Errors in splicing have been linked to many diseases, including cancer. The spliceosome is responsible for precisely joining the protein coding sequences that will be translated to protein. It is composed of five small nuclear RNA (U1, U2, U4, U5, and U6) along with over 100 unique proteins. Understanding the RNA/RNA and RNA/protein interactions that position the pre-mRNA substrate in the spliceosome for the two catalytic steps of splicing is key to determining the molecular mechanisms of this dynamic complex.

In this thesis, we compared two states of the spliceosome complexes, catalytic and post-catalytic. We investigated the role of pre-mRNA sequences in the spliceosome complex stalled prior to the second step of splicing. We next developed a new strategy that traps the spliceosome after the second step of splicing, but before the mRNA is released. We show that structural rearrangements of the RNA substrate exist as the spliceosome transitions from the first to second step of splicing. Using mass spectrometry assays, we identify proteins that stably associate with affinity-purified catalytic and post-catalytic complexes. We show enrichment of SF3B components and loss of an ATPase, DHX35, in the post-catalytic complex. In conjunction with biochemical studies, we use electron microscopy to determine structures of both complexes to further gain insights into the spliceosome's role in splicing. This study serves as a foundation for understanding the mechanisms underlying the later stages of spliceosomal progression.

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Cover page: Structural Analysis of Catalytic and Post-catalytic Spliceosome Complexes
  • Article
  • Peer Reviewed
UC Santa Cruz Previously Published Works (2014)
Isolating spliceosomes at a specific assembly stage requires a means to stall or enrich for one of the intermediate splicing complexes. We describe strategies to arrest spliceosomes at different points of complex formation and provide a detailed protocol developed for isolating intact splicing complexes arrested between the first and second chemical steps of splicing. Briefly, spliceosomes are assembled on a radiolabeled in vitro-transcribed splicing substrate from components present in nuclear extract of HeLa cells. Spliceosome progression is arrested after the first step of splicing chemistry by mutating the pre-mRNA substrate at the 3' splice site. The substrate also contains binding sites for the MS2 protein, which serve as an affinity tag. Purification of arrested spliceosomes is carried out in two steps: (1) size exclusion chromatography and (2) affinity selection via a fusion of MS2 and maltose-binding protein (MBP). Complex assembly and purification are analyzed by denaturing polyacrylamide gel electrophoresis.
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2013)
    In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3' exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3' exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5' and 3' exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.
      Cover page: Rearrangements within human spliceosomes captured after exon ligation
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