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From Spliceosome Assembly to Catalysis: Multiple Roles for DEAD-box ATPase Prp28 and Master Regulator Prp8

  • Author(s): Price, Argenta Margaret
  • Advisor(s): Guthrie, Christine
  • et al.

The spliceosome is a dynamic ribonucleoprotein machine that identifies and removes introns from pre-mRNA molecules. It is composed of five small RNAs and over 100 proteins that assemble de-novo on each intron, through an ordered cascade of recognition, rearrangement, and re-recognition events. At its catalytic core, the spliceosome is thought to be a ribozyme, like the ribosome and group II introns. What then are the functions of the many protein components of the spliceosome? One group of proteins, the family of DExD/H-box ATPase, catalyzes conformational rearrangements to enhance fidelity and drive the ordered cascade of splicing events. Regulation of these events is critical for proper spliceosome function. One particularly interesting protein, Prp8, is thought to affect the structure of the catalytic core of the spliceosome and also serve as a master regulator of spliceosome assembly and activity. Yet how Prp8 interacts with other components of the spliceosome, to regulate their activity or to affect the structure of the catalytic core, is poorly understood.

We began by exploring how Prp8 regulates Prp28, the DEAD-box ATPase that promotes U1 release from and U6 association with the 5' splice site during spliceosome assembly. We found that a cluster of mutations in the N-terminus of Prp8 suppressed the growth defect of the cold-sensitive prp28-1 allele. Surprisingly, Prp28 was required not only for U1 release but also for earlier steps including ATP-independent formation of commitment complex 2. The prp28-1 suppressors in Prp8 also suppressed defects in early this ATP-independent step, suggesting that Prp8 and the U5 snRNP also participate in the very earliest steps of spliceosome assembly (Chapter 1). Next we explored the dynamics of commitment complex 2 formation and its dependence on Prp28 using single molecule FRET to probe pre-mRNA conformations (Chapter 2).

Prp8 is critical for splicing catalysis as well as assembly, and it sits at the catalytic core of the spliceosome. Two classes of Prp8 alleles have been characterized that bias the spliceosome toward two opposing conformations. We propose a model, based on comparisons to the group II ribozyme, that these conformations represent catalytic and transitional (rearrangement) conformations of the spliceosome. We used a cluster of mutations that stabilize two different structures in Prp8 to investigate how a structural toggle in Prp8 affects the catalytic and transitional conformations of the spliceosome. Supporting our model, we found that Prp8 alleles predicted to stabilize the catalytic conformation were error prone and catalyzed in the 1st step of splicing efficiently in vitro, while alleles predicted to stabilize the transitional conformation were hyper-accurate and inefficient at the 1st step of splicing (Chapter 3).

Both spliceosome assembly and catalysis are points at which the fidelity or activity of the spliceosome could be regulated. My goal in this research has been to understand how individual steps are controlled by proteins such as Prp28 and Prp8 in hopes that this will give us a better context in which to study how cells could regulate these components to regulate splicing. New structural studies are giving the field the power to generate more informed hypotheses, based on structure, as a starting point for future genetic and biochemical studies.

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