Structural and Functional Investigation of Four Essential pre-mRNA Splicing Proteins

• Author / Creator

  Garside, Erin

• Exons, protein coding regions in eukaryotic genes, are interrupted by non-coding introns that must be removed from transcribed pre-mRNA prior to translation into protein. Introns are removed, and exons ligated, in a process called pre-mRNA splicing. Conserved sites within the intron, the 5′ and 3′ splice sites (SS) and the branch point sequence, define the boundaries of the intron. Intron removal and exon ligation at correct splice sites is essential for the translation of functional protein from the resulting mRNA.Chemically, pre-mRNA splicing occurs via two transesterification reactions. In the first step, branching, the 2′ hydroxyl of an adenosine within the branch point sequence attacks the 5′ SS to generate a 2′-5′ phosphodiester linkage and a free 5′ exon with a 3′ hydroxyl. In the second step, exon ligation, the 3′ hydroxyl of the 5′ exon attacks the 3′ SS to generate ligated exons and a branched intron.Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megaDalton complex composed of five distinct small nuclear ribonucleoprotein particles (snRNPs) and other protein complexes, which assembles on every intron to be removed. Each snRNP features a U-rich small RNA stabilized within a ring of Sm proteins and a set of snRNP-specific proteins. U1 snRNP recognizes the 5′ SS via base-pairing with its small nuclear RNA (snRNA). U2 snRNA forms an imperfect duplex with the branch point sequence. The un-base-paired branch A is excluded from the duplex and selected as the nucleophile for the first step of splicing. SF3b, a subcomplex of U2 snRNP that dissociates from the spliceosome prior to the first step, stabilizes this duplex and keeps the branch A sequestered prior to the branching reaction. U4 snRNA in the U4/U6.U5 tri-snRNP base-pairs with the catalytic residues of U6 snRNA to prevent premature formation of the spliceosome active site. U5 snRNP, the largest snRNP, contains many regulatory proteins required for the progression of the spliceosome through the splicing cycle.This thesis investigates the role of four essential snRNP proteins: U1C, Sap49, Snu13, and Prp8.U1C is a component of the U1 snRNP. It features an N-terminal zinc finger and stabilizes the duplex between the 5′ SS and U1 snRNA. Positively charged residues of U1C are thought to interact with the 5′ SS during its initial recognition. We designed constructs to cross-link U1C to the 5′ SS to study the nature of this interaction.Sap49, part of the SF3b complex of U2 snRNP, is shown to cross-link to the intron upstream of the branch A. Mutations in Sap49 that cause haplo-insufficiency are associated with the acrofacial dysostosis Nager syndrome. A recently described I84R mutation has been linked to a mild form of Nager syndrome. We failed to express recombinant I84R Sap49, and the corresponding mutation is not viable in S. cerevisiae cells. We propose that this mutant is not stable, leading to degradation and haplo-insufficiency resulting in Nager syndrome.Snu13 is a small, highly conserved protein found in both the U4 snRNP and the non-spliceosomal box C/D snoRNP. It is maintained in the highly reduced spliceosome of the red alga C. merolae. Our work shows this Snu13 is very similar to other Snu13s, both in its structure and its binding to U4 snRNA. These results suggest that results from studies of the C. merolae are relevant to the human spliceosome.Prp8 is the largest, most highly conserved spliceosomal protein. It features an RNase H-like (RH) domain that regulates progression through the splicing cycle. Sequence analysis of the C. merolae Prp8 predicts the absence of a highly conserved 17 amino acid insertion in the RH domain, which we confirmed by X-ray crystallography. Other proteins observed interacting with this insertion in structures of the spliceosome throughout the splicing cycle are also predicted to be absent. Organisms with few introns, including C. merolae, and fewer spliceosome components also lack the insertion.RH metal binding in the human RH domain has been visualized crystallographically. Analysis of this metal binding suggests it is not required for splicing; however, a positive charge, either a metal or a nearby arginine, may play a role.Taken together, these findings increase our understanding of pre-mRNA splicing and the spliceosome.

• Subjects / Keywords

  • Splicing
  • Snu13
  • Sap49
  • Spliceosome
  • Prp8
  • U1C

• Graduation date

  Spring 2019

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-x2ka-vj03

• License

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