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Structural Insights Into RNA Splicing

  • Author(s): Peters, Jessica
  • Advisor(s): Toor, Navtej
  • et al.
Abstract

The formation of branched lariat RNA is an evolutionarily conserved feature of RNA splicing reactions for both group II and spliceosomal introns. In both splicing systems, the lariat is formed in the first of two transesterification reactions as a result of the 2' hydroxyl of a conserved bulged adenosine within the intron attacking the phosphate backbone at the 5' splice site. In the second step, the 3' hydroxyl of the 5' exon attacks the 3' splice site, resulting in ligation of the exons and release of the intron as a lariat. Due to the mechanistic and structural similarities, it is believed that group II introns and the spliceosome share a common ancestor. Though many structures have been published of both group II introns and the spliceosome, the precise structural requirements for splicing in both systems remain unknown. In the case of group II introns, all of the structures published prior to the work in this dissertation are of a primitive group IIC intron from the bacterium Oceanobacillus iheyensis (O.i.). This intron splices through a hydrolytic pathway and therefore does not provide a comprehensive understanding of branch formation, which severely limits the conclusions that can be applied to our understanding of the spliceosome. This dissertation describes various experiments that have been performed to better understand how group II introns and the RNA components of the spliceosome active site are able to properly fold and place substrates within the single active site for the two subsequent reactions.

Two new crystal structures of the lariat-forming IIB intron from the brown algae Pylaiella litoralis, P.li.LSUI2, at different stages of splicing are outlined in this dissertation: the intermediate lariat–3' exon and post-catalytic lariat. Two different arrangements of the catalytic triplex, the residues which create the active site scaffold, are observed in these structures. However, examination of all published O.i. crystal structures reveals the same catalytic triplex arrangement throughout splicing. Therefore, the rearrangement that occurs in the more evolved P.li.LSUI2 intron is likely facilitating the transition between the stages of splicing and relevant to branch formation and exon ligation in the spliceosome. These structures are also the first in which domain VI is visualized. Domain VI is integral to the branching pathway as it contains the first-step nucleophilic residue. For the first time, domain II can be seen forming two tetraloop-receptor interactions, -' and -', with domain VI proximal to the bulged adenosine. These tertiary interactions appear to stabilize domain VI. However, disrupting either or both interactions simultaneously results in a stalling of the second step. Additionally, iridium hexamine binds in the major groove of domain VI and promotes exon ligation. Taken together, this suggests that domain VI is dynamic and plays an important role in facilitating both steps of splicing.

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