Mechanistic and structural studies of spliceosome active site formation
The spliceosome is a eukaryotic molecular machine composed of uridine-rich small nuclear RNAs (U snRNA) and dozens of proteins that facilitate an RNA processing event known as pre-RNA splicing. By catalyzing the removal of intronic RNA the spliceosome plays an critical role in determining the final RNA sequence from pre-RNA transcripts. Proper removal of introns in pre-mRNA maintains the coding register of mature mRNAs that are eventually translated into functional proteins by the ribosome. Accurate splicing is necessary to repeatedly generate mRNAs coding for the same protein across multiple cells. Additionally, alternative splicing, the regulated inclusion and exclusion of specific exons in the final mRNA, greatly expands the coding potential of eukaryotic genomes. Introns and spliced non-coding RNAs also serve important cellular roles. During splicing the spliceosome undergoes multiple timed rearrangements to properly define the boundaries of each intron and generate the spliceosome active site. Due to the spliceosome’s structurally transient nature the precise functions and mechanisms driving many rearrangements remains undefined.
Chemical probing is a powerful tool used for biomolecule structural research and is capable of characterizing spliceosome structural features and changes during splicing. During my thesis I used a new protein probing protocol designed by the Jurica lab to globally map lysine reactivity within multiple conformations of the spliceosome. From our data and published cryo-electron microscopy structures of the spliceosome we predicted how the core spliceosome scaffold Prp8 interacts with U5 snRNA and its 5 exon binding partner during active site formation. We then used our model to guide genetic experiments and tested the necessary amino acids within Prp8 for putative roles in 5 splice site selection. Our genetic data suggests a subset of lysines that change in probe reactivity between the pre-activated and catalytic spliceosome work together with specific spliceosome protein components (Isy1 and Snu114) to position the 5 splice site.
In addition to chemical probing, I designed experiments to purify a more conformationally homogenous spliceosome sample for cryo-electron microscopy structure determination. During this work I added a lithium salt wash to my spliceosome purification procedure hoping the salt would wash away loosely associated proteins that bind the spliceosome sub-stoichiometrically. If correct, then 2D particle reconstruction from negative stain electron micrographs will show a more clear average of spliceosomes with higher molecular detail than spliceosomes purified without lithium washes. During the course of this project I successfully purified catalytic spliceosomes, washed them with lithium acetate then subject them to analysis via negative stain electron microscopy. From electron micrographs I generated two dimensional averaged particles that exhibited highly similar features to spliceosome purified without lithium. Lightly crosslinking the spliceosome complexes together prior to imaging was additionally used to determine if lithium exposure washed away spliceosome components destabilized during sample preparation. Unfortunately due to difficulties in cryo-EM sample preparation and imaging I never was able to generate a high resolution spliceosome structure from cryo-EM data.