UC Santa Cruz

Pre-mRNA splicing is critical for controlling normal gene expression within the cell. Several splicing associated RNA helicases and their binding partners are misregulated in a variety of cancers including leukemia, prostate, glioma, and non-small-cell lung cancer. Some of the RNA helicases from the DHX-family are involved in multiple cellular pathways, and to define their specific activity they rely on binding partners that contain G-patch domains. For instance, DHX15 has been characterized for its role in spliceosome disassembly after splicing and ribosome biogenesis directed by G-patch proteins TFIP11 and NKRF, respectively. My dissertation focuses on a new model for DHX15 in which it ensures fidelity of early spliceosome assembly. I hypothesize that DHX15 acts in a quality-control (QC) mechanism to disassemble aberrant spliceosomes. Due to the dynamic nature of the spliceosome, in that intronic landmarks are divergent as well as the multitude of rearrangements required to remove an intron, mistakes are bound to happen and the lack of a QC step during early assembly has long been a mystery. To address the role that DHX15 may be playing during early spliceosome assembly, I utilized a combination of RNA substrates and energy sources (ATP, GTP and AMP-PNP) to parse apart the different energy-dependent steps of A-complex assembly. Then through immunodepletion asked how removal of DHX15 affects the system. In attempting to determine where DHX15 binds within our complexes, I utilized a system of truncated RNA substrates and endogenous RNase H digestion of the U2 snRNA. Through this work, I found that depletion of DHX15 results in an accumulation of A- and B-complexes yet results in a decrease in splicing efficiency. Further, certain regions of the U2 snRNA are prone to NTP-dependent rearrangements, whereas others are protected or unaffected. In addition, the work in this thesis solely relied on HeLa cell nuclear extract and protein levels including DHX15 vary across cell types. Therefore, I worked to develop protocols and understand why other cell lines have yet to yield robust activity in nuclear extract in addition to why we observe inconsistent activity between HeLa nuclear extract preps. Finally, to understand how DHX15 may be impacting early complex formation, I devised a purification scheme to allow for early complexes to be purified for further study. Taken together, the data produced during my graduate career and described in this dissertation has yielded another path of study within the spliceosome and may provide a scaffold for future therapeutics in targeting diseases such as cancers and myelodysplastic syndromes.