Melanie Ohi, Ph.D.
2015 Grant Recipient
Understanding How Mutations in hSF3B1 Isolated from MDS Patients Affect the Structural Integrity and Function of the Spliceosome
Basic Science Research Grant 2015
In cells, genes, composed of DNA, are transcribed into RNAs and then the RNAs are translated into proteins. In this manner our genes provide the blueprint for all of the proteins needed by cells to function properly. However, genes contain sequences that need to be removed before a protein can be made, a process termed RNA splicing. The regions of the RNA transcript that contains meaningful protein-coding regions are called exons, and the “junk” regions, are called introns. In RNA splicing, the introns are precisely clipped out and the remaining exons are glued together to produce a mature message (mRNA). The mRNA is then transcribed into a functional protein.
The spliceosome is the cellular machine responsible for recognizing and removing the introns from RNA and then ligating the exons together to form the mRNA. The spliceosome is composed of 100’s of proteins and five RNAs. It is a dynamic nano-machine that requires the precise coordination and regulation of many moving parts in order to correctly recognize and remove introns. Mistakes made by the spliceosome lead to proteins that do not fold properly or have altered functions in the cell. Splicing mistakes can cause human disease.
$ Myelodysplastic Syndrome (MDS) is a disease that occurs when blood-forming cells in the bone marrow stop dividing properly. Over half of MDS patients have mutations in spliceosome components, suggesting that altered RNA splicing is a major cause of disease progression. However, how these MDS mutations change spliceosome function is not understood hindering the development of new and more effective treatment strategies for MDS patients. Our focus is to study the ways common mutations found in MDS patients alter spliceosome organization and function. Our research is focused on studying mutations in a core spliceosome protein called SF3B1. Approximately 30% of MDS patients have mutations in SF3B1.
$ SF3B1 mutations found in MDS patients change specific amino acids found in a region of the protein called HEAT repeats. HEAT repeats are predicted to act as a scaffold that binds other spliceosome proteins and perhaps RNA. Our hypothesis is that SF3B1 MDS mutations alter the ability of SF3B1 to interact with binding partners, changing the organization and function of the spliceosome. We have used the fission yeast Schizosaccharomyces pombe as a model organism to make and purify mutant spliceosomes containing MDS mutations. We have analyzed these mutant spliceosomes using a structural approach called single particle electron microscopy. From these analyses we have found that many of the SF3B1 MDS mutations cause the spliceosome to be unstable and “fall apart” during purification. The MDS mutant spliceosomes appear to be especially fragile. These results were surprising, since the yeast carrying MDS mutations do not have any obvious growth phenotypes. We are continuing our structural studies to understand the specific molecular connections within the spliceosome that are being altered by the MDS mutations. Additionally we have also done functional studies in the MDS mutant yeast. From this work, we have found unexpected connections between the MDS mutations and spliceosome motors involved in spliceosome activation. The overall goal of this project is to improve the fundamental knowledge of how the MDS mutations physically alter spliceosome function, information needed for understanding MDS disease progression.
Takizawa, Y., Binshtein, E., Erwin, A.E., Pyburn, T.M., Mittendorf, K.F., Ohi, M.D. (2017) While the Revolution Will Not Be Crystallized, Biochemistry Reigns Supreme. Protein Science 2017, 26(1): 69-81