Center for Matrix Biology - Vanderbilt University and Medical Center

Melanie D. Ohi, Ph.D.
Assistant Professor in the Department of Cell and Developmental Biology

	Office Address:	4160 MRB III
	Mailing Address:	4160 MRB III Nashville, TN 37232
	Lab address:	Office: 4160A MRBIII
	Phone:	936-7780
	FAX:	936-5673
		email \| website

Research Summary

Proteins carry out most cellular processes as members of dynamic multi-protein assemblies. Although progress has been made cataloging the constituents of specific complexes, we have only limited knowledge of how proteins assemble into macromolecular machines and how these machines perform their cellular functions. We are using yeast genetics, biochemistry and single particle cryo-electron microscopy (EM) to explore the structural and functional organization of complexes involved in pre-mRNA splicing and complexes involved in the protein ubiquitination pathway.

Single Particle Cryo-Electron Microscopy
Single particle cryo-EM is a powerful technique for determining the structures of large, dynamic complexes that are too difficult to crystallize. In this structural approach, purified complexes are applied to grids covered with holey carbon film and quickly frozen by plunging the grids into liquid ethane. The rapid freezing prevents water from forming ice crystals and embeds the molecules in a layer of vitrified (or amorphous) ice preserving the specimen in a near-native environment. Images of the preserved particles are taken using an electron microscope. Digital image processing methods are then used to produce 3D models from the images of complex particles trapped in vitrified ice.

The Spliceosome
Although the human genome contains ~25,000 genes, it is estimated that we make over 90,000 proteins. The disparity between our genome and our proteome can be explained by the activity of the spliceosome, a large macromolecular machine composed of RNA and protein components. This complex catalyzes the excision of non-coding introns from a pre-messenger RNA (pre-mRNA) to create a mature message (mRNA). Although the composition of the spliceosome is known, it remains a mystery how this dynamic machine functions. To understand spliceosome function and regulation it will be essential to develop three-dimensional (3D) pictures of how the numerous spliceosomal proteins and RNA components organize into one machine. A number of projects in the lab focus on the functional and structural characterization of stable spliceosomal complexes isolated from the fission yeast S. pombe.

The Anaphase Promoting Complex or Cyclosome (APC/C)
Ubiquitin (Ub) becomes covalently attached to substrate proteins via an enzyme cascade consisting of activating (E1), conjugating (E2), and ligating (E3) enzymes. E3 ubiquitin ligases vary widely in size, composition, and enzymology reflecting their regulatory role in substrate recognition. The APC/C is an ubiquitin ligase composed of 13 polypeptides that facilitates the transfer of Ub from E2s to specific substrates. The APC/C is regulated at different stages of the cell cycle by both phosphorylation events and by the binding of specific activators or inhibitors to the core complex. Projects in the lab focus on structurally characterizing the APC/C purified from different stages of the cell cycle.

Research Keywords

Biochemistry, Protein Structure, Structural Biology, Yeast,

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Associated initiative(s):

Career opportunities:
None currently available

Selected Publications

» Ohi, MD, Ren, L, Wall, JS, Gould, KL, Walz, T Structural characterization of the fission yeast U5.U2/U6 spliceosome complex Proc Natl Acad Sci U S A 2007. 104:3195-200

» Huang, DT, Hunt, HW, Zhuang, M, Ohi, MD, Holton, JM, Schulman, BA Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity Nature 2007. 445:394-8

» Ohi, MD, Feoktistova, A, Ren, L, Yip, C, Cheng, Y, Chen, JS, Yoon, HJ, Wall, JS, Huang, Z, Penczek, PA, Gould, KL, Walz, T Structural organization of the anaphase-promoting complex bound to the mitotic activator Slp1 Mol Cell 2007. 28:871-85

» Vander Kooi, CW, Ohi, MD, Rosenberg, JA, Oldham, ML, Newcomer, ME, Gould, KL, Chazin, WJ The Prp19 U-box crystal structure suggests a common dimeric architecture for a class of oligomeric E3 ubiquitin ligases Biochemistry 2006. 45:121-30

» Crampton, D.J., Ohi, M.D., Qimron,E., Lee,S.J., Walz, T., and Richardson, C. Oligomeric states of bacteriophage T7 gene 4 primase/helicase Journal of Molecular Biology 2006. 360:667-677

» Ohi, MD, Vander Kooi, CW, Rosenberg, JA, Ren, L, Hirsch, JP, Chazin, WJ, Walz, T, Gould, KL Structural and functional analysis of essential pre-mRNA splicing factor Prp19p Mol Cell Biol 2005. 25:451-60

» Wilkinson, CR, Dittmar, GA, Ohi, MD, Uetz, P, Jones, N, Finley, D Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast Curr Biol 2004. 14:2283-8

» Ohi, M.D., Li, Y., Cheng, Y. and Walz, T. Negative staining and image classification-powerful tools in modern electron microscopy Biol. Proc. Online 2004. 6:23-34

» Ohi, MD, Vander Kooi, CW, Rosenberg, JA, Chazin, WJ, Gould, KL Structural insights into the U-box, a domain associated with multi-ubiquitination Nat Struct Biol 2003. 10:250-5

» Ohi, MD, Gould, KL Characterization of interactions among the Cef1p-Prp19p-associated splicing complex RNA 2002. 8:798-815

» Ohi, MD, Link, AJ, Ren, L, Jennings, JL, McDonald, WH, Gould, KL Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs Mol Cell Biol 2002. 22:2011-24

» Berry, LD, Feoktistova, A, Wright, MD, Gould, KL The schizosaccharomyces pombe dim1(+) gene interacts with the anaphase-promoting complex or cyclosome (APC/C) component lid1(+) and is required for APC/C function Mol Cell Biol 1999. 19:2535-46