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Vanderbilt-Ingram Cancer CenterVanderbilt-Ingram Cancer Center

 

Melanie D. Ohi, Ph.D.

Associate Professor of Cell and Developmental Biology
Researcher

Contact Information:

Vanderbilt University Medical Center
465 21st Ave So, 3160-A MRB 3
Nashville, TN 37232-8240
615-936-7780
Fax: 615-936-5673

Research Description

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.

Pore Forming Toxins
There are a number of projects in the lab that focus on studying the structure and function of pore forming bacterial toxins. We work with a multidisciplinary team of researchers to understand how these toxins function as both soluble and membrane inserted proteins, with the main goal of gaining a mechanistic understanding of how these toxins contribute to pathogenesis.

Publications
  • González-Rivera C, Campbell AM, Rutherford SA, Pyburn TM, Foegeding NJ, Barke TL, Spiller BW, McClain MS, Ohi MD, Lacy DB, Cover TL. A non-oligomerizing mutant form of Helicobacter pylori VacA allows structural analysis of the p33 domain. Infect. Immun [print-electronic]. 2016 Jul 7/5/2016; PMID: 27382020, PII: IAI.00254-16, DOI: 10.1128/IAI.00254-16, ISSN: 1098-5522.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/27382020.
  • Pyburn TM, Foegeding NJ, González-Rivera C, McDonald NA, Gould KL, Cover TL, Ohi MD. Structural organization of membrane-inserted hexamers formed by Helicobacter pylori VacA toxin. Mol. Microbiol [print-electronic]. 2016 Jun 6/16/2016; PMID: 27309820, DOI: 10.1111/mmi.13443, ISSN: 1365-2958.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/27309820.
  • McDonald NA, Takizawa Y, Feoktistova A, Xu P, Ohi MD, Vander Kooi CW, Gould KL. The Tubulation Activity of a Fission Yeast F-BAR Protein Is Dispensable for Its Function in Cytokinesis. Cell Rep [print-electronic]. 2016 Jan 1/26/2016; 14(3): 534-46. PMID: 26776521, PMCID: PMC4731314, PII: S2211-1247(15)01503-X, DOI: 10.1016/j.celrep.2015.12.062, ISSN: 2211-1247.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/26776521.
  • Foegeding NJ, Caston RR, McClain MS, Ohi MD, Cover TL. An Overview of Helicobacter pylori VacA Toxin Biology. Toxins (Basel). 2016; 8(6): PMID: 27271669, PMCID: PMC4926140, PII: toxins8060173, DOI: 10.3390/toxins8060173, ISSN: 2072-6651.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/27271669.
  • Frick-Cheng AE, Pyburn TM, Voss BJ, McDonald WH, Ohi MD, Cover TL. Molecular and Structural Analysis of the Helicobacter pylori cag Type IV Secretion System Core Complex. MBio. 2016; 7(1): PMID: 26758182, PII: mBio.02001-15, DOI: 10.1128/mBio.02001-15, ISSN: 2150-7511.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/26758182.
  • McDonald NA, Vander Kooi CW, Ohi MD, Gould KL. Oligomerization but Not Membrane Bending Underlies the Function of Certain F-BAR Proteins in Cell Motility and Cytokinesis. Dev. Cell. 2015 Dec 12/21/2015; 35(6): 725-36. PMID: 26702831, PMCID: PMC4691284, PII: S1534-5807(15)00758-3, DOI: 10.1016/j.devcel.2015.11.023, ISSN: 1878-1551.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/26702831.
  • Shin Y, Du Y, Collier SE, Ohi MD, Lang MJ, Ohi R. Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8. Proc. Natl. Acad. Sci. U.S.A [print-electronic]. 2015 Jul 7/21/2015; 112(29): E3826-35. PMID: 26150501, PMCID: PMC4517267, PII: 1500272112, DOI: 10.1073/pnas.1500272112, ISSN: 1091-6490.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/26150501.
  • Binshtein E, Ohi MD. Cryo-electron microscopy and the amazing race to atomic resolution. Biochemistry [print-electronic]. 2015 May 5/26/2015; 54(20): 3133-41. PMID: 25955078, DOI: 10.1021/acs.biochem.5b00114, ISSN: 1520-4995.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/25955078.
  • Gao M, Thomson TC, Creed TM, Tu S, Loganathan SN, Jackson CA, McCluskey P, Lin Y, Collier SE, Weng Z, Lasko P, Ohi MD, Arkov AL. Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells, bind Tudor and protect from transposable elements. EMBO Rep [print-electronic]. 2015 Mar; 16(3): 379-86. PMID: 25600116, PMCID: PMC4364877, PII: embr.201439694, DOI: 10.15252/embr.201439694, ISSN: 1469-3178.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/25600116.
  • Collier SE, Voehler M, Peng D, Ohi R, Gould KL, Reiter NJ, Ohi MD. Structural and functional insights into the N-terminus of Schizosaccharomyces pombe Cdc5. Biochemistry [print-electronic]. 2014 Oct 10/21/2014; 53(41): 6439-51. PMID: 25263959, PMCID: PMC4204884, DOI: 10.1021/bi5008639, ISSN: 1520-4995.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/25263959.
  • Sturgill EG, Das DK, Takizawa Y, Shin Y, Collier SE, Ohi MD, Hwang W, Lang MJ, Ohi R. Kinesin-12 Kif15 targets kinetochore fibers through an intrinsic two-step mechanism. Curr. Biol [print-electronic]. 2014 Oct 10/6/2014; 24(19): 2307-13. PMID: 25264249, PMCID: PMC4207087, PII: S0960-9822(14)01035-5, DOI: 10.1016/j.cub.2014.08.022, ISSN: 1879-0445.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/25264249.
  • Livesay SB, Collier SE, Bitton DA, Bähler J, Ohi MD. Structural and functional characterization of the N terminus of Schizosaccharomyces pombe Cwf10. Eukaryotic Cell [print-electronic]. 2013 Nov; 12(11): 1472-89. PMID: 24014766, PMCID: PMC3837936, PII: EC.00140-13, DOI: 10.1128/EC.00140-13, ISSN: 1535-9786.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/24014766.
  • Folkmann AW, Collier SE, Zhan X, Aditi, Ohi MD, Wente SR. Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease. Cell [print-electronic]. 2013 Oct 10/24/2013; 155(3): 582-93. PMID: 24243016, PMCID: PMC3855398, PII: S0092-8674(13)01160-4, DOI: 10.1016/j.cell.2013.09.023, ISSN: 1097-4172.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/24243016.
  • Benefield DA, Dessain SK, Shine N, Ohi MD, Lacy DB. Molecular assembly of botulinum neurotoxin progenitor complexes. Proc. Natl. Acad. Sci. U.S.A [print-electronic]. 2013 Apr 4/2/2013; 110(14): 5630-5. PMID: 23509303, PMCID: PMC3619295, PII: 1222139110, DOI: 10.1073/pnas.1222139110, ISSN: 1091-6490.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/23509303.
  • Pattanayek R, Yadagiri KK, Ohi MD, Egli M. Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator. Cell Cycle [print-electronic]. 2013 Mar 3/1/2013; 12(5): 810-7. PMID: 23388462, PMCID: PMC3610728, PII: 23757, DOI: 10.4161/cc.23757, ISSN: 1551-4005.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/23388462.
  • Chambers MG, Pyburn TM, González-Rivera C, Collier SE, Eli I, Yip CK, Takizawa Y, Lacy DB, Cover TL, Ohi MD. Structural analysis of the oligomeric states of Helicobacter pylori VacA toxin. J. Mol. Biol [print-electronic]. 2013 Feb 2/8/2013; 425(3): 524-35. PMID: 23178866, PMCID: PMC3612943, PII: S0022-2836(12)00892-3, DOI: 10.1016/j.jmb.2012.11.020, ISSN: 1089-8638.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/23178866.
  • Cox RG, Livesay SB, Johnson M, Ohi MD, Williams JV. The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins. J. Virol [print-electronic]. 2012 Nov; 86(22): 12148-60. PMID: 22933271, PMCID: PMC3486500, PII: JVI.01133-12, DOI: 10.1128/JVI.01133-12, ISSN: 1098-5514.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/22933271.
  • Johnson AE, Collier SE, Ohi MD, Gould KL. Fission yeast Dma1 requires RING domain dimerization for its ubiquitin ligase activity and mitotic checkpoint function. J. Biol. Chem [print-electronic]. 2012 Jul 7/27/2012; 287(31): 25741-8. PMID: 22669973, PMCID: PMC3406662, PII: M112.349712, DOI: 10.1074/jbc.M112.349712, ISSN: 1083-351X.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/22669973.
  • Mittendorf KF, Deatherage CL, Ohi MD, Sanders CR. Tailoring of membrane proteins by alternative splicing of pre-mRNA. Biochemistry [print-electronic]. 2012 Jul 7/17/2012; 51(28): 5541-56. PMID: 22708632, PMCID: PMC3448030, DOI: 10.1021/bi3007065, ISSN: 1520-4995.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/22708632.
  • Chen JS, Lu LX, Ohi MD, Creamer KM, English C, Partridge JF, Ohi R, Gould KL. Cdk1 phosphorylation of the kinetochore protein Nsk1 prevents error-prone chromosome segregation. J. Cell Biol [print-electronic]. 2011 Nov 11/14/2011; 195(4): 583-93. PMID: 22065639, PMCID: PMC3257533, PII: jcb.201105074, DOI: 10.1083/jcb.201105074, ISSN: 1540-8140.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/22065639.
  • Archuleta TL, Du Y, English CA, Lory S, Lesser C, Ohi MD, Ohi R, Spiller BW. The Chlamydia effector chlamydial outer protein N (CopN) sequesters tubulin and prevents microtubule assembly. J. Biol. Chem [print-electronic]. 2011 Sep 9/30/2011; 286(39): 33992-8. PMID: 21841198, PMCID: PMC3190796, PII: M111.258426, DOI: 10.1074/jbc.M111.258426, ISSN: 1083-351X.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/21841198.
  • McLean JR, Chaix D, Ohi MD, Gould KL. State of the APC/C: organization, function, and structure. Crit. Rev. Biochem. Mol. Biol [print-electronic]. 2011 Apr; 46(2): 118-36. PMID: 21261459, DOI: 10.3109/10409238.2010.541420, ISSN: 1549-7798.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/21261459.
  • Ren L, McLean JR, Hazbun TR, Fields S, Vander Kooi C, Ohi MD, Gould KL. Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19. PLoS ONE. 2011; 6(2): e16719. PMID: 21386897, PMCID: PMC3046128, DOI: 10.1371/journal.pone.0016719, ISSN: 1932-6203.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/21386897.
  • Pruitt RN, Chambers MG, Ng KK, Ohi MD, Lacy DB. Structural organization of the functional domains of Clostridium difficile toxins A and B. Proc. Natl. Acad. Sci. U.S.A [print-electronic]. 2010 Jul 7/27/2010; 107(30): 13467-72. PMID: 20624955, PMCID: PMC2922184, PII: 1002199107, DOI: 10.1073/pnas.1002199107, ISSN: 1091-6490.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/20624955.
  • González-Rivera C, Gangwer KA, McClain MS, Eli IM, Chambers MG, Ohi MD, Lacy DB, Cover TL. Reconstitution of Helicobacter pylori VacA toxin from purified components. Biochemistry. 2010 Jul 7/13/2010; 49(27): 5743-52. PMID: 20527875, PMCID: PMC2910095, DOI: 10.1021/bi100618g, ISSN: 1520-4995.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/20527875.
  • Roberts-Galbraith RH, Ohi MD, Ballif BA, Chen JS, McLeod I, McDonald WH, Gygi SP, Yates JR, Gould KL. Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site. Mol. Cell. 2010 Jul 7/9/2010; 39(1): 86-99. PMID: 20603077, PMCID: PMC2916701, PII: S1097-2765(10)00453-3, DOI: 10.1016/j.molcel.2010.06.012, ISSN: 1097-4164.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/20603077.
  • Vander Kooi CW, Ren L, Xu P, Ohi MD, Gould KL, Chazin WJ. The Prp19 WD40 domain contains a conserved protein interaction region essential for its function. Structure. 2010 May 5/12/2010; 18(5): 584-93. PMID: 20462492, PMCID: PMC2871033, PII: S0969-2126(10)00109-7, DOI: 10.1016/j.str.2010.02.015, ISSN: 1878-4186.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/20462492.
  • 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 Dec 12/14/2007; 28(5): 871-85. PMID: 18082611, PMCID: PMC2197158, PII: S1097-2765(07)00664-8, DOI: 10.1016/j.molcel.2007.10.003, ISSN: 1097-2765.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/18082611.
  • 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 [print-electronic]. 2007 Feb 2/27/2007; 104(9): 3195-200. PMID: 17360628, PMCID: PMC1805518, PII: 0611591104, DOI: 10.1073/pnas.0611591104, ISSN: 0027-8424.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/17360628.
  • 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 [print-electronic]. 2007 Jan 1/25/2007; 445(7126): 394-8. PMID: 17220875, PMCID: PMC2821831, PII: nature05490, DOI: 10.1038/nature05490, ISSN: 1476-4687.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/17220875.
  • 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 Jan 1/10/2006; 45(1): 121-30. PMID: 16388587, PMCID: PMC2570371, DOI: 10.1021/bi051787e, ISSN: 0006-2960.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/16388587.
  • 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: 667-77.
  • 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 Jan; 25(1): 451-60. PMID: 15601865, PMCID: PMC538785, PII: 25/1/451, DOI: 10.1128/MCB.25.1.451-460.2005, ISSN: 0270-7306.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/15601865.
  • 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 Dec 12/29/2004; 14(24): 2283-8. PMID: 15620657, PII: S096098220400870X, DOI: 10.1016/j.cub.2004.11.058, ISSN: 0960-9822.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/15620657.
  • 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((1)): 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 Apr; 10(4): 250-5. PMID: 12627222, PII: nsb906, DOI: 10.1038/nsb906, ISSN: 1072-8368.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/12627222.
  • Ohi MD, Gould KL. Characterization of interactions among the Cef1p-Prp19p-associated splicing complex. RNA. 2002 Jun; 8(6): 798-815. PMID: 12088152, PMCID: PMC1370298, ISSN: 1355-8382.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/12088152.
  • 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 Apr; 22(7): 2011-24. PMID: 11884590, PMCID: PMC133674, ISSN: 0270-7306.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/11884590.
  • 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 Apr; 19(4): 2535-46. PMID: 10082519, PMCID: PMC84046, ISSN: 0270-7306.
    Available from: http://www.ncbi.nlm.nih.gov/pubmed/10082519.