Professor, Department of Cell Biology Adjunct Professor, Graduate Faculty, Instructional Leadership & Academic Curriculum, The Jeannine Rainbolt College of Education
Fellow of the American Association for the Advancement of Science
Ph.D., The Hahnemann Medical College B.S., Union College
Office Phone: (405) 271-2377 Fax Number: (405) 271-3548
Email: Lawrence-Rothblum@ouhsc.edu
University of Oklahoma Health Sciences Center Department of Cell Biology 940 Stanton L.Young Blvd., BMS 564 Oklahoma City, OK 73104
Our laboratory has focused upon the mechanism of transcription by RNA polymerase I and its regulation. It has long been our hypothesis that understanding the mechanism of transcription would 1) provide the basis for understanding its regulation and 2) provide a mechanism for its inhibition under conditions where it is dysregulated or pathological.
1. The laboratory's first studies focused on the definition of the cis-acting elements and trans-acting factors of rDNA transcription and focused on the rat rDNA repeat. These studies contributed to our understanding of the mechanism of transcription of the mammalian ribosomal genes. We found that the promoter consisted of two domains a core promoter element required for transcription and an upstream element required for stable preinitiation complex formation (a) and that they must be stereochemically aligned (b). Subsequently we demonstrated that rat rDNA transcription required three components in vitro, competent RNA polymerase I, a species specific transcription factor (similar to human SL1) and a factor that increased the efficiency of transcription, similar to human UBF (c), and that UBF was not species specific (d) in contrast to other reports.
a. Xie, W.Q., Rothblum, L.I. Domains of the rat rDNA promoter must be aligned stereospecifically. Mol Cell Biol. 1992 Mar;12(3):1266-75. PMID: 1545808 PMCID: PMC369559
b. Cassidy, B., Haglund, R., Rothblum, L.I. Regions upstream from the core promoter of the rat ribosomal gene are required for the formation of a stable transcription initiation complex by RNA polymerase I in vitro. Biochim Biophys Acta. 1987 Jul 14;909(2):133-44. PMID: 3593729
c. Smith, S.D., Oriahi, E., Lowe, D., Yang-Yen, H.F., O'Mahony, D., Rose, K., Chen, C., and Rothblum, L.I. Characterization of factors that direct transcription of rat ribosomal DNA. Mol. Cell. Biol. 10: 3105-3116, 1990. PMID: 2342470 PMCID: PMC360675
d. Pikaard, C.S., Smith, S.D., Reeder, R.H., Rothblum. L. rUBF, an RNA polymerase I transcription factor from rats, produces DNase I footprints identical to those produced by xUBF, its homolog from frogs. Mol Cell Biol. 1990 Jul;10(7):3810-2. PMID:2355924 PMCID:PMC360842
2. Our understanding of the role(s) of one of the transcription factors, UBF, led to two important discoveries. First, in collaboration with Howard Morgan and Bruce Sells we demonstrated that rDNA transcription was the central mechanism through which the accumulation of ribosomes was regulated in hypertrophic growth (a) and development (b). This was contrary to dogma and established new paradigms for understanding both mitotic and hypertrophic growth. Secondly, we found that the protein product of the retinoblastoma susceptibility gene, Rb, a known anti-oncogene and regulator of transcription by RNA polymerase II also regulated transcription by RNA polymerase I. Again, this was a paradigm shift and has led to a greater understanding of how cells regulate transcription by all three nuclear transcription systems (c,d).
a. McDermott, P.J., Rothblum, L.I., Smith, S.D., Morgan, H.E.: Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture. J. Biol. Chem., 264:18220-18227, 1989. PMID: 2808374
b. Larson DE, Xie W, Glibetic M, O'Mahony D, Sells BH, Rothblum LI. Coordinated decreases in rRNA gene transcription factors and rRNA synthesis during muscle cell differentiation. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7933-6. PMID: 8396256 PMCID: PMC47261
c. Cavanaugh, A.H., Hempel, W. M., Taylor, L.J., Rogalsky, V., Todorov, G., Rothblum, L.I. Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product. Nature, 374: 177-180, 1995. PMID: 7877691
d. Hannan, K.M., Hannan, R.D., Smith, S.D., Jefferson,L.S., Lun, M., Rothblum, L.I. Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction between UBF and SL-1. Oncogene 19:4988-4999, 2000. PMID: 11042686
3. One recent project has focused on the role of a polymerase associated initiation factor, Rrn3 in rDNA transcription, its function and its regulation. We were the first to demonstrate this was the rDNA transcription factor first identified by Fu Li-Yu and Feigelson that was cycleheximide sensitive. We showed that cycloheximide inhibited its phosphorylation and its association with Pol I (a), and that it must be phosphorylated to function in recruitment of the polymerase to the committed template. We demonstrated that Rrn3 functioned stoichiometrically and not catalytically (b), that it was required for the formation of the heparin-resistant open promoter complex (c) and that it itself was a DNA-binding protein (d). Most recently, we have found that the inhibition of the association of Rrn3 with Pol I, using a peptide based on the sequence of rpa43, is capable of inhibiting rDNA transcription and causes the death of tumor cells in tissue culture (e).
a. Cavanaugh, A.H., Hirschler-Laszkiewicz, I., Hu, Q., Dundr, M., Smink, T., Misteli, T., Rothblum, L.I. Rrn3 phosphorylation is a regulatory checkpoint for ribosome biogenesis. J. Biol. Chem. 277: 27423-27432, 2002. PMID: 12015311
b. Hirschler-Laszkiewicz, I., Cavanaugh, A., Mirza, A., Lun, M., Hu, Q., Smink, T., Rothblum, L.I. Rrn3 becomes inactivated in the process of ribosomal DNA transcription.. J. Biol. Chem. 278: 18953–18959, 2003. PMID: 12646563
c. Cavanaugh, A. H., Evans, A. Rothblum, L.I. Mammalian Rrn3 Is Required for the formation of a Transcription Competent Preinitiation Complex Containing RNA Polymerase I. Gene Expression 14:131-147, 2008. PMID: 18590050 PMCID: PMC2526047
d. Stepanchick, A. Zhi, H-J., Cavanaugh, A., Rothblum, K., Schneider, D.A., Rothblum, L.I. DNA-binding by the ribosomal DNA transcription factor Rrn3 is essential for ribosomal DNA Transcription. J Biol Chem.;288:9135-44, 2013 PMID: 23393135 PMCID: PMC3610986
e. Rothblum, K., Hu, Q., Penrod, Y., Rothblum L.I. Selective Inhibition of rDNA Transcription by a Small-Molecule Peptide that Targets the Interface Between RNA Polymerase I and Rrn3. Mol Cancer Res. 12:1586-1596.2014 PMID: 25033839 PMCID: PMC4233170
4. Another recent project has been to pursue the mechanism of action of two Polymerase Associated Factors that in mammalian cells are referred to as PAF53 and PAF49. These two proteins are the orthologs of two yeast proteins RPA49 and RPA34, respectively. We found that they were the targets of regulation by growth factors and that their regulation appears to be cell-type specific. Studies in yeast made possible by combining genetics and biochemistry have established that these two proteins are important for rDNA transcription. However, there are differences between yeast and mammalian rDNA transcription, such that it is necessary to come up with methods for combining genetics and biochemistry in mammalian cells.
We have used CRISPR/Cas9 technology to introduce an inducible degron into the genes that encode the mammalian rDNA transcription factors (a). This allows us to rapidly knockdown a targeted protein and replace it with wild-type or mutant forms of the protein in order to do both cell biological and biochemical studies of the factors. We have used this technique to study the role of PAF53 in rDNA transcription and cell proliferation. We have found a novel, helix-turn-helix, DNA-binding domain in the “linker” that has a very significant role in rDNA transcription. Our colleague, Dr. Bruce Knutson confirmed that this same structure was essential for function by the yeast homolog, A49, of PAF53. Our studies on the role(s) of PAF53 are summarized in the following figures. These studies are now being extended to PAF49 (b,c,d).
a. Conditional depletion of the RNA polymerase I subunit PAF53 reveals that it is essential for mitosis and enables identification of functional domains. McNamar R, Abu-Adas Z, Rothblum K, Knutson B.A., Rothblum, L.I. J Biol Chem. 2019 Dec 27;294(52):19907-19922.
b. Dynamics of the RNA polymerase I TFIIF/TFIIE-like subcomplex: a mini-review. Knutson B.A., McNamar R, Rothblum, L.I. Biochem Soc Trans. 2020 Oct 30;48(5):1917-1927.
c. The Mammalian and Yeast A49 and A34 Heterodimers: Homologous but Not the Same. McNamar R, Rothblum K., Rothblum, L.I. Genes (Basel). 2021 Apr 22;12(5):620.
d. PAF49: An RNA Polymerase I subunit essential for rDNA transcription and stabilization of PAF53. Rachel McNamar , Emma Freeman , Kairo N Baylor , Aula M Fakhouri, Sui Huang , Bruce A Knutson , Lawrence I Rothblum J. Biol. Chem.. 2023 Aug;299 (8):104951. doi: 10.1016/j.jbc.2023.104951.
Cavanaugh AH, Hempel WM, Taylor LJ, Rogalsky V, Todorov G, and Rothblum LI. Activity of rNA polymerase I transcription factor UBF blocked by Rb gene product. Nature, 374:177-180. 1995.
Hannan RD, Luyken J, and Rothblum LI. Regulation of rDNA transcription factors during cardiomyocyte hypertrophy induced by adrenergic agents. J. Biol. Chem. 270:8290-8297. 1995.
Hannan K, Kennedy BK, Cavanaugh A, Hannan RD, Hirschler-Laszkiewicz I, Jefferson L, Rothblum LI. RNA polymerase I transcription in Confluent Cells: Rb downregulates rDNA transcription during confluence-induced cell cycle arrest. Oncogene. 19:3487-3497, 2000.
Cavanaugh AH, Hirschler-Laszkiewicz I, Hu Q, Dundr M, Smink T, Misteli T, and Rothblum LI. Rrn3 phosphorylation is a regulatory checkpoint for ribosome biogenesis. J. Biol. Chem. 277:27423-27432, 2002.
Dundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI, Phair RD, Misteli T. A kinetic framework for a mammalian RNA polymerase in vivo. Science, 298:1623-626, 2002.
Hirschler-Laszkiewicz I, Cavanaugh A, Mirza A, Lun M, Hu Q, Smink T, and Rothblum LI. Rrn3 functions stoichiometrically in ribosomal DNA transcription. J. Biol. Chem. 278:18953-18959, 2003.
Brandenburger Y, Arthur JF, Woodcock EA, Du, X-J, Gao, X-M, Autelitano, DJ, Rothblum, LI, and Hannan RD. Cardiac hypertrophy in vivo is associated with increased expression of the ribosomal gene transcription factor UBF. FEBS Letters 548:79-84, 2003.
Stefanovsky V, Langlois F, Gagnon-Kugler T, Rothblum LI, and Moss T. Growth factor signaling regulates elongation of RNA polymerase I transcription in mammals via UBF phosphorylation and r-chromatin remodeling. Mol. Cell. 21:629-639, 2006.
Cavanaugh AH, Evans A, Rothblum LI. Mammalian Rrn3 is required for the formation of a transcription competent preinitiation complex containing RNA polymerase I. Gene Expr. 14:131-147, 2008.
Sanij E, Poortinga G, Sharkey K, Hung S, Holloway TP, Quin J, Robb E, Wong LH, Thomas WG, Stefanovsky V, Moss T, Rothblum L, Hannan KM, McArthur GA, Pearson RB, Hannan RD. UBF levels determine the number of active ribosomal RNA genes in mammals. J Cell Biol. 183:1259-1274, 2008.
Penrod Y, Rothblum K, Rothblum LI. (2012) Characterization of the interactions of mammalian RNA polymerase I associated proteins PAF53 and PAF49. Biochemistry 51:6519-6526.
Hannan KM, Sanij E, Rothblum LI, Hannan RD, Pearson RB.(2013) Dysregulation of RNA polymerase I transcription during disease. Biochim Biophys Acta. 1829(3-4):342-360.
Stepanchick A, Zhi H, Cavanaugh AH, Rothblum K, Schneider DA, Rothblum LI. (2013) DNA binding by the ribosomal DNA transcription factor Rrn3 is essential for ribosomal DNA transcription. J Biol Chem. 288:9135-9144.
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