Juan D. Alfonzo, Ph.D.                        

The Alfonzo Lab

alfonzo.1@osu.edu

Office: 614-292-0004

   Lab: 614-292-0141

   Fax: 614-292-8120

Assistant Professor
B.S., Indiana University- Bloomington, IN
Ph.D., Indiana University- Bloomington, IN
Post-doctoral Research, University of California, Los Angeles

Editing & Modification of tRNA: Roles in Mitochondrial Biogenesis and Disease.

We are interested in RNA processing events that are unique to trypanosomes and could be exploited as targets for the design of therapies against protozoan diseases. To date, the Protozoa are responsible for the infection and death of millions of people worldwide. I am especially interested in two facets of RNA processing in trypanosomes: tRNA editing and tRNA modification.

The Mechanism of tRNA Editing

RNA editing involves any process by which the information content of a given transcript (mRNA, tRNA, etc.) is changed so that it will differ from that encoded in the genome. RNA editing occurs by a variety of chemically distinct post-transcriptional mechanisms including nucleotide insertion, deletions, base deaminations, and nucleotide exchange (to name a few) and plays an important role in the regulation of gene expression. In trypanosomes, I discovered that the nucleus-encoded tRNATrp undergoes an essential cytosine to uridine editing of the wobble position of the anticodon upon importation into the mitochondrion. Most notable, the deamination of cytosine to uridine at the anticodon allows the decoding of UGA codons as tryptophan. We are interested in the mechanism of tRNA editing and plan to use a genetic and biochemical approach to achieve a complete structure/function analysis of this process in trypanosomes

The Modifications of tRNA and Contribution to Mitochondrial Function

The functional importance of RNA modifications is evident from the large number of phylogenetically conserved modifications that occur in various cellular RNAs. Little is known about the role and extent of RNA modifications in early divergent eukaryotes. In addition, there are increasing numbers of reports linking tRNA modifications with mitochondrial diseases. I plan to use trypanosomes as a model system to study the role that specific tRNA modifications play in mitochondrial function. These studies will be the basis for examining the role site-specific tRNA modifications plays in mitochondrial function. The long-term goal is to utilize the exquisite specificity of these editing and modification enzymes for the long-term applications in gene therapy in humans.

In general, the emphasis of my laboratory will focus on the translation of RNA structure into RNA function in a biological system. Specifically, I hope to dissect the pathways involved in tRNA maturation, including editing and modification, to address issues involving molecular recognition. I firmly believe this research will not only uncover useful knowledge of basic cellular processes, but also provides the added incentive of great overall implications towards applications in medicine.

The Alfonzo Lab

                  THE RESEARCH TEAM:

              Juan Alfonzo

              Yadwinder Deol            

              Kirk Gaston

              Cecill J. Howard

              Frank Ragone

              Mary Anne Rubio

              Jess Spears

              Ashlie Tseng

              Jessica Wohlgamuth-Benedum

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ALFONZOITES

(past lab members who have left the nest, but are not forgotten...)   

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We are grateful to our wonderful collaborators:

Michael Ibba

F. Nina Papavasiliou

Larry Simpson

George Cross

Dieter Soll & Jesse Reinhart

Otavio H. Thiemann

Frederic Allain

Henri Grosjean

Valerie de Crecy-Legard

Jiunn- Liang Chen

Andreas Reichert & Stephane Duvezin-Caubet

Alex Huttenhofer

David Rueda & Krishanthi Karunatilaka

Venkat Gopalan

Julius Lukes

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ALL MADE POSSIBLE BY RESEARCH FUNDING FROM:

New in 2007 ! 

Garcia LT, Leite NR, Alfonzo, J.D., Thiemann OH.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference.
Mem Inst Oswaldo Cruz. 2007 Sep;102(6):757-62. (Abstract) (PDF)

Gaston KW, Rubio MA, Spears JL, Pastar I, Nina Papavasiliou F, Alfonzo, J.D.

C to U editing at position 32 of the anticodon loop precedes tRNA 5' leader removal in trypanosomatids.
Nucleic Acids Res. 2007 Oct 4; [Epub ahead of print] (Abstract) (PDF)

Garcia LT, Leite NR, Alfonzo, J.D., Thiemann OH.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference.
Mem Inst Oswaldo Cruz. 2007 Sep 5; [Epub ahead of print] (Abstract) (PDF)

Ling J, Roy H, Qin D, Rubio MA, Alfonzo, J.D., Fredrick K, Ibba M.

Pathogenic mechanism of a human mitochondrial tRNAPhe mutation associated with myoclonic epilepsy with ragged red fibers syndrome.Proc Natl Acad Sci U S A. 2007 Sep 25;104(39):15299-304. Epub 2007 Sep 18. (Abstract) (PDF)

Rubio MA, Pastar I, Gaston KW, Ragone FL, Janzen CJ, Cross GA, Papavasiliou FN, Alfonzo, J.D.  An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U deamination of DNA. Proc Natl Acad Sci U S A. 2007 May 8;104(19):7821-6. Epub 2007 May 1. (Abstract) (PDF)

Madej MJ, Alfonzo, J.D., Huttenhofer A. Small ncRNA transcriptome analysis from kinetoplast mitochondria of Leishmania tarentolae. Nucleic Acids Res. 2007;35(5):1544-54. Epub 2007 Feb 7. (Abstract) (PDF)

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Cassago, A., Rodrigues E.M., Prieto, E.L., Gaston, K.W., Alfonzo, J.D., Iribar, M.P., Berry, M.J., Cruz, A.K. and Thiemann, O.H. 2006.  Identification of Leishmania selenoproteins and SECIS element. Mol. Biochem. Parasitol. 149:128-134. (Abstract) (PDF)

Yakovich, A.J., Ragone, F.L., Alfonzo, J.D., Sackett, D.L., Werbovetz, K.A. 2006.  Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening. Exp. Parasitol. 114:289-296. (Abstract) (PDF)

Rubio, M.A., Ragone, F., Gaston, K., Ibba, M. and Alfonzo, J.D. 2006.  C to U editing stimulates A to I editing in the anticodon loop of a threonyl tRNA in trypanosomatids. J. Biol. Chem. 281:115-120. (Abstract) (PDF)

Rinehart J., Krett B., Rubio M.A., Alfonzo J.D., and Söll, D. 2005. Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion. Genes and Development. 19:583-592. (Abstract) (PDF)

Roy H., Ling J., Alfonzo J., and Ibba M. 2005. Loss of editing activity during the evolution of mitochondrial phenyalanyl-tRNA synthetase. J. Biol. Chem. 280:38186-92. (Abstract) (PDF)

Rubio MA. T., and Alfonzo J.D. 2005. Editing and modification in trypanosomatids: the reshaping of non-coding RNAs. Topics in Current Genetics. 12:71-86.

Sbicego S, Alfonzo JD, Estevez AM, Rubio MA, Kang X, Turck CW, Peris M, Simpson L.  RBP38, a novel RNA-binding protein from trypanosomatid mitochondria, modulates RNA stability. Eukaryot Cell. 2003 Jun;2(3):560-8. (Abstract) (PDF)

Monzani PS, Alfonzo JD, Simpson L, Oliva G, Thiemann OH. Cloning, characterization and preliminary crystallographic analysis of Leishmania hypoxanthine-guanine phosphoribosyltransferase. Biochim Biophys Acta. 2002 Jul 29;1598(1-2):3-9. (Abstract) (PDF)

Crain PF*, Alfonzo JD*, Rozenski J, Kapushoc ST, McCloskey JA, Simpson L.  Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency. RNA. 2002 Jun;8(6):752-61. (* equal contribution) (Abstract) (PDF)

Kapushoc ST, Alfonzo JD, Simpson L. Differential localization of nuclear-encoded tRNAs between the cytosol and mitochondrion in Leishmania tarentolae. RNA. 2002 Jan;8(1):57-68. (PDF)

Carrillo R, Thiemann OH, Alfonzo JD, Simpson L. Uridine insertion/deletion RNA editing in Leishmania tarentolae mitochondria shows cell cycle dependence. Mol Biochem Parasitol. 2001 Mar;113(1):175-81.

Kapushoc ST, Alfonzo JD, Rubio MA, Simpson L. End processing precedes mitochondrial importation and editing of tRNAs in Leishmania tarentolae. J Biol Chem. 2000 Dec 1;275(48):37907-14. (Abstract) (PDF)

Rubio MA, Liu X, Yuzawa H, Alfonzo JD, Simpson L. Selective importation of RNA into isolated mitochondria from Leishmania tarentolae. RNA. 2000 Jul;6(7):988-1003. (PDF)

Simpson L, Thiemann OH, Savill NJ, Alfonzo JD, Maslov DA. Evolution of RNA editing in trypanosome mitochondria. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):6986-93. Review. (Abstract) (PDF)

Alfonzo JD, Blanc V, Estevez AM, Rubio MA, Simpson L. C to U editing of the anticodon of imported mitochondrial tRNA(Trp) allows decoding of the UGA stop codon in Leishmania tarentolae. EMBO J. 1999 Dec 15;18(24):7056-62. (Abstract) (PDF)

Estevez AM, Thiemann OH, Alfonzo JD, Simpson L. T7 RNA polymerase-driven transcription in mitochondria of Leishmania tarentolae and Trypanosoma brucei. Mol Biochem Parasitol. 1999 Oct 15;103(2):251-9. (Abstract) (PDF)

Blanc V, Alfonzo JD, Aphasizhev R, Simpson L. The mitochondrial RNA ligase from Leishmania tarentolae can join RNA molecules bridged by a complementary RNA. J Biol Chem. 1999 Aug 20;274(34):24289-96. (Abstract) (PDF)

Alfonzo JD, Crother TR, Guetsova ML, Daignan-Fornier B, Taylor MW. APT1, but not APT2, codes for a functional adenine phosphoribosyltransferase in Saccharomyces cerevisiae.J Bacteriol. 1999 Jan;181(1):347-52. (Abstract) (PDF)

Thiemann OH, Alfonzo JD, Simpson L. Cloning and characterization of Leishmania tarentolae adenine phosphoribosyltransferase. Mol Biochem Parasitol. 1998 Sep 1;95(1):141-6. (Abstract) (PDF)

Alfonzo JD, Thiemann OH, Simpson L. Purification and characterization of MAR1. A mitochondrial associated ribonuclease from Leishmania tarentolae. J Biol Chem. 1998 Nov 6;273(45):30003-11. (Abstract) (PDF)

Simpson L, Wang SH, Thiemann OH, Alfonzo JD, Maslov DA, Avila HA.U-insertion/deletion Edited Sequence Database. Nucleic Acids Res. 1998 Jan 1;26(1):170-6. (Abstract) (PDF)

Alfonzo JD, Sahota A, Taylor MW. Purification and characterization of adenine phosphoribosyltransferase from Saccharomyces cerevisiae. Biochim Biophys Acta. 1997 Sep 5;1341(2):173-82. (Abstract) (PDF)

Alfonzo JD, Thiemann O, Simpson L. The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria. Nucleic Acids Res. 1997 Oct 1;25(19):3751-9. Review. (Abstract) (PDF)

Alfonzo JD, Sahota A, Deeley MC, Ranjekar P, Taylor MW. Cloning and characterization of the adenine phosphoribosyltransferase-encoding gene (APT1) from Saccharomyces cerevisiae. Gene. 1995 Aug 8;161(1):81-5. (Abstract) (PDF)