| Associate
Professor
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.
New
in 2009!
Wohlgamuth-Benedum
JM, Rubio MA, Paris Z, Long S, Poliak P,
Lukes J, Alfonzo J.D. Thiolation
controls cytoplasmic tRNA stability and
acts as a negative determinant for tRNA
editing in mitochondria. J Biol Chem. 2009
Jul 2. (Abstract)
(PDF)
Alfonzo
J.D., Söll D. Mitochondrial
tRNA import - the challenge to understand
has just begun. Biol Chem. 2009 Jun 27.
(Abstract)
(PDF)
Paris
Z, Rubio MA, Lukes J, Alfonzo J.D..
Mitochondrial tRNA import in Trypanosoma
brucei is independent of thiolation
and the Rieske protein. RNA. 2009 Jul;15(7):1398-406.
Epub 2009 May 22. (Abstract)
(PDF)
_______________________________________________________________________________________________
Simoes-Barbosa
A, Louly C, Franco OL, Rubio MA, Alfonzo
J.D., Johnson PJ. The divergent
eukaryote Trichomonas vaginalis
has an m7G cap methyltransferase capable
of a single N2 methylation. Nucleic Acids
Res. 2008 Dec;36(21):6848-58. Epub 2008
Oct 28. (Abstract)
(PDF)
Rubio M.A., Rinehart J., Krett B., Duvezin-Caubet
S, Reichert AS, Söll, D, Alfonzo
J.D.
Mammalian
mitochondria have the innate ability to
import tRNAs by a mechanism distinct from
protein import.
Proc Natl Acad Sci U S A. 2008 Jul 8;105(27):9186-91.
Epub 2008 Jun 27. (Abstract)
(PDF)
Gaston
KW, Rubio MA, Alfonzo, J.D.
OXOPAP
assay: for selective amplification of aminoacylated
tRNAs from total cellular fractions.
Methods. 2008 Feb;44(2):170-5.
_______________________________________________________________________________________________
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;102(6):757-62.
(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)
___________________________________________________________________________________________
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)
__________________________________________________________________________________
The
Alfonzo Lab
Rishav Aggarwal
Juan Alfonzo
Kirk Gaston
Cecil J. Howard
Zdenek
Paris
Mary Anne Rubio
Petr
Ruzicka
Jess Spears
__________________________________________________________________________________
January
11-16, 2009
Gordon
Research Conference: RNA EDITING
Roles of RNA And DNA Editing
and Modification
in
Cellular Function
Hotel
Galvez
Galveston, TX
__________________________________________________________________________________
__________________________________________________________________________________
We
are grateful to our wonderful collaborators:
F.
Nina Papavasiliou
Julius
Lukes &
Zdenek Paris
Larry
Simpson
George
Cross
Michael
Ibba
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
The
Alfonzo Lab
__________________________________________________________________________________
Thanks
for making our research possible...
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