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Dr. Laurie Read
Microbiology and Immunology
Associate Professor
249 Biomedical Research Building
Buffalo NY, 14214
Phone: (716) 829-3307
Email: lread@buffalo.edu
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DESCRIPTION OF INTERESTS
Posttranscriptional Regulation of Gene Expression in Parasitic Protozoa
Trypanosomes are members of the kinetoplastid protozoa, which cause enormous medical and economic distress in Third World countries. They are eukaryotic parasites which are the causative agents for diseases such as Sleeping Sickness, Leishmaniasis, and Chagas disease. In our laboratory, we study the parasitic trypanosome, Trypanosoma brucei. In addition to being of great medical and economic importance, T. brucei is an excellent model system for the study of posttranscriptional gene regulation, because regulation at the level of transcription is essentially absent in this organism. Our primary focus is on two RNA processing events in T. brucei: RNA editing and RNA turnover. A third related area of research is the mechanism by which posttranslational modification of RNA binding proteins by arginine methylation regulates RNA editing, trafficking, turnover, and trans-splicing.
RNA editing
RNA editing is a novel mechanism for regulating gene expression in which sequence information is added to mRNAs after transcription by specific uridine addition and deletion. The editing of mRNAs in T. brucei is so extensive that uridine insertions can double the size of the transcript. Editing generates translatable messages by creating the open reading frames as well as proper initiation and termination signals. The phenomenon is of fundamental importance in understanding how genetic information can be stored and processed. We are studying the mechanisms used by T. brucei to regulate editing of specific RNAs, particularly as they are differentially edited between life cycle stages. We identified the first RNA editing regulatory factor, a mitochondrial RNA binding protein termed RBP16. Genetic disruption of RBP16 in insect stage trypanosomes causes massive down-regulation of a specific subset of editing events. Currently, we are using a combination of biochemical and genetic approaches to elucidate the mechanisms by which RBP16 regulates editing of specific RNAs and to determine its regulatory scope throughout the trypanosome life cycle. Our approaches include gene knock-down of RBP16 and RBP16-associated proteins in both insect and mammalian life cycle stages, analysis of the biochemical effects of RBP16 on RNA editing in vitro, and yeast-two hybrid and TAP affinity chromatography approaches to identification of RBP16 binding partners. Future directions will involve the identification and characterization of additional RNA editing regulatory proteins.
RNA turnover
The levels of translatable mRNAs are dictated by the balance between transcription rates and mRNA turnover rates. Because transcription is largely unregulated in T. brucei, the mechanisms by which mRNA turnover is controlled take on enhanced importance. We have identified two pathways for mRNA turnover in T. brucei mitochondria. One pathway is specific for polyadenylated RNAs and depends on the UTP concentration, while the second pathway is independent of the polyadenylation state of the RNA and nucleotide concentrations. We developed an in vitro RNA turnover system that allows us to directly examine the effects of specific 3? sequences on RNA degradation. We are also using this system as a starting point for biochemical purification of the proteins that catalyze and regulate RNA turnover pathways. In addition, we used a bioinformatics approach to identify trypanosome homologs of the yeast mitochondrial degradosome proteins DSS1 (an exoribonuclease) and SUV3 (and RNA helicase). Biochemical and genetic studies are underway to determine the roles of these proteins in turnover of the various classes of mitochondrial RNAs.
Protein arginine methylation
Methylation of arginine residues in proteins is a posttranscriptional modification whose important in areas such as signal transduction, RNA trafficking, mRNA splicing, and transcription is just recently becoming apparent. Interestingly, a very large percentage of proteins that undergo arginine methylation are RNA binding proteins. Given that gene regulation in trypanosomes relies so heavily on RNA processing, our hypothesis is that arginine methylation is especially important in these organisms. We showed that multiple proteins in T. brucei are subject to arginine methylation (including the mitochondrial RNA binding protein, RBP16). In addition, we identified two genes encoding the protein arginine methyltransferases (PRMTs) that catalyze this modification. Studies are currently underway to determine the effect of PRMT down-regulation in trypanosomes on growth rate as well as on specific RNA processing events. We are identifying novel PRMT substrates in T. brucei using both yeast two-hybrid and affinity chromatography methods. Finally, mutation of the methylated arginine residues in RBP16 will allow us to determine how this modification modulates the function and macromolecular interactions of this protein.
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EDUCATION
| 1990 |
Ph.D., Cellular and Molecular Physiology
Tufts University School of Medicine
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| 1985 |
M.A., Cell Biology and Physiology
University of California at Santa Barbara
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| 1982 |
Bachelor of Arts, Biology
Boston University
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EXPERTISE
RNA editing,RNA turnover and Protein arginine methylation
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PUBLICATIONS
| Miller MM, Read LK; Trypanosoma brucei: functions of RBP16 cold shock and RGG domains in macromolecular interactions.; Exp Parasitol; 2003 Oct; 105(2); 140-148 |
| Ryan CM, Militello KT, Read LK; Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs.; J Biol Chem; 2003 Aug; 278(35); 32753-32762 |
| RYAN, CM,MILITELLO KT, and READ, LK; Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs; J. Biol. Chem.; 2003; 278; 32753-32762 |
| Michel Pelletier, Read L; RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei; RNA; 2003 Jan; 9; 457-468 |
| MILLER, MM and READ LK; Trypanosoma brucei: Functions of RBP16 cold shock and RGG domains in macromolecular interactions; Exp. Parasitol.; 2003; 105; 140-148 |
| Hayman ML, Miller MM, Chandler DM, Goulah CC, Read LK; The trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity.; Nucleic Acids Res; 2001 Dec; 29(24); 5216-5225 |
| Pelletier M, Xu Y, Wang X, Zahariev S, Pongor S, Aletta JM, Read LK; Arginine methylation of a mitochondrial guide RNA binding protein from Trypanosoma
brucei.; Mol Biochem Parasitol; 2001 Nov; 118(1); 49-59 |
| Pelletier M, Xu Y, Wang X, Zahariev S, Pongor S, Aletta JM, Read LK; Arginine methylation of a mitochondrial guide RNA binding protein from Trypanosoma brucei.; Mol Biochem Parasitol; 2001 Nov; 118(1); 49-59 |
| Pelletier, M., Xu, Y., Wang, X., Zahariev, S., Pongor, S., Aletta, J.M., Laurie Read; Arginine methylation of a mitochondrial guide RNA binding protein from Trypanosoma brucei; Mol. Biochem. Parasitol.; 2001 Jan; 118; 49-59 |
| Hayman, M.L., Miller, M.M., Chandler, D.M., Goulah, C.C., Laurie Read; The trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity; Nucl. Acids Res.; 2001 Jan; 29; 5216-5225 |
| Militello KT, Hayman ML, Read LK; Transcriptional and post-transcriptional in organello labelling of Trypanosoma brucei mitochondrial RNA.; Int J Parasitol; 2000 Apr; 30(5); 643-647 |
| Militello KT, Read LK; UTP-dependent and -independent pathways of mRNA turnover in Trypanosoma brucei mitochondria.; Mol Cell Biol; 2000 Apr; 20(7); 2308-2316 |
| Pelletier M, Miller MM, Read LK; RNA-binding properties of the mitochondrial Y-box protein RBP16.; Nucleic Acids Res; 2000 Mar; 28(5); 1266-1275 |
| Read LK, Militello KT, Nerantzakis GE; Cloning and characterisation of cDNA encoding the Trypanosoma brucei ribosomal protein L24.; Int J Parasitol; 1999 Apr; 29(4); 601-605 |
| Hayman ML, Read LK; Trypanosoma brucei RBP16 is a mitochondrial Y-box family protein with guide RNA binding activity.; J Biol Chem; 1999 Apr; 274(17); 12067-12074 |
| Militello KT, Read LK; Coordination of kRNA editing and polyadenylation in Trypanosoma brucei mitochondria: complete editing is not required for long poly(A) tract addition.; Nucleic Acids Res; 1999 Mar; 27(5); 1377-1385 |
| Hotchkiss TL, Nerantzakis GE, Dills SC, Shang L, Read LK; Trypanosoma brucei poly(A) binding protein I cDNA cloning, expression, and binding to 5 untranslated region sequence elements.; Mol Biochem Parasitol; 1999 Jan; 98(1); 117-129 |
| Schmid B, Read LK, Stuart K, Göringer HU; Experimental verification of the secondary structures of guide RNA-pre-mRNA chimaeric molecules in Trypanosoma brucei.; Eur J Biochem; 1996 Sep; 240(3); 721-731 |
| Corell RA, Read LK, Riley GR, Nellissery JK, Allen TE, Kable ML, Wachal MD, Seiwert SD, Myler PJ, Stuart KD; Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties.; Mol Cell Biol; 1996 Apr; 16(4); 1410-1418 |
| Read LK, Stankey KA, Fish WR, Muthiani AM, Stuart K; Developmental regulation of RNA editing and polyadenylation in four life cycle stages of Trypanosoma congolense.; Mol Biochem Parasitol; 1994 Dec; 68(2); 297-306 |
| Read LK, Göringer HU, Stuart K; Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.; Mol Cell Biol; 1994 Apr; 14(4); 2629-2639 |
| Read LK, Wilson KD, Myler PJ, Stuart K; Editing of Trypanosoma brucei maxicircle CR5 mRNA generates variable carboxy terminal predicted protein sequences.; Nucleic Acids Res; 1994 Apr; 22(8); 1489-1495 |
| Souza AE, Shu HH, Read LK, Myler PJ, Stuart KD; Extensive editing of CR2 maxicircle transcripts of Trypanosoma brucei predicts a protein with homology to a subunit of NADH dehydrogenase.; Mol Cell Biol; 1993 Nov; 13(11); 6832-6840 |
| Read LK, Stuart K; Conservation of gRNA gene cassette structure in African trypanosomes despite divergence in the defining flanking repeats.; Mol Biochem Parasitol; 1993 Aug; 60(2); 333-335 |
| Read LK, Fish WR, Muthiani AM, Stuart K; Maxicircle DNA and edited mRNA sequences of closely related trypanosome species: implications of kRNA editing for evolution of maxicircle genomes.; Nucleic Acids Res; 1993 Aug; 21(17); 4073-4078 |
| Read LK, Jacob AN, Fish WR, Muthiani AM, Stuart K; Sequences of three Trypanosoma congolense maxicircle genes allow prediction of regions encoding transcripts that undergo extensive RNA editing.; Mol Biochem Parasitol; 1993 Aug; 60(2); 337-341 |
| Stuart, K., Corell, R.A., Goringer, H.U., Koslowsky, D.J., Myler, P.J., Laurie Read, Riley, G.R., Shu, H.-H., Souza, A.E.; RNA editing in Trypanosoma brucei: gRNA diversity and redundancy; Plant Mitochondria; 1993 Jan; |
| Read LK, Corell RA, Stuart K; Chimeric and truncated RNAs in Trypanosoma brucei suggest transesterifications at non-consecutive sites during RNA editing.; Nucleic Acids Res; 1992 May; 20(9); 2341-2347 |
| Read LK, Myler PJ, Stuart K; Extensive editing of both processed and preprocessed maxicircle CR6 transcripts in Trypanosoma brucei.; J Biol Chem; 1992 Jan; 267(2); 1123-1128 |
| Koslowsky DJ, Bhat GJ, Read LK, Stuart K; Cycles of progressive realignment of gRNA with mRNA in RNA editing.; Cell; 1991 Nov; 67(3); 537-546 |
| Read LK, Mikkelsen RB; Comparison of adenylate cyclase and cAMP-dependent protein kinase in gametocytogenic and nongametocytogenic clones of Plasmodium falciparum.; J Parasitol; 1991 Jun; 77(3); 346-352 |
| Read LK, Mikkelsen RB; Plasmodium falciparum-infected erythrocytes contain an adenylate cyclase with properties which differ from those of the host enzyme.; Mol Biochem Parasitol; 1991 Mar; 45(1); 109-119 |
| Read LK, Mikkelsen RB; Cyclic AMP- and Ca2(+)-dependent protein kinases in Plasmodium falciparum.; Exp Parasitol; 1990 Jul; 71(1); 39-48 |
| Laurie Read, Margulis, L., Stolz, R., Obar, R., Sawyer, T.K.; A new strain of Paratetramitus jugusus from Laguna Figueroa, Baja California, Mexico; Biol. Bull.; 1983 Jan; 165; 241-264 |
GRANTS
April 2002 to March 2007
Proteins Involved in kRNA Editing
National Institute of General Medical Sciences
Laurie Read
$1,250,000
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April 2000 to March 2005
Mitochondrial RNA Metabolism in trypanosoma brucei
National Institute of Allergy and Infectious Diseases
Laurie Read
$1,000,000
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October 1998 to September 2001
RNA binding proteins in Plasmodium falciparum
NIH
Noreen Williams, Laurie Read
$230,499
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June 1997 to December 1998
RNA Binding Proteins in Plasmodium falciparum
UB Multidisciplinary Pilot Project Program
Laurie Read, Margaret Hollingsworth, Noreen Williams
$20,000
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July 1996 to June 2001
Mitochondrial RNA Processing in Trypanosoma brucei
Burroughs Wellcome Fund New Investigator in Molecular Parasitology award
Laurie Read
$195,000
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