Laurie K. Read, Ph.D.
Department of Microbiology & Immunology
Phone: (716) 829-3307
Fax: (716) 829-2158
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 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.
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 are using both in organello and in vitro systems to identify cis-acting RNA sequences that regulate the turnover rates of specific mitochondrial RNA populations. We find that very small edited RNA elements can destabilize non-adenylated RNAs. Further, we have demonstrated that mRNA polyadenylation has opposite effects on the stability of unedited and edited RNAs. Unedited RNAs are destabilized by the addition of a 3’ poly(A) tail; conversely, edited RNAs are stabilized by 3’ polyadenylation. We are currently using our in vitro turnover system to determine the mechanisms by which edited cis-acting sequences and polyadenylation regulate mitochondrial RNA turnover. The in vitro system is also being used 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. These studies will provide insight into mechanisms used by the parasite to regulate gene expression as it cycles between its mammalian host and tse tse fly insect vector.
Protein arginine methylation
Methylation of arginine residues in proteins is a posttranslational 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 is allowing us to determine how this modification modulates the function and macromolecular interactions of this protein. See also: http://www.acsu.buffalo.edu/~lread/
Pelletier, M., Y. Xu, X. Wang, S. Zahariev, S. Pongor, J.M. Aletta, and L.K. Read. 2001. Arginine methylation of a mitochondrial guide RNA binding protein from Trypanosoma brucei. Mol. Biochem. Parasitol. 118:49-59.
Hayman, M.L., M.M. Miller, D.M. Chandler, C.C. Goulah, and L.K. Read. 2001. Trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity. Nucl. Acids Res. 29:5216-5225.
Pelletier, M. and L.K. Read. 2003. RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial RNAs. RNA 9:457-468.
Ryan, C.M., K.T. Militello, and L.K. Read. 2003. Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs. J. Biol. Chem. 278:32753-32762.
Miller, M.M. and L.K. Read. 2003. Trypanosoma brucei: Functions of RBP16 cold shock and RGG domains in macromolecular interactions. Exp. Parasitol. 105:140-148.
Penschow, J.L., D.A. Sleve, C.M. Ryan, and L.K. Read. 2004. TbDSS-1 is an essential Trypanosoma brucei exoribonuclease homolog that has pleitropic effects on mitochondrial RNA metabolism. Eukaryot. Cell. 3:1206-1216.
Kao, C-Y, and L.K. Read. 2005. Opposing effects of polyadenylation on the stability of edited and unedited RNAs in Trypanosoma brucei. Mol. Cell. Biol. 25:1634-1644.
Ryan, C.M. and L.K. Read, 2005. UTP-stimulated turnover of polyadenylated mitrochondrial RNA requires RNA polymerization and involves the RET1 TUTase. RNA 11:763-773.
Pelletier, M., D.A. Pasternack, and L.K. Read. 2005. In vitro and in vivo characterization of the major type I protein arginine methyltransferase from Trypnaosoma brucei. Mol. Biochem. Parastiol. 144:206-217.
Ryan, C.M., C.-Y. Kao, D.A. Sleve, and L.K. Read. 2006. Biphasic decay of guide RNAs in Trypanosoma brucei. Mol. Biochem. Parasitol. 146:68-77.
Miller, M.M., K. Halbig, J. Cruz-Reyes, and L.K. Read. 2006. RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction. RNA 12:1292-1303.
Goulah, C.G., M. Pelletier, and L.K. Read. 2006. Arginine methylation regulates mitochondrial gene expression in Trypanosoma brucei through multiple effector proteins. RNA 12:1545-1555.
Duan P. Xu Y. Birkaya B. Myers J. Pelletier M. Read LK. Guarnaccia C. Pongor S. Denman RB. Aletta JM. Generation of polyclonal antiserum for the detection of methylarginine proteins. Journal of Immunological Methods. 320(1-2):132-42, 2007 Mar 30.
Goulah CC. Read LK. Differential effects of arginine methylation on RBP16 mRNA binding, guide RNA (gRNA) binding, and gRNA-containing ribonucleoprotein complex formation. Journal of Biological Chemistry. 282(10):7181-90, 2007 Mar 9.
Pelletier M. Read LK. Aphasizhev R. Isolation of RNA bidning proteins involved in insertion/deletion editing. Methods in Enzymology. 424:75-105, 2007.
Mattiacio, J.L. and L.K. Read. 2008. Roles for TbDSS-1 in RNA surveillance and decay of maturation by products from the 12S rRNA locus. Nucl. Acids Res. 36:319-329.
Ammerman, M.L., J.C. Fisk, and L.K. Read. 2008. gRNA/pre-mRNA annealing and RNA chaperone activity of RBP16. RNA 14:1069-1080.
Fisk, J.C., M.L. Ammerman, V. Presnyak, and L.K. Read. 2008. TbRGG2, an essential RNA editing accessory factor in two Trypanosoma brucei life cycle stages. J. Biol. Chem. 283:23016-23025.
Fisk, J.C., J. Sayegh, C. Zurita-Lopez, S.G. Menon, V. Presnyak, S. Clarke, and L.K. Read. 2009. A type III protein arginine methyltransferase from the protozoan parasite, Trypanosoma brucei. J. Biol. Chem. 284:11590-11600.
Mattiacio, J.L. and L.K. Read. 2009. Evidence for a degradosome-like complex in the mitochondria of Trypanosoma brucei. FEBS Lett. 583:2333-2338.
Fisk, J.C., V. Presnyak, M.L. Ammerman, and L.K. Read. 2009. Distinct and overlapping functions of MRP1/2 and RBP16 in mitochondrial RNA metabolism. Mol. Cell. Biol. 29:5214-5225.
Sprehe, M., J.C. Fisk, S.E. McEvoy, L.K. Read, and M.A. Schumacher. 2010. Structure of the T. brucei p22 protein, a COII-specific RNA editing accessory factor. J. Biol. Chem. 85:18899-18908.
Fisk, J.C., C. Zurita-Lopez, J. Sayegh, D. Tomasello, S. Clarke, and L.K. Read. 2010. TbPRMT6 is a Type I protein arginine methyltransferase that contributes to cytokinesis in Trypanosoma brucei. Eukaryot. Cell. 9:866-877.
Ammerman, M.L., V. Presnyak, J.C, Fisk, B.M. Foda, and L.K. Read. 2010. TbRGG2 facilitates kinetoplastid RNA editing initiation and progression through intrinsic pause sites. RNA 16:2239-2251.