Molecular Recognition in Biological Systems and Bioinformatics

RNA processing; RNA editing; RNA turnover; RNA-protein interactions; protein arginine methylation.

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 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 importance 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.

Trypanosoma brucei

• RNA binding protein, RBP16
• Exoribonuclease
• TbDSS-1
• RNA helicase
• TbSUV3
• Protein arginine methyltransferases TbPRMT1 and TbPRMT1

African sleeping sickness

• RNA interference
• Bacterial protein expression
• RNA gel shift
• Primer extension
• In vitro RNA synthesis
• All basic molecular biology techniques
• In vitro enzymatic assays
• Immunoprecipitation

• Bacterial expression vectors for 6Xhis
• MBP
• GST tags

• S. cerevisiae strain AH109 for yeast two-hybrid analysis
• Trypanosoma brucei EATRO 164 procyclic forms
• T. brucei strains for genetic manipulation (procyclic and bloodstream form)

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