Regulation of Gene Expression in Parasitic Protozoa

The quality of life of millions is compromised by parasitic infection. Trypanosomes, spread by tsetse flies to humans and grazing animals in sub- Saharan Africa or by reduviid bugs in many areas of South and Central America, cause debilitating and lethal disease. These unicellular, eukaryotic organisms reside extracellularly, in the bloodstream and tissue spaces of the host or intracellularly, within smooth muscle cells. As parasites, they readily differentiate during their complex life cycle to adapt to numerous nutritional conditions and immunological challenges. Scientific research has uncovered a multitude of unusual genetic, biochemical and structural properties of trypanosomes. We hope that by exploiting some of these parasite-specific traits we can devise ways to alleviate disease.

Messenger RNA molecules in eukaryotic cells usually begin as large precursor forms that are cut and spliced to produce stable and translatable mature mRNAs. In most cases, a single molecule of RNA is the starting material. However, trypanosomes synthesize messenger RNAs in a unique way. All trypanosome mRNAs are produced by a splicing reaction that fuses a short, capped RNA (called the spliced leader) to each protein coding pre-mRNA. The spliced leader is essential for gene expression; however, its synthesis, regulation and role in mRNA maturation and translation remain enigmas. Our work is focused on understanding the nucleic acid and protein signals responsible for these processes.

To dissect the components of the spliced leader gene essential for its transcription, we are using a combination of genetic analysis and DNA-protein binding studies. Protein purification and subsequent genetic analysis will enable us to identify unique trans-acting factors of the spliced leader transcription pathway. This work has lead to our discovery of PBP-1, a basal transcription factor that is an essential component of the spliced leader RNA gene expression pathway.

Since the 5' cap present on each mature mRNA is added in trans in trypanosomes, the "one pre-mRN -one mRNA" rule observed in other eukaryotes does not apply here. Thus, trypanosomes synthesize polycistronic pre-mRNAs that are cleaved into multiple mature mRNAs via RNA trans- splicing. Post- transcriptional regulation of gene expression is probably orchestrated in part by differential spliced leader addition to specific sites within polycistronic pre-mRNAs. We are investigating the signals that direct the spliced leader to specific precursor transcripts whose mRNA levels appear to be post-transcriptionally regulated.

Finally, we have developed an in vitro RNA turnover system that is enabling us to explore and define the signals that regulate differential mRNA turnover as a function of life cycle stage in trypanosomes.

Selected Publications

• Tyler Weisbarth R, Das A, Castellano P, Fisher MA, Wu H, Bellofatto V. The Trypanosoma cruzi RNA-binding protein RBP42 is expressed in the cytoplasm throughout the life cycle of the parasite. Parasitol Res. 2018 Apr;117(4):1095-1104. doi: 10.1007/s00436-018-5787-9. Epub 2018 Feb 23. PMID: 29473141

• Das A, Banday M, Fisher MA, Chang YJ, Rosenfeld J, Bellofatto V. An essential domain of an early-diverged RNA polymerase II functions to accurately decode a primitive chromatin landscape. Nucleic Acids Res. 2017 Jul 27;45(13):7886-7896. doi: 10.1093/nar/gkx486. PMID: 28575287

• Liu W, Das A, Morales R, Banday M, Aris V, Lukac DM, Soteropoulos P, Wah DA, Palenchar J, Bellofatto V., Chromatin immunoprecipitation and microarray analysis reveal that TFIIB occupies the SL RNA gene promoter region in Trypanosoma brucei chromosomes. Mol Biochem Parasitol. 2012 Dec;186(2):139-42. doi: 10.1016/j.molbiopara.2012.09.003. Epub 2012 Sep 19. PMID: 22999857

• Das A, Morales R, Banday M, Garcia S, Hao L, Cross GA, Estevez AM, Bellofatto V. The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism. RNA. 2012 Nov;18(11):1968-83. doi: 10.1261/rna.033829.112. Epub 2012 Sep 10. PMID: 22966087

• Utter, C. J., Garcia, S. A., Milone, J., & Bellofatto, V. (2011). Poly (A)-specific Ribonuclease (PARN-1) function in stage-specific mRNA turnover in Trypanosoma brucei. Eukaryot Cell. 2011 Sep;10(9):1230-40. doi: 10.1128/EC.05097-11. Epub 2011 Jul 8. PMID: 21743004

• Das, A., & Bellofatto, V. (2009). The non-canonical CTD of RNAP-II is essential for productive RNA synthesis in Trypanosoma brucei. PLoS One. 2009 Sep 9;4(9):e6959. doi: 10.1371/journal.pone.0006959. PMID: 19742309

• Ibrahim BS, Kanneganti N, Rieckhof GE, Das A, Laurents DV, Palenchar JB, Bellofatto V, Wah DA. (2009). Structure of the C-terminal domain of transcription factor IIB from Trypanosoma brucei. Proceedings of the National Academy of Sciences, 106(32), 13242-13247. PMID: 19666603

• Banerjee H, Palenchar JB, Lukaszewicz M, Bojarska E, Stepinski J, Jemielity J, Guranowski A, Ng S, Wah DA, Darzynkiewicz E, Bellofatto V. (2009). Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins.RNA,15(8), 1554-1564. PMID: 19541768

• Das, A., Banday, M., & Bellofatto, V. (2008). RNA polymerase transcription machinery in trypanosomes. Eukaryotic cell, 7(3), 429-434. PMID: 17951525

* Full list of publications on PubMed

Awards and Honors

Honors:

Named fellow of the American Association for the Advancement of Science (AAAS), 2017

Faculty of the Month, Group on Women In Medicine and Science (GWIMS), New Jersey Medical School, March 2016

Fellow, Executive Leadership in Academic Medicine (ELAM), Drexel University College of Medicine, 2010

Site visitor of Intramural Research Programs, Food and Drug Administration, Center for Biologics Evaluation and Research, 2006

Invitee, 10th Annual IUBMB conference on Infectious disease: biochemistry of parasites, vectors and host, 2006

Chair of Foundation of UMDNJ study section, 2006

Co-organizer, International Meeting on Kinetoplastid Cell Biology and Molecular Genetics, 2005, 2007 and 2009

Invitee, International Meeting on the Cellular and Molecular Biology of Parasitic, Protozoa (invitation only), Germany, 2003

Permanent Member of NIH Study Section (Tropical Medicine & Parasitology), 1999-2003

Member, American Heart Association, Study Section, 1996

Participant in EMBO course in Molecular Genetics, Pavia, Italy, 1981

High honors in Biological Sciences, Rutgers University, 1977

Rutgers University Scholarship Award, 1976

Awards:

Award for Excellence in Research, Foundation of UMDNJ, 2009

New Investigators Award in Molecular Parasitology, Burroughs Wellcome Fund, 1998-2001

Irma T. Hirschl Trust Award, 1990-1995

Education

1984 Ph.D. in Molecular Biology, Albert Einstein College of Medicine of Yeshiva University

1977 B.S. with high honors in Biological Sciences, Douglass College of Rutgers University

Positions

2014-Present. Interim Chair, Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School

2005-2014. Vice Chair for Research, Department of Microbiology and Molecular Genetics, Rutgers New Jersey Medical School (former UMDNJ NJMS)

2004-Present. Professor, Department of Microbiology and Molecular Genetics, Rutgers New Jersey Medical School (former UMDNJ NJMS)