Vivian Bellofatto

Rutgers New Jersey Medical School, Newark, New Jersey, United States

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Publications (51)291.49 Total impact

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    ABSTRACT: RNA polymerase II (RNAP-II) synthesizes the m7G-capped Spliced Leader (SL) RNA and most protein-coding mRNAs in trypanosomes. RNAP-II recruitment to DNA usually requires a set of transcription factors that make sequence-specific contacts near transcriptional start sites within chromosomes. In trypanosomes, the transcription factor TFIIB is necessary for RNAP-II-dependent SL RNA transcription. However, the trypanosomal TFIIB (tTFIIB) lacks the highly basic DNA binding region normally found in the C-terminal region of TFIIB proteins. To assess the precise pattern of tTFIIB binding within the SL RNA gene locus, as well as within several other loci, we performed chromatin immunoprecipitation/microarray analysis using a tiled gene array with a probe spacing of 10 nucleotides. We found that tTFIIB binds non-randomly within the SL RNA gene locus mainly within a 220-nt long region that straddles the transcription start site. tTFIIB does not bind within the small subunit (SSU) rRNA locus, indicating that trypanosomal TFIIB is not a component of an RNAP-I transcriptional complex. Interestingly, discrete binding sites were observed within the putative promoter regions of two loci on different chromosomes. These data suggest that although trypanosomal TFIIB lacks a highly basic DNA binding region, it nevertheless localizes to discrete regions of chromatin that include the SL RNA gene promoter.
    Molecular and Biochemical Parasitology 12/2012; 186(2):139–142. · 2.73 Impact Factor
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    ABSTRACT: RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein-RNA interactions in vivo using UV light. Specific RBP42-mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.
    RNA 09/2012; 18(11):1968-83. · 5.09 Impact Factor
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    Vivian Bellofatto, Jeffrey Wilusz
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    ABSTRACT: The level of an mRNA within a cell depends on both its rate of synthesis and rate of decay. Now, independent studies by Bregman et al. and Trcek et al. provide evidence that these two processes are integrated. They show that transcription factors and DNA promoters can directly influence the relative stability of transcripts that they produce.
    Cell 12/2011; 147(7):1438-9. · 31.96 Impact Factor
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    ABSTRACT: Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5' to 3' exonuclease decay or 3' to 5' exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher plants. Trypanosomatids possess three different PARN genes, PARN-1, -2, and -3, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite Trypanosoma brucei. Here we show that T. brucei PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability in vivo, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production.
    Eukaryotic Cell 07/2011; 10(9):1230-40. · 3.59 Impact Factor
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    ABSTRACT: In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor IIB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 A resolution structure of the C-terminal domain of tTFIIB (tTFIIB(C)). The tTFIIB(C) structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, tTFIIB(C) contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosome-specific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms.
    Proceedings of the National Academy of Sciences 09/2009; 106(32):13242-7. · 9.81 Impact Factor
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    ABSTRACT: A new member of the FHIT protein family, designated HIT-45, has been identified in the African trypanosome Trypanosoma brucei. Recombinant HIT-45 proteins were purified from trypanosomal and bacterial protein expression systems and analyzed for substrate specificity using various dinucleoside polyphosphates, including those that contain the 5'-mRNA cap, i.e., m(7)GMP. This enzyme exhibited typical dinucleoside triphosphatase activity (EC 3.6.1.29), having its highest specificity for diadenosine triphosphate (ApppA). However, the trypanosome enzyme contains a unique amino-terminal extension, and hydrolysis of cap dinucleotides with monomethylated guanosine or dimethylated guanosine always yielded m(7)GMP (or m(2,7)GMP) as one of the reaction products. Interestingly, m(7)Gpppm(3)(N6, N6, 2'O)A was preferred among the methylated substrates. This hypermethylated dinucleotide is unique to trypanosomes and may be an intermediate in the decay of cap 4, i.e., m(7)Gpppm(3)(N6, N6, 2'O)Apm(2'O)Apm(2'O)Cpm(2)(N3, 2'O)U, that occurs in these organisms.
    RNA 07/2009; 15(8):1554-64. · 5.09 Impact Factor
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    Anish Das, Vivian Bellofatto
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    ABSTRACT: The carboxy-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II (RNAP-II) is essential for gene expression in metazoa and yeast. The canonical CTD is characterized by heptapeptide repeats. Differential phosphorylation of canonical CTD orchestrates transcriptional and co-transcriptional maturation of mRNA and snRNA. Many organisms, including trypanosomes, lack a canonical CTD. In these organisms, the CTD is called a non-canonical CTD or pseudo-CTD (PsiCTD. In the African trypanosome, Trypanosoma brucei, the PsiCTD is approximately 285 amino acids long, rich in serines and prolines, and phosphorylated. We report that T. brucei RNAP-II lacking the entire PsiCTD or containing only a 95-amino-acid-long PsiCTD failed to support cell viability. In contrast, RNAP-II with a 186-amino-acid-long PsiCTD maintained cellular growth. RNAP-II with PsiCTD truncations resulted in abortive initiation of transcription. These data establish that non-canonical CTDs play an important role in gene expression.
    PLoS ONE 02/2009; 4(9):e6959. · 3.53 Impact Factor
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    ABSTRACT: Thermodynamic processes with free energy parameters are often used in algorithms that solve the free energy minimization problem to predict secondary structures of single RNA sequences. While results from these algorithms are promising, an observation is that single sequence-based methods have moderate accuracy and more information is needed to improve on RNA secondary structure prediction, such as covariance scores obtained from multiple sequence alignments. We present in this paper a new approach to predicting the consensus secondary structure of a set of aligned RNA sequences via pseudo-energy minimization. Our tool, called RSpredict, takes into account sequence covariation and employs effective heuristics for accuracy improvement. RSpredict accepts, as input data, a multiple sequence alignment in FASTA or ClustalW format and outputs the consensus secondary structure of the input sequences in both the Vienna style Dot Bracket format and the Connectivity Table format. Our method was compared with some widely used tools including KNetFold, Pfold and RNAalifold. A comprehensive test on different datasets including Rfam sequence alignments and a multiple sequence alignment obtained from our study on the Drosophila X chromosome reveals that RSpredict is competitive with the existing tools on the tested datasets. RSpredict is freely available online as a web server and also as a jar file for download at http://datalab.njit.edu/biology/RSpredict.
    Bioinformatics and biology insights 01/2009; 3:51-69.
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    ABSTRACT: Constrained sequence alignment has been studied extensively in the past. Different forms of constraints have been investigated, where a constraint can be a subsequence, a regular expression, or a probability matrix of symbols and positions. However, constrained structural alignment has been investigated to a much lesser extent. In this paper, we present an efficient method for constrained structural alignment and apply the method to detecting conserved secondary structures, or structural motifs, in a set of RNA molecules. The proposed method combines both sequence and structural information of RNAs to find an optimal local alignment between two RNA secondary structures, one of which is a query and the other is a subject structure in the given set. The method allows a biologist to annotate conserved regions, or constraints, in the query RNA structure and incorporates these regions into the alignment process to obtain biologically more meaningful alignment scores. A statistical measure is developed to assess the significance of the scores. Experimental results based on detecting internal ribosome entry sites in the RNA molecules of hepatitis C virus and Trypanosoma brucei demonstrate the effectiveness of the proposed method and its superiority over existing techniques.
    Computational biology and chemistry 09/2008; 32(4):264-72. · 1.37 Impact Factor
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    Anish Das, Mahrukh Banday, Vivian Bellofatto
    Eukaryotic Cell 04/2008; 7(3):429-34. · 3.59 Impact Factor
  • Vivian Bellofatto, Jennifer B Palenchar
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    ABSTRACT: RNA interference (RNAi) is a cellular mechanism that is often exploited as a technique for quelling the expression of a specific gene. RNAi studies are carried out in vivo, making this a powerful means for the study of protein function in situ Several trypanosomatids, including those organisms responsible for human and animal diseases, naturally possess the machinery necessary for RNAi manipulations. This allows for the use of RNAi in unraveling many of the pressing questions regarding the parasite's unique biology. The completion of the Trypanosoma brucei genome sequence, coupled with several powerful genetic tools, has resulted in widespread utilization of RNAi in this organism. The key steps for RNAi-based reduction of gene expression, including parasite cell culture, DNA transfection, RNAi expression, and experimental execution, are discussed with a focus on procyclic forms of Trypanosoma brucei.
    Methods in molecular biology (Clifton, N.J.) 02/2008; 442:83-94. · 1.29 Impact Factor
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    ABSTRACT: In this paper we propose a new method for aligning two RNA secondary structures by taking into account the presence of conserved RNA substructures, or constraints, in the alignment process. Our method allows the incorporation of specific knowledge about the RNA structures being analyzed while computing their alignment and thus is very useful for RNA motif detection. We develop statistical measures to assess the significance of alignment scores. Experimental results obtained by using the constrained alignment method to search for structural motifs in the untranslated regions of Trypanosoma brucei mRNAs demonstrate the good performance of the proposed method and its superiority over existing methods.
    Bioinformatics and Bioengineering, 2007. BIBE 2007. Proceedings of the 7th IEEE International Conference on; 11/2007
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    ABSTRACT: RADAR is a web server that provides a multitude of functionality for RNA data analysis and research. It can align structure-annotated RNA sequences so that both sequence and structure information are taken into consideration during the alignment process. This server is capable of performing pairwise structure alignment, multiple structure alignment, database search and clustering. In addition, RADAR provides two salient features: (i) constrained alignment of RNA secondary structures, and (ii) prediction of the consensus structure for a set of RNA sequences. RADAR will be able to assist scientists in performing many important RNA mining operations, including the understanding of the functionality of RNA sequences, the detection of RNA structural motifs and the clustering of RNA molecules, among others. The web server together with a software package for download is freely accessible at http://datalab.njit.edu/biodata/rna/RSmatch/server.htm and http://www.ccrnp.ncifcrf.gov/~bshapiro/
    Nucleic Acids Research 08/2007; 35(Web Server issue):W300-4. · 8.81 Impact Factor
  • Vivian Bellofatto
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    ABSTRACT: Parasites of the Trypanosomatidae family are unable to synthesize purines. Instead, they rely on their hosts to supply these necessary compounds. The article by Gudin et al. identifies three transport mechanisms of the equilibrative nucleoside transporter family by which nucleosides and nucleobases are transported in this medically important family of organisms. The work by Gudin et al. characterizes the dynamics of these transporters and points to further areas for future genetic and therapeutic experiments.
    Trends in Parasitology 06/2007; 23(5):187-9; discussion 190. · 5.51 Impact Factor
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    ABSTRACT: The signal-recognition particle (SRP) mediates the translocation of membrane and secretory proteins across the endoplasmic reticulum upon interaction with the SRP receptor. In trypanosomes, the main RNA molecule is the spliced-leader (SL) RNA, which donates the SL sequence to all messenger RNA through trans-splicing. Here, we show that RNA interference silencing of the SRP receptor (SRalpha) in Trypanosoma brucei caused the accumulation of SRP on ribosomes and triggered silencing of SL RNA (SLS). SLS was elicited due to the failure of the SL RNA-specific transcription factor tSNAP42 to bind to its promoter. SL RNA reduction, in turn, eliminated mRNA processing and resulted in a significant reduction of all mRNA tested. SLS was also induced under pH stress and might function as a master regulator in trypanosomes. SLS is reminiscent of, but distinct from, the unfolded protein response and can potentially act as a new target for parasite eradication.
    EMBO Reports 05/2007; 8(4):408-13. · 7.19 Impact Factor
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    ABSTRACT: In eukaryotes the seven Sm core proteins bind to U1, U2, U4, and U5 snRNAs. In Trypanosoma brucei, Sm proteins have been implicated in binding both spliced leader (SL) and U snRNAs. In this study, we examined the function of these Sm proteins using RNAi silencing and protein purification. RNAi silencing of each of the seven Sm genes resulted in accumulation of SL RNA as well as reduction of several U snRNAs. Interestingly, U2 was unaffected by the loss of SmB, and both U2 and U4 snRNAs were unaffected by the loss of SmD3, suggesting that these snRNAs are not bound by the heptameric Sm complex that binds to U1, U5, and SL RNA. RNAi silencing and protein purification showed that U2 and U4 snRNAs were bound by a unique set of Sm proteins that we termed SSm (specific spliceosomal Sm proteins). This is the first study that identifies specific core Sm proteins that bind only to a subset of spliceosomal snRNAs.
    RNA 02/2007; 13(1):30-43. · 5.09 Impact Factor
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    Anish Das, Hong Li, Tong Liu, Vivian Bellofatto
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    ABSTRACT: In Trypanosoma brucei, transcription by RNA polymerase II accounts for the expression of the spliced leader (SL) RNA and most protein coding mRNAs. To understand the regulation of RNA polymerase II transcription in these parasites, we have purified a transcriptionally active enzyme through affinity chromatography of its essential subunit, RPB4. The enzyme preparation is active in both promoter-independent and promoter-dependent in vitro transcription assays. Importantly, the enzyme is sensitive to alpha-amanitin inhibition, a hallmark of eukaryotic RNA polymerase II enzymes. Using mass spectrometric analysis we have identified the previously unobserved RPB12 subunit of T. brucei RNA polymerase II. TbRPB12 contains a conserved CX(2)CX(10-15)CX(2)C zinc binding motif that is characteristic of other eukaryotic RPB12 polypeptides. We also identified seven proteins that associate with T. brucei RNA polymerase II. While both bioinformatics and biochemical analysis have focused on the subunit structure of trypanosome RNA polymerases, this is the first study that reveals a functional RNA polymerase II enzyme.
    Molecular and Biochemical Parasitology 01/2007; 150(2):201-10. · 2.73 Impact Factor
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    Jennifer B Palenchar, Vivian Bellofatto
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    ABSTRACT: Trypanosoma brucei and the other members of the trypanosomatid family of parasitic protozoa, contain an unusual RNA polymerase II enzyme, uncoordinated mRNA 5' capping and transcription initiation events, and most likely contain an abridged set of transcription factors. Pre-mRNA start sites remain elusive. In addition, two important life cycle stage-specific mRNAs are transcribed by RNA polymerase I. This review interprets these unusual transcription traits in the context of parasite biology.
    Molecular and Biochemical Parasitology 05/2006; 146(2):135-41. · 2.73 Impact Factor
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    ABSTRACT: Transcription by RNA polymerase II in trypanosomes deviates from the standard eukaryotic paradigm. Genes are transcribed polycistronically and subsequently cleaved into functional mRNAs, requiring trans splicing of a capped 39-nucleotide leader RNA derived from a short transcript, the spliced leader (SL) RNA. The only identified trypanosome RNA polymerase II promoter is that of the SL RNA gene. We have previously shown that transcription of SL RNA requires divergent trypanosome homologs of RNA polymerase II, TATA binding protein, and the small nuclear RNA (snRNA)-activating protein complex. In other eukaryotes, TFIIB is an additional key component of transcription for both mRNAs and polymerase II-dependent snRNAs. We have identified a divergent homolog of the usually highly conserved basal transcription factor, TFIIB, from the pathogenic parasite Trypanosoma brucei. T. brucei TFIIB (TbTFIIB) interacted directly with the trypanosome TATA binding protein and RNA polymerase II, confirming its identity. Functionally, in vitro transcription studies demonstrated that TbTFIIB is indispensable in SL RNA gene transcription. RNA interference (RNAi) studies corroborated the essential nature of TbTFIIB, as depletion of this protein led to growth arrest of parasites. Furthermore, nuclear extracts prepared from parasites depleted of TbTFIIB, after the induction of RNAi, required recombinant TbTFIIB to support spliced leader transcription. The information gleaned from TbTFIIB studies furthers our understanding of SL RNA gene transcription and the elusive overall transcriptional processes in trypanosomes.
    Eukaryotic Cell 03/2006; 5(2):293-300. · 3.59 Impact Factor
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    ABSTRACT: In this paper we present RADAR, a web-based toolkit for RNA data analysis and research. The toolkit is capable of performing database search, multiple structure alignment, and pairwise structure comparison. In addition, RADAR provides two salient features: (1) constrained alignment of RNA secondary structures, and (2) prediction of the consensus for a set of RNA secondary structures. RADAR assists scientists in performing many important RNA mining operations, including understanding the functionality of RNA sequences, the detection of structural RNA motifs and the clustering of RNA molecules, among others. The toolkit is fully operational and accessible on the Web at http://datalab.njit.edu/biodata/rna/RSmatch/server.htm
    Sixth IEEE International Symposium on BioInformatics and BioEngineering (BIBE 2006), 16-18 October 2006, Arlington, Virginia, USA; 01/2006

Publication Stats

990 Citations
291.49 Total Impact Points

Institutions

  • 1994–2012
    • Rutgers New Jersey Medical School
      • • Department of Microbiology and Molecular Genetics
      • • Department of Microbiology and Molecular Genetics (RWJ Medical School)
      Newark, New Jersey, United States
  • 2006
    • Rutgers, The State University of New Jersey
      New Brunswick, New Jersey, United States
  • 2003
    • Netherlands Cancer Institute
      Amsterdamo, North Holland, Netherlands
  • 1987–1994
    • The Rockefeller University
      New York City, New York, United States