Vivian Bellofatto

Trypanosomal TBP Functions with the Multisubunit Transcription Factor tSNAP To Direct Spliced-Leader RNA Gene Expression

ABSTRACT Protein-coding genes of trypanosomes are mainly transcribed polycistronically and cleaved into functional mRNAs in a process that requires trans splicing of a capped 39-nucleotide RNA derived from a short transcript, the spliced-leader (SL) RNA. SL RNA genes are individually transcribed from the only identified trypanosome RNA polymerase II promoter. We have purified and characterized a sequence-specific SL RNA promoter-binding complex, tSNAPc, from the pathogenic parasite Trypanosoma brucei, which induces robust transcriptional activity within the SL RNA gene. Two tSNAPc subunits resemble essential components of the metazoan transcription factor SNAPc, which directs small nuclear RNA transcription. A third subunit is unrelated to any eukaryotic protein and identifies tSNAPc as a unique trypanosomal transcription factor. Intriguingly, the unusual trypanosome TATA-binding protein (TBP) tightly associates with tSNAPc and is essential for SL RNA gene transcription. These findings provide the first view of the architecture of a transcriptional complex that assembles at an RNA polymerase II-dependent gene promoter in a highly divergent eukaryote.

Spliced‐leader RNA silencing: a novel stress‐induced mechanism in Trypanosoma brucei

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 (SRα) 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.

RNA polymerase II-dependent transcription in trypanosomes is associated with a SNAP complex-like transcription factor

Spliced leader RNA transcription is essential for cell viability in trypanosomes. The SL RNA genes are expressed from the only defined RNA polymerase II-dependent promoter identified to date in the trypanosome genome. The SL RNA gene promoter has been shown by in vitro and in vivo analyses to have a tripartite architecture. The upstream most cis-acting element, called PBP-1E, is located between 70 and 60 bp upstream from the transcription start site. This essential element functions along with two downstream elements to direct efficient and proper initiation of transcription. Electrophoretic mobility-shift studies detected a 122-kDa protein, called PBP-1, which interacts with PBP-1E. This protein is the first sequence-specific, double-stranded DNA-binding protein isolated in trypanosomes. Three polypeptides copurify with PBP-1 activity, suggesting that PBP-1 is composed of 57-, 46-, and 36-kDa subunits. We have cloned the genes that encode the 57- and 46-kDa subunits. The 46-kDa protein is a previously uncharacterized protein and may be unique to trypanosomes. Its predicted tertiary structure suggests it binds DNA as part of a complex. The 57-kDa subunit is orthologous to the human small nuclear RNA-activating protein (SNAP)50, which is an essential subunit of the SNAP complex (SNAPc). In human cells, SNAPc binds to the proximal sequence element in both RNA polymerase II- and III-dependent small nuclear RNA gene promoters. These findings identify a surprising link in the transcriptional machinery across a large evolutionary distance in the regulation of small nuclear RNA genes in eukaryotes.

Transcription Initiation at the TATA-less Spliced Leader RNA Gene Promoter Requires at Least Two DNA-binding Proteins and a Tripartite Architecture That Includes an Initiator Element

Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the “universal” transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m7G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical double-stranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (+1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans,Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence–TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.

A Divergent Transcription Factor TFIIB in Trypanosomes Is Required for RNA Polymerase II-Dependent Spliced Leader RNA Transcription and Cell Viability

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.

Essential components of the mini-exon gene promoter in the trypanosomatid Leptomonas seymouri.

In members of the Trypanosomatidae family of parasitic protozoa, the mini-exon (MX) genes encode the mini-exon donor RNA (medRNA) that contributes a small, 39-nt exon to all pre-mRNAs during mRNA maturation. Previously we have shown that a single copy of a MX gene can be expressed continuously from a stable episome transfected into the monogenetic trypanosomatid Leptomonas seymouri. We now identify components of the MX gene promoter. A series of 10-bp block substitution mutations in a tagged MX gene were transfected into Leptomonas on an episomal vector. Expression of tagged and endogenous medRNA was assessed in stably transformed clonal cell populations. Results show that less than half of the 757-bp MX gene is necessary for medRNA transcription and that the key components of the MX gene promoter lie within the proximal 70-bp sequence upstream from the transcription initiation site. Transcription requires several sequence-specific blocks within this 70-bp region. Leptomonas cell extracts contain protein(s) that appear to interact with a subset of these sequences in gel mobility shift assays. All trypanosomatid MX genes contain an AT-rich region at the +10 to +20 position within the transcribed region of the MX gene. Mutagenesis of this region within an episomal copy of the MX gene did not block tagged medRNA synthesis but did cause a 10-fold increase in the steady-state amount of endogenous medRNA.

The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism

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 RBP42s function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.

CHARACTERIZATION OF TWO PROTEIN ACTIVITIES THAT INTERACT AT THE PROMOTER OF THE TRYPANOSOMATID SPLICED LEADER RNA

All trypanosome mRNAs have a spliced leader (SL). The SL RNA gene in Leptomonas seymouri is a member of the small nuclear RNA gene family. However, the SL RNA is required in stoichiometric amounts for trans-splicing during mRNA formation. Expression of the SL RNA gene requires sequence elements at bp −60 to −70 and bp −30 to −40 upstream from the transcription initiation site. Using conventional and affinity chromatography, we have identified and characterized an ∼122-kDa protein, promoter-binding protein (PBP) −1, that binds to double-strand DNA. The PBP-1-binding site is within the bp −60 to −70 element determined by DNase I footprinting. Therefore, PBP-1 is the first characterized double-strand DNA binding activity that interacts with a trypanosome gene promoter. A second protein, PBP-2, interacts with the PBP-1:DNA complex and its DNase I footprint extends to include the second promoter element (bp −30 to −40). An alteration of the spacing between the two promoter elements or mutation of the second element decreases PBP-2/PBP-1:DNA stability. Taken together, these data suggest that PBP-1 and PBP-2 are components of a transcription initiation complex that assembles within the SL RNA gene promoter.

RNA Polymerase Transcription Machinery in Trypanosomes

Transcription is a fundamental biological process employed by all living organisms to decode their genetic information. The information stored in genomic DNA is copied into RNA molecules by polymerization of ribonucleotide building blocks, which ultimately gives rise to different classes of transcripts. mRNAs encode polypeptides, rRNAs drive the macromolecular protein-synthesis machinery, and tRNAs act as adaptor molecules to assemble amino acids into proteins. Synthesis of specific transcripts is influenced by environmental and internal cell signals, which in turn are pivotal for the control of cellular regulatory networks. Trypanosomes are unicellular parasitic protozoa, members of the order Kinetoplastidae, which diverged early during evolution. They cause a wide range of debilitating diseases in humans and domestic animals. Trypanosoma brucei, known as the African trypanosome, is transmitted by tsetse flies in subSaharan Africa (15). Infection fulminates into African sleeping sickness in humans and nagana in animals (3). T. brucei is a digenetic parasite that cycles as a procyclic form in the digestive tract of the tsetse vector and as an extracellular bloodstream form in its mammalian host. During its complex life cycle, the parasite passes through five successive morphologically distinct forms (39). Parasites change from the procyclic form, which is characterized by a procyclic-specific surface coat, through two morphologically distinct forms in the fly and then they emerge as long, slender bloodstream forms, covered with a variant surface glycoprotein coat. Once inside the mammalian host, the long slender bloodstream form actively divides and establishes parasitemia. In the late phases of infection, the morphology of the parasite changes to nondividing short stumpy forms, which are ready to be taken up by the insect during a blood meal. The bloodstream form, with a rudimentary mitochondrion, is perfectly adapted to utilize the abundant supply of glucose from the mammalian blood and generate sufficient energy by glycolysis. The insect form, on the other hand, has a functional mitochondrion and generates most of its energy by respiration. These necessary metabolic adaptations depend upon a precise orchestration of numerous metabolic and cell biological activities. Studies of trypanosomes have uncovered several unusual biological phenomena (6). Notable among them are trans splicing and RNA editing (reviewed in references 7, 35, 46, 48, and 59). Protein-coding genes in trypanosomes are transcribed as long polycistronic precursor RNAs. Individual mature mRNAs are formed by trans splicing of a 39-nucleotide spliced leader (SL) RNA at the 5 end and subsequent 3 end maturation. RNA editing, used to produce mitochondrial mRNA, requires extensive alterations of primary transcripts by guide RNAs. Although guide RNA-dependent RNA editing is uniquely observed in trypanosomes, trans splicing has subsequently been observed in several other lower eukaryotes, including nematodes, trematodes, euglenoids, and chordates. Therefore, studies of trypanosomes are expected to uncover cryptic mechanistic components of eukaryotic biology and reveal exotic cellular processes.

RADAR: a web server for RNA data analysis and research

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/