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.

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 5Ј end and subsequent 3Ј 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. The...

A unique feature of RNA polymerase II (RNA pol II) is its long C-terminal extension, called the carboxy-terminal domain (CTD). The well-studied eukaryotes possess a tandemly repeated 7-amino-acid sequence, called the canonical CTD, which orchestrates various steps in mRNA synthesis. Many eukaryotes possess a CTD devoid of repeats, appropriately called a non-canonical CTD, which performs completely unknown functions. Trypanosoma brucei, the etiologic agent of African Sleeping Sickness, deploys an RNA pol II that contains a non-canonical CTD to accomplish an unusual transcriptional program; all protein-coding genes are transcribed as part of a polygenic precursor mRNA (pre-mRNA) that is initiated within a several-kilobase-long region, called the transcription start site (TSS), which is upstream of the first protein-coding gene in the polygenic array. In this report, we show that the non-canonical CTD of T. brucei RNA pol II is important for normal protein-coding gene expression, likely directing RNA pol II to the TSSs within the genome. Our work reveals the presence of a primordial CTD code within eukarya and indicates that proper recognition of the chromatin landscape is a central function of this RNA pol II-distinguishing domain.

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.