Trypanosoma brucei TBRGG1, a Mitochondrial Oligo(U)-binding Protein That Co-localizes with an in VitroRNA Editing Activity

Abstract

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV crosslinking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780 -786).Trypanosomes are primitive eukaryotes whose parasitic life cycle involves the differentiation into several successive adaptive forms in different hosts and environments. The main developmental stages are the bloodstream form in the mammalian host and the procyclic form in the tsetse fly vector. Each of these stages is characterized by a major surface protein, the variant surface glycoprotein in the bloodstream form and procyclin in the procyclic form (1, 2). Another major difference between these forms is the energy metabolism. Bloodstream forms possess an inactive mitochondrion and respire through the catabolism of glucose in specialized organelles termed glycosomes, whereas procyclic forms utilize a fully functional mitochondrion for oxidative phosphorylation, and amino acids probably serve as the major carbon source in vivo (3). In the bloodstream, the mitochondrion is reactivated when the trypanosomes differentiate from the proliferative slender form into the quiescent stumpy form through several intermediate stages.The nuclear genome of these organisms appears to be organized in long polycistronic transcription units and probably contains only a few promoters (4 -7). The expression of many genes analyzed so far appears to be stage-specific and reflects the developmental stage of the parasite. Interestingly, genes belonging to the same transcription unit are often differentially stage-regulated, indicating that post-transcriptional processes operating at the levels of RNA maturation, stability, and translation are primarily responsible for controlling cellular differentiation (4 -7). The primary polycistronic transcripts of trypanosomes are rapidly processed into mature mRNAs by trans-splicing and polyadenylation. These processing events appear to be coupled, the choice of a polyadenylation site being apparently dictated by the position of the downstream splice site, probably through the scanning of transcripts by a multifactorial complex enc...

mRNA maturation by two-step trans-splicing/polyadenylation processing in trypanosomes

“…Previously, two dicistrons encoding RBPs have been reported in African trypanosomes, although it is not known whether they can be further processed (8,32). Moreover, we found in the literature that several protein-coding genes related to mRNA metabolism in trypanosomes are expressed as transcripts longer than expected from the prediction of their cDNA length, e.g., poly(A)-binding protein 1 (PABP1) and TSR1 (see SI Table 2).…”

A tale of two TUTases

“…The nature of the RNA linker is unknown, but bound gRNA is a likely candidate. This model is almost certainly incomplete, because there are a number of proteins, such as the putative RNA helicase, mHEL61 (21), the mRNA-binding protein, REAP1 (22), and the oligo[U]-binding proteins RBP16 (23), and TBRGG1 (24), which are also possibly involved in editing. …”

A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

“…Plasmids encoding other genes suggested to be involved in trypanosome RNA editing were also included. These are MRP1 and MRP2 (37), mHel61 (38), RBP16 (39), REAP-1 (40), TbRGG1 (16,41), and KRET1 (7,42). The plasmid pools were partially digested with five restriction enzymes that recognize 4-bp sequences, all generating 5Ј-CG overhangs (AciI, HinPII, MaeII, MspI, and TaqI), and fragments ranging up to 2 kb were gel-isolated and ligated into ClaI-digested yeast two-hybrid plasmids pGAD-C1, -C2, and -C3 and pGBD-C1, -C2, and -C3, which are identical except for the translational reading frame of the polylinker region (36).…”