RBP38, a Novel RNA-Binding Protein from Trypanosomatid Mitochondria, Modulates RNA Stability

Abstract

We describe here the isolation and characterization of a novel RNA-binding protein, RBP38, from Leishmania tarentolae mitochondria. This protein does not contain any known RNA-binding motifs and is highly conserved among the trypanosomatids, but no homologues were found in other organisms. Recombinant LtRBP38 binds single and double-stranded (ds) RNA substrates with dissociation constants in the 100 nM range, as determined by fluorescence polarization analysis. Downregulation of expression of the homologous gene, TbRBP38, in procyclic Trypanosoma brucei by using conditional dsRNA interference resulted in 80% reduction of steady-state levels of RNAs transcribed from both maxicircle and minicircle DNA. In organello pulse-chase labeling experiments were used to determine the stability of RNAs in mitochondria that were depleted of TbRBP38. The half-life of metabolically labeled RNA decreased from ϳ160 to ϳ60 min after depletion. In contrast, there was no change in transcriptional activity. These observations suggest a role of RBP38 in stabilizing mitochondrial RNA.RNA-binding proteins are involved in the synthesis, processing, transport, translation, degradation, and stabilization of RNA (7, 24). The mitochondrial genome of trypanosomatids is composed of thousands of circular DNA molecules, which are catenated and form a dense body of DNA, called the kinetoplast DNA network. This consists of two types of molecules: maxicircles and minicircles. The maxicircle encodes two rRNAs, 18 mRNAs, and a few gRNAs (2). The minicircle encodes the majority of the gRNAs (28). The maxicircle protein coding regions are very compact, and many of the genes overlap extensively, leaving little room for regulatory sequences that might control transcription and RNA processing. It was shown previously that maxicircle transcription produces polycistronic RNAs that are processed to mature RNAs (16,27). The presence of such precursors indicates that the mature RNAs are generated by 3Ј-and 5Ј-end processing, which, for several of the overlapping genes, eliminates a portion of the coding regions of the adjacent transcripts, which then must be degraded. Mitochondrial biogenesis in Trypanosoma brucei is developmentally regulated (23,26,29). In long slender bloodstream forms mitochondrial function is strongly repressed.During differentiation, the mitochondrion is partially activated in the short stumpy bloodstream form, but only in the insect midgut procyclic form is the mitochondrion fully active. This change in physiology is reflected in a stage-specific regulation of mitochondrial gene expression (15, 18). The levels of some mitochondrial transcripts in short stumpy cells are intermediate between the long slender and procyclic forms, whereas other transcripts are already at the procyclic levels. The transcription rates and processing of the rRNA genes are the same in the bloodstream and the procyclic forms, suggesting that the difference in abundance of the mature rRNAs is due to differential stability in the two forms of the life cycle (19). T...

Naphthalene-based RNA Editing Inhibitor Blocks RNA Editing Activities and Editosome Assembly in Trypanosoma brucei

“…This supports our observation that C35 inhibits the formation of high molecular weight RNA-containing editosome complexes. Another candidate target of C35 can be RBP38, a previously identified essential protein that has been shown to be able to bind to both single-stranded and double-stranded RNA and is important for the stability of mitochondrial RNA (50). The properties of this protein match the observation that C35 abolishes the interaction of its target with both single-stranded gRNA and double-stranded gRNA-mRNA complex (Fig.…”

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

“…In contrast, L. tarentolae's natural host is the Gecko lizard (Tarentola mauritanica) (Wenyon, 1921), and this parasite species does not infect humans. Furthermore, L. tarentolae can be cultured to higher cell densities than L. amazonensis; typical achievable cell densities are 1.8 × 10 8 parasites/ml (Sbicego et al, 2003) and 5 × 10 7 parasites/ml (Werbovetz et al, 1999), respectively. In addition, L. amazonensis requires supplemented Schneider's Drosophila medium for high density growth (Werbovetz et al, 1999), whereas L. tarentolae can be grown in Brain Heart Infusion supplemented with hemin (Sbicego et al, 2003), with the latter medium being considerably less expensive.…”

“…Furthermore, L. tarentolae can be cultured to higher cell densities than L. amazonensis; typical achievable cell densities are 1.8 × 10 8 parasites/ml (Sbicego et al, 2003) and 5 × 10 7 parasites/ml (Werbovetz et al, 1999), respectively. In addition, L. amazonensis requires supplemented Schneider's Drosophila medium for high density growth (Werbovetz et al, 1999), whereas L. tarentolae can be grown in Brain Heart Infusion supplemented with hemin (Sbicego et al, 2003), with the latter medium being considerably less expensive. Herein we report the purification and amino acid sequence of tubulin from L. tarentolae, along with a comparison of the effects of selected antimicrotubule agents on tubulin from L. tarentolae and L. amazonensis.…”

TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex

“…However, a recent study of REAP-1 knock-out cells implies a role in RNA stability rather than editing (Hans et al 2007). Although RBP38 was first identified as a protein that binds gRNA-mRNA duplexes (Sbicego et al 2003), its function seems to be in the initiation of kinetoplast DNA replication (Liu et al 2006).…”