Dataset Information

Kinetoplastid Specific RNA-Protein Interactions in Trypanosoma cruzi Ribosome Biogenesis.

ABSTRACT: RNA binding proteins (RBP) play essential roles in the highly conserved and coordinated process of ribosome biogenesis. Our laboratory has previously characterized two essential and abundant RBPs, P34 and P37, in Trypanosoma brucei which are required for several critical steps in ribosome biogenesis. The genes for these proteins have only been identified in kinetoplastid organisms but not in the host genome. We have identified a homolog of the TbP34 and TbP37 in a T. cruzi strain (termed TcP37/NRBD). Although the N-terminal APK-rich domain and RNA recognition motifs are conserved, the C-terminal region which contains putative nuclear and nucleolar localization signals in TbP34 and TbP37 is almost entirely missing from TcP37/NRBD. We have shown that TcP37/NRBD is expressed in T. cruzi epimastigotes at the level of mature mRNA and protein. Despite the loss of the C-terminal domain, TcP37/NRBD is present in the nucleus, including the nucleolus, and the cytoplasm. TcP37/NRBD interacts directly with Tc 5S rRNA, but does not associate with polyadenylated RNA. TcP37/NRBD also associates in vivo and in vitro with large ribosomal protein TcL5 and, unlike the case of T. brucei, this association is strongly enhanced by the presence of 5S rRNA, suggesting that the loss of the C-terminal domain of TcP37/NRBD may alter the interactions within the complex. These results indicate that the unique preribosomal complex comprised of L5, 5S rRNA, and the trypanosome-specific TcP37/NRBD or TbP34 and TbP37 is functionally conserved in trypanosomes despite the differences in the C-termini of the trypanosome-specific protein components.

SUBMITTER: Umaer K

PROVIDER: S-EPMC4488245 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Kinetoplastid Specific RNA-Protein Interactions in Trypanosoma cruzi Ribosome Biogenesis.

Umaer Khan K Williams Noreen N

PloS one 20150629 6

RNA binding proteins (RBP) play essential roles in the highly conserved and coordinated process of ribosome biogenesis. Our laboratory has previously characterized two essential and abundant RBPs, P34 and P37, in Trypanosoma brucei which are required for several critical steps in ribosome biogenesis. The genes for these proteins have only been identified in kinetoplastid organisms but not in the host genome. We have identified a homolog of the TbP34 and TbP37 in a T. cruzi strain (termed TcP37/N ...[more]

PMID: 26121669

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Project description:Eukaryotic ribosome biogenesis is an essential cellular process involving tightly coordinated assembly of multiple rRNA and protein components. Much of our understanding of this pathway has come from studies performed with yeast model systems. These studies have identified critical checkpoints in the maturation of the large ribosomal subunit (LSU/60S), one of which is the proper formation and incorporation of the 5S ribonucleoprotein complex (5S RNP). Research on the 5S RNP has identified a complex containing the four proteins L5, L11, Rpf2, and Rrs1 as well as 5S rRNA. Our laboratory has studied the 5S RNP in Trypanosoma brucei, a eukaryotic parasite, and identified the proteins P34 and P37 as essential, parasite-specific members of this complex. We have additionally identified homologues of L5, Rpf2, Rrs1, and 5S rRNA in T. brucei and characterized their roles in this essential process. In this study, we examined the T. brucei homologue of ribosomal protein L11 as a member of the 5S RNP. We showed that TbL11 is essential and that it is important for proper ribosome subunit formation and 60S rRNA processing. Additionally, we identified TbL11 interactions with TbL5 and TbRpf2, as well as novel interactions with the kinetoplast-specific proteins P34 and P37. These findings expand our understanding of a crucial process outside the context of model yeast organisms and highlight differences in an otherwise highly conserved process that could be used to develop future treatments against T. brucei IMPORTANCE The human-pathogenic, eukaryotic parasite Trypanosoma brucei causes human and animal African trypanosomiases. Treatments for T. brucei suffer from numerous hurdles, including adverse side effects and developing resistance. Ribosome biogenesis is one critical process for T. brucei survival that could be targeted for new drug development. A critical checkpoint in ribosome biogenesis is formation of the 5S RNP, which we have shown involves the trypanosome-specific proteins P34 and P37 as well as homologues of Rpf2, Rrs1, and L5. We have identified parasite-specific characteristics of these proteins and involvement in key parts of ribosome biogenesis, making them candidates for future drug development. In this work, we characterized the T. brucei homologue of ribosomal protein L11. We show that it is essential for parasite survival and is involved in ribosome biogenesis and rRNA processing. Furthermore, we identified novel interactions with P34 and P37, characteristics that make this protein a potential target for novel chemotherapeutics.

Project description:Protein geranylgeranyltransferase type I (PGGT-I) and protein farnesyltransferase (PFT) occur in many eukaryotic cells. Both consist of two subunits, the common alpha subunit and a distinct beta subunit. In the gene database of protozoa Trypanosoma cruzi, the causative agent of Chagas' disease, a putative protein that consists of 401 amino acids with approximately 20% amino acid sequence identity to the PGGT-I beta of other species was identified, cloned, and characterized. Multiple sequence alignments show that the T. cruzi ortholog contains all three of the zinc-binding residues and several residues uniquely conserved in the beta subunit of PGGT-I. Co-expression of this protein and the alpha subunit of T. cruzi PFT in Sf9 insect cells yielded a dimeric protein that forms a tight complex selectively with [(3)H]geranylgeranyl pyrophosphate, indicating a key characteristic of a functional PGGT-I. Recombinant T. cruzi PGGT-I ortholog showed geranylgeranyltransferase activity with distinct specificity toward the C-terminal CaaX motif of protein substrates compared to that of the mammalian PGGT-I and T. cruzi PFT. Most of the CaaX-containing proteins with X=Leu are good substrates of T. cruzi PGGT-I, and those with X=Met are substrates for both T. cruzi PFT and PGGT-I, whereas unlike mammalian PGGT-I, those with X=Phe are poor substrates for T. cruzi PGGT-I. Several candidates for T. cruzi PGGT-I or PFT substrates containing the C-terminal CaaX motif are found in the T. cruzi gene database. Among five C-terminal peptides of those tested, a peptide of a Ras-like protein ending with CVLL was selectively geranylgeranylated by T. cruzi PGGT-I. Other peptides with CTQQ (Tcj2 DNAJ protein), CAVM (TcPRL-1 protein tyrosine phosphatase), CHFM (a small GTPase like protein), and CQLF (TcRho1 GTPase) were specific substrates for T. cruzi PFT but not for PGGT-I. The mRNA and protein of the T. cruzi PGGT-I beta ortholog were detected in three life-cycle stages of T. cruzi. Cytosol fractions from trypomastigotes (infectious mammalian stage) and epimastigotes (insect stage) were shown to contain levels of PGGT-I activity that are approximately 100-fold lower than PFT activity. The CaaX mimetics known as PGGT-I inhibitors show very low potency against T. cruzi PGGT-I compared to the mammalian enzyme, suggesting the potential to develop selective inhibitors against the parasite enzyme.

Dataset Information

Kinetoplastid Specific RNA-Protein Interactions in Trypanosoma cruzi Ribosome Biogenesis.

Publications

Kinetoplastid Specific RNA-Protein Interactions in Trypanosoma cruzi Ribosome Biogenesis.

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