Trypanosoma brucei mitochondrial 

ribonucleoprotein complexes which contain 12S and 9S 

ribosomal RNAs

Antibiotics have been widely used to identify ribosomal activity in Trypanosoma brucei mitochondria. The validity of some of the results has been questioned because the permeability of the trypanosome cell membrane for some antibiotics was not adequately addressed. Here we describe translation inhibition experiments with digitonin-permeabilized trypanosomes to exclude diffusion barriers through the cell membrane. Using this system we were able to confirm, next to the eukaryotic and thus cycloheximide-sensitive translation system, the existence of a prokaryotic-type translational activity being cycloheximide resistant, chloramphenicol sensitive and streptomycin dependent. We interpret this observation analogous to what has been found for other eukarya as the independent protein synthesis activity of the mitochondrial organelle. We further examined the putative translational apparatus by using isokinetic density-gradient analysis of mitochondrial extracts. The 2 mitochondrially encoded rRNAs, the 9S and 12S rRNAs, were found to co-fractionate in a single RNP complex, approximately 80S in size. This complex disassembled at reduced MgCl2 concentrations into 2 unusually small complexes of 17·5S, containing the 9S rRNA, and 20S containing the 12S rRNA. A preliminary stoichiometry determination suggested a multicopy assembly of these putative subunits in a 2[ratio ]3 ratio (20S[ratio ]17·5S).

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Genus-specific recruitment of filovirus ribonucleoprotein complexes into budding particles

Journal of General Virology ◽

10.1099/vir.0.036863-0 ◽

2011 ◽

Vol 92 (12) ◽

pp. 2900-2905 ◽

Cited By ~ 15

Author(s):

Larissa Spiegelberg ◽

Victoria Wahl-Jensen ◽

Larissa Kolesnikova ◽

Heinz Feldmann ◽

Stephan Becker ◽

...

Keyword(s):

Matrix Protein ◽

Spherical Particles ◽

Target Cells ◽

Genome Replication ◽

Replication Competent Virus ◽

Ribonucleoprotein Complexes ◽

Virus Morphogenesis ◽

Rnp Complex ◽

Model Virus ◽

Transcription And Replication

The filoviral matrix protein VP40 orchestrates virus morphogenesis and budding. To do this it interacts with both the glycoprotein (GP1,2) and the ribonucleoprotein (RNP) complex components; however, these interactions are still not well understood. Here we show that for efficient VP40-driven formation of transcription and replication-competent virus-like particles (trVLPs), which contain both an RNP complex and GP1,2, the RNP components and VP40, but not GP1,2 and VP40, must be from the same genus. trVLP preparations contained both spherical and filamentous particles, but only the latter were able to infect target cells and to lead to genome replication and transcription. Interestingly, the genus specificity of the VP40–RNP interactions was specific to the formation of filamentous trVLPs, but not to spherical particles. These results not only further our understanding of VP40 interactions, but also suggest that special care is required when using trVLP or VLP systems to model virus morphogenesis.

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A partially self-regenerating synthetic cell

Nature Communications ◽

10.1038/s41467-020-20180-6 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Barbora Lavickova ◽

Nadanai Laohakunakorn ◽

Sebastian J. Maerkl

Keyword(s):

Resource Competition ◽

Living Systems ◽

Translation System ◽

System Function ◽

Fundamental Function ◽

Dna Templates ◽

Synthetic Cell ◽

Synthesis Activity ◽

Robust System ◽

Protein Components

AbstractSelf-regeneration is a fundamental function of all living systems. Here we demonstrate partial molecular self-regeneration in a synthetic cell. By implementing a minimal transcription-translation system within microfluidic reactors, the system is able to regenerate essential protein components from DNA templates and sustain synthesis activity for over a day. By quantitating genotype-phenotype relationships combined with computational modeling we find that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieve simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.

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An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2)

Microbiology ◽

10.1099/mic.0.26490-0 ◽

2003 ◽

Vol 149 (11) ◽

pp. 3299-3309 ◽

Cited By ~ 39

Author(s):

Yoshiko Okamoto-Hosoya ◽

Takeshi Hosaka ◽

Kozo Ochi

Keyword(s):

Protein Synthesis ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Translation System ◽

In The Wild ◽

Synthesis Activity ◽

Aberrant Protein ◽

Ribosomal Protein S12 ◽

High Level ◽

Actinorhodin Production

Certain mutations in the rpsL gene (encoding the ribosomal protein S12) activate or enhance antibiotic production in various bacteria. K88E and P91S rpsL mutants of Streptomyces coelicolor A3(2), with an enhanced actinorhodin production, were found to exhibit an aberrant protein synthesis activity. While a high level of this activity (as determined by the incorporation of labelled leucine) was detected at the late stationary phase in the mutants, it decreased with age of the cells in the wild-type strain. In addition, the aberrant protein synthesis was particularly pronounced when cells were subjected to amino acid shift-down, and was independent of their ability to accumulate ppGpp. Ribosomes of K88E and P91S mutants displayed an increased accuracy in protein synthesis as demonstrated by the poly(U)-directed cell-free translation system, but so did K43N, K43T, K43R and K88R mutants, which were streptomycin resistant but showed no effect on actinorhodin production. This eliminates the possibility that the increased accuracy level is a cause of the antibiotic overproduction in the K88E and P91S mutants. The K88E and P91S mutant ribosomes exhibited an increased stability of the 70S complex under low concentrations of magnesium. The authors propose that the aberrant activation of protein synthesis caused by the increased stability of the ribosome is responsible for the remarkable enhancement of antibiotic production in the K88E and P91S mutants.

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Translation inhibition during the induction of apoptosis: RNA or protein degradation?

Biochemical Society Transactions ◽

10.1042/bst0320606 ◽

2004 ◽

Vol 32 (4) ◽

pp. 606-610 ◽

Cited By ~ 37

Author(s):

M. Bushell ◽

M. Stoneley ◽

P. Sarnow ◽

A.E. Willis

Keyword(s):

Protein Synthesis ◽

Cellular Level ◽

Translation Inhibition ◽

Initiation Factors ◽

Translation Initiation Factors ◽

Inhibition Of Protein Synthesis ◽

Induction Of Apoptosis ◽

Global Increase ◽

Translational Apparatus ◽

Temporally Correlated

The induction of apoptosis leads to a substantial inhibition of protein synthesis. During this process changes to the translation-initiation factors, the ribosome and the cellular level of mRNA have been documented. However, it is by no means clear which of these events are necessary to achieve translational shutdown. In this article, we discuss modifications to the translational apparatus that occur during apoptosis and examine the potential contributions that they make to the inhibition of protein synthesis. Moreover, we present evidence that suggests that a global increase in the rate of mRNA degradation occurs before the caspase-dependent cleavage of initiation factors. Increased mRNA decay is temporally correlated with the shutdown of translation and therefore plays a major role in the inhibition of protein synthesis in apoptotic cells.

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Isoproterenol induces a ribosomal modification in the heart and the skeletal muscle of hamsters

Bioscience Reports ◽

10.1007/bf01117436 ◽

1985 ◽

Vol 5 (1) ◽

pp. 13-19

Author(s):

Josette Noël ◽

Léa Brakier-Gingras

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Magnesium Concentration ◽

Translation System ◽

Synthesis Activity ◽

And Control

The protein synthesis activity of heart, skeletal muscle and liver polysomes from isoprotenerol-treated and control hamsters has been compared in an in vitro non-inititating translation system. Heart and skeletal muscle polysomes from treated hamsters were less active than controls and required a higher magnesium concentration for optimal protein synthesis. These results suggest that there is a conformational modification in heart and skeletal muscle ribosomes from isoprotenerol-treated hamsters. No such change was observed with ribosomes from the liver of isoproterenol-treated hamsters.

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Trypanosoma brucei Homologue of Regulator of Ribosome Synthesis 1 (Rrs1) Has Direct Interactions with Essential Trypanosome-Specific Proteins

mSphere ◽

10.1128/msphere.00453-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 1

Author(s):

Daniel Jaremko ◽

Martin Ciganda ◽

Noreen Williams

Keyword(s):

Drug Development ◽

Trypanosoma Brucei ◽

Ribosome Biogenesis ◽

5S Rrna ◽

Content Type ◽

Animal African Trypanosomiasis ◽

Rnp Complex ◽

Future Drug ◽

Specific Proteins ◽

Additional Member

ABSTRACT Studies in eukaryotic ribosome biogenesis have largely been performed in yeast, where they have described a highly complex process involving numerous protein and RNA components. Due to the complexity and crucial nature of this process, a number of checkpoints are necessary to ensure that only properly assembled ribosomes are released into the cytoplasm. Assembly of the 5S ribonucleoprotein (RNP) complex is one of these checkpoints for late-stage 60S subunit maturation. Studies in Saccharomyces cerevisiae have identified the 5S rRNA and four proteins, L5, L11, Rpf2, and Rrs1, as comprising the ribosome-associated 5S RNP. Work from our laboratory has shown that in the eukaryotic pathogen Trypanosoma brucei, the 5S RNP includes trypanosome-specific proteins P34/P37, as well as homologues of L5, Rpf2, and 5S rRNA. In this study, we examine a homologue of Rrs1 and identify it as an additional member of the T. brucei 5S RNP. Using RNA interference, we show that TbRrs1 is essential for the survival of T. brucei and has an important role in ribosome subunit formation and, together with TbRpf2, plays a role in 25/28S and 5.8S rRNA processing. We further show that TbRrs1 is a member of the T. brucei 5S RNP through the identification of novel direct interactions with P34/P37 and 5S rRNA as well as with TbL5 and TbRpf2. These unique characteristics of TbRrs1 highlight the importance of studying ribosome biogenesis in the context of diverse organisms and identify interactions that could be targeted for future drug development. IMPORTANCE Trypanosoma brucei is a parasite responsible for human and animal African trypanosomiasis. Current treatments for these diseases have numerous problems, and the development of novel chemotherapeutics can be achieved by identifying targets that are parasite specific and part of essential processes. Ribosome biogenesis is the process of generating translation-competent ribosomes and is critical for survival in all organisms. Work from our laboratory has shown that the formation of the 5S RNP, a crucial checkpoint in ribosome biogenesis, requires trypanosome-specific proteins P34/P37 and homologues of Rpf2 and L5 which possess parasite-specific characteristics. In this study, we characterize TbRrs1, an additional member of the T. brucei 5S RNP, and show that it is essential for parasite survival and has unique interactions with P34/P37 and 5S rRNA. This expands our understanding of the 5S RNP in T. brucei and identifies new targets for future drug development.

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Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1410 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1410-1418 ◽

Cited By ~ 55

Author(s):

R A Corell ◽

L K Read ◽

G R Riley ◽

J K Nellissery ◽

T E Allen ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Uv Treatment ◽

Rna Ligase ◽

Guide Rna ◽

Ribonucleoprotein Complexes ◽

Multicomponent Complex ◽

Editing Activity ◽

Atpase 6

Transcripts from many mitochondrial genes in kinetoplastids undergo RNA editing, a posttranscriptional process which inserts and deletes uridines. By assaying for deletion editing in vitro, we found that the editing activity from Trypanosoma brucei mitochondrial lysates (S.D. Seiwert and K.D. Stuart), Science 266:114-117,1994) sediments with a peak of approximately 20S. RNA helicase, terminal uridylyl transferase, RNA ligase, and adenylation activities, which may have a role in editing, cosediment in a broad distribution, with most of each activity at 35 to 40S. Most ATPase 6 (A6) guide RNA and unedited A6 mRNA sediments at 20 to 30S, with some sedimenting further into the gradient, while most edited A6 mRNA sediments at >35S. Several mitochondrial proteins which cross-link specifically with guide RNA upon UV treatment also sediment in glycerol gradients. Notably, a 65-kDa protein sediments primarily at approximately 20S, a 90-kDa protein sediments at 35 to 40S, and a 25-kDa protein is present at <10S. Most ribonucleoprotein complexes that form with gRNA in vitro sediment at 10 to 20S, except for one, which sediments at 30 to 45S. These results suggest that RNA editing takes place within a multicomponent complex. The potential functions of and relationships between the 20S and 35 to 40S complexes are discussed.

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Characterization of RBP9 and RBP10, two developmentally regulated RNA-binding proteins in Trypanosoma brucei

Open Biology ◽

10.1098/rsob.160159 ◽

2017 ◽

Vol 7 (4) ◽

pp. 160159 ◽

Cited By ~ 4

Author(s):

Luis Miguel De Pablos ◽

Steve Kelly ◽

Janaina de Freitas Nascimento ◽

Jack Sunter ◽

Mark Carrington

Keyword(s):

Trypanosoma Brucei ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Ectopic Expression ◽

Developmentally Regulated ◽

Mrna Metabolism ◽

Ribonucleoprotein Complexes ◽

Processing Body ◽

External Signals

The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.

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