Dynamics of Mitochondrial RNA-Binding Protein Complex in Trypanosoma brucei and Its Petite Mutant under Optimized Immobilization Conditions

ABSTRACT There are a variety of complex metabolic processes ongoing simultaneously in the single, large mitochondrion of Trypanosoma brucei . Understanding the organellar environment and dynamics of mitochondrial proteins requires quantitative measurement in vivo . In this study, we have validated a method for immobilizing both procyclic stage (PS) and bloodstream stage (BS) T. brucei brucei with a high level of cell viability over several hours and verified its suitability for undertaking fluorescence recovery after photobleaching (FRAP), with mitochondrion-targeted yellow fluorescent protein (YFP). Next, we used this method for comparative analysis of the translational diffusion of mitochondrial RNA-binding protein 1 (MRP1) in the BS and in T. b. evansi . The latter flagellate is like petite mutant Saccharomyces cerevisiae because it lacks organelle-encoded nucleic acids. FRAP measurement of YFP-tagged MRP1 in both cell lines illuminated from a new perspective how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism. This work represents the first attempt to examine this process in live trypanosomes.

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Characterization of MRP RNA–protein interactions within the perinucleolar compartment

Molecular Biology of the Cell ◽

10.1091/mbc.e10-09-0768 ◽

2011 ◽

Vol 22 (6) ◽

pp. 858-866 ◽

Cited By ~ 15

Author(s):

Callie Pollock ◽

Kelly Daily ◽

Van Trung Nguyen ◽

Chen Wang ◽

Marzena Anna Lewandowska ◽

...

Keyword(s):

Cancer Cells ◽

Rna Binding ◽

Fluorescent Protein ◽

Gene Expression Regulation ◽

Rna Binding Proteins ◽

Binding Protein ◽

Mitochondrial Rna ◽

Human Telomerase Reverse Transcriptase ◽

Perinucleolar Compartment

The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA–processing (MRP) RNA, pyrimidine tract–binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA–containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)–PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA–protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC.

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An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.00232-14 ◽

2014 ◽

Vol 14 (2) ◽

pp. 149-157 ◽

Cited By ~ 7

Author(s):

Natalie M. McAdams ◽

Michelle L. Ammerman ◽

Julee Nanduri ◽

Kaylen Lott ◽

John C. Fisk ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Rna Binding ◽

Mitochondrial Gene ◽

Mitochondrial Rna ◽

Insect Vector ◽

Sequence Specificity ◽

Content Type

ABSTRACT In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo , inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei . TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.

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Trypanosoma brucei RRM1 Is a Nuclear RNA-Binding Protein and Modulator of Chromatin Structure

mBio ◽

10.1128/mbio.00114-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 20

Author(s):

Arunasalam Naguleswaran ◽

Kapila Gunasekera ◽

Bernd Schimanski ◽

Manfred Heller ◽

Andrew Hemphill ◽

...

Keyword(s):

Gene Expression ◽

Rna Interference ◽

Heat Shock ◽

Trypanosoma Brucei ◽

Chromatin Structure ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Content Type ◽

Nuclear Rna

ABSTRACT TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure. IMPORTANCE Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.

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LRP130, a Pentatricopeptide Motif Protein with a Noncanonical RNA-Binding Domain, Is Bound In Vivo to Mitochondrial and Nuclear RNAs

Molecular and Cellular Biology ◽

10.1128/mcb.23.14.4972-4982.2003 ◽

2003 ◽

Vol 23 (14) ◽

pp. 4972-4982 ◽

Cited By ~ 107

Author(s):

Stavroula Mili ◽

Serafín Piñol-Roma

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Mitochondrial Gene ◽

Rna Binding Protein ◽

Mitochondrial Rna ◽

Potential Candidate ◽

Pentatricopeptide Repeat ◽

Oxidase Deficiency

ABSTRACT LRP130 (also known as LRPPRC) is an RNA-binding protein that is a constituent of postsplicing nuclear RNP complexes associated with mature mRNA. It belongs to a growing family of pentatricopeptide repeat (PPR) motif-containing proteins, several of which have been implicated in organellar RNA metabolism. We show here that only a fraction of LRP130 proteins are in nuclei and are directly bound in vivo to at least some of the same RNA molecules as the nucleocytoplasmic shuttle protein hnRNP A1. The majority of LRP130 proteins are located within mitochondria, where they are directly bound to polyadenylated RNAs in vivo. In vitro, LRP130 binds preferentially to polypyrimidines. This RNA-binding activity maps to a domain in its C-terminal region that does not contain any previously described RNA-binding motifs and that contains only 2 of the 11 predicted PPR motifs. Therefore, LRP130 is a novel type of RNA-binding protein that associates with both nuclear and mitochondrial mRNAs and as such is a potential candidate for coordinating nuclear and mitochondrial gene expression. These findings provide the first identification of a mammalian protein directly bound to mitochondrial RNA in vivo and provide a possible molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.

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Faculty Opinions recommendation of Developmental progression to infectivity in Trypanosoma brucei triggered by an RNA-binding protein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717967962.793469395 ◽

2013 ◽

Author(s):

Gloria Rudenko

Keyword(s):

Trypanosoma Brucei ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Developmental Progression

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Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

Nucleic Acids Research ◽

10.1093/nar/gkaa144 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4507-4520 ◽

Cited By ~ 6

Author(s):

Smriti Pandey ◽

Chandra M Gravel ◽

Oliver M Stockert ◽

Clara D Wang ◽

Courtney L Hegner ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Site Directed Mutagenesis ◽

Genetic Identification ◽

Messenger Rnas ◽

Functional Surface

Abstract The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5′ and 3′ untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ’s interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ’s NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.

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A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014346 ◽

2020 ◽

Vol 295 (42) ◽

pp. 14291-14304

Author(s):

Kathrin Bajak ◽

Kevin Leiss ◽

Christine Clayton ◽

Esteban Erben

Keyword(s):

Gene Expression ◽

Trypanosoma Brucei ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Affinity Purification ◽

Critical Role ◽

Rna Binding Protein ◽

Mrna Translation

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5′-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.

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