scholarly journals RNA-Binding Proteins and Their Targets in Trypanosoma brucei: Single Nucleotide Resolution Using iCLIP and iCLAP

2020 ◽  
pp. 303-323
Author(s):  
Sameer Dixit ◽  
Juan D. Alfonzo ◽  
Julius Lukeš
Keyword(s):  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals Differential Binding of Mitochondrial Transcripts by MRB8170 and MRB4160 Regulates Distinct Editing Fates of Mitochondrial mRNA in Trypanosomes

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Sameer Dixit ◽  
Michaela Müller-McNicoll ◽  
Vojtěch David ◽  
Kathi Zarnack ◽  
Jernej Ule ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Affinity Purification ◽  
Mitochondrial Rna ◽  
Nucleotide Resolution ◽  
Additional Role

ABSTRACT A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei . Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome. IMPORTANCE Trypanosoma brucei mitochondrial mRNAs undergo maturation by RNA editing, a unique process involving decrypting open reading frames by the precise deletion and/or insertion of uridine (U) residues at specific positions on an mRNA. This process is catalyzed by multiprotein complexes, such as the RNA editing core complex, which provides the enzymatic activities needed for U insertion/deletion at a single editing site. Less well understood is how RNA editing occurs throughout an mRNA bearing multiple sites. To address this question, we mapped at single-nucleotide resolution the RNA interactions of two unique RNA-binding proteins (RBPs). These RBPs are part of the mitochondrial RNA-binding complex 1, hypothesized to mediate multiple rounds of RNA editing. Both RBPs were shown to mark mRNAs for the process in correlation with the number of editing sites on the transcript. Surprisingly, one also binds mRNAs that bypass RNA editing, indicating that it may have an additional role outside RNA editing.

scholarly journals Profiling the binding sites of RNA-binding protein by LACE-seq

2021 ◽  
Author(s):  
Ruibao Su ◽  
Di Wang ◽  
Changchang Cao ◽  
Yuanchao Xue
Keyword(s):  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Transcription Termination ◽  
Early Embryo ◽  
Linear Amplification ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Abstract RNA-binding proteins (RBPs) directly interact with various RNAs in living cells to regulate their processing, translation, and stability. Identifying the precise binding sites of RBPs is critical for appreciating their physiological or pathological roles in germline and early embryo development. Current methods typically need millions of cells to map RBP binding positions, which prevents us from appreciating the crucial role of RBPs in early development. Here, we present the LACE-seq method for unbiased mapping of RBP-binding sites at single-nucleotide resolution in fewer cells or even single oocytes. LACE-seq depends on RBP-mediated reverse transcription termination, and linear amplification of the cDNA ends for deep sequencing. To further promote its application, we describe a step-by-step protocol about how to construct a successful LACE-seq library.

scholarly journals The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-form Trypanosoma brucei

2019 ◽  
Author(s):  
Kathrin Bajak ◽  
Kevin Leiss ◽  
Christine Clayton ◽  
Esteban Erben
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosomal Protein ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Bloodstream Form ◽  
Control Of Gene Expression ◽  
Mrna Binding Proteins ◽  
Mrna Binding

AbstractKinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We here show that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Results from a pull-down of tagged ERBP1 suggest that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.

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

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.

scholarly journals Trypanosoma brucei RNA Binding Proteins p34 and p37 Mediate NOPP44/46 Cellular Localization via the Exportin 1 Nuclear Export Pathway

2007 ◽  
Vol 6 (12) ◽  
pp. 2206-2213 ◽  
Author(s):  
Kristina Hellman ◽  
Kimberly Prohaska ◽  
Noreen Williams
Keyword(s):  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Nuclear Export ◽  
Cellular Localization ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Export Signal ◽  
Amino Acid Sequences ◽  
Protein Levels

ABSTRACT We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.

scholarly journals SEQing: web-based visualization of iCLIP and RNA-seq data in an interactive python framework

2019 ◽  
Author(s):  
Martin Lewinski ◽  
Yannik Bramkamp ◽  
Tino Köster ◽  
Dorothee Staiger
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Seq ◽  
Web Based ◽  
Genome Wide ◽  
Functional Relevance ◽  
Nucleotide Resolution

AbstractBackgroundRNA-binding proteins interact with their target RNAs at specific sites. These binding sites can be determined genome-wide through individual nucleotide resolution crosslinking immunoprecipitation (iCLIP). Subsequently, the binding sites have to be visualized. So far, no visualization tool exists that is easily accessible but also supports restricted access so that data can be shared among collaborators.ResultsHere we present SEQing, a customizable interactive dashboard to visualize crosslink sites on target genes of RNA-binding proteins that have been obtained by iCLIP. Moreover, SEQing supports RNA-seq data that can be displayed in a diffrerent window tab. This allows, e.g. crossreferencing the iCLIP data with genes differentially expressed in mutants of the RBP and thus obtain some insights into a potential functional relevance of the binding sites. Additionally, detailed information on the target genes can be incorporated in another tab.ConclusionSEQing is written in Python3 and runs on Linux. The web-based access makes iCLIP data easily accessible, even with mobile devices. SEQing is customizable in many ways and has also the option to be secured by a password. The source code is available at https://github.com/malewins/SEQing.

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

Open Biology ◽  
2017 ◽  
Vol 7 (4) ◽  
pp. 160159 ◽  
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.

scholarly journals Trypanosoma brucei PRMT1 Is a Nucleic Acid Binding Protein with a Role in Energy Metabolism and the Starvation Stress Response

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Lucie Kafková ◽  
Chengjian Tu ◽  
Kyle L. Pazzo ◽  
Kyle P. Smith ◽  
Erik W. Debler ◽  
...  
Keyword(s):  
Life Cycle ◽  
Energy Metabolism ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Starvation Stress ◽  
Cycle Progression ◽  
Arginine Methyltransferase ◽  
Content Type

ABSTRACT In Trypanosoma brucei and related kinetoplastid parasites, transcription of protein coding genes is largely unregulated. Rather, mRNA binding proteins, which impact processes such as transcript stability and translation efficiency, are the predominant regulators of gene expression. Arginine methylation is a posttranslational modification that preferentially targets RNA binding proteins and is, therefore, likely to have a substantial impact on T. brucei biology. The data presented here demonstrate that cells depleted of T. brucei PRMT1 (TbPRMT1), a major type I protein arginine methyltransferase, exhibit decreased virulence in an animal model. To understand the basis of this phenotype, quantitative global proteomics was employed to measure protein steady-state levels in cells lacking TbPRMT1. The approach revealed striking changes in proteins involved in energy metabolism. Most prominent were a decrease in glycolytic enzyme abundance and an increase in proline degradation pathway components, changes that resemble the metabolic remodeling that occurs during T. brucei life cycle progression. The work describes several RNA binding proteins whose association with mRNA was altered in TbPRMT1-depleted cells, and a large number of TbPRMT1-interacting proteins, thereby highlighting potential TbPRMT1 substrates. Many proteins involved in the T. brucei starvation stress response were found to interact with TbPRMT1, prompting analysis of the response of TbPRMT1-depleted cells to nutrient deprivation. Indeed, depletion of TbPRMT1 strongly hinders the ability of T. brucei to form cytoplasmic mRNA granules under starvation conditions. Finally, this work shows that TbPRMT1 itself binds nucleic acids in vitro and in vivo, a feature completely novel to protein arginine methyltransferases. IMPORTANCE Trypanosoma brucei infection causes human African trypanosomiasis, also known as sleeping sickness, a disease with a nearly 100% fatality rate when untreated. Current drugs are expensive, toxic, and highly impractical to administer, prompting the community to explore various unique aspects of T. brucei biology in search of better treatments. In this study, we identified the protein arginine methyltransferase (PRMT), TbPRMT1, as a factor that modulates numerous aspects of T. brucei biology. These include glycolysis and life cycle progression signaling, both of which are being intensely researched toward identification of potential drug targets. Our data will aid research in those fields. Furthermore, we demonstrate for the first time a direct association of a PRMT with nucleic acids, a finding we believe could translate to other organisms, including humans, thereby impacting research in fields as distant as human cancer biology and immune response modulation.

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