Roles for TbDSS-1 in RNA surveillance and decay of maturation by-products from the 12S rRNA locus

The Trypanosoma brucei exoribonuclease, TbDSS-1, has been implicated in multiple aspects of mitochondrial RNA metabolism. Here, we investigate the role of TbDSS-1 in RNA processing and surveillance by analyzing 12S rRNA processing intermediates in TbDSS-1 RNAi cells. RNA fragments corresponding to leader sequence upstream of 12S rRNA accumulate upon TbDSS-1 depletion. The 5′ extremity of 12S rRNA is generated by endonucleolytic cleavage, and TbDSS-1 degrades resulting upstream maturation by-products. RNAs with 5′ ends at position −141 and 3′ ends adjacent to the mature 5′ end of 12S rRNA are common and invariably possess oligo(U) tails. 12S rRNAs with mature 3′ ends and unprocessed 5′ ends also accumulate in TbDSS-1 depleted cells, suggesting that these RNAs represent dead-end products normally destined for decay by TbDSS-1 in an RNA surveillance pathway. Together, these data indicate dual roles for TbDSS-1 in degradation of 12S rRNA maturation by-products and as part of a mitochondrial RNA surveillance pathway that eliminates stalled 12S processing intermediates. We further provide evidence that TbDSS-1 degrades RNAs originating upstream of the first gene on the minor strand of the mitochondrial maxicircle suggesting that TbDSS-1 also removes non-functional RNAs generated from other regions of the mitochondrial genome

Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs

Polyadenylation of RNAs plays a critical role in modulating rates of RNA turnover and ultimately in controlling gene expression in all systems examined to date. In mitochondria, the precise mechanisms by which RNAs are degraded, including the role of polyadenylation, are not well understood. Our previous in organello pulse-chase experiments suggest that poly(A) tails stimulate degradation of mRNAs in the mitochondria of the protozoan parasite Trypanosoma brucei (Militello, K. T., and Read, L. K. (2000) Mol. Cell. Biol. 21, 731-742). In this report, we developed an in vitro assay to directly examine the effects of specific 3′-sequences on RNA degradation. We found that a salt-extracted mitochondrial membrane fraction preferentially degraded polyadenylated mitochondrially and non-mitochondrially encoded RNAs over their non-adenylated counterparts. A poly(A) tail as short as 5 nucleotides was sufficient to stimulate rapid degradation, although an in vivo tail length of 20 adenosines supported the most rapid decay. A poly(U) extension did not promote rapid RNA degradation, and RNA turnover was slowed by the addition of uridine residues to the poly(A) tail. To stimulate degradation, the poly(A) element must be located at the 3′ terminus of the RNA. Finally, we demonstrate that degradation of polyadenylated RNAs occurs in the 3′ to 5′ direction through the action of a hydrolytic exonuclease. These experiments demonstrate that the poly(A) tail can act as a cis-acting element to facilitate degradation of T. brucei mitochondrial mRNAs

Additive and Transcript-Specific Effects of KPAP1 and TbRND Activities on 3′ Non-Encoded Tail Characteristics and mRNA Stability in Trypanosoma brucei

Short, non-encoded oligo(A), oligo(U), or A/U tails can impact mRNA stability in kinetoplastid mitochondria. However, a comprehensive picture of the relative effects of these modifications in RNA stability is lacking. Furthermore, while the U-preferring exoribonuclease TbRND acts on U-tailed gRNAs, its role in decay of uridylated mRNAs has only been cursorily investigated. Here, we analyzed the roles of mRNA 3′ tail composition and TbRND in RNA decay using cells harbouring single or double knockdown of TbRND and the KPAP1 poly(A) polymerase. Analysis of mRNA abundance and tail composition reveals dramatic and transcript-specific effects of adenylation and uridylation on mitochondrial RNAs. Oligo(A) and A-rich tails can stabilize a proportion of edited and never-edited RNAs. However, non-tailed RNAs are not inherently unstable, implicating additional stability determinants and/or spatial segregation of sub-populations of a given RNA in regulation of RNA decay. Oligo(U) tails, which have been shown to contribute to decay of some never-edited RNAs, are not universally destabilizing. We also show that RNAs display very different susceptibility to uridylation in the absence of KPAP1, a factor that may contribute to regulation of decay. Finally, 3′ tail composition apparently impacts the ability of an RNA to be edited

Developmental dynamics of mitochondrial mRNA abundance and editing reveal roles for temperature and the differentiation-repressive kinase RDK1 in cytochrome oxidase subunit II mRNA editing.

Developmental regulation of mitochondrial uridine insertion/deletion editing in Trypanosoma brucei is necessary to modulate parasite metabolism as it shifts from dependence on glycolysis for ATP production in the mammalian bloodstream form (BSF) to oxidative phosphorylation in the insect procyclic form (PCF). However, the timing and stimuli that regulate mRNA editing have been poorly characterized. Here, we utilized a pleomorphic T. brucei strain and quantitative RT-PCR and droplet digital PCR analyses to evaluate the changes in total mRNA abundance and editing as parasites progressively differentiate from slender BSF to PCF and investigate the effect of individual stimuli on mitochondrial gene expression. We observed little change during the slender-to-stumpy BSF transition. Rather, we found that mainly the mitochondrial cytochrome (COI, COII, COIII, and CYb) mRNAs are upregulated within 24 h after stumpy BSF is stimulated to differentiate to PCF in vitro and during in vivo tsetse fly infections. Temperature reduction from 37°C to 27°C is a critical factor for increasing the editing of COII and COIII mRNAs and COIV protein expression but not the editing of CYb mRNA or RISP protein expression. We further demonstrate that the depletion of the differentiation-repressive kinase RDK1 couples with temperature reduction to stimulate COII mRNA editing, and the accessory factor p22 is required for the cold-responsive upregulation of COII mRNA editing. Overall, we show that cytochrome mRNAs are regulated during development by distinct stimuli through a variety of methods to increase their abundance and/or editing. IMPORTANCE Trypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock. The parasite has a complex life cycle consisting of several developmental forms in the human and tsetse fly insect vector. Both the mammalian and insect hosts provide different nutritional environments, so T. brucei must adapt its metabolism to promote its survival and to complete its life cycle. As T. brucei is transmitted from the human host to the fly, the parasite must regulate its mitochondrial gene expression through a process called uridine insertion/deletion editing to achieve mRNAs capable of being translated into functional respiratory chain proteins required for energy production in the insect host. Therefore, it is essential to understand the mechanisms by which T. brucei regulates mitochondrial gene expression during transmission from the mammalian host to the insect vector

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

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.IMPORTANCETrypanosoma 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. Copyright © 2018 Kafková et al

Trypanosoma brucei poly(A) binding protein I cDNA cloning, expression, and binding to 5 untranslated region sequence elements

Abstract Poly(A) binding protein I (PABPI) is a highly conserved eukaryotic protein that binds mRNA poly(A) tails and functions in the regulation of translational efficiency and mRNA stability. As a first step in our investigation of the role(s) of mRNA poly(A) tails in posttranscriptional gene regulation in Trypanosoma brucei, we have cloned the cDNA encoding PABPI from this organism. The cDNA predicts a protein homologous to PABPI from other organisms and displaying conserved features of these proteins, including four RNA binding domains that span the N-terminal two-thirds of the protein. Comparison of northern blot data with the cDNA sequence indicates an unusually long 3% untranslated region (UTR) of approximately three kilobases. The 5% UTR contains both A-rich and AU repeat regions, the former being a ubiquitous property of PABPI 5' UTRs. T. brucei PABPI, expressed as a glutathione-S-transferase fusion protein, bound to RNA comprised of its full length 5% UTR in UV cross-linking experiments. This suggests that PABPI may play an autoregulatory role in its own expression. Competition experiments indicate that the A-rich region, but not the AU repeats, are involved in this binding

Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation

Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes. Biological significance T. brucei is a protozoan parasite that causes lethal African sleeping sickness in humans and nagana in livestock, thereby imposing a significant medical and economic burden on sub-Saharan Africa. The parasite encounters very different environments as it cycles between mammalian and insect hosts, and must exert cellular responses to these varyin

WISE/NEOWISE observations of Active Bodies in the Main Belt

We report results based on mid-infrared photometry of 5 active main belt objects (AMBOs) detected by the Wide-field Infrared Survey Explorer (WISE) spacecraft. Four of these bodies, P/2010 R2 (La Sagra), 133P/Elst-Pizarro, (596) Scheila, and 176P/LINEAR, showed no signs of activity at the time of the observations, allowing the WISE detections to place firm constraints on their diameters and albedos. Geometric albedos were in the range of a few percent, and on the order of other measured comet nuclei. P/2010 A2 was observed on April 2-3, 2010, three months after its peak activity. Photometry of the coma at 12 and 22 {\mu}m combined with ground-based visible-wavelength measurements provides constraints on the dust particle mass distribution (PMD), dlogn/dlogm, yielding power-law slope values of {\alpha} = -0.5 +/- 0.1. This PMD is considerably more shallow than that found for other comets, in particular inbound particle fluence during the Stardust encounter of comet 81P/Wild 2. It is similar to the PMD seen for 9P/Tempel 1 in the immediate aftermath of the Deep Impact experiment. Upper limits for CO2 & CO production are also provided for each AMBO and compared with revised production numbers for WISE observations of 103P/Hartley 2.Comment: 32 Pages, including 5 Figure

Lexis and grammar of mitochondrial RNA processing in Trypanosomes

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover

Architecture of the trypanosome RNA editing accessory complex, MRB1

Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation