Editosome Accessory Factors KREPB9 and KREPB10 in Trypanosoma brucei

Multiprotein complexes, called editosomes, catalyze the uridine insertion and deletion RNA editing that forms translatable mitochondrial mRNAs in kinetoplastid parasites. We have identified here two new U1-like zinc finger proteins that associate with editosomes and have shown that they are related to KREPB6, KREPB7, and KREPB8, and thus we have named them Kinetoplastid RNA Editing Proteins, KREPB9 and KREPB10. They are conserved and syntenic in trypanosomatids although KREPB10 is absent in Trypanosoma vivax and both are absent in Leishmania. Tandem affinity purification (TAP)-tagged KREPB9 and KREPB10 incorporate into ∼20S editosomes and/or subcomplexes thereof and preferentially associate with deletion subcomplexes, as do KREPB6, KREPB7, and KREPB8. KREPB10 also associates with editosomes that are isolated via a chimeric endonuclease, KREN1 in KREPB8 RNA interference (RNAi) cells, or MEAT1. The purified complexes have precleaved editing activities and endonuclease cleavage activity that appears to leave a 5′ OH on the 3′ product. RNAi knockdowns did not affect growth but resulted in relative reductions of both edited and unedited mitochondrial mRNAs. The similarity of KREPB9 and KREPB10 to KREPB6, KREPB7, and KREPB8 suggests they may be accessory factors that affect editing endonuclease activity and as a consequence may affect mitochondrial mRNA stability. KREPB9 and KREPB10, along with KREPB6, KREPB7, and KREPB8, may enable the endonucleases to discriminate among and accurately cleave hundreds of different editing sites and may be involved in the control of differential editing during the life cycle of T. brucei

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

Novel organization of mitochondrial minicircles and guide RNAs in the zoonotic pathogen Trypanosoma lewisi

Abstract Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented

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

The chicken–egg scenario of protein folding revisited

AbstractWhat is the first step in protein folding – hydrophobic collapse (compaction) or secondary structure formation? It is still not clear if the major driving force in protein folding is hydrogen bonding or hydrophobic interactions or both. We analyzed data on the conformational characteristics of 41 globular proteins in native and partially folded conformational states. Our analysis shows that a good correlation exists between relative decrease in hydrodynamic volume and increase in secondary structure content. No compact equilibrium intermediates lacking secondary structure, or highly ordered non-compact species, were found. This correlation provides experimental support for the hypothesis that hydrophobic collapse occurs simultaneously with formation of secondary structure in the early stages of the protein folding

The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster

The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation

Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing

We want to thank Kathy Kyler for editing this manuscript, Ken Stuart for supplying monoclonal antisera against RECC subunits, and Laurie K. Read for her gift of polyclonal antisera against GAP1 and RGG2. Funding: National Science Foundation Grant No. NSF1122109 (PI: J.Cruz-Reyes.). NIH/National Institute of Allergies and Infectious Diseases R01 AI088011 (PI: Blaine Mooers). Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103640. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.Yeshttp://www.plosone.org/static/editorial#pee