Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems. However, since a lack of tRNA genes in T. brucei mitochondria makes it essential to import a complete set from the cytoplasm, we surprisingly found that Q-modified tRNAs are preferentially imported over their unmodified counterparts. In turn, their absence from mitochondria has a pronounced effect on organellar translation and affects function. Although Q modification in T. brucei is globally important for codon selection, it is more so for mitochondrial protein synthesis. These results provide a unique example of the combined regulatory effect of codon usage and wobble modifications on protein synthesis; all driven by tRNA intracellular transport dynamics.

• MeSH
• antikodon genetika   MeSH
• buněčné jádro genetika   ultrastruktura   MeSH
• cytoplazma genetika   ultrastruktura   MeSH
• guanosin genetika   MeSH
• kodon genetika   MeSH
• konformace nukleové kyseliny * MeSH
• mitochondrie genetika   MeSH
• nukleosid Q genetika   MeSH
• posttranskripční úpravy RNA genetika   MeSH
• proteosyntéza genetika   MeSH
• RNA transferová genetika   ultrastruktura   MeSH
• Trypanosoma brucei brucei genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antikodon MeSH
• guanosin MeSH
• kodon MeSH
• nukleosid Q MeSH
• RNA transferová MeSH

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.

• MeSH
• adenosintrifosfatasy genetika   MeSH
• editace RNA MeSH
• fylogeneze MeSH
• genom mitochondriální MeSH
• guide RNA, Kinetoplastida genetika   MeSH
• mitochondrie genetika   MeSH
• podjednotky proteinů genetika   MeSH
• protozoální DNA genetika   MeSH
• RNA protozoální genetika   MeSH
• Trypanosoma lewisi genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• guide RNA, Kinetoplastida MeSH
• podjednotky proteinů MeSH
• protozoální DNA MeSH
• RNA protozoální MeSH

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.

• Klíčová slova
• RNA decay, RNA editing, Trypanosoma, kinetoplast, mitochondria, polyadenylation,
• MeSH
• editace RNA fyziologie   MeSH
• RNA mitochondriální genetika   metabolismus   MeSH
• RNA protozoální genetika   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• RNA mitochondriální MeSH
• RNA protozoální MeSH

Diplonemids are highly abundant heterotrophic marine protists. Previous studies showed that their strikingly bloated mitochondrial genome is unique because of systematic gene fragmentation and manifold RNA editing. Here we report a comparative study of mitochondrial genome architecture, gene structure and RNA editing of six recently isolated, phylogenetically diverse diplonemid species. Mitochondrial gene fragmentation and modes of RNA editing, which include cytidine-to-uridine (C-to-U) and adenosine-to-inosine (A-to-I) substitutions and 3' uridine additions (U-appendage), are conserved across diplonemids. Yet as we show here, all these features have been pushed to their extremes in the Hemistasiidae lineage. For example, Namystynia karyoxenos has its genes fragmented into more than twice as many modules than other diplonemids, with modules as short as four nucleotides. Furthermore, we detected in this group multiple A-appendage and guanosine-to-adenosine (G-to-A) substitution editing events not observed before in diplonemids and found very rarely elsewhere. With >1,000 sites, C-to-U and A-to-I editing in Namystynia is nearly 10 times more frequent than in other diplonemids. The editing density of 12% in coding regions makes Namystynia's the most extensively edited transcriptome described so far. Diplonemid mitochondrial genome architecture, gene structure and post-transcriptional processes display such high complexity that they challenge all other currently known systems.

• MeSH
• chromozomy genetika   MeSH
• editace RNA genetika   MeSH
• Euglenozoa genetika   MeSH
• fylogeneze MeSH
• genom mitochondriální * MeSH
• geny * MeSH
• konzervovaná sekvence MeSH
• mitochondriální DNA genetika   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

Most eukaryotic RNAs are posttranscriptionally modified. The majority of modifications promote RNA maturation, others may regulate function and stability. The 3' terminal non-templated oligouridylation is a widespread modification affecting many cellular RNAs at some stage of their life cycle. It has diverse roles in RNA metabolism. The most prevalent is the regulation of stability and quality control. On the cellular and organismal level, it plays a critical role in a number of pathways, such as cell cycle regulation, cell death, development or viral infection. Defects in uridylation have been linked to several diseases. This review summarizes the current knowledge about the role of the 3' terminal oligo(U)-tailing in biology of various RNAs in eukaryotes and describes key factors involved in these pathways.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

• Klíčová slova
• RNA degradation, RNA modification, RNA processing, RNA surveillance, RNA uridylation, tutase,
• MeSH
• Eukaryota MeSH
• eukaryotické buňky fyziologie   MeSH
• lidé MeSH
• RNA metabolismus   MeSH
• úpravy 3' konce RNA * MeSH
• uridin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• RNA MeSH
• uridin MeSH

UNLABELLED: 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.

• MeSH
• editace RNA * MeSH
• mitochondrie metabolismus   MeSH
• prekurzory RNA metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• prekurzory RNA MeSH
• protozoální proteiny MeSH

RNA editing, which adds sequence information to RNAs post-transcriptionally, is a widespread phenomenon throughout eukaryotes. The most complex form of this process is the uridine (U) insertion/deletion editing that occurs in the mitochondria of kinetoplastid protists. RNA editing in these flagellates is specified by trans-acting guide RNAs and entails the insertion of hundreds and deletion of dozens of U residues from mitochondrial RNAs to produce mature, translatable mRNAs. An emerging model indicates that the machinery required for trypanosome RNA editing is much more complicated than previously appreciated. A family of RNA editing core complexes (RECCs), which contain the required enzymes and several structural proteins, catalyze cycles of U insertion and deletion. A second, dynamic multiprotein complex, the Mitochondrial RNA Binding 1 (MRB1) complex, has recently come to light as another essential component of the trypanosome RNA editing machinery. MRB1 likely serves as the platform for kinetoplastid RNA editing, and plays critical roles in RNA utilization and editing processivity. MRB1 also appears to act as a hub for coordination of RNA editing with additional mitochondrial RNA processing events. This review highlights the current knowledge regarding the complex molecular machinery involved in trypanosome RNA editing. WIREs RNA 2016, 7:33-51. doi: 10.1002/wrna.1313 For further resources related to this article, please visit the WIREs website.

• MeSH
• editace RNA * MeSH
• protozoální proteiny genetika   MeSH
• RNA protozoální genetika   MeSH
• Trypanosoma brucei brucei genetika   MeSH
• uridin genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• protozoální proteiny MeSH
• RNA protozoální MeSH
• uridin MeSH

Trypanosoma brucei is the causative agent of the human and veterinarian diseases African sleeping sickness and nagana. A majority of its mitochondrial-encoded transcripts undergo RNA editing, an essential process of post-transcriptional uridine insertion and deletion to produce translatable mRNA. Besides the well-characterized RNA editing core complex, the mitochondrial RNA-binding 1 (MRB1) complex is one of the key players. It comprises a core complex of about six proteins, guide RNA-associated proteins (GAPs) 1/2, which form a heterotetramer that binds and stabilizes gRNAs, plus MRB5390, MRB3010, and MRB11870, which play roles in initial stages of RNA editing, presumably guided by the first gRNA:mRNA duplex in the case of the latter two proteins. To better understand all functions of the MRB1 complex, we performed a functional analysis of the MRB8620 core subunit, the only one not characterized so far. Here we show that MRB8620 plays a role in RNA editing in both procyclic and bloodstream stages of T. brucei, which reside in the tsetse fly vector and mammalian circulatory system, respectively. While RNAi silencing of MRB8620 does not affect procyclic T. brucei fitness when grown in glucose-containing media, it is somewhat compromised in cells grown in the absence of this carbon source. MRB8620 is crucial for integrity of the MRB1 core, such as its association with GAP1/2, which presumably acts to deliver gRNAs to this complex. In contrast, GAP1/2 is not required for the fabrication of the MRB1 core. Disruption of the MRB1 core assembly is followed by the accumulation of mRNAs associated with GAP1/2.

• Klíčová slova
• RNA editing, mitochondrion, trypanosome,
• MeSH
• buněčné linie MeSH
• editace RNA * MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondriální proteiny fyziologie   MeSH
• mitochondrie MeSH
• protozoální proteiny fyziologie   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• messenger RNA MeSH
• mitochondriální proteiny MeSH
• protozoální proteiny MeSH

UNLABELLED: Perkinsela is an enigmatic early-branching kinetoplastid protist that lives as an obligate endosymbiont inside Paramoeba (Amoebozoa). We have sequenced the highly reduced mitochondrial genome of Perkinsela, which possesses only six protein-coding genes (cox1, cox2, cox3, cob, atp6, and rps12), despite the fact that the organelle itself contains more DNA than is present in either the host or endosymbiont nuclear genomes. An in silico analysis of two Perkinsela strains showed that mitochondrial RNA editing and processing machineries typical of kinetoplastid flagellates are generally conserved, and all mitochondrial transcripts undergo U-insertion/deletion editing. Canonical kinetoplastid mitochondrial ribosomes are also present. We have developed software tools for accurate and exhaustive mapping of transcriptome sequencing (RNA-seq) reads with extensive U-insertions/deletions, which allows detailed investigation of RNA editing via deep sequencing. With these methods, we show that up to 50% of reads for a given edited region contain errors of the editing system or, less likely, correspond to alternatively edited transcripts. IMPORTANCE: Uridine insertion/deletion-type RNA editing, which occurs in the mitochondrion of kinetoplastid protists, has been well-studied in the model parasite genera Trypanosoma, Leishmania, and Crithidia. Perkinsela provides a unique opportunity to broaden our knowledge of RNA editing machinery from an evolutionary perspective, as it represents the earliest kinetoplastid branch and is an obligatory endosymbiont with extensive reductive trends. Interestingly, up to 50% of mitochondrial transcripts in Perkinsela contain errors. Our study was complemented by use of newly developed software designed for accurate mapping of extensively edited RNA-seq reads obtained by deep sequencing.

• MeSH
• Amoebozoa parazitologie   MeSH
• delece genu * MeSH
• editace RNA * MeSH
• Kinetoplastida genetika   růst a vývoj   MeSH
• mitochondriální DNA chemie   genetika   MeSH
• mitochondrie genetika   MeSH
• sekvenční analýza DNA MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

Our understanding of kinetoplastid RNA (kRNA) editing has centered on this paradigm: guide RNAs (gRNAs) provide a blueprint for uridine insertion/deletion into mitochondrial mRNAs by the RNA editing core complex (RECC). The characterization of constituent subunits of the mitochondrial RNA-binding complex 1 (MRB1) implies that it too is vital to the editing process. The recently elucidated MRB1 architecture will be instrumental in putting functional data from individual subunits into context. Our model depicts two functions for MRB1: mediating multi-round kRNA editing by coordinating the exchange of multiple gRNAs required by RECC to edit lengthy regions of mRNAs, and then linking kRNA editing with other RNA processing events.

• MeSH
• editace RNA * MeSH
• guide RNA, Kinetoplastida metabolismus   MeSH
• Kinetoplastida genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• RNA protozoální genetika   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• uridin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• guide RNA, Kinetoplastida MeSH
• messenger RNA MeSH
• protozoální proteiny MeSH
• RNA protozoální MeSH
• uridin MeSH