Identification of novel guide RNAs from the mitochondria of<i>Trypanosoma brucei</i>

Madej, M; Niemann, Moritz; Hüttenhofer, Alexander; Göringer, H. Ulrich

doi:10.4161/rna.5.2.6043

Cited by 17 publications

(16 citation statements)

References 29 publications

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“…The majority of gRNAs were identified by comparing edited mRNAs and the corresponding pseudogenes. On the other hand, direct sequencing of short RNAs from mitochondria of Leishmania tarentolae (24) and T. brucei (25,30) showed that gRNAs specifying known editing sites constitute only a fraction of the total short RNA population and that most short RNAs are uridylylated. The two established components of gRNA biogenesis, RET1 and the GRBC1/2 complex, interact in an RNA-dependent manner, and their knockdowns ablate mature gRNAs (4,44).…”

Section: Discussionmentioning

confidence: 99%

RET1-Catalyzed Uridylylation Shapes the Mitochondrial Transcriptome in Trypanosoma brucei

Aphasizheva

Aphasizhev

2010

Molecular and Cellular Biology

157

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RNA uridylylation is critical for the expression of the mitochondrial genome in trypanosomes. Short U tails are added to guide RNAs and rRNAs, while long A/U heteropolymers mark 3 ends of most mRNAs. Three divergent mitochondrial terminal uridylyl transferases (TUTases) are known: RET1 catalyzes guide RNA (gRNA) uridylylation, RET2 executes U insertion mRNA editing, and MEAT1 associates with the editosomelike complex. However, the activities responsible for 3 uridylylation of rRNAs and mRNAs, and the roles of these modifications, are unclear. To dissect the functions of mitochondrial TUTases, we investigated the effects of their repression and overexpression on abundance, processing, 3-end status, and in vivo stability of major mitochondrially encoded RNA classes. We show that RET1 adds U tails to gRNAs, rRNAs, and select mRNAs and contributes U's into A/U heteropolymers. Furthermore, RET1's TUTase activity is required for the nucleolytic processing of gRNA, rRNA, and mRNA precursors. The U tail's presence does not affect the stability of gRNAs and rRNAs, while transcript-specific uridylylation triggers 3 to 5 mRNA decay. We propose that the minicircle-encoded antisense transcripts, which are stabilized by RET1-catalyzed uridylylation, may direct a nucleolytic cleavage of multicistronic precursors.

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Section: Discussionmentioning

confidence: 99%

RET1-Catalyzed Uridylylation Shapes the Mitochondrial Transcriptome in Trypanosoma brucei

Aphasizheva

Aphasizhev

2010

Molecular and Cellular Biology

157

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“…Their highly localized unwindin g mode prevents large-scale unravelling of carefully assembled RNA or RNP structures, and the efficient unwinding of short duplexes is adapted to the separation of duplexes in physiological RNAs and RNPs, which rarely exceed one helical turn. Typical examples of local unwinding are thought to occur during processes that involve guide RNAs (gRNAs), such as the unwinding of small nuclear RNA (snRNA) in pre-mRNA splicing, small nucleola r RNA (snoRNA) in ribosome biogenesis or gRNA in mitochondria l RNA editing [35][36][37][38] .…”

Section: Mitochondrial Rna Editingmentioning

confidence: 99%

From unwinding to clamping — the DEAD box RNA helicase family

Linder

Jankowsky

2011

Nat Rev Mol Cell Biol

956

1,069

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Nearly all aspects of RNA metabolism, from transcription and translation to mRNA decay, involve RNA helicases, which are enzymes that use ATP to bind or remodel RNA and RNA-protein complexes (ribonucleoprotein (RNP) complexes) 1 . RNA helicases are found in all three domains of life, and many viruses also encode one or more of these proteins 2,3 . Together with the structurally related DNA helicases that function in replication, recombination and repair, the RNA helicases are classified into superfamilies and families, based on sequence and structural features 3,4 . DEAD box proteins form the largest helicase family, with 37 members in humans and 26 in Saccharomyces cerevisiae 3 , and are characterized by the presence of an Asp-Glu-Ala-Asp (DEAD) motif.DEAD box helicases have central and, in many cases, essential physiological roles in cellular RNA metabolism 5 (FIG. 1). The proteins generally function as part of larger multicomponent assemblies, such as the spliceosom e or the eukaryotic translation initiation machinery 6 . Mutations and deregulation of several DEAD box proteins have been linked to disease states, including cancer 7 . Although all DEAD box proteins contain a structurally highly conserved core with conserved ATP-binding and RNA-binding sites, different proteins have been associated with diverse and seemingly unrelated functions, including the disassembly of RNPs, chaperoning during RNA folding and even stabil ization of protein complexes on RNA 5,6 . How these highly conserved proteins fulfil such an array of different functions has been a long-standing question.Research has now started to illuminate the molecular basis for this functional diversity. In this Review, we summarize how structural data, together with biochemical and biophysical studies, have revealed unexpected modes by which these proteins function. We also discuss how novel molecular and cell biological approaches have better defined the cellular roles of DEAD box helicases. We outline our current view on common structural and mechan istic themes that have emerged, and on physical models of how these 'unusual' RNA helicases work.Structures of DEAD box RNA helicases A number of structural studies have universally shown that members of the DEAD box family contain a highly conserved helicase core that harbours the binding sites for ATP and RNA 3,4,8 . The core is surrounded by variable auxiliary domains, which are thought to be critical for the diverse functions of these enzymes.Structure of the helicase core. DEAD box proteins belong to helicase superfamily 2 (SF2) 2 . Similarly to all SF2 helicases, DEAD box proteins are built around a highly conserved helicase core of two virtually identical domains that resemble the bacterial recombination protein recombinase A (RecA) 3,4,8 (FIG. 2a,b). Within this helicase core, at least 12 characteristic sequence motifs are located at conserved positions (FIG. 2a,b). Some of these motifs are conserved across the entire SF2 family, whereas others are found only in the DEAD box family 3 ....

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“…Finally, we asked if there is evidence that published alternative gRNAs are utilized in these cells and whether editing progresses beyond these regions in a way that would allow for translation of the alternative sequence (Madej et al 2008;Koslowsky et al 2014;Madina et al 2014). The published gRNAs we include here are likely only a small fraction of total alternative gRNAs in any given cell.…”

Section: Evidence For Utilization Of Alternative Grnasmentioning

confidence: 99%

“…The sequence matching the alternative gRNA must be edited with full fidelity up to the 5 ′ end of the region covered by that alternative gRNA. Three published alternative gRNAs for RPS12 and one for ND7-5 ′ were examined in this manner (Madej et al 2008;Koslowsky et al 2014;Madina et al 2014). This analysis revealed no evidence for utilization of the ND7-5 ′ gRNA.…”

Section: Evidence For Utilization Of Alternative Grnasmentioning

confidence: 99%

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High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing

Simpson

Bruno

Bard

et al. 2016

RNA

145

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Uridine insertion/deletion RNA editing in kinetoplastids entails the addition and deletion of uridine residues throughout the length of mitochondrial transcripts to generate translatable mRNAs. This complex process requires the coordinated use of several multiprotein complexes as well as the sequential use of noncoding template RNAs called guide RNAs. The majority of steadystate mitochondrial mRNAs are partially edited and often contain regions of mis-editing, termed junctions, whose role is unclear. Here, we report a novel method for sequencing entire populations of pre-edited partially edited, and fully edited RNAs and analyzing editing characteristics across populations using a new bioinformatics tool, the Trypanosome RNA Editing Alignment Tool (TREAT). Using TREAT, we examined populations of two transcripts, RPS12 and ND7-5 ′ , in wild-type Trypanosoma brucei. We provide evidence that the majority of partially edited sequences contain junctions, that intrinsic pause sites arise during the progression of editing, and that the mechanisms that mediate pausing in the generation of canonical fully edited sequences are distinct from those that mediate the ends of junction regions. Furthermore, we identify alternatively edited sequences that constitute plausible alternative open reading frames and identify substantial variability in the 5 ′ UTRs of both canonical and alternatively edited sequences. This work is the first to use high-throughput sequencing to examine full-length sequences of whole populations of partially edited transcripts. Our method is highly applicable to current questions in the RNA editing field, including defining mechanisms of action for editing factors and identifying potential alternatively edited sequences.

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Identification of novel guide RNAs from the mitochondria ofTrypanosoma brucei

Cited by 17 publications

References 29 publications

RET1-Catalyzed Uridylylation Shapes the Mitochondrial Transcriptome in Trypanosoma brucei

RET1-Catalyzed Uridylylation Shapes the Mitochondrial Transcriptome in Trypanosoma brucei

From unwinding to clamping — the DEAD box RNA helicase family

High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing

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