<i>Trypanosoma brucei</i> ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

Reifur, Larissa; Koslowsky, Donna J.

doi:10.1261/rna.1144508

Cited by 17 publications

(10 citation statements)

References 81 publications

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“…To ensure that any observed differences in binding are not caused by varying numbers of nucleotides in assayed binding substrates, a molar excess of torula yeast RNA was included during the incubation of the tested substrate RNAs. Not much is known about the structure of mt mRNAs, in contrast to studies demonstrating threehelical structures in mRNA:gRNA duplexes (Reifur and Koslowsky 2008) and the presence of two hairpin loops in gRNAs, including gA6[14] (Schmid et al 1995). While our data do not indicate which structural features of the mRNAs are exploited by the 100-kDa MRB8170 for binding, one possible structural determinant of the apparent preference for mRNAs is that they may have significant stretches that are single-stranded compared with gRNA.…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

Kafková

Ammerman

Faktorová

et al. 2012

RNA

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A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

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Section: Discussionmentioning

confidence: 99%

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

Kafková

Ammerman

Faktorová

et al. 2012

RNA

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“…In this process, the initiating gRNA anchors to a short never-edited region at the 3 ′ end of the transcript. Early in the process, this gRNA will contain intramolecular hairpins, and it does not fully align to the pre-edited mRNA (Koslowsky et al 1991(Koslowsky et al , 2004Schmid et al 1995;Koslowsky 1999, 2001a,b;Reifur and Koslowsky 2008;Reifur et al 2010). Once the editing guided by a gRNA is complete, that gRNA is complementary to the fully edited mRNA through a combination of Watson-Crick and G-U base-pairing.…”

Section: Introductionmentioning

confidence: 99%

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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“…Some editing substrates initially form a three-helical structure with the editing site at or near the three-way helical junction (Reifur and Koslowsky 2008). The U-helix and the anchor helix flank the editing site, and the template sequence of the gRNA forms a stem-loop opposite the editing site that serves as the third helix of the three-way junction.…”

Section: Introductionmentioning

confidence: 99%

The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate

Mooers¹,

Singh²

2011

RNA

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Guide RNAs bind antiparallel to their target pre-mRNAs to form editing substrates in reaction cycles that insert or delete uridylates (Us) in most mitochondrial transcripts of trypanosomes. The 59 end of each guide RNA has an anchor sequence that binds to the pre-mRNA by base-pair complementarity. The template sequence in the middle of the guide RNA directs the editing reactions. The 39 ends of most guide RNAs have~15 contiguous Us that bind to the purine-rich unedited pre-mRNA upstream of the editing site. The resulting U-helix is rich in G d U wobble base pairs. To gain insights into the structure of the U-helix, we crystallized 8 bp of the U-helix in one editing substrate for the A6 mRNA of Trypanosoma brucei. The fragment provides three samples of the 59-AGA-39/59-UUU-39 base-pair triple. The fusion of two identical U-helices head-to-head promoted crystallization. We obtained X-ray diffraction data with a resolution limit of 1.37 Å . The U-helix had low and high twist angles before and after each G d U wobble base pair; this variation was partly due to shearing of the wobble base pairs as revealed in comparisons with a crystal structure of a 16-nt RNA with all Watson-Crick base pairs. Both crystal structures had wider major grooves at the junction between the poly(U) and polypurine tracts. This junction mimics the junction between the template helix and the U-helix in RNA-editing substrates and may be a site of major groove invasion by RNA editing proteins.

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Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

Cited by 17 publications

References 81 publications

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

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

The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate

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