Site-specific and substrate-specific control of accurate mRNA editing by a helicase complex in trypanosomes

Kumar, Vikas; Ivens, Alasdair; Goodall, Zachary; Meehan, Joshua; Doharey, Pawan Kumar; Hillhouse, Andrew; Osorio, Daniel; Cai, James J.; Zhang, Xiuren; Schnaufer, Achim; Cruz‐Reyes, Jorge

doi:10.1261/rna.076513.120

Cited by 13 publications

(29 citation statements)

References 58 publications

(150 reference statements)

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“…As argued in Cooper et al (2019) , the former hypothesis is controversial, despite accumulating evidence for the persistence of noncanonical editing patterns in mRNA populations ( Ochsenreiter et al 2008a ; Kirby et al 2016 ; Simpson et al 2016 ; Gerasimov et al 2018 ; Kumar et al 2020 ) and evidence that at least one alternatively edited mRNA is translated into a protein, AEP-1 ( Ochsenreiter and Hajduk 2006 ; Ochsenreiter et al 2008b ). An alternatively edited variant of the A6 mRNA has been documented for different strains, although in this case the predicted protein product is unchanged ( Koslowsky et al 2014 ; Madina et al 2014 ; Cooper et al 2019 ; Kumar et al 2020 ). The two variants can be explained by the use of two different initiator gRNAs ( Cooper et al 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Organization of minicircle cassettes and guide RNA genes in Trypanosoma brucei

Cooper

Wadsworth

Schnaufer

et al. 2022

RNA

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Mitochondrial DNA of protists of order Kinetoplastida comprises thousands of interlinked circular molecules arranged in a disk-like network. It composes of two types of molecules called maxicircles and minicircles. Minicircles encode so called guide RNA (gRNA) genes that mediate post-transcriptional editing of maxicircle encoded genes, many extensively so along their full length. There are thousands of minicircles per network. They are diverse and vary in copy number. The human sleeping sickness parasite Trypanosoma brucei has one of the most diverse sets of minicircle classes of all studied trypanosomatids with hundreds of different classes, each encoding one to four gRNA genes within cassettes defined by 18 bp imperfect inverted repeats. About a third of cassettes in T. brucei have no identifiable gRNA genes even though their sequence structures are similar to cassettes with identifiable gRNAs. These so called non-canonical gRNA genes have remained a mystery for many years. It is only until recently that the sequences of almost all minicircle classes for some subspecies and isolates of T. brucei have been sequenced and annotated with corresponding verification of gRNA expression by small-RNA transcriptome data. These datasets provide a rich resource for understanding the structure of minicircle classes, cassettes and gRNA genes and their transcription, and for studying the evolution and maintenance of the diverse, and often redundant, classes and genes. Here we provide a statistical description of the functionality, expression, structure and sequence of gRNA genes in a differentiation-competent, laboratory-adapted strain of T. brucei. We obtain a clearer definition of what is a gRNA gene in this species. Our analysis supports the idea that many, if not all, of the cassettes without an identifiable gRNA gene contain decaying remnants of once functional gRNA genes. We also report several new, unexplained discoveries such as the association between cassette position on the minicircle and gene expression and functionality, and the association between a gene's initiation sequence and the position of its anchor sequence.

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Section: Discussionmentioning

confidence: 99%

Organization of minicircle cassettes and guide RNA genes in Trypanosoma brucei

Cooper

Wadsworth

Schnaufer

et al. 2022

RNA

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“…The Kinetoplast RNA Editing Endonuclease1 (KREN1) containing module functions in the deletion of U residues, whereas RECC variants containing KREN2 and KREN3 have somewhat overlapping function and are involved in U insertion ( 13–15 ). REH2C was recently reported to affect total editing and accuracy of editing in a site-specific and substrate-specific manner on RESC-associated transcripts ( 16 ). Interestingly, purified RECC lacks detectable RNA, while RESC and REH2C bind editing substrates, intermediates, and products, and interact with RECC through RNA linkers ( 6 , 17–19 ).…”

Section: Introductionmentioning

confidence: 99%

Trypanosome RNAEditing Substrate Binding Complex integrity and function depends on the upstream action of RESC10

Dubey

Tylec

McAdams

et al. 2021

Nucleic Acids Research

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Uridine insertion/deletion editing of mitochondrial mRNAs is a characteristic feature of kinetoplastids, including Trypanosoma brucei. Editing is directed by trans-acting gRNAs and catalyzed by related RNA Editing Core Complexes (RECCs). The non-catalytic RNA Editing Substrate Binding Complex (RESC) coordinates interactions between RECC, gRNA and mRNA. RESC is a dynamic complex comprising GRBC (Guide RNA Binding Complex) and heterogeneous REMCs (RNA Editing Mediator Complexes). Here, we show that RESC10 is an essential, low abundance, RNA binding protein that exhibits RNase-sensitive and RNase-insensitive interactions with RESC proteins, albeit its minimal in vivo interaction with RESC13. RESC10 RNAi causes extensive RESC disorganization, including disruption of intra-GRBC protein–protein interactions, as well as mRNA depletion from GRBC and accumulation on REMCs. Analysis of mitochondrial RNAs at single nucleotide resolution reveals transcript-specific effects: RESC10 dramatically impacts editing progression in pan-edited RPS12 mRNA, but is critical for editing initiation in mRNAs with internally initiating gRNAs, pointing to distinct initiation mechanisms for these RNA classes. Correlations between sites at which editing pauses in RESC10 depleted cells and those in knockdowns of previously studied RESC proteins suggest that RESC10 acts upstream of these factors and that RESC is particularly important in promoting transitions between uridine insertion and deletion RECCs.

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“…The copyright holder for this preprint (which this version posted July 27, 2022. ; https://doi.org/10.1101/2022.07.27.501750 doi: bioRxiv preprint Sortino process continues in the general 3' to 5' direction until the mRNA is fully edited. U-indel RNA editing is considered to be relatively inefficient because the bulk of steady-state mitochondrial mRNAs are partially edited, with only a very small number of fully edited mRNAs detectable (Koslowsky et al 1991;Simpson et al 2016;Gerasimov et al 2018;Kumar et al 2020;Smith et al 2020;Dubey et al 2021). Complicating matters, greater than 90% of these partially edited mRNAs contain regions of mis-editing termed junctions at the leading edge of a correctly sequence.…”

Section: Introductionmentioning

confidence: 99%

“…The RECCs interact transiently with REH2C and RESC through RNA interactions (Aphasizheva et al 2014;Kumar et al 2016;Aphasizheva et al 2020). REH2C contains a DEAH/RHA type RNA helicase and two cofactors, and facilitates substrate-specific and site-preferential changes in total editing on transcripts associated with RESC (Kumar et al 2016;Kumar et al 2020). RESC contains ~20 proteins arranged into modules: the Guide RNA Binding Complex (GRBC), the heterogeneous RNA Editing Mediator Complexes (REMCs), and in some models, the Polyadenylation Mediator complex (PAMC) (Aphasizheva et al 2020;Dubey et al 2021).…”

Section: Introductionmentioning

confidence: 99%

Conserved and transcript-specific functions of the RESC factors, RESC13 and RESC14, in kinetoplastid RNA editing

Sortino

Tylec

Chen

et al. 2022

RNA

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Uridine insertion/deletion RNA editing is an extensive post-transcriptional modification of mitochondrial mRNAs in kinetoplastid organisms, including Trypanosoma brucei. This process is carried out using trans-acting gRNAs and complex protein machinery. The essential RNA Editing Substrate Binding Complex (RESC) serves as the scaffold that modulates protein and RNA interactions during editing, and contains the Guide RNA Binding Complex (GRBC), the RNA Editing Mediator Complexes (REMCs), and organizer proteins. Despite the importance of RESC in editing, the functions of each protein comprising this complex are not completely understood. Here, we further define the roles of a REMC protein, RESC13, and a RESC organizer, RESC14, using high-throughput sequencing on two large pan-edited mRNAs, A6 and COIII. When comparing our analyses to that of a previously published small pan-edited mRNA, RPS12, we find that RESC13 has conserved functions across the three transcripts with regards to editing initiation, gRNA utilization, gRNA exchange, and restricting the formation of long mis-edited junctions that likely arise from its ability to modulate RNA structure. However, RESC13 does have transcript-specific effects on the types of long junctions whose formation it restricts. RESC14 has a conserved effect on gRNA utilization across the three transcripts analyzed, but has transcript-specific effects on editing initiation, gRNA exchange, and junction formation. Our data suggest that transcript-specific effects of both proteins are due to differences in transcript length and sequences as well as transcript-specific protein interactions. These findings highlight the importance of studying multiple transcripts to determine the function of editing factors.

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Site-specific and substrate-specific control of accurate mRNA editing by a helicase complex in trypanosomes

Cited by 13 publications

References 58 publications

Organization of minicircle cassettes and guide RNA genes in Trypanosoma brucei

Organization of minicircle cassettes and guide RNA genes in Trypanosoma brucei

Trypanosome RNAEditing Substrate Binding Complex integrity and function depends on the upstream action of RESC10

Conserved and transcript-specific functions of the RESC factors, RESC13 and RESC14, in kinetoplastid RNA editing

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