An official website of the United States government
Abstract U-insertion/deletion (U-indel) RNA editing in trypanosome mitochondria is directed by guide RNAs (gRNAs). This editing may developmentally control respiration in bloodstream forms (BSF) and insect procyclic forms (PCF). Holo-editosomes include the accessory RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C), but the specific proteins controlling differential editing remain unknown. Also, RNA editing appears highly error prone because most U-indels do not match the canonical pattern. However, despite extensive non-canonical editing of unknown functions, accurate canonical editing is required for normal cell growth. In PCF, REH2C controls editing fidelity in RESC-bound mRNAs. Here, we report that KREH2, a REH2C-associated helicase, developmentally controls programmed non-canonical editing, including an abundant 3′ element in ATPase subunit 6 (A6) mRNA. The 3′ element sequence is directed by a proposed novel regulatory gRNA. In PCF, KREH2 RNAi-knockdown up-regulates the 3′ element, which establishes a stable structure hindering element removal by canonical initiator-gRNA-directed editing. In BSF, KREH2-knockdown does not up-regulate the 3′ element but reduces its high abundance. Thus, KREH2 differentially controls extensive non-canonical editing and associated RNA structure via a novel regulatory gRNA, potentially hijacking factors as a ‘molecular sponge’. Furthermore, this gRNA is bifunctional, serving in canonical CR4 mRNA editing whilst installing a structural element in A6 mRNA.
more »
« less
KREH2 helicase represses ND7 mRNA editing in procyclic-stage Trypanosoma brucei by opposite modulation of canonical and ‘moonlighting’ gRNA utilization creating a proposed mRNA structure
Abstract Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) T. brucei. This editing, directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs and may developmentally control respiration. Canonical editing by gRNAs that specify protein-encoding mRNA sequences occurs amid massive non-canonical editing of unclear sources and biological significance. We found PCF-specific repression at a major early checkpoint in mRNA ND7, involving helicase KREH2-dependent opposite modulation of canonical and non-canonical ‘terminator’ gRNA utilization. Terminator-programmed editing derails canonical editing and installs proposed repressive structure in 30% of the ND7 transcriptome. BSF-to-PCF differentiation in vitro recreated this negative control. Remarkably, KREH2-RNAi knockdown relieved repression and increased editing progression by reverting canonical/terminator gRNA utilization. ND7 transcripts lacking early terminator-directed editing in PCF exhibited similar negative editing control along the mRNA sequence, suggesting global modulation of gRNA utilization fidelity. The terminator is a ‘moonlighting’ gRNA also associated with mRNA COX3 canonical editing, so the gRNA transcriptome seems multifunctional. Thus, KREH2 is the first identified repressor in developmental editing control. This and our prior work support a model whereby KREH2 activates or represses editing in a stage and substrate-specific manner. KREH2’s novel dual role tunes mitochondrial gene expression in either direction during development.
more »
« less
- PAR ID:
- 10533560
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 52
- Issue:
- 19
- ISSN:
- 0305-1048
- Format(s):
- Medium: X Size: p. 11940-11959
- Size(s):
- p. 11940-11959
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Adenosine-to-inosine (A-to-I) messenger RNA (mRNA) editing can affect the sequence and function of translated proteins and has been extensively investigated in eukaryotes. However, the prevalence of A-to-I mRNA editing in bacteria, its governing regulatory principles, and its biological significance are poorly understood. Here, we show that A-to-I mRNA editing occurs in hundreds of transcripts across dozens of gammaproteobacterial species, with most edits predicted to recode protein sequences. Furthermore, we reveal conserved regulatory determinants controlling editing across gammaproteobacterial species. Using Acinetobacter baylyi as a model, we show that mutating TadA, the mediating enzyme, reduces editing across all sites. Conversely, overexpressing TadA resulted in the editing of >300 transcripts, attesting to the editing potential of TadA. Notably, we show for the first time, at the protein level, that normal levels of A-to-I mRNA editing lead to wild-type bacteria expressing two protein isoforms from a single gene. Finally, we show that a TadA mutant with deficient editing activity does not grow at high temperatures, suggesting that RNA editing has a functional role in bacteria. Our work reveals that A-to-I mRNA editing in bacteria is widespread and has the potential to reshape the bacterial transcriptome and proteome.more » « less
-
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spa- tiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized trans- gene-free and multiplex genome editing. In this review, we discuss current strategies and future per- spectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.more » « less
-
Abstract Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the “double tap” method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a “second chance” at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.more » « less
-
Abstract Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.more » « less
