How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs

ABSTRACT In trypanosome RNA editing, uridylate (U) residues are inserted and deleted at numerous sites within mitochondrial pre-mRNAs by an ∼20S protein complex that catalyzes cycles of cleavage, U addition/U removal, and ligation. We used RNA interference to deplete TbMP18 (band VII), the last unexamined major protein of our purified editing complex, showing it is essential. TbMP18 is critical for the U-deletional and U-insertional cleavages and for integrity of the ∼20S editing complex, whose other major components, TbMP99, TbMP81, TbMP63, TbMP52, TbMP48, TbMP42 (bands I through VI), and TbMP57, instead sediment as ∼10S associations. Additionally, TbMP18 augments editing substrate recognition by the TbMP57 terminal U transferase, possibly aiding the recognition component, TbMP81. The other editing activities and their coordination in precleaved editing remain active in the absence of TbMP18. These data are reminiscent of the data on editing subcomplexes reported by A. Schnaufer et al. (Mol. Cell 12:307-319, 2003) and suggest that these subcomplexes are held together in the ∼20S complex by TbMP18, as was proposed previously. Our data additionally imply that the proteins are less long-lived in these subcomplexes than they are when held in the complete editing complex. The editing endonucleolytic cleavages being lost when the editing complex becomes fragmented, as upon TbMP18 depletion, should be advantageous to the trypanosome, minimizing broken mRNAs.

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A-to-I RNA Editing Affects lncRNAs Expression after Heat Shock

Genes ◽

10.3390/genes9120627 ◽

2018 ◽

Vol 9 (12) ◽

pp. 627 ◽

Cited By ~ 2

Author(s):

Roni Haas ◽

Nabeel Ganem ◽

Ayya Keshet ◽

Angela Orlov ◽

Alla Fishman ◽

...

Keyword(s):

Heat Shock ◽

Rna Editing ◽

Rna Structures ◽

Wild Type ◽

Double Stranded Rna ◽

Adenosine Deaminases ◽

Heat Shock Stress ◽

Non Coding Rnas ◽

Upregulated Genes ◽

Shock Stress

Adenosine to inosine (A-to-I) RNA editing is a highly conserved regulatory process carried out by adenosine-deaminases (ADARs) on double-stranded RNA (dsRNAs). Although a considerable fraction of the transcriptome is edited, the function of most editing sites is unknown. Previous studies indicate changes in A-to-I RNA editing frequencies following exposure to several stress types. However, the overall effect of stress on the expression of ADAR targets is not fully understood. Here, we performed high-throughput RNA sequencing of wild-type and ADAR mutant Caenorhabditis elegans worms after heat-shock to analyze the effect of heat-shock stress on the expression pattern of genes. We found that ADAR regulation following heat-shock does not directly involve heat-shock related genes. Our analysis also revealed that long non-coding RNAs (lncRNAs) and pseudogenes, which have a tendency for secondary RNA structures, are enriched among upregulated genes following heat-shock in ADAR mutant worms. The same group of genes is downregulated in ADAR mutant worms under permissive conditions, which is likely, considering that A-to-I editing protects endogenous dsRNA from RNA-interference (RNAi). Therefore, temperature increases may destabilize dsRNA structures and protect them from RNAi degradation, despite the lack of ADAR function. These findings shed new light on the dynamics of gene expression under heat-shock in relation to ADAR function.

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A protein–protein interaction underlies the molecular basis for substrate recognition by an adenosine-to-inosine RNA-editing enzyme

Nucleic Acids Research ◽

10.1093/nar/gky800 ◽

2018 ◽

Vol 46 (18) ◽

pp. 9647-9659 ◽

Cited By ~ 12

Author(s):

Suba Rajendren ◽

Aidan C Manning ◽

Haider Al-Awadi ◽

Kentaro Yamada ◽

Yuichiro Takagi ◽

...

Keyword(s):

Rna Editing ◽

Protein Interaction ◽

Molecular Basis ◽

Substrate Recognition ◽

Protein Protein Interaction ◽

Editing Enzyme

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Substrate Determinants for RNA Editing and Editing Complex Interactions at a Site for Full-round U Insertion

Journal of Biological Chemistry ◽

10.1074/jbc.m605554200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4265-4276 ◽

Cited By ~ 6

Author(s):

Catherine Cifuentes-Rojas ◽

Paula Pavia ◽

Alfredo Hernandez ◽

Daniel Osterwisch ◽

Concepcion Puerta ◽

...

Keyword(s):

Rna Editing ◽

Molecular Mechanisms ◽

Substrate Recognition ◽

Cross Linking ◽

Internal Loop ◽

Complex Interactions ◽

Loop Region ◽

Mrna Cleavage ◽

Hybrid Substrates ◽

Helical Turn

Multisubunit RNA editing complexes catalyze uridylate insertion/deletion RNA editing directed by complementary guide RNAs (gRNAs). Editing in trypanosome mitochondria is transcript-specific and developmentally controlled, but the molecular mechanisms of substrate specificity remain unknown. Here we used a minimal A6 pre-mRNA/gRNA substrate to define functional determinants for full-round insertion and editing complex interactions at the editing site 2 (ES2). Editing begins with pre-mRNA cleavage within an internal loop flanked by upstream and downstream duplexes with gRNA. We found that substrate recognition around the internal loop is sequence-independent and that completely artificial duplexes spanning a single helical turn are functional. Furthermore, after our report of cross-linking interactions at the deletion ES1 (35), we show for the first time editing complex contacts at an insertion ES. Our studies using site-specific ribose 2′ substitutions defined 2′-hydroxyls within the (a) gRNA loop region and (b) flanking helixes that markedly stimulate both pre-mRNA cleavage and editing complex interactions at ES2. Modification of the downstream helix affected scissile bond specificity. Notably, a single 2′-hydroxyl at ES2 is essential for cleavage but dispensable for editing complex cross-linking. This study provides new insights on substrate recognition during full-round editing, including the relevance of secondary structure and the first functional association of specific (pre-mRNA and gRNA) riboses with both endonuclease cleavage and cross-linking activities of editing complexes at an ES. Importantly, most observed cross-linking interactions are both conserved and relatively stable at ES2 and ES1 in hybrid substrates. However, they were also detected as transient low-stability contacts in a non-edited transcript.

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Structural Pattern Differences in Unbranched Rod-like RNA of Hepatitis Delta Virus affect RNA Editing

Viruses ◽

10.3390/v11100934 ◽

2019 ◽

Vol 11 (10) ◽

pp. 934 ◽

Cited By ~ 2

Author(s):

Hsu ◽

Juang ◽

Kuo ◽

Li ◽

Iang ◽

...

Keyword(s):

Rna Editing ◽

Rna Structure ◽

Hepatitis Delta Virus ◽

Rna Structures ◽

Base Pairing ◽

Hepatitis Delta ◽

Virion Assembly ◽

Editing Efficiency ◽

Naturally Occurring ◽

Delta Virus

Hepatitis delta virus (HDV) RNA forms an unbranched rod-like structure and complexes with the delta antigen (HDAg). Host ADAR1-catalyzed RNA editing at the amber/W site of the small HDAg leads to the production of the large HDAg, which inhibits replication and is required for virion assembly. For HDV genotype 1, amber/W editing is controlled by HDAg and the RNA structure immediate vicinity and downstream of the editing site. Here, the effects of 20 mutants carrying an increased length of consecutive base-pairing at various sites in HDV RNA on amber/W site editing were examined. All nine mutants carrying genomic regions that formed up to 15 consecutive base pairs, which is also the maximum length observed in 41 naturally occurring HDV genomes, showed normal editing rate. However, mutants carrying a 16 or 17 consecutive base-paired antigenomic segment located as far as 114 nt upstream could increase editing efficiency, possibly by interfering with HDAg binding. These data show for the first time that extended base-pairing upstream of the amber/W site could increase HDV RNA editing efficiency. Furthermore, it appears that the naturally occurring HDV RNA structures have been selected for suboptimal amber/W RNA editing, which favors the HDV replication cycle.

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A-to-I RNA Editing Affects lncRNAs Expression after Heat Shock

10.20944/preprints201811.0177.v1 ◽

2018 ◽

Author(s):

Roni Haas ◽

Nabeel S. Ganem ◽

Ayya Keshet ◽

Angela Orlov ◽

Alla Fishman ◽

...

Keyword(s):

Gene Expression ◽

Heat Shock ◽

Rna Editing ◽

Rna Structures ◽

Wild Type ◽

Adenosine Deaminases ◽

C Elegans ◽

Heat Shock Stress ◽

Upregulated Genes ◽

Shock Stress

Adenosine to inosine (A-to-I) RNA editing is a highly conserved regulatory process carried out by adenosine-deaminases (ADARs) on dsRNAs. Although a considerable fraction of the transcriptome is edited, the function of most editing sites is unknown. Previous studies indicate changes in A-to-I RNA editing frequencies following exposure to several stress types. However, the overall effect of stress on the expression of ADAR targets is not fully understood. Here, we performed high-throughput RNA sequencing of wild-type and ADAR mutant C. elegans worms after heat-shock to analyze the effect of heat-shock stress on the expression pattern of genes. We found that ADAR regulation following heat-shock does not directly involve heat-shock related genes. Our analysis also revealed that lncRNAs and pseudogenes, which have a tendency for secondary RNA structures, are enriched among upregulated genes following heat-shock in ADAR mutant worms. The same group of genes is downregulated in ADAR mutant worms under permissive conditions, which is likely, considering that A-to-I editing protects endogenous dsRNA from RNA-interference (RNAi). Therefore, temperature increases may destabilize dsRNA structures and protect them from RNAi degradation, despite the lack of ADAR function. These findings shed new light on the dynamics of gene expression under heat-shock in relation to ADAR function.

Download Full-text