@article{22772, abstract = {The cytoplasmic antiviral sensor MDA5 is activated by double-stranded RNAs. Endogenous double-stranded RNAs are modified by the A-to-I RNA-editing ADAR family to prevent activation of MDA5. In vivo, cytoplasmic ADAR1p150 is critically required to suppress MDA5 activation, yet the editing signature of all ADAR isoforms is strongly overlapping in mice. Further, it is not clear how A-to-I modifications in dsRNA prevent MDA5 activation. Here we show that 3′ UTRs harboring inverted repeats activate MDA5 in vitro and in cells. In vitro editing by either ADAR isoform leads to editing at overlapping hotspot regions and prevents MDA5 activation in vitro and in cells. Remarkably, only inosines introduced by RNA editing are capable of suppressing MDA5 activation, while replacing guanosines with inosines during in vitro transcription has no impact on MDA5 activation. A comparison of inosines introduced by ADAR1p150 in vitro, in cells, and in vivo suggests that a small number of A-to-I conversions may be critically required to suppress MDA5 activation. As those critical editing events are predominantly altering A:U basepairs into I:U wobble basepairs, we suggest that the helical distortion introduced by those wobble pairs may prevent MDA5 polymerization and thus downstream activation of the type I interferon response.}, author = {Varada, Rajagopal and Leuchtenberger, Alina F. and Vesely, Cornelia and Kaczmarek, Beata M and Mansouri Khosravi, Hamid and Mandl, Therese C. and Milanovic, Katarina and Honarmand Tamizkar, Kasra and Rajendra, Vinod and Senoner, Hannes and Steinbichl, Linda and Borojevic, Marija and Sombke, Andy and Schmidt, Katy and Eckhard, Margret and Hofacker, Ivo L. and Walkley, Carl and Heraud-Farlow, Jacki E. and Picardi, Ernesto and Bernecky, Carrie A and Jantsch, Michael F.}, issn = {1362-4962}, journal = {Nucleic Acids Research}, number = {16}, publisher = {Oxford University Press}, title = {{Distinguishing self from non-self RNA by editing-specific inosine patterns}}, doi = {10.1093/nar/gkag845}, volume = {54}, year = {2026}, } @article{15277, abstract = {Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.}, author = {Fuchs, Armin and Riegler, Stefan and Ayatollahi, Zahra and Cavallari, Nicola and Giono, Luciana E and Nimeth, Barbara A and Mutanwad, Krishna V and Schweighofer, Alois and Lucyshyn, Doris and Barta, Andrea and Petrillo, Ezequiel and Kalyna, Maria}, issn = {1362-4962}, journal = {Nucleic Acids Research}, keywords = {Genetics}, number = {2}, pages = {1133--1151}, publisher = {Oxford University Press}, title = {{Targeting alternative splicing by RNAi: From the differential impact on splice variants to triggering artificial pre-mRNA splicing}}, doi = {10.1093/nar/gkaa1260}, volume = {49}, year = {2021}, } @article{21095, abstract = {Inspired by DNA mimic proteins, we have introduced aromatic foldamers bearing phosphonate groups as synthetic mimics of the charge surface of B-DNA and competitive inhibitors of some therapeutically relevant DNA-binding enzymes: the human DNA Topoisomerase 1 (Top1) and the human HIV-1 integrase (HIV-1 IN). We now report on variants of these anionic foldamers bearing carboxylates instead of phosphonates. Several new monomers have been synthesized with protecting groups suitable for solid phase synthesis (SPS). Six hexadecaamides have been prepared using SPS. Proof of their resemblance to B-DNA was brought by the first crystal structure of one of these DNA-mimic foldamers in its polyanionic form. While some of the foldamers were found to be as active as, or even more active than, the original phosphonate oligomers, others had no activity at all or could even stimulate enzyme activity in vitro. Some foldamers were found to have differential inhibitory effects on the two enzymes. These results demonstrate a strong dependence of inhibitory activity on foldamer structure and charge distribution. They open broad avenues for the development of new classes of derivatives that could inhibit the interaction of specific proteins with their DNA target thereby influencing the cellular pathways in which they are involved.}, author = {Corvaglia, Valentina and Carbajo, Daniel and Prabhakaran, Panchami and Ziach, Krzysztof and Mandal, Pradeep K and Santos, Victor Dos and Legeay, Carole and Vogel, Rachel and Parissi, Vincent and Pourquier, Philippe and Huc, Ivan}, issn = {1362-4962}, journal = {Nucleic Acids Research}, number = {11}, pages = {5511--5521}, publisher = {Oxford University Press}, title = {{Carboxylate-functionalized foldamer inhibitors of HIV-1 integrase and Topoisomerase 1: Artificial analogues of DNA mimic proteins}}, doi = {10.1093/nar/gkz352}, volume = {47}, year = {2019}, } @article{15143, abstract = {To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA–RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA–RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.}, author = {Bravo, Jack Peter Kelly and Borodavka, Alexander and Barth, Anders and Calabrese, Antonio N and Mojzes, Peter and Cockburn, Joseph J B and Lamb, Don C and Tuma, Roman}, issn = {1362-4962}, journal = {Nucleic Acids Research}, keywords = {Genetics}, number = {15}, pages = {7924--7937}, publisher = {Oxford University Press}, title = {{Stability of local secondary structure determines selectivity of viral RNA chaperones}}, doi = {10.1093/nar/gky394}, volume = {46}, year = {2018}, } @article{21101, abstract = {It has previously been shown that the use of racemic mixtures of naturally chiral macromolecules such as protein and DNA can significantly aid the crystallogenesis process, thereby addressing one of the major bottlenecks to structure determination by X-ray crystallographic methods—that of crystal growth. Although previous studies have provided convincing evidence of the applicability of the racemic crystallization technique to DNA through the study of well-characterized DNA structures, we sought to apply this method to a historically challenging DNA sequence. For this purpose we chose a non-self-complementary DNA duplex containing the biologically-relevant Pribnow box consensus sequence ‘TATAAT’. Four racemic crystal structures of this previously un-crystallizable DNA target are reported (with resolutions in the range of 1.65–2.3 Å), with further crystallographic studies and structural analysis providing insight into the racemic crystallization process as well as structural details of this highly pertinent DNA sequence.}, author = {Mandal, Pradeep K and Collie, Gavin W. and Srivastava, Suresh C. and Kauffmann, Brice and Huc, Ivan}, issn = {1362-4962}, journal = {Nucleic Acids Research}, number = {12}, pages = {5936--5943}, publisher = {Oxford University Press}, title = {{Structure elucidation of the Pribnow box consensus promoter sequence by racemic DNA crystallography}}, doi = {10.1093/nar/gkw367}, volume = {44}, year = {2016}, } @article{6130, abstract = {Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.}, author = {Chen, Changchun and Fenk, Lorenz A. and de Bono, Mario}, issn = {1362-4962}, journal = {Nucleic Acids Research}, number = {20}, publisher = {Oxford University Press}, title = {{Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination}}, doi = {10.1093/nar/gkt805}, volume = {41}, year = {2013}, } @article{3754, abstract = {Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in real-time [mRNA] down to similar to 40 nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of mRNA concentration in individual living bacteria.}, author = {Guet, Calin C and Bruneaux, Luke and Min, Taejin and Siegal Gaskins, Dan and Figueroa, Israel and Emonet, Thierry and Cluzel, Philippe}, issn = {1362-4962}, journal = {Nucleic Acids Research}, number = {12}, publisher = {Oxford University Press}, title = {{Minimally invasive determination of mRNA concentration in single living bacteria}}, doi = {10.1093/nar/gkn329}, volume = {36}, year = {2008}, } @article{870, abstract = {Only a fraction of eukaryotic genes affect the phenotype drastically. We compared 18 parameters in 1273 human morbid genes, known to cause diseases, and in the remaining 16 580 unambiguous human genes. Morbid genes evolve more slowly, have wider phylogenetic distributions, are more similar to essential genes of Drosophila melanogaster, code for longer proteins containing more alanine and glycine and less histidine, lysine and methionine, possess larger numbers of longer introns with more accurate splicing signals and have higher and broader expressions. These differences make it possible to classify as non-morbid 34% of human genes with unknown morbidity, when only 5% of known morbid genes are incorrectly classified as non-morbid. This classification can help to identify disease-causing genes among multiple candidates.}, author = {Kondrashov, Fyodor and Ogurtsov, Aleksey and Kondrashov, Alexey}, issn = {1362-4962}, journal = {Nucleic Acids Research}, number = {5}, pages = {1731 -- 1737}, publisher = {Oxford University Press}, title = {{Bioinformatical assay of human gene morbidity}}, doi = {10.1093/nar/gkh330}, volume = {32}, year = {2004}, }