Browsing by Subject "RNA structure"

Now showing 1 - 3 of 3
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    A CRISPR mis-insertion in the Zic3 5'UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin
    (Company of Biologists, 2025) Bellchambers, Helen M.; Padua, Maria B.; Ware, Stephanie M.; Pediatrics, School of Medicine
    Zic3 loss of function is associated with a range of congenital defects, including heterotaxy and isolated heart defects in humans, as well as neural tube defects, situs anomalies, and tail kinks in model organisms. Here, we describe a novel Zic3ins5V mouse line generated due to a mis-insertion during the CRISPR genome editing process, which altered the Zic3 5'UTR structure. Mice with this insertion developed similar phenotypes to Zic3LacZ null mice, including heterotaxy, isolated heart defects, neural tube defects and tail kinks. Surprisingly, gene expression analysis revealed that the novel Zic3ins5V line displays higher levels of Zic3 mRNA, but western blot analysis confirmed that levels of ZIC3 were greatly reduced in vivo. RNAfold, an RNA secondary structure prediction tool, showed that this mis-insertion may cause the formation of a large stem-loop hairpin incorporating some of the 5'UTR and first exon of Zic3, and the insertion of similar hairpins in a cell-based assay caused the loss of ZIC3 expression. Thus, this mouse line displays a loss of ZIC3 protein consistent with the inhibitory effects of 5'UTR stem-loop hairpin structures.
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    Experimental and computational methods for studying the dynamics of RNA-RNA interactions in SARS-COV2 genomes
    (Oxford University Press, 2024) Srivastava, Mansi; Dukeshire, Matthew R.; Mir, Quoseena; Omoru, Okiemute Beatrice; Manzourolajdad, Amirhossein; Janga, Sarath Chandra; Biomedical Engineering and Informatics, Luddy School of Informatics, Computing, and Engineering
    Long-range ribonucleic acid (RNA)–RNA interactions (RRI) are prevalent in positive-strand RNA viruses, including Beta-coronaviruses, and these take part in regulatory roles, including the regulation of sub-genomic RNA production rates. Crosslinking of interacting RNAs and short read-based deep sequencing of resulting RNA–RNA hybrids have shown that these long-range structures exist in severe acute respiratory syndrome coronavirus (SARS-CoV)-2 on both genomic and sub-genomic levels and in dynamic topologies. Furthermore, co-evolution of coronaviruses with their hosts is navigated by genetic variations made possible by its large genome, high recombination frequency and a high mutation rate. SARS-CoV-2’s mutations are known to occur spontaneously during replication, and thousands of aggregate mutations have been reported since the emergence of the virus. Although many long-range RRIs have been experimentally identified using high-throughput methods for the wild-type SARS-CoV-2 strain, evolutionary trajectory of these RRIs across variants, impact of mutations on RRIs and interaction of SARS-CoV-2 RNAs with the host have been largely open questions in the field. In this review, we summarize recent computational tools and experimental methods that have been enabling the mapping of RRIs in viral genomes, with a specific focus on SARS-CoV-2. We also present available informatics resources to navigate the RRI maps and shed light on the impact of mutations on the RRI space in viral genomes. Investigating the evolution of long-range RNA interactions and that of virus–host interactions can contribute to the understanding of new and emerging variants as well as aid in developing improved RNA therapeutics critical for combating future outbreaks.
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    Mg2+ Effect on Argonaute and RNA Duplex by Molecular Dynamics and Bioinformatics Implications
    (PLOS (Public Library of Science), 2014-10-17) Nam, Seungyoon; Ryu, Hyojung; Son, Won-joon; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Nephew, Kenneth P.; Lee, Jinhyuk; Medical Sciences Program at Indiana University Bloomington
    RNA interference (RNAi), mediated by small non-coding RNAs (e.g., miRNAs, siRNAs), influences diverse cellular functions. Highly complementary miRNA-target RNA (or siRNA-target RNA) duplexes are recognized by an Argonaute family protein (Ago2), and recent observations indicate that the concentration of Mg2+ ions influences miRNA targeting of specific mRNAs, thereby modulating miRNA-mRNA networks. In the present report, we studied the thermodynamic effects of differential [Mg2+] on slicing (RNA silencing cycle) through molecular dynamics simulation analysis, and its subsequent statistical analysis. Those analyses revealed different structural conformations of the RNA duplex in Ago2, depending on Mg2+ concentration. We also demonstrate that cation effects on Ago2 structural flexibility are critical to its catalytic/functional activity, with low [Mg2+] favoring greater Ago2 flexibility (e.g., greater entropy) and less miRNA/mRNA duplex stability, thus favoring slicing. The latter finding was supported by a negative correlation between expression of an Mg2+ influx channel, TRPM7, and one miRNA’s (miR-378) ability to downregulate its mRNA target, TMEM245. These results imply that thermodynamics could be applied to siRNA-based therapeutic strategies, using highly complementary binding targets, because Ago2 is also involved in RNAi slicing by exogenous siRNAs. However, the efficacy of a siRNA-based approach will differ, to some extent, based on the Mg2+ concentration even within the same disease type; therefore, different siRNA-based approaches might be considered for patient-to-patient needs.