Investigating RNA-RNA interactions through computational and biophysical analysis

Mrozowich, Tyler; Park, Sean M; Waldl, Maria; Henrickson, Amy; Nelson, Corey R; Demeler, Borries; Hofacker, Ivo L.; Wolfinger, Michael T.; Patel, Trushar R.

doi:10.1101/2022.02.01.478553

Cited by 3 publications

(2 citation statements)

References 75 publications

(104 reference statements)

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“…Plasmids containing cDNA for ZIKV 5’ and 3’ NTRs were transformed and cultured in E. coli NEBα (NEB, Canada) competent cells and further recovered using GeneJET Plasmid Maxiprep Kit (ThermoFisher Scientific, Canada) as per manufacturer’s protocol, followed by a 4-hour linearization with XbaI endonuclease (NEB, Canada) at 37 °C. ZIKV NTRs RNAs were synthesized by in vitro transcription using an in-house purified T7 polymerase, as previously described (75,76). Subsequently, RNAs were purified on a Superdex TM 200 10/300 GL column (Global Life Science Solutions, USA) equilibrated with RNA buffer (10 mM Bis-tris pH 6.0, 100 mM NaCl, 15 mM KCl, 5 mM MgCl 2 , 5% glycerol).…”

Section: Methodsmentioning

confidence: 99%

Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

Mazeaud,

Pfister,

Owen

et al. 2023

Preprint

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SUMMARYZika virus (ZIKV) infection causes significant human disease that, with no approved treatment or vaccine, constitutes a major public health concern. Its life cycle entirely relies on the cytoplasmic fate of the viral RNA genome (vRNA) through a fine-tuned equilibrium between vRNA translation, replication and packaging into new virions, all within virus-induced replication organelles (vRO). In this study, with an RNAi mini-screening and subsequent functional characterization, we have identified insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a new host dependency factor that regulates vRNA synthesis. In infected cells, IGF2BP2 associates with viral NS5 polymerase and redistributes to the perinuclear viral replication compartment. Combined fluorescencein situhybridization-based confocal imaging,in vitrobinding assays, and immunoprecipitation coupled to RT-qPCR, showed that IGF2BP2 directly interacts with ZIKV vRNA 3’-nontranslated region. Using ZIKV sub-genomic replicons and a replication-independent vRO induction system, we demonstrated that IGF2BP2 knockdown impairsde novoviral organelle biogenesis and, consistently, vRNA synthesis. Finally, the analysis of immunopurified IGF2BP2 complex using quantitative mass spectrometry and RT-qPCR, revealed that ZIKV infection alters the protein and RNA interactomes of IGF2BP2. Altogether, our data support that ZIKV hijacks and remodels the IGF2BP2 ribonucleoprotein complex to regulate vRO biogenesis and vRNA neosynthesis.

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Section: Methodsmentioning

confidence: 99%

Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

Mazeaud,

Pfister,

Owen

et al. 2023

Preprint

Self Cite

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“…Therefore, SAXS is an excellent complementary approach to solving RNA structure synergistically with other biophysical methods. Numerous studies have used SAXS to help determine several RNA [31][32][33] and RNA-protein complexes structures 34 .…”

Section: Introductionmentioning

confidence: 99%

Cleavage region organizes the structural architecture of the B2 SINE ribozyme

Singhal,

Mrozowich,

Chaudhury

et al. 2024

Preprint

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The SINE-encoded B2 retrotransposon is an RNA Polymerase III transcript that gets upregulated during various cellular stress responses. The B2 noncoding RNA can directly inhibit RNA Polymerase II, leading to a significant downregulation of transcripts during stress. Our recent findings have shown that B2 is a self-cleaving epigenetic ribozyme and that cleavage can be induced by interactions with epigenetic factors, co-regulating its function across distinct chromatin-binding target loci. Here, by integrating RNA chemical probing, small angle X-ray scattering, and 3D motif modeling, we determine structural ensemble-to-function relations for the B2 SINE ribozyme RNA. Perturbations of the RNA suggest that the B2 SINE ribozyme has a well-defined secondary structure and dynamic tertiary structure that both critically depend on the presence of the active site of cleavage. In an RNA engineering approach, we examine the effect of point mutations, deletions of the main cleavage site and deletions of the cleavage domain on the structural ensemble of the RNA. By combining this with functional data, we obtain relations of structural ensembles to various functional states. This perturbative approach serves as a template to unravel the relation of structural ensembles to functional states for other ncRNA and mRNA systems.

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A comprehensive review of methods to study lncRNA–protein interactions in solution

Badmalia

Pereira

Siddiqui

et al. 2022

Biochemical Society Transactions

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The long non-coding RNAs (lncRNAs) other than rRNA and tRNA were earlier assumed to be ‘junk genomic material’. However, recent advancements in genomics methods have highlighted their roles not only in housekeeping but also in the progression of diseases like cancer as well as viral infections. lncRNAs owing to their length, have both short-range and long-range interactions resulting in complex folded structures that recruit various biomolecules enabling lncRNAs to undertake their various biological functions. Using cell lysate pull-down assays increasing number of lnRNAs-interacting proteins are being identified. These interactions can be further exploited to develop targeted novel therapeutic strategies to inhibit lncRNA–protein interactions. This review attempts to succinctly techniques that can identify and characterize the lnRNAs–protein interactions (i.e. affinity, stoichiometry, and thermodynamics). Furthermore, using other sophisticated biophysical techniques, one can also perform size estimations, and determine low-resolution structures. Since these methods study the biomolecules in solution, large-scale structural observations can be performed in real-time. This review attempts to briefly introduce the readers to biochemical and biophysical techniques, such that they can utilize these methods to obtain a holistic characterization of the biomolecules of interest. Additionally, it should be noted that the use of these methods is not limited to the characterization of the interacting molecules but can also be used to determine the efficacy of the therapeutic molecules to disrupt these interactions.

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Investigating RNA-RNA interactions through computational and biophysical analysis

Cited by 3 publications

References 75 publications

Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

Cleavage region organizes the structural architecture of the B2 SINE ribozyme

A comprehensive review of methods to study lncRNA–protein interactions in solution

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