Conserved RNA Sequences Research Articles

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

Abstract MP219: Hnrnpa2b1-dependent Selective Sorting Of Mir-486a-5p Into Msc-derived Exosomes Contributes To Cardioprotection

Exosomes (Exo) are a class of extracellular vesicles and involvement of stem cell-derived Exo in cardiac repair and cardioprotection is thought to be an important in the heart. Our HYPOTHESIS Is that specific microRNAs (miRs) from mesenchymal stem cell (MSC)-derived exosomes are actively and selectively sorted into Exo by RNA binding proteins and motifs on the miR, to serve specific functions of the Exo, including Cardioprotection. Methods: We characterized the miR populations of parental MSCs and their Exo via RNA Seq and confirmed by QRT-PCR the subpopulation of miRs that is increased in Exo vs . MSC cells. We then used Multiple Em for Motif Elicitation (MEME) Version 5.3.3 and determined the predicted conserved motifs. From these, we predicted RNA binding protein sites from the literature. In parallel, we performed mass spectrometry and western blot analyses to determine RNA binding proteins in MSC and Exo. Predicting that hnRNPA2B1 was a likely RNA binding protein for the new motif, we knocked out the cognate gene (CRISPR) in MSC and evaluated the KO Exo vs. the WT Exo by RNA Seq and QRT-PCR. We performed protein and RNA pulldowns, and EMSA to validate binding of hnRNPA2B1 to several of the miRs, and investigated the effects of these miRs on cell survival after simIR and in an in vivo mouse model of MI. Results: We found a set of eight miRs that are selectively concentrated in the MSC Exo. MEME software predicted a conserved binding motif of gAGu, which is close to canonical sites for binding of hnRNPA2B1 and hnRNPA1. We determined hnRNPA2B1 was in MSC and Exo and showed KO hnRNPA2B1 cells and Exo had no compensatory perturbation of other RNA binding proteins. The KO MSC Exo show reduction of the selective sorting of the miRs of interest. Pulldowns and binding assay results verify binding of hnRNPA2B1 to both miR-486a-5p and miR-122a. Finally, we showed that miR-486a-5p is protective in H9C2 cells submitted to simIR and results in significant 68% reduction of infarct size (n=7, P=0.0175) in vivo in association with repression of PDCD4 expression and apoptosis. Conclusions: We determined that a set of miRs is selectively concentrated in MSC Exo and demonstrated the necessity of hnRNPA2B1 in that process. This appears to involve a conserved RNA sequence motif (mutational analysis underway). A major miR affected is miR-486a-5p, which is strongly cardioprotective. Our results support that miR-486a-5p is selectively concentrated in MSC Exo and contributes to cardioprotection by reducing PDCD4 activity in apoptosis.

RNA, Genome Output and Input.

RNA, the transcriptional output of genomes, not only templates protein synthesis or directly engages in catalytic functions, but can feed back to the genome and serve as regulatory input for gene expression. Transcripts affecting the RNA abundance of other genes act by mechanisms similar to and in concert with protein factors that control transcription. Through recruitment or blocking of activating and silencing complexes to specific genomic loci, RNA and protein factors can favor transcription or lower the local gene expression potential. Most regulatory proteins enter nuclei from all directions to start the search for increased affinity to specific DNA sequences or to other proteins nearby genuine gene targets. In contrast, RNAs emerge from spatial point sources within nuclei, their encoding genes. A transcriptional burst can result in the local appearance of multiple nascent RNA copies at once, in turn increasing local nucleic acid density and RNA motif abundance before diffusion into the nuclear neighborhood. The confined initial localization of regulatory RNAs causing accumulation of protein co-factors raises the intriguing possibility that target specificity of non-coding, and probably coding, RNAs is achieved through gene/RNA positioning and spatial proximity to regulated genomic regions. Here we review examples of positional cis conservation of regulatory RNAs with respect to target genes, spatial proximity of enhancer RNAs to promoters through DNA looping and RNA-mediated formation of membrane-less structures to control chromatin structure and expression. We speculate that linear and spatial proximity between regulatory RNA-encoding genes and gene targets could possibly ease the evolutionary pressure on maintaining regulatory RNA sequence conservation.

In silico identification of conserved cis-acting RNA elements in the SARS-CoV-2 genome.

Aim:The aim of this study was to computationally predict conserved RNA sequences and structures known as cis-acting RNA elements (CREs) in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome.Materials & methods:Bioinformatics tools were used to analyze and predict CREs by obtaining viral sequences from available databases.Results:Computational analysis revealed the presence of RNA stem-loop structures within the 3′ end of the ORF1ab region analogous to previously identified SARS-CoV genomic packaging signals. Alignment-based RNA secondary structure predictions of the 5′ end of the SARS-CoV-2 genome also identified conserved CREs.Conclusion:These CREs may be potential vaccine and/or antiviral therapeutic targets; however, further studies are warranted to confirm their roles in the SARS-CoV-2 life cycle.

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look.

As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Here we present a first look at RNA sequence conservation and structural propensities in the SARS-CoV-2 genome. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nt as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences subsequently reported from the COVID-19 outbreak, and we present a curated list of 30 “SARS-related-conserved” regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 “SARS-CoV-2-conserved-structured” regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the extended 5′ UTR, frameshifting stimulation element, and 3′ UTR. Lastly, we predict regions of the SARS-CoV-2 viral genome that have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 “SARS-CoV-2-conserved-unstructured” genomic regions may be most easily accessible by hybridization in primer-based diagnostic strategies.

Examination of specific binding activity of packaging sequence RNAs to the SARS‐CoV nucleocapsid by using cell‐based in vivo assay for RNA‐Protein interaction

Severe acute respiratory syndrome coronavirus (SARS‐CoV) is the etiological agent of SARS, an emerging disease characterized by atypical pneumonia. The SARS‐CoV nucleocapsid protein serves multiple functions in viral replication, transcription and the formation of viral genome complex. Coronaviruses specifically package genomic RNA into assembled virions, little is known about the specific interaction between Nucleocapsid protein and packaging RNA sequence in previously. Herein, we exploited a cell‐based translation inhibition assay to elucidate the protein‐RNA interaction in living cells. Our results demonstrated that the packaging signal is pivotal in the specific interaction of the nucleocapsid and viral genomic RNA for incorporation into virions. We identified that the specific conserved RNA sequence of the nsp12 encoding vRNA of SARS‐CoV, and conserved RNAs of other corona viruses are major determinant sequences for the N‐protein interaction, respectively. In addition, using deletion mapping of the N‐protein, we also found that the specific in vivo RNA binding activity of the Nucleocapsid protein of the SARS‐CoV requires both the N‐terminal and C‐terminal domain. These results provide the first evidence for a specific interaction of the SARS‐CoV Nucleocapsid protein with the its viral packaging RNA sequence in vivo that will be useful as a valuable research tool for further detailed studies and providing new targets for antiviral therapies.Support or Funding InformationThis work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University. In addition, this work was supported by a grant from the National Research Foundation of Korea (NRF‐2017R1D1A1B03030315).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synchronous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment based on a zwitterionic copper (II) metal–organic framework

From a three-dimensional (3D) metal–organic framework (MOF) of {[Cu(Cmdcp)(phen)(H2O)]2·9H2O}n (1, H3CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide, phen = phenanthroline), a sensitive and selective fluorescence sensor has been developed for the simultaneous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded microRNA-like (miRNA-like) fragment. The results from molecular dynamics simulation confirmed that MOF 1 absorbs carboxyfluorescein (FAM)-tagged and 5(6)-carboxyrhodamine, triethylammonium salt (ROX)-tagged probe ss-DNA (probe DNA, P-DNA) by π…π stacking and hydrogen bonding, as well as additional electrostatic interactions to form a sensing platform of P-DNAs@1 with quenched FAM and ROX fluorescence. In the presence of targeted ebolavirus conserved RNA sequences or ebolavirus-encoded miRNA-like fragment, the fluorophore-labeled P-DNA hybridizes with the analyte to give a P-DNA@RNA duplex and released from MOF 1, triggering a fluorescence recovery. Simultaneous detection of two target RNAs has also been realized by single and synchronous fluorescence analysis. The formed sensing platform shows high sensitivity for ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment with detection limits at the picomolar level and high selectivity without cross-reaction between the two probes. MOF 1 thus shows the potential as an effective fluorescent sensing platform for the synchronous detection of two ebolavirus-related sequences, and offer improved diagnostic accuracy of Ebola virus disease.

Exploitation of microRNAs by Japanese Encephalitis virus in human microglial cells.

JEV infection in CNS leads to the JE neuroinflammation. Children and old age individual have been reported to be more prone to JEV infection. MicroRNAs are endogenous, small non-coding RNAs, which regulate the gene expression. These are ∼22 nucleotide long, conserved RNA sequence that binds at the 3'UTR of a target mRNA and regulate the post-transcriptional gene expression. The role of microRNAs has been reported in several diseases like cancer, viral infection, neuro-degeneration, diabetes etc. In the present study, the human microglial cells were infected with JEV (JaOArS982). The control and infected samples were subject to microarray profiling for microRNA expression. The microarray profile yielded differentially expressed microRNAs from JEV infected samples. The microRNA gene targets, gene ontology, annotations, and pathways were identified through various bioinformatics tools. Additionally, the pathways were mostly found common to "ubiquitin mediated proteolysis," "cytokine signaling," "maintenance of barrier function/cell junctions," JAK/STAT pathway" "Toll-like receptor signaling," "Wnt-signaling," "adhesion molecules," "apoptosis," "endocytosis," "vesicle mediated transport" etc.

Identification of 15 candidate structured noncoding RNA motifs in fungi by comparative genomics

BackgroundWith the development of rapid and inexpensive DNA sequencing, the genome sequences of more than 100 fungal species have been made available. This dataset provides an excellent resource for comparative genomics analyses, which can be used to discover genetic elements, including noncoding RNAs (ncRNAs). Bioinformatics tools similar to those used to uncover novel ncRNAs in bacteria, likewise, should be useful for searching fungal genomic sequences, and the relative ease of genetic experiments with some model fungal species could facilitate experimental validation studies.ResultsWe have adapted a bioinformatics pipeline for discovering bacterial ncRNAs to systematically analyze many fungal genomes. This comparative genomics pipeline integrates information on conserved RNA sequence and structural features with alternative splicing information to reveal fungal RNA motifs that are candidate regulatory domains, or that might have other possible functions. A total of 15 prominent classes of structured ncRNA candidates were identified, including variant HDV self-cleaving ribozyme representatives, atypical snoRNA candidates, and possible structured antisense RNA motifs. Candidate regulatory motifs were also found associated with genes for ribosomal proteins, S-adenosylmethionine decarboxylase (SDC), amidase, and HexA protein involved in Woronin body formation. We experimentally confirm that the variant HDV ribozymes undergo rapid self-cleavage, and we demonstrate that the SDC RNA motif reduces the expression of SAM decarboxylase by translational repression. Furthermore, we provide evidence that several other motifs discovered in this study are likely to be functional ncRNA elements.ConclusionsSystematic screening of fungal genomes using a computational discovery pipeline has revealed the existence of a variety of novel structured ncRNAs. Genome contexts and similarities to known ncRNA motifs provide strong evidence for the biological and biochemical functions of some newly found ncRNA motifs. Although initial examinations of several motifs provide evidence for their likely functions, other motifs will require more in-depth analysis to reveal their functions.

Melting temperature highlights functionally important RNA structure and sequence elements in yeast mRNA coding regions.

Secondary structure elements in the coding regions of mRNAs play an important role in gene expression and regulation, but distinguishing functional from non-functional structures remains challenging. Here we investigate the dependence of sequence–structure relationships in the coding regions on temperature based on the recent PARTE data by Wan et al. Our main finding is that the regions with high and low thermostability (high Tm and low Tm regions) are under evolutionary pressure to preserve RNA secondary structure and primary sequence, respectively. Sequences of low Tm regions display a higher degree of evolutionary conservation compared to high Tm regions. Low Tm regions are under strong synonymous constraint, while high Tm regions are not. These findings imply that high Tm regions contain thermo-stable functionally important RNA structures, which impose relaxed evolutionary constraint on sequence as long as the base-pairing patterns remain intact. By contrast, low thermostability regions contain single-stranded functionally important conserved RNA sequence elements accessible for binding by other molecules. We also find that theoretically predicted structures of paralogous mRNA pairs become more similar with growing temperature, while experimentally measured structures tend to diverge, which implies that the melting pathways of RNA structures cannot be fully captured by current computational approaches.

A Review on Recent Computational Methods for Predicting Noncoding RNAs.

Noncoding RNAs (ncRNAs) play important roles in various cellular activities and diseases. In this paper, we presented a comprehensive review on computational methods for ncRNA prediction, which are generally grouped into four categories: (1) homology-based methods, that is, comparative methods involving evolutionarily conserved RNA sequences and structures, (2) de novo methods using RNA sequence and structure features, (3) transcriptional sequencing and assembling based methods, that is, methods designed for single and pair-ended reads generated from next-generation RNA sequencing, and (4) RNA family specific methods, for example, methods specific for microRNAs and long noncoding RNAs. In the end, we summarized the advantages and limitations of these methods and pointed out a few possible future directions for ncRNA prediction. In conclusion, many computational methods have been demonstrated to be effective in predicting ncRNAs for further experimental validation. They are critical in reducing the huge number of potential ncRNAs and pointing the community to high confidence candidates. In the future, high efficient mapping technology and more intrinsic sequence features (e.g., motif and k-mer frequencies) and structure features (e.g., minimum free energy, conserved stem-loop, or graph structures) are suggested to be combined with the next- and third-generation sequencing platforms to improve ncRNA prediction.

Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site.

Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5′ UTR of the HIV genomic RNA (vRNA) and a host primer tRNALys3. Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription.

Human immunodeficiency virus type 1 splicing at the major splice donor site is controlled by highly conserved RNA sequence and structural elements.

Human immunodeficiency virus type 1 (HIV-1) splicing has to be strictly controlled to ensure the balanced production of the unspliced and all differently spliced viral RNAs. Splicing at the major 59 splice site (59ss) that is used for the synthesis of all spliced RNAs is modulated by the local RNA structure and binding of regulatory SR proteins. Here, we demonstrate that the suboptimal sequence complementarity between this 59ss and U1 small nuclear RNA (snRNA) also contributes to prevent excessive splicing. Analysis of a large set of HIV-1 sequences revealed that all three regulatory features of the 59ss region (RNA structure, SR protein binding and sequence complementarity with U1 snRNA) are highly conserved amongst virus isolates, which supports their importance. Combined mutations that destabilize the local RNA structure, remove binding sites for inhibitory SR proteins and optimize the U1 snRNA complementarity resulted in almost complete splicing and accordingly reduced virus replication.

Abstract 46: Identification of Novel Alternate Splicing Events in Humans With RBFOX2 Mutations and Hypoplastic Left Heart Syndrome

Alternative splicing (AS) of protein isoforms is an integral mechanism for cardiac development. RNA Binding Protein, Fox-1 Homolog (C. Elegans) 2 (RBFOX2) is an RNA binding protein preferentially expressed in muscle and neuronal cells and regulates tissue-specific alternate exon splicing in ~2,100 target genes by binding the conserved RNA sequence motif (U)GCAUG. RBFOX2 was recently implicated in the pathogenesis of abnormal cardiac and cerebral development via loss-of-function studies in zebrafish and mouse. However, convincing evidence remains incomplete, as the full complement of RBFOX2 target genes and differential exon usage (DEU) in human cardiovascular cell lines are incompletely defined. We identified de novo mutations in RBFOX2 from four human cases of congenital heart disease (CHD) with hypoplastic left heart syndrome (HLHS) via whole exome sequencing. To test the hypothesis that RBFOX2 mutations alter DEU in known target genes, we performed RNA-seq on ductus arteriosus tissue from human CHD cases with and without RBFOX2 mutation. Analysis of RNA-seq for DEU was performed with DEXSeq. To limit the effect of differential gene expression, we restricted analysis to subjects with high global gene expression correlation (r2 > 0.9, case=1 vs. control=5). DEU in known RBFOX2 target genes were highly enriched compared to all known genes (115/2,100 vs. 589/26,310, p=5.78e-15). A high percentage of the DEU genes (60.0%, 69/115) have high heart expression (HHE) in the developing mouse (mean expression of four cardiac chambers at e14.5). DEU genes with HHE include VCL, TPM1, FN1, ACTN1, and CALD1. Functional annotation clustering reveals enrichment for several actin, cytoskeletal, and contractile Gene Ontology terms, suggesting a possible role in the epithelial-mesenchymal transition (EMT) developmental processes active during early cardiac formation. We also identified enrichment from genes implicated in CHD by allelic specific expression or de novo mutations (44/1,263 vs. 589/26,310, p=0.007). These results are the first in humans to identify differentially expressed exons associated with RBFOX2 mutations in CHD and suggests RBFOX2-mediated alternate splicing may influence EMT pathways implicated in the pathogenesis of HLHS.

SiRNA-induced mutation in HIV-1 polypurine tract region and its influence on viral fitness.

Converting single-stranded viral RNA into double stranded DNA for integration is an essential step in HIV-1 replication. Initial polymerization of minus-strand DNA is primed from a host derived tRNA, whereas subsequent plus-strand synthesis requires viral primers derived from the 3′ and central polypurine tracts (3′ and cPPTs). The 5′ and 3′ termini of these conserved RNA sequence elements are precisely cleaved by RT-associated RNase H to generate specific primers that are used to initiate plus-strand DNA synthesis. In this study, siRNA wad used to produce a replicative HIV-1 variant contained G(-1)A and T(-16)A substitutions within/adjacent to the 3′PPT sequence. Introducing either or both mutations into the 3′PPT region or only the G(-1)A substitution in the cPPT region of NL4-3 produced infectious virus with decreased fitness relative to the wild-type virus. In contrast, introducing the T(-16)A or both mutations into the cPPT rendered the virus(es) incapable of replication, most likely due to the F185L integrase mutation produced by this nucleotide substitution. Finally, the effects of G(-1)A and T(-16)A mutations on cleavage of the 3′PPT were examined using an in vitro RNase H cleavage assay. Substrate containing both mutations was mis-cleaved to a greater extent than either wild-type substrate or substrate containing the T(-16)A mutation alone, which is consistent with the observed effects of the equivalent nucleotide substitutions on the replication fitness of NL4-3 virus. In conclusion, siRNA targeting of the HIV-1 3′PPT region can substantially suppress virus replication, and this selective pressure can be used to generate infectious virus containing mutations within or near the HIV-1 PPT. Moreover, in-depth analysis of the resistance mutations demonstrates that although virus containing a G(-1)A mutation within the 3′PPT is capable of replication, this nucleotide substitution shifts the 3′-terminal cleavage site in the 3′PPT by one nucleotide (nt) and significantly reduces viral fitness.

Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex

Doublesex (dsx) is a downstream key regulator in insect sex determination pathway. In Drosophila, alternative splicing of Dm-dsx gene is sex-specifically regulated by transformer (tra), in which the functional TRA promotes female-specific Dm-dsx. However, the sex determination pathway in Lepidoptera is not well understood; here we focused on alternative splicing of doublesex (dsx) in two agricultural pests, Asian corn borer (Ostrinia furnacalis) and cotton bollworm (Helicoverpa armigera), as well as the silkworm (Bombyx mori). More than a dozen new alternative splicing isoforms of dsx were found in the Lepidopteran females, which exist in all tested developmental stages and differentiated tissues. Alignment of mRNA and protein sequences of doublesex revealed high conservation of this gene in Lepidoptera. Strength analysis of splice sites revealed a weak 5′ splice site at intron 3 in Lepidopteran dsx, which was experimentally confirmed. Furthermore, we identified highly conserved RNA sequences in the Lepidopteran dsx, including RNA elements I (14 nt), II (11 nt), III (26 nt), IV (17 nt), 3E-1 (8 nt) and 3E-2 (8 nt). The RNA elements III and IV were previously found in exon 4 of B. mori dsx and bound with Bm-PSI, which suppressed the inclusion of exons 3 & 4 into the male-specific Bm-dsx. Then we identified and analyzed the homologous genes of Bm-psi in the two Lepidopteran pests, which expressed at similar levels and exhibited a unique isoform in the males and females from each Lepidoptera. Importantly, mutagenesis of Bm-dsx mini-genes and their expression in BmN cell line demonstrated that three RNA elements are involved in the female-specific alternative splicing of Bm-dsx. Mutations in the RNA cis-elements 3E-1 and 3E-2 resulted in decreased inclusion of exon 3 into the female-specific dsx mRNA, suggesting that these two elements would be exonic splicing enhancers that facilitate the recognition of the weak 5′ splice site at intron 3 of Lepidopteran dsx. We propose that the 5′ splice sites at intron 3 are weak, resulting in multiple alternative splicing events in intron 3 of female Lepidoptera dsx. Activation of the 5′ splice site requires regulatory cis-elements in exons 3 for female-specific splicing of Lepidoptera dsx.

Bone Morphogenetic Protein/SMAD Signaling Orients Cell Fate Decision by Impairing KSRP-Dependent MicroRNA Maturation

MicroRNAs (miRNAs) are essential regulators of development, physiology, and evolution, and their biogenesis is strictly controlled at multiple levels. Regulatory proteins, such as KSRP, modulate rates and timing of enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Here, we show that KSRP silencing in mesenchymal C2C12 cells produces a change in the transcriptome largely overlapping that induced by bone morphogenetic protein 2 (BMP2) signaling activation. This induces osteoblastic differentiation while preventing myogenic differentiation. KSRP silencing- and BMP2-dependent myogenic miRNA (myomiR) maturation blockade is required for osteoblastic differentiation of C2C12 cells. Our results demonstrate that phosphorylated R-SMAD proteins, the transducers of BMP2 signal, associate with phosphorylated KSRP and block its interaction with primary myomiRs. This abrogates KSRP-dependent myomiR maturation, with SMAD4, SMAD5, and SMAD9 silencing being able to rescue KSRP function. Thus, SMAD-induced blockade of KSRP-dependent myomiR maturation is critical for orienting C2C12 cell differentiation toward osteoblastic lineage.

Reprint of: Core protein-mediated 5′–3′ annealing of the West Nile virus genomic RNA in vitro

Genome cyclization through conserved RNA sequences located in the 5′ and 3′ terminal regions of flavivirus genomic RNA is essential for virus replication. Although the role of various cis-acting RNA elements in panhandle formation is well characterized, almost nothing is known about the potential contribution of protein cofactors to viral RNA cyclization. Proteins with nucleic acid chaperone activities are encoded by many viruses (e.g., retroviruses, coronaviruses) to facilitate RNA structural rearrangements and RNA–RNA interactions during the viral replicative cycle. Since the core protein of flaviviruses is also endowed with potent RNA chaperone activities, we decided to examine the effect of West Nile virus (WNV) core on 5′–3′ genomic RNA annealing in vitro. Core protein binding resulted in a dramatic, dose-dependent increase in 5′–3′ complex formation. Mutations introduced in either the UAR (upstream AUG region) or CS (conserved sequence) elements of the viral RNA diminished core protein-dependent annealing, while compensatory mutations restored the 5′–3′ RNA interaction. The activity responsible for stimulating RNA annealing was mapped to the C-terminal RNA-binding region of WNV core protein. These results indicate that core protein – besides its function in viral particle formation – might be involved in the regulation of flavivirus genomic RNA cyclization, and thus virus replication.

Core protein-mediated 5′–3′ annealing of the West Nile virus genomic RNA in vitro

Genome cyclization through conserved RNA sequences located in the 5′ and 3′ terminal regions of flavivirus genomic RNA is essential for virus replication. Although the role of various cis-acting RNA elements in panhandle formation is well characterized, almost nothing is known about the potential contribution of protein cofactors to viral RNA cyclization. Proteins with nucleic acid chaperone activities are encoded by many viruses (e.g., retroviruses, coronaviruses) to facilitate RNA structural rearrangements and RNA–RNA interactions during the viral replicative cycle. Since the core protein of flaviviruses is also endowed with potent RNA chaperone activities, we decided to examine the effect of West Nile virus (WNV) core on 5′–3′ genomic RNA annealing in vitro. Core protein binding resulted in a dramatic, dose-dependent increase in 5′–3′ complex formation. Mutations introduced in either the UAR (upstream AUG region) or CS (conserved sequence) elements of the viral RNA diminished core protein-dependent annealing, while compensatory mutations restored the 5′–3′ RNA interaction. The activity responsible for stimulating RNA annealing was mapped to the C-terminal RNA-binding region of WNV core protein. These results indicate that core protein – besides its function in viral particle formation – might be involved in the regulation of flavivirus genomic RNA cyclization, and thus virus replication.

A liver‐specific microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication

Regulated gene expression and progeny production are essential for persistent and chronic infection by human pathogens, such as hepatitis B virus (HBV), which affects >400 million people worldwide and is a major cause of liver disease. In this study, we provide the first direct evidence that a liver-specific microRNA, miR-122, binds to a highly conserved HBV pregenomic RNA sequence via base-pairing interactions and inhibits HBV gene expression and replication. The miR-122 target sequence is located at the coding region of the mRNA for the viral polymerase and the 3' untranslated region of the mRNA for the core protein. In cultured cells, HBV gene expression and replication reduces with increased expression of miR-122, and the expression of miR-122 decreases in the presence of HBV infection and replication. Furthermore, analyses of clinical samples demonstrated an inverse linear correlation in vivo between the miR-122 level and the viral loads in the peripheral blood mononuclear cells of HBV-positive patients. Our results suggest that miR-122 may down-regulate HBV replication by binding to the viral target sequence, contributing to the persistent/chronic infection of HBV, and that HBV-induced modulation of miR-122 expression may represent a mechanism to facilitate viral pathogenesis.