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PloS One 2019The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical...
The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com-RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.
Topics: Bacteriophage mu; Base Pairing; Binding Sites; DNA; Genes, Viral; Haemophilus; Nucleic Acid Conformation; Open Reading Frames; Protein Biosynthesis; RNA, Complementary; RNA, Messenger; SELEX Aptamer Technique; Solvents; Transcription, Genetic; Viral Proteins; Zinc Fingers
PubMed: 31022205
DOI: 10.1371/journal.pone.0214481 -
Handbook of Experimental Pharmacology 2006Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar... (Review)
Review
Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation. This conformational restriction is translated into unprecedented hybridization affinity towards complementary single-stranded RNA molecules. That makes fully modified LNAs, LNA/DNA mixmers, or LNA/RNA mixmers uniquely suited for mimicking RNA structures and for RNA targeting in vitro or in vivo. The focus of this chapter is on LNA antisense, LNA-modified DNAzymes (LNAzymes), LNA-modified small interfering (si)RNA (siLNA), LNA-enhanced expression profiling by real-time RT-PCR and detection and analysis of microRNAs by LNA-modified probes.
Topics: Animals; Humans; Nucleic Acids; RNA; RNA, Antisense; RNA, Complementary; RNA, Small Interfering
PubMed: 16594628
DOI: 10.1007/3-540-27262-3_21 -
Nucleic Acids Research Jul 2002Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides...
Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.
Topics: Alternative Splicing; Animals; Cattle; DNA; DNA, Antisense; Fetal Blood; Globins; HeLa Cells; Humans; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligonucleotides; RNA, Complementary; RNA, Messenger; Ribonuclease H; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transfection
PubMed: 12087157
DOI: 10.1093/nar/gkf412 -
Journal of the American Pharmacists... 2022A conceptual framework has been developed specifically for cost-related nonadherence (CRNA) that differs from models proposed for general medication nonadherence. (Review)
Review
BACKGROUND
A conceptual framework has been developed specifically for cost-related nonadherence (CRNA) that differs from models proposed for general medication nonadherence.
OBJECTIVE
This study aimed to demonstrate that CRNA studies are best explained by a conceptual framework developed for general medication nonadherence.
METHODS
A systematic literature review was conducted using MEDLINE via PubMed, CINAHL, ScienceDirect, and Google Scholar databases from 2008 to 2020. Articles were considered for inclusion if they were research studies, used a self-reported measure for CRNA, and provided self-reported data on factors associated with CRNA.
RESULTS
A total of 58 studies were identified and included in the review. Factors related to financial pressures were consistently associated with CRNA corresponding to conceptual frameworks for both CRNA and general medication nonadherence. However, noneconomic factors, classified as moderators in the CRNA framework (i.e., patient factors, disease factors, clinician factors), consistently demonstrated independent effects, often with similar strength of association compared with economic factors. Overall, the pattern of risk factors identified in CRNA studies was consistent with general nonadherence except for indicators of poor health. Poor health was often associated with an increased risk of CRNA, whereas the inverse association is generally observed in general nonadherence studies (i.e., nonadherence higher in primary prevention vs. secondary prevention). However, the apparent disagreement was likely caused by the general population studied rather than a unique causal pathway for CRNA.
CONCLUSION
Financial difficulties are extremely common among people who take prescription medications. However, current evidence is insufficient to support a conceptual framework that differs from general medication nonadherence.
Topics: Humans; Medication Adherence; Prescription Drugs; RNA, Complementary; Risk Factors; Self Report
PubMed: 35131189
DOI: 10.1016/j.japh.2022.01.011 -
The Journal of Biological Chemistry Dec 2010Influenza virus transcription is a prototype of primer-dependent initiation. Its replication mechanism is thought to be primer-independent. The internal initiation and...
Influenza virus transcription is a prototype of primer-dependent initiation. Its replication mechanism is thought to be primer-independent. The internal initiation and realignment model for influenza virus genome replication has been recently proposed (Deng, T., Vreede, F. T., and Brownlee, G. G. (2006) J. Virol. 80, 2337-2348). We obtained new results, which led us to propose a novel model for the initiation of viral RNA (vRNA) replication. In our study, we analyzed the initiation mechanisms of influenza virus vRNA and complementary RNA (cRNA) synthesis in vitro, using purified RNA polymerase (RdRp) and 84-nt model RNA templates. We found that, for vRNA → cRNA →, RdRp initiated replication from the second nucleotide of the 3'-end. Therefore, host RNA-specific ribonucleotidyltransferases are required to add one nucleotide (purine residues are preferred) to the 3'-end of vRNA to make the complete copy of vRNA. This hypothesis was experimentally proven using poly(A) polymerase. For cRNA → vRNA, the dinucleotide primer AG was synthesized from UC (fourth and fifth from the 3'-end) by RdRp pausing at the sixth U of UUU and realigning at the 3'-end of cRNA template; then RdRp was able to read through the entire template RNA. The RdRp initiation complex was not stable until it had read through the UUU of cRNA and the UUUU of vRNA at their respective 3'-ends. This was because primers overlapping with the first U of the clusters did not initiate transcription efficiently, and the initiation product of v84+G (the v84 template with an extra G at its 3'-end), AGC, realigned to the 3'-end.
Topics: Oligoribonucleotides; Orthomyxoviridae; RNA, Complementary; RNA, Viral; RNA-Dependent RNA Polymerase; Viral Proteins; Virus Replication
PubMed: 20858902
DOI: 10.1074/jbc.M110.130062 -
BMC Bioinformatics Nov 2019Vast amounts of next generation sequencing RNA data has been deposited in archives, accompanying very diverse original studies. The data is readily available also for...
BACKGROUND
Vast amounts of next generation sequencing RNA data has been deposited in archives, accompanying very diverse original studies. The data is readily available also for other purposes such as genome annotation or transcriptome assembly. However, selecting a subset of available experiments, sequencing runs and reads for this purpose is a nontrivial task and complicated by the inhomogeneity of the data.
RESULTS
This article presents the software VARUS that selects, downloads and aligns reads from NCBI's Sequence Read Archive, given only the species' binomial name and genome. VARUS automatically chooses runs from among all archived runs to randomly select subsets of reads. The objective of its online algorithm is to cover a large number of transcripts adequately when network bandwidth and computing resources are limited. For most tested species VARUS achieved both a higher sensitivity and specificity with a lower number of downloaded reads than when runs were manually selected. At the example of twelve eukaryotic genomes, we show that RNA-Seq that was sampled with VARUS is well-suited for fully-automatic genome annotation with BRAKER.
CONCLUSIONS
With VARUS, genome annotation can be automatized to the extent that not even the selection and quality control of RNA-Seq has to be done manually. This introduces the possibility to have fully automatized genome annotation loops over potentially many species without incurring a loss of accuracy over a manually supervised annotation process.
Topics: Algorithms; Animals; Databases, Genetic; Drosophila melanogaster; Eukaryota; High-Throughput Nucleotide Sequencing; Introns; Molecular Sequence Annotation; RNA, Complementary; Sequence Analysis, RNA; Software; Transcriptome
PubMed: 31703556
DOI: 10.1186/s12859-019-3182-x -
Cell Reports Jan 2013Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate...
Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate mitochondrial restructuring and the cellular processes that drive this adaptive response are largely obscure. To better define these systems, we performed matched quantitative genomic and proteomic analyses of mouse muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in cellular iron homeostasis are highly coordinated with this process and that depletion of cellular iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and oxidative capacity. We further show that this process is universal across a broad range of cell types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be leveraged to explore posttranscriptional and posttranslational processes that are essential for mitochondrial adaptation.
Topics: Animals; Cell Respiration; DNA, Mitochondrial; Gene Expression Regulation; HEK293 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Iron; Iron Chelating Agents; Mice; Mitochondrial Proteins; Mitochondrial Turnover; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Proteomics; RNA, Complementary; RNA, Messenger; Rats; Time Factors; Trans-Activators; Transcription Factors
PubMed: 23318259
DOI: 10.1016/j.celrep.2012.11.029 -
Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice.Nucleic Acids Research Jun 2018Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the...
Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.
Topics: Animals; Apolipoproteins B; Binding Sites; Cells, Cultured; Female; Genetic Therapy; Liver; Mice; Mice, Inbred C57BL; Nucleic Acid Heteroduplexes; Oligonucleotides, Antisense; Proprotein Convertase 9; RNA, Complementary; RNA, Messenger; Ribonuclease H
PubMed: 29790953
DOI: 10.1093/nar/gky397 -
RNA Biology May 2021Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5' and 3' untranslated regions (UTRs). All segments have highly...
Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5' and 3' untranslated regions (UTRs). All segments have highly conserved extremities of 13 and 12 nucleotides at the 5' and 3' UTRs, respectively, constructing the viral RNA (vRNA) promoter. Adjacent to the duplex stem of 3 base pairs (bps) between the two conserved strands, additional 1-4 bps are existing in a segment-specific manner. We investigated the roles of the matrix (M) segment-specific base pair between the 14 nucleotide uridine (U14') of the 5' UTR and the 13 nucleotide adenosine (A13) of the 3' UTR by preparing possible vRNA promoters, named vXY, as well as cRNA promoters, named cYX. We analysed their RNA-dependent RNA replication efficiency using the minigenome replicon system and an enzyme assay system with synthetic RNA promoters. Notably, in contrast to vAC(s) that is a synthetic vRNA promoter with A14' and C13, base-pair disruption at the complementary RNA (cRNA) promoter in cAC(s), which has A13' and C14, not only reduced viral RNA replication in cells but also impaired initiation of unprimed vRNA synthesis. Reverse genetics experiments confirmatively exhibited that this breakage in the cRNA promoter affected the rescue of infectious virus. The present study suggests that the first segment-specific base pair plays an essential role in generating infectious viruses by regulating the promoter activity of cRNA rather than vRNA. It could provide insights into the role of the segment-specific nucleotides in viral genome replication for sustainable infection.
Topics: 3' Untranslated Regions; Animals; Dogs; Gene Expression Regulation, Viral; HEK293 Cells; Humans; Influenza A virus; Madin Darby Canine Kidney Cells; Nucleic Acid Conformation; Nucleotides; Promoter Regions, Genetic; RNA, Complementary; RNA, Viral; Transcription, Genetic
PubMed: 33317417
DOI: 10.1080/15476286.2020.1864182 -
Nucleic Acids Research Dec 1996A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing...
A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.
Topics: Base Composition; Base Sequence; Circular Dichroism; DNA; Drug Stability; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes; Phosphoric Diester Hydrolases; Purines; Pyrimidines; RNA, Complementary; Thermodynamics
PubMed: 8972851
DOI: 10.1093/nar/24.23.4660