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RNA (New York, N.Y.) Aug 2022Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification...
Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass analysis. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.
Topics: Liposomes; Nanoparticles; RNA Caps; RNA, Messenger; Ribonuclease H
PubMed: 35680168
DOI: 10.1261/rna.079173.122 -
The FEBS Journal Mar 2009
Topics: Animals; Biocatalysis; Databases, Protein; Mice; Mice, Knockout; Ribonuclease H
PubMed: 19228198
DOI: 10.1111/j.1742-4658.2009.06906.x -
Molecular Cell Mar 2017R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1,...
R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.
Topics: Binding Sites; DNA; Genomic Instability; HEK293 Cells; HeLa Cells; Humans; Nucleic Acid Conformation; Protein Binding; Protein Interaction Domains and Motifs; RNA; RNA Interference; Replication Protein A; Ribonuclease H; Structure-Activity Relationship; Time Factors; Transcription, Genetic; Transfection
PubMed: 28257700
DOI: 10.1016/j.molcel.2017.01.029 -
The Journal of Cell Biology Jun 2021The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has...
The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.
Topics: Antibodies, Monoclonal; Antibody Affinity; Artifacts; DNA; Humans; Nucleic Acid Heteroduplexes; RNA; Ribonuclease H
PubMed: 33830170
DOI: 10.1083/jcb.202004079 -
The Enzymes 2021All retroviruses encode the enzyme, reverse transcriptase (RT), which is involved in the conversion of the single-stranded viral RNA genome into double-stranded DNA. RT... (Review)
Review
All retroviruses encode the enzyme, reverse transcriptase (RT), which is involved in the conversion of the single-stranded viral RNA genome into double-stranded DNA. RT is a multifunctional enzyme and exhibits DNA polymerase and ribonuclease H (RNH) activities, both of which are essential to the reverse-transcription process. Despite the successful development of polymerase-targeting antiviral drugs over the last three decades, no bona fide inhibitor against the RNH activity of HIV-1 RT has progressed to clinical evaluation. In this review article, we describe the retroviral RNH function and inhibition, with primary consideration of the structural aspects of inhibition.
Topics: DNA-Directed DNA Polymerase; HIV-1; Reverse Transcription; Ribonuclease H
PubMed: 34861939
DOI: 10.1016/bs.enz.2021.07.007 -
The FEBS Journal Mar 2009Ribonucleases H are enzymes that cleave the RNA of RNA/DNA hybrids that form during replication and repair and which could lead to DNA instability if they were not... (Review)
Review
Ribonucleases H are enzymes that cleave the RNA of RNA/DNA hybrids that form during replication and repair and which could lead to DNA instability if they were not processed. There are two main types of RNase H, and at least one of them is present in most organisms. Eukaryotic RNases H are larger and more complex than their prokaryotic counterparts. Eukaryotic RNase H1 has acquired a hybrid binding domain that confers processivity and affinity for the substrate, whereas eukaryotic RNase H2 is composed of three different proteins: the catalytic subunit (2A), similar to the monomeric prokaryotic RNase HII, and two other subunits (2B and 2C) that have no prokaryotic counterparts and as yet unknown functions, but that are necessary for catalysis. In this minireview, we discuss some of the most recent findings on eukaryotic RNases H1 and H2, focusing on the structural data on complexes between human RNase H1 and RNA/DNA hybrids that had provided great detail of how the hybrid binding- and RNase H-domains recognize and cleave the RNA strand of the hybrid substrates. We also describe the progress made in understanding the in vivo function of eukaryotic RNases H. Although prokayotes and some single-cell eukaryotes do not require RNases H for viability, in higher eukaryotes RNases H are essential. Rnaseh1 null mice arrest development around E8.5 because RNase H1 is necessary during embryogenesis for mitochondrial DNA replication. Mutations in any of the three subunits of human RNase H2 cause Aicardi-Goutières syndrome, a human neurological disorder with devastating consequences.
Topics: Amino Acid Sequence; Animals; Humans; Mice; Mitochondria; Molecular Sequence Data; Protein Conformation; Ribonuclease H; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 19228196
DOI: 10.1111/j.1742-4658.2009.06908.x -
The Journal of Biological Chemistry Aug 2020The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a...
The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.
Topics: Enzyme Stability; Escherichia coli; Escherichia coli Proteins; Protein Folding; Protein Unfolding; Proteolysis; Ribonuclease H; Ribosomes
PubMed: 32527724
DOI: 10.1074/jbc.RA120.013909 -
The FEBS Journal Mar 2009Retroviral reverse transcriptases possess both a DNA polymerase and an RNase H activity. The linkage with the DNA polymerase activity endows the retroviral RNases H with... (Review)
Review
Retroviral reverse transcriptases possess both a DNA polymerase and an RNase H activity. The linkage with the DNA polymerase activity endows the retroviral RNases H with unique properties not found in the cellular counterparts. In addition to the typical endonuclease activity on a DNA/RNA hybrid, cleavage by the retroviral enzymes is also directed by both DNA 3' recessed and RNA 5' recessed ends, and by certain nucleotide sequence preferences in the vicinity of the cleavage site. This spectrum of specificities enables retroviral RNases H to carry out a series of cleavage reactions during reverse transcription that degrade the viral RNA genome after minus-strand synthesis, precisely generate the primer for the initiation of plus strands, facilitate the initiation of plus-strand synthesis and remove both plus- and minus-strand primers after they have been extended.
Topics: Biocatalysis; Models, Molecular; Protein Conformation; Retroviridae; Ribonuclease H; Structure-Activity Relationship; Substrate Specificity; Transcription, Genetic
PubMed: 19228195
DOI: 10.1111/j.1742-4658.2009.06909.x -
Nucleic Acids Research Jun 2020Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically...
Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.
Topics: Cell Membrane; DNA-Binding Proteins; Humans; Intracellular Space; Ligands; Phosphorothioate Oligonucleotides; Protein Binding; Protein Domains; Proteins; RNA-Binding Proteins; Ribonuclease H; Structure-Activity Relationship; Transcription Factors
PubMed: 32356888
DOI: 10.1093/nar/gkaa299 -
Nucleic Acid Therapeutics Apr 2017In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides... (Review)
Review
In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.
Topics: Biological Transport; Cell Membrane; Eukaryotic Cells; Gene Expression Regulation; Humans; Kinetics; Membrane Glycoproteins; Nucleic Acid Hybridization; Oligonucleotides, Antisense; RNA, Messenger; Receptors, Complement; Ribonuclease H
PubMed: 28080221
DOI: 10.1089/nat.2016.0656