ABSTRACT
The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.
Subject(s)
Drug Resistance, Viral/genetics , Exons/genetics , HIV-1/genetics , Mutation/genetics , RNA Splicing/genetics , RNA, Messenger/genetics , Cell Line , Drug Resistance, Viral/drug effects , Exons/drug effects , HEK293 Cells , HIV Integrase/genetics , HIV Integrase Inhibitors/pharmacology , HIV-1/drug effects , HeLa Cells , Humans , Mutation/drug effects , RNA Splice Sites/drug effects , RNA Splice Sites/genetics , RNA Splicing/drug effects , Regulatory Sequences, Nucleic Acid/genetics , Virus Replication/drug effects , Virus Replication/geneticsABSTRACT
Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness (e.g. use of cryptic splice sites or downstream AUGs). When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5' untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.
Subject(s)
Binding Sites/genetics , Morpholinos/pharmacology , Zebrafish Proteins/genetics , Zebrafish/genetics , 5' Untranslated Regions/drug effects , 5' Untranslated Regions/genetics , Alleles , Animals , Embryo, Nonmammalian , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Gene Knockdown Techniques/methods , Genetic Techniques , Mutation/drug effects , Mutation/genetics , Phenotype , RNA Splice Sites/drug effects , RNA Splice Sites/genetics , Sensitivity and Specificity , Zygote/drug effectsABSTRACT
The recognition of RNA functions beyond canonical protein synthesis has challenged the central dogma of molecular biology. Indeed, RNA is now known to directly regulate many important cellular processes, including transcription, splicing, translation, and epigenetic modifications. The misregulation of these processes in disease has led to an appreciation of RNA as a therapeutic target. This potential was first recognized in bacteria and viruses, but discoveries of new RNA classes following the sequencing of the human genome have invigorated exploration of its disease-related functions in mammals. As stable structure formation is evolving as a hallmark of mammalian RNAs, the prospect of utilizing small molecules to specifically probe the function of RNA structural domains and their interactions is gaining increased recognition. To date, researchers have discovered bioactive small molecules that modulate phenotypes by binding to expanded repeats, microRNAs, G-quadruplex structures, and RNA splice sites in neurological disorders, cancers, and other diseases. The lessons learned from achieving these successes both call for additional studies and encourage exploration of the plethora of mammalian RNAs whose precise mechanisms of action remain to be elucidated. Efforts toward understanding fundamental principles of small molecule-RNA recognition combined with advances in methodology development should pave the way toward targeting emerging RNA classes such as long noncoding RNAs. Together, these endeavors can unlock the full potential of small molecule-based probing of RNA-regulated processes and enable us to discover new biology and underexplored avenues for therapeutic intervention in human disease. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA in Disease and Development > RNA in Disease.
Subject(s)
RNA Probes/chemistry , RNA Probes/pharmacology , RNA/chemistry , RNA/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Animals , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Nervous System Diseases/genetics , Nervous System Diseases/metabolism , Phenotype , RNA/genetics , RNA Splice Sites/drug effects , RNA Splice Sites/geneticsABSTRACT
There have been many attempts to unveil the therapeutic potential of antisense molecules during the last decade. Due to its specific role in canonical Wnt signalling, ß-catenin is a potential target for an antisense-based antitumour therapy. In order to establish such a strategy with peptide nucleic acids, we developed a reporter assay for quantification of antisense effects. The luciferase-based assay detects splice blocking with high sensitivity. Using this assay, we show that the splice donor of exon 13 of ß-catenin is particularly suitable for an antisense strategy, as it results in a truncated protein which lacks transactivating functions. Since the truncated proteins retain the interactions with Tcf/Lef proteins, they act in a dominant negative fashion competing with wild-type proteins and thus blocking the transcriptional activity of ß-catenin. Furthermore, we show that the truncation does not interfere with binding of cadherin and α-catenin, both essential for its function in cell adhesion. Therefore, the antisense strategy blocks Wnt signalling with high efficiency but retains other important functions of ß-catenin.
Subject(s)
Gene Knockdown Techniques/methods , Peptide Nucleic Acids/pharmacology , Wnt Signaling Pathway/drug effects , beta Catenin/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Exons , HEK293 Cells , HeLa Cells , Humans , RNA Splice Sites/drug effects , TCF Transcription Factors/metabolism , beta Catenin/antagonists & inhibitors , beta Catenin/metabolismABSTRACT
See Fratta and Isaacs (doi:10.1093/brain/awy091) for a scientific commentary on this article.The RNA binding proteins TDP-43 (encoded by TARDBP) and hnRNP A1 (HNRNPA1) are each mutated in certain amyotrophic lateral sclerosis cases and are often mislocalized in cytoplasmic aggregates within motor neurons of affected patients. Cytoplasmic inclusions of TDP-43, which are accompanied by a depletion of nuclear TDP-43, are observed in most amyotrophic lateral sclerosis cases and nearly half of frontotemporal dementia cases. Here, we report that TDP-43 binds HNRNPA1 pre-mRNA and modulates its splicing, and that depletion of nuclear TDP-43 results in increased inclusion of a cassette exon in the HNRNPA1 transcript, and consequently elevated protein levels of an isoform containing an elongated prion-like domain, referred to as hnRNP A1B. Combined in vivo and in vitro approaches demonstrated greater fibrillization propensity for hnRNP A1B, which drives protein aggregation and is toxic to cells. Moreover, amyotrophic lateral sclerosis patients with documented TDP-43 pathology showed neuronal hnRNP A1B cytoplasmic accumulation, indicating that TDP-43 mislocalization may contribute to neuronal vulnerability and loss via altered HNRNPA1 pre-mRNA splicing and function. Given that TDP-43 and hnRNP A1 each bind, and thus modulate, a third of the transcriptome, our data suggest a much broader disruption in RNA metabolism than previously considered.
Subject(s)
Alternative Splicing/genetics , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , DNA-Binding Proteins/metabolism , Heterogeneous Nuclear Ribonucleoprotein A1/genetics , Protein Aggregation, Pathological/metabolism , Alternative Splicing/drug effects , Cell Death/drug effects , Cell Death/genetics , Cytoplasm/drug effects , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , Dactinomycin/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , HEK293 Cells , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Humans , Immunoprecipitation , Motor Neurons/drug effects , Motor Neurons/metabolism , Mutation/genetics , Nucleic Acid Synthesis Inhibitors/pharmacology , Oligopeptides/genetics , Oligopeptides/metabolism , RNA Splice Sites/drug effects , RNA Splice Sites/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Spinal Cord/pathology , TransfectionABSTRACT
GU-AG consensus sequences are used for intron recognition in the majority of cases of pre-mRNA splicing in eukaryotes. Mutations at splice junctions often cause exon skipping, short deletions, or insertions in the mature mRNA, underlying one common molecular mechanism of genetic diseases. Using N-ethyl-N-nitrosourea, a novel recessive mutation named seal was produced, associated with fragile bones and susceptibility to fractures (spine and limbs). A single nucleotide transversion (T â A) at the second position of intron 36 of the Col1a1 gene, encoding the type I collagen, α1 chain, was responsible for the phenotype. Col1a1 seal mRNA expression occurred at greatly reduced levels compared to the wild-type transcript, resulting in reduced and aberrant collagen fibers in tibiae of seal homozygous mice. Unexpectedly, splicing of Col1a1 seal mRNA followed the normal pattern despite the presence of the donor splice site mutation, likely due to the action of a putative intronic splicing enhancer present in intron 25, which appeared to function redundantly with the splice donor site of intron 36. Seal mice represent a model of human osteogenesis imperfecta, and reveal a previously unknown mechanism for splicing "rescue."
Subject(s)
Collagen Type I/genetics , Ethylnitrosourea/pharmacology , Mutation , Osteogenesis Imperfecta/genetics , RNA Splice Sites/drug effects , Animals , Collagen Type I, alpha 1 Chain , Disease Models, Animal , Humans , Introns/genetics , Male , Mice , RNA Splicing/geneticsABSTRACT
Phosphorylation of the spliceosome is essential for RNA splicing, yet how and to what extent kinase signaling affects splicing have not been defined on a genome-wide basis. Using a chemical genetic approach, we show in Schizosaccharomyces pombe that the SR protein kinase Dsk1 is required for efficient splicing of introns with suboptimal splice sites. Systematic substrate mapping in fission yeast and human cells revealed that SRPKs target evolutionarily conserved spliceosomal proteins, including the branchpoint-binding protein Bpb1 (SF1 in humans), by using an RXXSP consensus motif for substrate recognition. Phosphorylation of SF1 increases SF1 binding to introns with nonconsensus splice sites in vitro, and mutation of such sites to consensus relieves the requirement for Dsk1 and phosphorylated Bpb1 in vivo. Modulation of splicing efficiency through kinase signaling pathways may allow tuning of gene expression in response to environmental and developmental cues.
Subject(s)
Introns/genetics , Protein Serine-Threonine Kinases/genetics , RNA Splicing , Schizosaccharomyces pombe Proteins/genetics , Amino Acid Motifs/genetics , Amino Acid Sequence , Blotting, Western , Mutation , Oligonucleotide Array Sequence Analysis , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , RNA Splice Sites/drug effects , RNA Splicing Factors , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Substrate SpecificityABSTRACT
Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5' of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3' splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition.
Subject(s)
Alternative Splicing/drug effects , Antineoplastic Agents/pharmacology , Pyrans/pharmacology , Spiro Compounds/pharmacology , Cell Division/drug effects , Down-Regulation/drug effects , HeLa Cells , Humans , Phosphoproteins/metabolism , Protein Binding/drug effects , Protein Stability/drug effects , RNA Precursors/metabolism , RNA Splice Sites/drug effects , RNA Splicing Factors , Ribonucleoprotein, U2 Small Nuclear/metabolism , Spliceosomes/drug effectsABSTRACT
The fibrillin-1 gene (FBN1) mutations are associated with a broad spectrum of disorders including Marfan syndrome (MFS) and show great clinical heterogeneity. An underrepresentation for mutations leading to premature termination codon (PTC) in FBN1 exons 24-32 was found in neonatal or severe MFS but the underlying cause was unclear. This study thoroughly examined two FBN1 mutations on exons 24-32 region to illustrate the molecular mechanisms underlying these FBN1 mutations on MFS etiology. Two nucleotide substitutions, c.3208G> C, the last nucleotide of exon 26, and c.3209A>G, the first nucleotide of exon 27, affecting the same amino acid, p.D1070H and p.D1070G, respectively, gave very different phenotypes. We demonstrate that c.3208G>C generates two alternatively spliced transcripts, while c.3209A>G does not affect the splicing. We further demonstrate that the aberrantly spliced transcripts do not go through nonsense-mediated decay, but rather produce unstable, premature protein peptides that are degraded by endoplasmic reticulum associated degradation. The molecular mechanism outlined here defines a model for the pathogenesis of PTC-containing mutation within the exons 24-32 of FBN1 in MFS. Furthermore, our data suggest that PTC mutation within this region may lead to early lethality in neonatal MFS.
Subject(s)
Exons/genetics , Marfan Syndrome/genetics , Nucleotides/genetics , RNA Splice Sites/genetics , Base Sequence , Blotting, Western , Cell Line , Codon, Nonsense/genetics , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Fatal Outcome , Female , Fibrillin-1 , Fibrillins , Humans , Infant, Newborn , Intracellular Space/drug effects , Intracellular Space/metabolism , Leupeptins/pharmacology , Microfilament Proteins/genetics , Microscopy, Fluorescence , Molecular Sequence Data , Mutant Proteins/metabolism , Mutation/genetics , Pregnancy , Protein Processing, Post-Translational/drug effects , RNA Splice Sites/drug effects , RNA Splicing/drug effects , RNA Splicing/genetics , RNA Stability/drug effects , RNA Stability/geneticsABSTRACT
Alternative pre-mRNA splicing is an important element in eukaryotic gene expression, as most of the protein-coding genes use this process to generate multiple protein isoforms from a single gene. An increasing number of human diseases are now recognized to be caused by the selection of 'wrong' alternative exons. Research during the last few years identified a number of low-molecular-mass chemical substances that can change alternative exon usage. Most of these substances act by either blocking histone deacetylases or by interfering with the phosphorylation of splicing factors. How the remaining large number of these substances affect splicing is not yet fully understood. The emergence of these low-molecular-mass substances provides not only probes for studying alternative pre-mRNA splicing, but also opens the door to the possible harnessing of these compounds as drugs to control diseases caused by the selection of 'wrong' splice sites.
Subject(s)
Alternative Splicing/drug effects , RNA Splice Sites/drug effects , RNA Splice Sites/genetics , Alternative Splicing/genetics , Animals , HumansABSTRACT
The efficient and non-toxic nuclear delivery of steric-block oligonucleotides (ON) is a prerequisite for therapeutic strategies involving splice correction or exon skipping. Cationic cell penetrating peptides (CPPs) have given rise to much interest for the intracellular delivery of biomolecules, but their efficiency in promoting cytoplasmic or nuclear delivery of oligonucleotides has been hampered by endocytic sequestration and subsequent degradation of most internalized material in endocytic compartments. In the present study, we compared the splice correction activity of three different CPPs conjugated to PMO(705), a steric-block ON targeted against the mutated splicing site of human beta-globin pre-mRNA in the HeLa pLuc705 splice correction model. In contrast to Tat48-60 (Tat) and oligoarginine (R(9)F(2)) PMO(705) conjugates, the 6-aminohexanoic-spaced oligoarginine (R-Ahx-R)(4)-PMO(705) conjugate was able to promote an efficient splice correction in the absence of endosomolytic agents. Our mechanistic investigations about its uptake mechanisms lead to the conclusion that these three vectors are internalized using the same endocytic route involving proteoglycans, but that the (R-Ahx-R)(4)-PMO(705) conjugate has the unique ability to escape from lysosomial fate and to access to the nuclear compartment. This vector, which has displays an extremely low cytotoxicity, the ability to function without chloroquine adjunction and in the presence of serum proteins. It thus offers a promising lead for the development of vectors able to enhance the delivery of therapeutic steric-block ON in clinically relevant models.
Subject(s)
Drug Delivery Systems/methods , Morpholines/administration & dosage , Oligonucleotides, Antisense/administration & dosage , Peptide Nucleic Acids/administration & dosage , Peptides/administration & dosage , RNA Splicing/drug effects , Animals , CHO Cells , Cell Culture Techniques , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cricetinae , Cricetulus , Endocytosis , Endosomes/drug effects , Endosomes/metabolism , HeLa Cells , Humans , Morpholines/chemistry , Morpholinos , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/pharmacokinetics , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/pharmacology , Peptides/chemistry , Peptides/pharmacology , RNA Splice Sites/drug effectsABSTRACT
Increased expression of cyclooxygenase-2 (COX-2) has been implicated in the onset of both term and preterm labor. In this context, both selective and nonselective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labor. However, recent evidence indicates that these tocolytics may have potentially adverse fetal and maternal side effects. Therefore, the development of more specific and nontoxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2 by specifically targeting both the 3' and 5' acceptor and donor sites of exon 4 of COX-2's pre-mRNA sequence. Confocal microscopy on "live" cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the Endo-Porter formula (Gene-Tools, LLC, Philomath, OR), with delivery efficiencies of 82 and 78%, respectively, in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by lipopolysaccharide administration, was observed at both the mRNA and protein levels. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay, which reflects the rate of conversion of arachidonic acid to prostaglandin H2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and nontoxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labor and other inflammatory conditions.