Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides.

Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro.

Control of backbone chemistry and chirality boost oligonucleotide splice switching activity.

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype.

Stereochemistry Enhances Potency, Efficacy, and Durability of Antisense Oligonucleotides In Vitro and In Vivo in Multiple Species.

Purpose: Antisense oligonucleotides have been under investigation as potential therapeutics for many diseases, including inherited retinal diseases. Chemical modifications, such as chiral phosphorothioate (PS) backbone modification, are often used to improve stability and pharmacokinetic properties of these molecules. We aimed to generate a stereopure (metastasis-associated lung adenocarcinoma transcript 1) antisense oligonucleotide as a tool to assess the impact stereochemistry has on potency, efficacy, and durability of oligonucleotide activity when delivered by intravitreal injection to eye.

Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides.

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers.

Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology.

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes.

An Antibody to Detect Alanine-Expanded PABPN1: A New Tool to Study Oculopharyngeal Muscular Dystrophy.

Background: Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs.

Inhibition of Glycolate Oxidase With Dicer-substrate siRNA Reduces Calcium Oxalate Deposition in a Mouse Model of Primary Hyperoxaluria Type 1.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure.

Creatine kinase B is necessary to limit myoblast fusion during myogenesis.

Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes.

Control of mRNA stability contributes to low levels of nuclear poly(A) binding protein 1 (PABPN1) in skeletal muscle.

Background: The nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays critical roles at multiple steps in post-transcriptional regulation of gene expression. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing, and weakness in the proximal limb muscles. Why alanine-expanded PABPN1 leads to muscle-specific pathology is unknown.

PABPN1: molecular function and muscle disease.

The polyadenosine RNA binding protein polyadenylate-binding nuclear protein 1 (PABPN1) plays key roles in post-transcriptional processing of RNA. Although PABPN1 is ubiquitously expressed and presumably contributes to control of gene expression in all tissues, mutation of the PABPN1 gene causes the disease oculopharyngeal muscular dystrophy (OPMD), in which a limited set of skeletal muscles are affected. A major goal in the field of OPMD research is to understand why mutation of a ubiquitously expressed gene leads to a muscle-specific disease.

Expression of interferon-γ, interferon-α and related genes in individuals with Down syndrome and periodontitis.

Background: Recently, attenuation of anti-inflammatory and increase of pro-inflammatory mediators was demonstrated in individuals with Down syndrome (DS) in comparison with euploid patients during periodontal disease (PD), suggesting a shift to a more aggressive inflammation in DS.

Aim: To determine the influence of DS in the modulation of interferons (IFNs) signaling pathway in PD.

Materials And Methods: Clinical periodontal assessment was performed and gingival tissue samples obtained from a total of 51 subjects, including 19 DS individuals with PD, 20 euploid individuals with PD and 12 euploid individuals without PD.

H2B ubiquitylation controls the formation of export-competent mRNP.

Histone H2B ubiquitylation is a transcription-dependent modification that not only regulates nucleosome dynamics but also controls the trimethylation of histone H3 on lysine 4 by promoting ubiquitylation of Swd2, a component of both the histone methyltransferase COMPASS complex and the cleavage and polyadenylation factor(CPF). We show that preventing either H2B ubiquitylation or H2B-dependent modification of Swd2 results in nuclear accumulation of poly(A) RNA due to a defect in the integrity and stability of APT, a subcomplex of the CPF. Ubiquitin-regulated APT complex dynamics is required for the correct recruitment of the mRNA export receptor Mex67 to nuclear mRNPs.

Expression of the interleukin-10 signaling pathway genes in individuals with Down syndrome and periodontitis.

Background: Individuals with Down syndrome (DS) have a higher prevalence and severity of periodontal disease, which cannot be explained by poor oral hygiene alone and is related to changes in the immune response. The aim of this study is to evaluate whether DS was associated with differential modulation of expression of genes associated with proinflammatory and anti-inflammatory responses in periodontal disease.

Methods: A total of 51 individuals were evaluated: 19 individuals with DS and periodontal disease (group 1), 20 euploid individuals with periodontal disease (group 2; positive control), and 12 euploid individuals without periodontal disease (group 3; negative control).

RNA-binding proteins and gene regulation in myogenesis.

Skeletal muscle development, repair and function are dependent on highly coordinated expression of many genes. RNA-binding proteins are crucial determinants of gene expression in the health and disease of various tissues, including skeletal muscle. A variety of RNA-binding proteins are associated with a transcript during its life cycle and define the lifetime, cellular localization, processing and rate at which that transcript is translated and ultimately degraded.

Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans.

Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies.

The mitogen-activated protein kinase Slt2 regulates nuclear retention of non-heat shock mRNAs during heat shock-induced stress.

Cellular adaptation to environmental stress conditions requires rapid and specific changes in gene expression. During heat shock, most polyadenylated mRNAs are retained in the nucleus, whereas the export of heat shock-induced mRNAs is allowed. Although essential mRNA export factors are known, the precise mechanism for regulating transport is not fully understood.

Chemokine expression and control of muscle cell migration during myogenesis.

Adult regenerative myogenesis is vital for restoring normal tissue structure after muscle injury. Muscle regeneration is dependent on progenitor satellite cells, which proliferate in response to injury, and their progeny differentiate and undergo cell-cell fusion to form regenerating myofibers. Myogenic progenitor cells must be precisely regulated and positioned for proper cell fusion to occur.

Recognition of polyadenosine RNA by the zinc finger domain of nuclear poly(A) RNA-binding protein 2 (Nab2) is required for correct mRNA 3'-end formation.

Proteins bound to the poly(A) tail of mRNA transcripts, called poly(A)-binding proteins (Pabs), play critical roles in regulating RNA stability, translation, and nuclear export. Like many mRNA-binding proteins that modulate post-transcriptional processing events, assigning specific functions to Pabs is challenging because these processing events are tightly coupled to one another. To investigate the role that a novel class of zinc finger-containing Pabs plays in these coupled processes, we defined the mode of polyadenosine RNA recognition for the conserved Saccharomyces cerevisiae Nab2 protein and assessed in vivo consequences caused by disruption of RNA binding.

Loss of nuclear poly(A)-binding protein 1 causes defects in myogenesis and mRNA biogenesis.

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein.

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts.

Expression and purification of human respiratory syncytial virus recombinant fusion protein.

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia coli BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism.

Synthetic lethality between eIF5A and Ypt1 reveals a connection between translation and the secretory pathway in yeast.

The putative translation initiation factor 5A (eIF5A) is a small protein, highly conserved and essential in all organisms from archaea to mammals. Although the involvement of eIF5A in translation initiation has been questioned, new evidence reestablished the connection between eIF5A and this cellular process. In order to better understand the function of elF5A, a screen for synthetic lethal gene using the tif51A-3 mutant was carried out and a new mutation (G80D) was found in the essential gene YPT1, encoding a protein involved in vesicular trafficking.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis.

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A.

An interaction between two RNA binding proteins, Nab2 and Pub1, links mRNA processing/export and mRNA stability.

mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein.

Actively transcribed GAL genes can be physically linked to the nuclear pore by the SAGA chromatin modifying complex.

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry.