APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages.

The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific C>U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C>U RNA editing in mammals.

Variables and strategies in development of therapeutic post-transcriptional gene silencing agents.

Post-transcriptional gene silencing (PTGS) agents such as ribozymes, RNAi and antisense have substantial potential for gene therapy of human retinal degenerations. These technologies are used to knockdown a specific target RNA and its cognate protein. The disease target mRNA may be a mutant mRNA causing an autosomal dominant retinal degeneration or a normal mRNA that is overexpressed in certain diseases. All PTGS technologies depend upon the initial critical annealing event of the PTGS ligand to the target RNA. This event requires that the PTGS agent is in a conformational state able to support hybridization and that the target have a large and accessible single-stranded platform to allow rapid annealing, although such platforms are rare. We address the biocomplexity that currently limits PTGS therapeutic development with particular emphasis on biophysical variables that influence cellular performance. We address the different strategies that can be used for development of PTGS agents intended for therapeutic translation. These issues apply generally to the development of PTGS agents for retinal, ocular, or systemic diseases. This review should assist the interested reader to rapidly appreciate critical variables in PTGS development and facilitate initial design and testing of such agents against new targets of clinical interest.

Bottlenecks in development of retinal therapeutic post-transcriptional gene silencing agents.

Development of post-transcriptional gene silencing (PTGS) agents for therapeutic purposes is an immense challenge in modern biology. Established technologies used to knockdown a specific target RNA and its cognate protein: antisense, ribozyme, RNAi, all conditionally depend upon an initial, critical annealing event of the PTGS ligand to a target RNA. In this review we address the nature of the bottlenecks, emphasizing the biocomplexity of target RNA structure, that currently limit PTGS therapeutic development. We briefly review existing and emerging technologies designed to release these constraints to realize the potential of PTGS agents in gene based therapies.

GJB2: the spectrum of deafness-causing allele variants and their phenotype.

Genetic testing was completed on 1,294 persons with deafness referred to the Molecular Otolaryngology Research Laboratories to establish a diagnosis of DFNB1. Exon 2 of GJB2 was screened for coding sequence allele variants by denaturing high-performance liquid chromatography (DHPLC) complemented by bidirectional sequencing. If two deafness-causing mutations of GJB2 (encoding Connexin 26) were identified, further screening was not performed. If only a single deafness-causing mutation was identified, we screened for the g.1777179_2085947del (hereafter called del(GJB6-D13S1830); GenBank NT_024524.13) and mutations in the noncoding region of GJB2. Phenotype-genotype correlations were evaluated by categorizing mutations as either protein truncating or nontruncating. A total of 205 persons carried two GJB2 exon 2 mutations and were diagnosed as having DFNB1; 100 persons carried only a single deafness-causing allele variant of exon 2. A total of 37 of these persons were c.35delG carriers, and 51 carried other allele variants of GJB2. Persons diagnosed with DFNB1 segregating two truncating/nonsense mutations had a more severe phenotype than persons carrying two missense mutations, with mean hearing impairments being 88 and 37%, respectively (P < 0.05). The number of deaf c.35delG carriers was greater than expected when compared to the c.35delG carrier frequency in normal-hearing controls (P < 0.05), suggesting the existence of at least one other mutation outside the GJB2 coding region that does not complement GJB2 deafness-causing allele variants.

Gene Expression Changes in Chinchilla Cochlea from Noise-Induced Temporary Threshold Shift.

Acoustic overstimulation produces many anatomical, biochemical and physiological changes in the inner ear. However, the changes in gene expression that underlie these biological changes are poorly understood. Our approach to investigating this problem is to use gene microarrays to measure the changes in gene expression in the chinchilla inner ear following a 3 h or 6 h noise exposure (95 dB SPL, 707-1414 Hz). This noise exposure causes a temporary threshold shift (~40 dB) and a temporary reduction in distortion product otoacoustic emissions (DPOAE), but no permanent hearing loss or hair cell loss. Here, we present data showing (1) the suitability of mouse and human complementary DNA (cDNA) clones for detecting chinchilla cochlear gene transcripts, and (2) the change in cochlear gene transcripts in noise exposed chinchillas. Chinchilla cochlear transcript probes exhibited strong and discrete signals on both mouse and human cDNA filter arrays. Since the strongest hybridization occurred with mouse clones, mouse cDNA microarrays were used to study noise-induced changes in gene expression. Chinchilla cDNA probes were differentially labelled with Cy3 (control) or Cy5 (noise exposed) by random primed synthesis, hybridized to 8750 mouse cDNAs arrayed on microscope slides and analysed by laser fluorescent microscopy. Several classes of genes exhibited time-dependent up regulation of transcription, including those involved in protein synthesis, metabolism, cytoskeletal proteins, and calcium binding proteins. The results are discussed in relationship to previous studies showing noise-induced changes in structural proteins, calcium binding proteins, metabolic enzymes and membrane bound vesicles.