Statistical Analysis of Readthrough Levels for Nonsense Mutations in Mammalian Cells Reveals a Major Determinant of Response to Gentamicin

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable

Genomic features and computational identification of human microRNAs under long-range developmental regulation

<p>Abstract</p> <p>Background</p> <p>Recent functional studies have demonstrated that many microRNAs (miRNAs) are expressed by RNA polymerase II in a specific spatiotemporal manner during the development of organisms and play a key role in cell-lineage decisions and morphogenesis. They are therefore functionally related to a number of key protein coding developmental genes, that form genomic regulatory blocks (GRBs) with arrays of highly conserved non-coding elements (HCNEs) functioning as long-range enhancers that collaboratively regulate the expression of their target genes. Given this functional similarity as well as recent zebrafish transgenesis assays showing that the miR-9 family is indeed regulated by HCNEs with enhancer activity, we hypothesized that this type of miRNA regulation is prevalent. In this paper, we therefore systematically investigate the regulatory landscape around conserved self-transcribed miRNAs (ST miRNAs), with their own known or computationally inferred promoters, by analyzing the hallmarks of GRB target genes. These include not only the density of HCNEs in their vicinity but also the presence of large CpG islands (CGIs) and distinct patterns of histone modification marks associated with developmental genes.</p> <p>Results</p> <p>Our results show that a subset of the conserved ST miRNAs we studied shares properties similar to those of protein-coding GRB target genes: they are located in regions of significantly higher HCNE/enhancer binding density and are more likely to be associated with CGIs. Furthermore, their putative promoters have both activating as well as silencing histone modification marks during development and differentiation. Based on these results we used both an elevated HCNE density in the genomic vicinity as well as the presence of a bivalent promoter to identify 29 putative GRB target miRNAs/miRNA clusters, over two-thirds of which are known to play a role during development and differentiation. Furthermore these predictions include miRNAs of the miR-9 family, which are the only experimentally verified GRB target miRNAs.</p> <p>Conclusions</p> <p>A subset of the conserved miRNA loci we investigated exhibits typical characteristics of GRB target genes, which may partially explain their complex expression profiles during development.</p