Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes) and ribosome density (the average number of ribosomes bound per unit length of coding sequence) were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay.

Flexibility and constraint in the nucleosome core landscape of Caenorhabditis elegans chromatin.

Nucleosome positions within the chromatin landscape are known to serve as a major determinant of DNA accessibility to transcription factors and other interacting components. To delineate nucleosomal patterns in a model genetic organism, Caenorhabditis elegans, we have carried out a genome-wide analysis in which DNA fragments corresponding to nucleosome cores were liberated using an enzyme (micrococcal nuclease) with a strong preference for cleavage in non-nucleosomal regions. Sequence analysis of 284,091 putative nucleosome cores obtained in this manner from a mixed-stage population of C. elegans reveals a combined picture of flexibility and constraint in nucleosome positioning. As has previously been observed in studies of individual loci in diverse biological systems, we observe areas in the genome where nucleosomes can adopt a wide variety of positions in a given region, areas with little or no nucleosome coverage, and areas where nucleosomes reproducibly adopt a specific positional pattern. In addition to illuminating numerous aspects of chromatin structure for C. elegans, this analysis provides a reference from which to begin an investigation of relationships between the nucleosomal pattern, chromosomal architecture, and lineage-based gene activity on a genome-wide scale.

Population genetic variation in gene expression is associated with phenotypic variation in Saccharomyces cerevisiae.

BACKGROUND: The relationship between genetic variation in gene expression and phenotypic variation observable in nature is not well understood. Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understanding the molecular basis and evolution of complex traits. RESULTS: We compared levels of gene expression among nine natural isolates of Saccharomyces cerevisiae grown either in the presence or absence of copper sulfate. Of the nine strains, two show a reduced growth rate and two others are rust colored in the presence of copper sulfate. We identified 633 genes that show significant differences in expression among strains. Of these genes, 20 were correlated with resistance to copper sulfate and 24 were correlated with rust coloration. The function of these genes in combination with their expression pattern suggests the presence of both correlative and causative expression differences. But the majority of differentially expressed genes were not correlated with either phenotype and showed the same expression pattern both in the presence and absence of copper sulfate. To determine whether these expression differences may contribute to phenotypic variation under other environmental conditions, we examined one phenotype, freeze tolerance, predicted by the differential expression of the aquaporin gene AQY2. We found freeze tolerance is associated with the expression of AQY2. CONCLUSIONS: Gene expression differences provide substantial insight into the molecular basis of naturally occurring traits and can be used to predict environment dependent phenotypic variation.