Identification of novel targets for miR-29a using miRNA proteomics.

MicroRNAs (miRNAs) are short regulatory RNA molecules that interfere with the expression of target mRNA by binding to complementary sequences. Currently, the most common method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and conservation. Such algorithms predict hundreds of targets for each miRNA, necessitating tedious experimentation to identify the few functional targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional miRNA targets. We used Stable Isotope Labeling by amino acids in cell culture (SILAC) based proteomics to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. Over-expression of miR-29a, a miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of a set of proteins. Gene Ontology based classification showed that a significant sub-set of these targets, including Voltage Dependent Anion Channel 1 and 2 (VDAC1 and VDAC2) and ATP synthetase, were mitochondrial proteins involved in apoptosis. Using reporter assays, we established that miR-29a targets the 3' Untranslated Regions (3' UTR) of VDAC1 and VDAC2. However, due to the limited number of proteins identified using this approach and the inability to differentiate between primary and secondary effects we conclude that miRNA-proteomics is of limited utility as a high-throughput alternative for sensitive and unbiased miRNA target identification. However, this approach was valuable for rapid assessment of the impact of the miRNAs on the cellular proteome and its biological role in apoptosis.

Proximity of H2A.Z containing nucleosome to the transcription start site influences gene expression levels in the mammalian liver and brain.

Nucleosome positioning maps of several organisms have shown that Transcription Start Sites (TSSs) are marked by nucleosome depleted regions flanked by strongly positioned nucleosomes. Using genome-wide nucleosome maps and histone variant occupancy in the mouse liver, we show that the majority of genes were associated with a single prominent H2A.Z containing nucleosome in their promoter region. We classified genes into clusters depending on the proximity of H2A.Z to the TSS. The genes with no detectable H2A.Z showed lowest expression level, whereas H2A.Z was positioned closer to the TSS of genes with higher expression levels. We confirmed this relation between the proximity of H2A.Z and expression level in the brain. The proximity of histone variant H2A.Z, but not H3.3 to the TSS, over seven consecutive nucleosomes, was correlated with expression. Further, a nucleosome was positioned over the TSS of silenced genes while it was displaced to expose the TSS in highly expressed genes. Our results suggest that gene expression levels in vivo are determined by accessibility of the TSS and proximity of H2A.Z.