MiR-595, RPL27A and ribosomal dysgenesis

The importance of miRNAs in regulating gene expression has been addressed in several haematological cancers including myelodysplastic syndrome (MDS). In MDS, miR-145 and miR-146a have been associated with some of phenotypic features of 5q- syndrome. Monosomy 7/7q deletion is the second most common chromosomal abnormality in MDS and identifies a subgroup of patients with poor prognosis. Thus far, no studies have examined the role of miRNA dysregulation in MDS patients with monosomy 7. In this study we examined the functional consequences of deletion of miRNAs localized to 7q with particular focus on miR-595, which is localized to 7q36.3, which is commonly deleted region in 7q- and -7. Most of the available methodologies for miRNA target identification are based on computational algorithms. Therefore, there is a relatively high incidence of false positive target identification. To enable the identification of biologically relevant microRNA targets, a novel functional assay was developed in our lab, which is based on positive/negative selection (Gaken et al, 2012). Using this assay, I identified functional targets of miR-595 and correlated this with the MDS phenotype. Applying the functional assay to identify targets for miR-595 resulted in the identification of several targets including RPL27A, a large ribosomal subunit protein that was validated as a target for miR-595 by qRT-PCR and western blot analysis. Given the recognised association of ribosomal protein (RPS14) with 5q- syndrome, subtype of MDS, I assessed the biological function of RPL27A in order to identify its contribution to the disease pathogenesis. Using Polysomes fractionation,. RPL27A knockdown showed a reduction in the large ribosomal subunit (60S). On day 6 of the knockdown, the depletion of RPL27A led to p53 activation and induced variable levels of apoptosis in all cell lines. Interestingly, like RPS14 knockdown, the effect of RPL27A knockdown in 48 h was p53 dependent. The p53 independent effect, which was observed on day 6, was attributed to a defect in ribosome biogenesis based on the disruption of nucleolar staining in HCT-116 and HCT-116 p53-/- with depleted RPL27A using fibrillarin antibody and similar effects were seen with RPS14.