Identification of MicroRNAs Associated with Erythropoiesis Through Deep Sequencing of Red Blood Cells,

Abstract 3164

We have recently found that human mature red blood cells (RBCs), though lacking large RNAs, possess a diverse and abundant set of microRNAs (miRNAs). MiRNAs are a class of small RNAs that inhibit translation and/or promote degradation of target mRNAs. Red blood cells are an ideal model for small RNA identification due to the relatively low amount of contaminating mRNA, tRNA, and rRNA fragments.

We performed deep sequencing of small RNAs in human RBCs to provide an unbiased set of both known and novel microRNAs, and to gain insight into the process of erythropoiesis. Small RNA libraries were applied to the Illumina GAII deep sequencing platform from both HbAA and HbSS individuals, as microRNAs have also been shown to be genetic determinants of sickle cell phenotypes (Sangokoya 2010). A modified version of the probabilistic modeling algorithm, miRDeep (Friedländer 2008), was used to identify several novel and known microRNAs. Genomic loci that overlapped with miRNAs described in miRBase were identified as known miRNAs. The remaining genomic loci were identified as encoding candidate novel miRNAs. Collectively, we identified 253 predicted miRNA precursor loci from the two samples. 226 miRNA precursor loci annotated in miRBase were identified as known miRNAs, whereas the remaining 27 precursor loci were identified as candidate novel miRNAs. In addition to miRNAs, we identified other small RNAs that did not pass miRDeep criteria. One of the most abundant sequences consisted of a distinct fragment of Ro-associated Y4 RNA (hY4). Y RNAs are involved in chromosomal DNA replication and cell proliferation, and specifically, RNAs hY1 and hY4 have been shown to be present in mature human erythrocytes (O'Brien 1990).

In addition, we also identified the relative expression of both novel and known microRNAs during erythropoiesis using both human and mouse erythroid differentiation models. We are currently investigating the relevant targets and regulatory functions of these microRNAs during erythropoiesis, as well as experimental validation of authentic microRNA fuction of novel microRNAs. This global analysis will enhance our understanding of events dictating red cell maintenance and development.