Using High-Throughput Sequencing of Red Blood Cells and Platelets to Identify Micrornas Associated with Hematopoiesis.

Abstract 2326

Human mature red blood cells (RBC) and platelets are both terminally differentiated cells lacking nuclei. However, these two cell types do possess a diverse and abundant set of microRNAs (miRNAs), a set of small, non-coding RNAs acting as post-transcriptional regulators. To identify novel microRNAs associated with differentiation of RBCs and platelets from common progenitors, we performed high-throughput sequencing of these differentiated cells types. In particular, these accessible cells may prove valuable to identify disease biomarkers.

We identified an unbiased set of both known and novel microRNAs by preparing small RNA libraries for application to the Illumina GAII high-throughput sequencing platform. We used a modified version of the probabilistic modeling algorithm, miRDeep (Friedländer 2008), to identify many 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. In RBCs we identified 253 predicted miRNA precursor loci, with 226 miRNA precursor loci annotated in miRBase (known miRNAs), whereas the remaining 27 precursor loci were identified as novel miRNAs. In platelets we identified 566 predicted miRNA precursor loci, with 488 known miRNAs and 78 novel miRNAs. Other small RNAs that did not pass miRDeep criteria were also analyzed. One of the most abundant RNA sequences in the RBC sample consisted of a distinct fragment of Ro-associated Y4 RNA (hY4). Y RNAs have been shown to be involved in chromosomal DNA replication, and Y1 and Y4 have been shown to be present in mature erythrocytes (O'Brien 1990). These distinct non-coding RNAs may possess a unique role in erythroid cell expansion.

In addition, we assessed dynamic changes in the expression level of several selected microRNAs during human erythropoiesis. We are currently investigating relevant targets and regulatory functions of these microRNAs during erythropoiesis and platelet development. This global analysis will enhance our understanding of events dictating red cell and platelet maintenance and development.