Recent studies have established the critical role of microRNA (miRs) in the development of myelodysplastic syndromes (MDS). By analyzing deep sequencing data from The Cancer Genome Atlas project, we revealed that a distinctive miR-181a variant (isomiR) with a single 5-nucleotide (nt) addition is the dominant isoform expressed in patients. Given this addition is predicted to alter the target spectrum of isomiR-181a compared to wild-type miR-181a, we hypothesize that the effect of miR-181a is secondary to its isomiR, and the overexpression of isomiR-181a alters signaling pathways in hematopoietic stem cells associated with marrow failure syndromes. We will overexpress miR-181a and its isomiR with vector control in CD34+ cells, and perform HITS-CLIP combined with bioinformatics to decode mRNA binding sequences, with validation by luciferase assays in a high-throughput system. Immunoblotting will be used for further validation on protein expression. Predicted mRNA targets will be analyzed by IPA and DAVID programs, and the impact of critical biological pathways will be identified by enforced expression of miR-181a, isomiR-181a or vector control in CD34+ cells, followed by quantification of apoptosis, proliferation and CFC assays. A LNA oligonucleotide may be used to knock down miR181 and the isomiR. To functionally validate their role in MDS in vivo, NOG mice will be transplanted with CD34+ cells lentivirally transduced with miR-181a and isomiR-181a, and hematopoietic effect will be examined by blood counts, smear morphology of blood and marrow and progenitor assays. We expected that the expression of target protein will be inversely correlated with miR-181a and its isomiR, but only overexpression of isomiR-181a will induce MDS-like findings. We believe that appreciating the mechanisms by which miR participate in the biology of marrow failure will lead to the identification of predictive biomarkers and potentially novel therapeutic approaches.