Expanding single-cell transcriptomics to chart the role of noncoding RNAs in normal hematopoiesis - PROJECT ABSTRACT Hematopoietic stem and progenitor cells (HSPCs) carry the important responsibility of producing an enormous number of myeloid and lymphoid cells. Defects in HSPC function can lead to benign and malignant neoplasms as well as bone marrow failure, often driven by somatic mutations in key regulators. Noncoding (nc)RNAs are emerging regulators of hematopoietic self-renewal and lineage specification. Long (l)ncRNAs specifically contribute by modulating genome topology, chromatin compaction, gene transcription, post-transcriptional processing, translation, and cellular signaling. In addition, transfer (t)RNAs, which shuttle amino acids to the ribosome, shape the cellular proteome by enhancing the translation of messenger RNAs enriched for cognate codons. When dysregulated, these ncRNAs contribute to the pathogenesis of hematologic neoplasms. Despite these advances, ncRNAs are not profiled in standard single-cell RNA sequencing (scRNAseq): ncRNAs are mostly not endogenously polyadenylated (EPA), preventing their capture by oligo-deoxythymidine primers. This limitation of scRNAseq critically prevents the study of how ncRNAs bias and commit cells toward hematopoietic lineages. We hypothesize that ncRNAs, particularly lncRNAs and tRNAs, bias HSPCs toward specific cellular fates. To test this hypothesis, we developed a method, single-cell total RNA sequencing (scTRS), that incorporates RNA de-modification and exogenous polyadenylation to extend RNA capture to modified and non- EPA ncRNAs, which are not captured by other methods. Here, first, I aim to develop a computational pipeline to enable the processing and analysis of scTRS data. Second, I will apply scTRS in primary HSPCs to identify candidate ncRNA drivers of normal hematopoiesis. Then, I propose a series of in vitro perturbation experiments to validate and examine the roles of these candidate drivers of normal differentiation. Finally, because the scTRS workflow is compatible with commercially available droplet-based platforms and the computational pipeline will be broadly shared, scTRS will be available to research groups across fields for broad impact. Collectively, this work will deliver novel and actionable insights into how ncRNAs help orchestrate human hematopoiesis.
