Transfer RNAs in Hematopoietic Stem Cell Function

ABSTRACT
Hematopoietic Stem Cells (HSCs) produce all cells of the blood lineage throughout life. Defects in HSC self-
renewal can lead to immunological defects, anemia, and bone marrow failure. Enhanced HSC self-renewal can
result in hematopoietic malignancies. Thus, precise regulation of HSC self-renewal is essential for maintaining
hematopoietic and human health. Our previous work has shown that adult HSCs tightly control protein synthesis
and that modest changes in protein synthesis impair HSC self-renewal and function. However, the mechanisms
that regulate mRNA translation in HSCs remain largely unknown. Transfer RNAs (tRNAs) are non-coding
adaptor RNAs critical for mRNA translation that are encoded by hundreds of genes in the mammalian genome,
with multiple functional genes capable of decoding virtually every codon. We previously showed that the tRNA
repertoire influences neuronal function but the effect of changes in tRNA expression on hematopoietic cells is
unknown. In preliminary studies, we found that loss of n-Tr22, a member of the five gene arginine UCU tRNA
family significantly impairs HSC maintenance and self-renewal, and this is exacerbated in a sensitized genetic
background lacking the ribosome rescue factor Gtpbp2, resulting in a complete loss of adult, but not fetal HSCs.
We hypothesize that the sensitivity of adult HSCs to the n-Tr22 mutation may be due to differences in the tRNA
repertoire between adult HSCs and restricted progenitors, and between HSCs at different developmental stages.
The impact of tRNA mutations may be further influenced by differential codon usage in the transcriptome of these
cell populations. Finally, there may be cell-type-specific differences in the signaling pathways activated by loss
of a tRNA. We propose to test this hypothesis by using chromatin immunoprecipitation and sequencing to
determine the tRNA repertoire in the hematopoietic system. We will also analyze how this tRNA mutation
influences the maintenance and function of HSCs in the presence and absence of the Gtpbp2 mutation using
flow cytometry and long-term multilineage reconstitution assays. Finally, we will determine the effects of the loss
of n-Tr22 and Gtpbp2 on protein synthesis and gene expression by incorporation of a puromycin analog,
ribosome profiling, and RNA-sequencing. The results from this grant will not only shed light on the role of tRNAs
in regulating mRNA translation in the hematopoietic system, but also provide a means to understand the role of
these genes in the phenotypic heterogeneity common to many human hematopoietic disorders.

Grant Number: 5R01HL165172-03
NIH Institute/Center: NIH
Principal Investigator: SUSAN ACKERMAN