Non-coding RNA profiling by high throughput sequencing
Summary
Leukemogenesis requires enhanced self-renewal activity, which is induced by specific oncogenes. The underlying molecular mechanisms remain incompletely understood. We transduced mouse lineage negative bone marrow cells (enriched for hematopoietic stem and progenitor cells) with retrovirus expressing leukemic oncogene AML1-ETO9a, MYC and MLL-AF9 as well as empty vector (MIG). We found that all three oncogenes enhanced snoRNA formation. High abundance of snoRNAs was observed in primary human AML specimens with the notable exception of NPM1 mutant AML. Leukemogenesis by AML1-ETO required expression of the groucho related Amino Enhancer of Split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, loss of C/D box snoRNAs with concomitant loss of rRNA 2’-O-methylation resulted in decreased leukemia self-renewal potential.In summary, we identified C/D box snoRNAs and rRNA 2’-O-methylation as critical determinants of leukemic stem cell activity.
Overall design
We used small RNA-Seq to determine the expression profile of small nucleolar RNA (snoRNA) in 63 primary AML patient samples. To further investigate the role of AES and DDX21 in snoRNA formation we also analyzed kasumi-1 cell lines with shRNA based downregulation of AES or DDX21. Furthermore, we show that mouse snoRNAs are induced by leukemia oncogene AML-ETO9a, MLL-AF9 and MYC in mouse lineage negative bone marrow cells.
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