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Germline NPM1 mutations lead to altered rRNA 2’-O-methylation and cause dyskeratosis congenita (HITS-CLIP)

ABSTRACT: We identify nucleophosmin (NPM1) as an essential regulator of 2’-O-methylation on rRNA by directly binding C/D box small nucleolar RNAs (snoRNAs), thereby modulating translation. We demonstrate the importance of 2’-O-Me regulated translation for cellular growth, differentiation and hematopoietic stem cells (HSC) maintenance, and show that Npm1-inactivation in adult HSCs results in bone marrow failure (BMF). We identify NPM1 germline mutations in DC patients presenting with BMF; and demonstrate that they are deficient in snoRNA binding. CRISPR knock-in mice harboring the DC germline NPM1 mutation recapitulate both hematological and non-hematological features of DC. Thus, our findings provide a direct demonstration of the role of 2’-O-Me in the pathogenesis of human disease

ORGANISM(S): Mus musculus

PROVIDER: GSE135724 | GEO | 2019/08/13

REPOSITORIES: GEO

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Project description:Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDR) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend lifespan in DC cells. Of the cellular clones isolated due to increased lifespan, 34% had a guide RNA (gRNA) targeting CEBPB while gRNAs targeting WSB1, MED28 and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA-Seq to compare DC and control cells. Expression of inflammatory genes were found to be significantly elevated (p<0.0001) in addition to a key subset of these inflammation-related genes (IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5) which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere-length dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB’s contribution in DC cells’ senescence is ROS-independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.

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Germline NPM1 mutations lead to altered rRNA 2’-O-methylation and cause dyskeratosis congenita (HITS-CLIP)

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