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3898 Ribosome-Associated Protein Ribonuclease Inhibitor (RNH1) Regulates Hematopoietic Specific Translation

Program: Oral and Poster Abstracts
Session: 509. Bone Marrow Failure and Cancer Predisposition Syndromes: Congenital: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Bone Marrow Failure Syndromes, Diseases, Biological Processes, molecular biology
Monday, December 12, 2022, 6:00 PM-8:00 PM

Mayuresh Anant Sarangdhar1,2*, Martina Stillinovic, PhD1,2*, Nicola Daniele Andina, MD, PhD1,2,3*, Aubry Tardivel1*, Anne Angelillo-Scherrer, MD-PhD1,2 and Ramanjaneyulu Allam, PhD1,2*

1Department for BioMedical Research, University of Bern, Bern, Switzerland
2Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, Bern, Switzerland
3Department of Biomedicine, University Hospital Basel, Basel, Switzerland

The hematopoietic system is central to the lifelong production of blood and persistent immune surveillance. However, it has a propensity for translational defects following ribosomal mutations/dysregulation in ribosomopathies such as Diamond-Blackfan anemia (DBA). Ribosomal proteins (RPs) are ubiquitously present and mutations in their genes should show systemic effect, but what makes a hematopoietic system especially vulnerable to the translational defects is a longstanding unanswered question. Here, we report a higher vertebrate specific, ribosome associated protein Ribonuclease inhibitor 1 (RNH1) as a hematopoietic specific translation regulator. RNH1 is known for its strong interaction with angiogenin (ANG) to inhibit its ribonucleolytic activity. Previously, we have detected RNH1 in polysome fractions and shown to regulate erythropoiesis by controlling GATA1 mRNA translation.

RNH1 is ubiquitously present in human cells and thus knockout of RNH1 should affect the mRNA translation in all cell-types. Surprisingly, RNH1 deletion in human hematopoietic origin cell-lines such as erythroid leukemia cells (K562), monocytic cells (THP1 and MOLM13) and T lymphocytes (Jurkat) decreased polysome levels but not in non-hematopoietic origin cell-lines such as HEK293, HaCat, HeLa and SH-SY5Y. Further, luciferase reporter assays, Click-iT AHA (l-azidohomoalanine) assays and OP–Puro incorporation assay also confirmed decreased translation in hematopoietic RNH1 KO cell-lines but not in non-hematopoietic origin cells. Interestingly, expression of full length RNH1 protein as well as mutants that are unable to bind RNases were able to rescue translation defects in RNH1-KO hematopoietic cells, excluding involvement of RNase mediated mRNA degradation in translation defects. Since complete deletion of RNH1 is embryonic lethal in mice, we generated hematopoietic-specific and liver-specific RNH1 deleted mice. Supporting above results, OP-Puro incorporation experiments in these mice revealed that RNH1-deficiency leads to reduced translation in hematopoietic cells but not in hepatocytes. To gain insight into how RNH1 regulates hematopoietic specific translation, we first focused on understanding how RNH1 regulates mRNA translation. Using RNA-seq, polysome-seq, biochemical experiments and bioinformatic analysis, we found that RNH1 regulates RPs gene expression at translation level selectively in hematopoietic cells. This control of translation by RNH1 was found independent of mTOR signalling. Further, we found that translation inhibition in RNH1-KO hematopoietic cells is not a stress response as the common marker of cellular stress eIF2α remains unphosphorylated in RNH1-KO cells. RNH1 binds to ANG and ANG is known to inhibit translation under stress via generating small RNAs (tiRNAs) by tRNA cleavage. Surprisingly, we did not find an increase in the tiRNAs in the RNH1-KO cells at steady state without stress. Further studies are required to understand how RNH1 regulates cell-type specific translation.

Interestingly, it has been shown that RNH1 is translationally down-regulated after RPS19 knockdown in primary human HSPCs. RPS19 is frequently mutated in DBA. Supporting RNH1 role in hematopoietic specific translation and RPs gene expression, overexpression of it rescued erythroid and translation defects in RPS19 knockdown cells. Collectively, our results unravel the existence of hematopoietic specific translation regulator and may partially explain cell-type specific defects caused by mutations in RPs genes.

Disclosures: Angelillo-Scherrer: SNSF: Research Funding; Vaderis: Consultancy; Pfizer: Other: Speaker fees; Silence Therapeutics: Research Funding.

*signifies non-member of ASH