Cap-Independent Protein Translation in Hematopoiesis

Protein synthesis is differentially regulated across hematopoietic stem and progenitor cells (HSPCs). HSCs have lower and more highly regulated rates of protein synthesis than corresponding progenitors (Signer et al., 2014) and have a weak correlation between mRNA expression and protein abundance (Zaro et al., 2019). We hypothesized that these trends may be explained, in part, by differences in HSPC utilization of specific subsets of translation initiation, the rate-limiting step of protein synthesis. Most mRNAs assemble initiation factors at the 5' m⁷G-cap to recruit ribosomes, but some have internal ribosome entry sites (IRES) in their 5' untranslated regions (UTRs) that assist ribosome complex loading independent of some or all of the cap-machinery. IRES-mediated cap-independent translation is best understood in viral genomes where they serve to enable viral protein translation, even when host translation is inhibited. Eukaryotic IRES have been associated with stress survival, and the deletion of the IRES in Runx1 5' UTR is embryonic lethal with aberrant hematopoietic development (Nagamachi et al., 2010). How IRES-mediated cap-independent translation affects adult hematopoiesis is not well defined.

We assessed translation initiation in adult hematopoiesis by generating a C57BL/6J mouse model expressing a bicistronic fluorescent reporter that simultaneously reflects the rates of cap-dependent and IRES-mediated cap-independent translation. Contray to expectation, the ratio of IRES-mediated cap-independent to cap-dependent translation increased with murine HSPC differentiation. More mature progenitors had higher rates of IRES translation, independent of cell-cycle status. Human HSPCs transplanted into NSG mice phenocopied the murine trend. This phenomenon was independent of the IRES sequence as it was observed with both the RUNX1 and ECMV IRES. Therefore, the ratio of IRES-mediated cap-independent to cap-dependent translation increases with both mouse and human HSPC differentiation.

To assess if differential use of translation initiation machinery associated with hematopoietic function, in vitro colony assays were performed on HSPCs of comparable immunophenotype that differed in their relative use of IRES-mediated cap-independent and cap-dependent translation. HSPCs with the lowest ratio of IRES-mediated to cap-dependent translation had significantly greater clonogenicity compared to those with the highest ratio. To identify regulators of the IRES-mediated cap-independent to cap-dependent translation process, we performed a genome-wide Crispr inhibition screen on K562 cells expressing the bicistronic translation initiation reporter. Positive regulators of IRES-mediated translation were enriched for ribosomal biogenesis and rRNA processing, including the splicing factor and ribosomal biogenesis regulator PTBP1.

Therefore, translation initiation is differentially regulated in HSPC differentiation, mediated in part by PTBP1. Increased IRES-mediated cap-independent translation restricts clonogenicity. Shifting translation initiation from cap-dependent to cap-independent may represent a mechanism beyond gene expression that governs the hematopoietic differentiation process.

Sykes:Clear Creek Bio: Current holder of stock options in a privately-held company. Sankaran:Branch Biosciences: Consultancy; Forma Therapeutics: Consultancy; Cellarity: Consultancy; Ensoma: Consultancy; Novartis: Consultancy; Sana Biotechnology: Consultancy. Scadden:Editas Tx: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Agios Tx: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Inzen: Consultancy; LifeVaultBio: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Fate Tx: Current equity holder in publicly-traded company; Clear Creek Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; VCanBio: Consultancy; Dianippon Sumitomo Pharma: Research Funding.