Background: Protein biogenesis is a complex process involving nucleoli and ribosomes. Alterations in any step could lead to alterations in ribosome functionality and protein synthesis. Hnrnpk is an RNA-binding protein (RBP) involve in these processes, finding that an overexpression (OE) produces nucleus and nucleolar stress (NS), decreases transcription, and drives an imbalance in ribosome biogenesis, causing a reduced translation. Aims: To elucidate how hnRNP K dysregulation affects the hematopoietic stem cell (HSCs) biology. Methods: To study the impact of Hnrnpk OE in vivo, we developed an inducible tamoxifen mouse model, HnrnpkTg/hUbc-CreERT2. Survival was evaluated by Kaplan-Meier, phenotype was described by symptoms/signs, CBC, bone marrow (BM) H/E, IHC and FCM analysis, and serum IL-6 ELISA. HSCs were cultured to study the impact of Hnrnpk OE in the HSCs dynamics. Hnrnpk OE was established in vitro using CRISPR/SAM. RNA-seq analysis was performed in a single read 85-base format and analyzed with DESeq2. TMT-based deep proteome profiling was also performed. Both were GSEA preranked. Transcription and translation were tested using Click-it RNA and HPG kit respectively, and translation efficiency by polysome assay. NS were analyzed by confocal microscopy and transmission electron microscopy (TEM). Protein-protein interaction between Hnrnpk and Ncl was studied by IP. Possible phenotype rescue was carried using HnrnpkTg/hUbc-CreERT2/ c-Myclox/wt, HnrnpkTg/hUbc-CreERT2/ Tp53lox/wt and HnrnpkTg/hUBC-CreERT2/ NclKDin vitro and in vivo models. Cell cycle FACS, senescence assays and karyotyping were performed. Molecular mechanism was elucidated by qRT-PCR and WB. Results: Hnrnpk Tg/hUbc-CreERT2 mice had widespread Hnrnpk OE and lifespan's reduction. By CBC, we found the development of leukopenia, lymphopenia, anaemia and thrombocytopenia (Fig.A). BM H/E, IHC and FCM showed a reduction of B220 + and CD34 + and Sca1 + HSCs, and an increment in myeloid cells (Fig. B). Also, we found higher senescent β-galactosidase expression in BM and IL-6 in vivo (Fig.C). Then, we found a decay in viability and an exhaustion in HSCs (Fig.D). To understand Hnrnpk implication in BM failure phenotype in vivo, we generated Hnrnpk OE cells (Fig.E). RNA-seq showed an upregulation in G2/M-checkpoint pathway related molecules (Fig.F), confirmed by FACS analysis, showing and increment of arrested G2/M phase-cells (Fig.G). Moreover, we showed a rise in β-galactosidase activity, polyploidy and genomic instability (Fig. H), linked to an increment in p21 and p16 (Fig.I). Then, TEM revealed nucleolar alterations in the in vitro model, including segregation, anormal accumulations or fragmentation of nucleolar components, alterations validated by confocal microscopy (Fig.J-K). Also, we found a Ncl increment in vitro, consistent with the protein-protein interaction between Hnrnpk and Ncl. Then, proteomics showed that Hnrnpk OE correlates with ribosome biogenesis regulators, c-Myc and mTOR dysregulation (Fig.L). Moreover, we found a decrease in transcription (Fig.M), consistent with rRNAs reduction, driving translation and protein synthesis deficiency (Fig.N). There was a partial reversion of NS hallmarks in HnrnpkTg/hUbc-CreERT2/ c-Myclox/wt, HnrnpkTg/hUbc-CreERT2/ Tp53lox/wt and HnrnpkTg/hUBC-CreERT2/ NclKD MEFs in vitro models and partially rescue phenotype in vivo (Fig.O). Finally, we focused on the nucleolus. Thus, Hnrnpk OE cells showed a reduction in Fbl, increase in Ncl and Ncl diffusion in nucleoplasm. All these data suggest the existence of a ribosompathy-like phenotype. Conclusions: this work found that RBPs dysregulation such as Hnrnpk OEdrives BM failure phenotype, promoting the exhaustion of HSCs by nucleoulus/ribosome dysregulation that triggers cell cycle arrest and apoptosis, dependent of p53. We therefore suggest that Hnrnpk induces ribosome dysfunction consistent with some types of ribosomopathies. This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295, PI21/00191), ISCIII Miguel Servet (CP19/00140), Cancer Research UK [C355/A26819], Sociedad Española de Hematología y Hemoterapia (SEHH), FC AECC and AIRC under the Accelerator Award Program and National Cancer Institutes of Health Award (R01CA207204, SMP) Leukemia and Lymphoma Society (6577-19, SMP).
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Dec 5, 2024
James M Fulcher + 14
Open Access
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