Hnrnpk Overexpression Drives Nucleolar Aberrancies Causing Ribosomopathies

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, polyploi