The Splicing Factor Heterogeneous Nuclear Ribonucleoprotein L (hnRNPL) Restricts p53 Dependent and p53 Independent Cell Death Pathways In Hematopoietic Stem Cells

• Published in: Blood 2013-11, Vol.122 (21), p.2445-2445
• Main Authors: Gaudreau, Marie-Claude, Grapton, Damien, Heyd, Florian, Vadnais, Charles, Wilhelm, Brian T, Moroy, Tarik
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V122.21.2445.2445

Hematopoiesis is sustained by a pool of multipotent hematopoietic stem cells (HSCs) that have the capacity to differentiate into cells of all blood cell lineages. The pool of long-lived HSCs is maintained throughout life by the self-renewal ability of HSCs. New evidence suggests the process of alternative splicing is an important regulator of the maturation and activation of blood and immune effector cells. It is presently estimated that almost all multi-exon genes in human genome undergo alternative pre-mRNA splicing, and aberrant splicing has been linked to a variety of human pathologies. However, the role that pre-mRNA splicing may have for HSCs behaviour remains largely unexplored. Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is an RNA-binding protein that regulates alternative splicing by binding exonic splicing silencers elements (ESS) resulting in exon exclusion from the mature mRNA. RT-PCR analyses showed that hnRNPL is expressed in early stages of hematopoiesis including HSCs and lineage restricted hematopoietic progenitors. To test the role of hnRNPL in hematopoietic differentiation, we have generated conditional deficient mice, since a constitutive deletion of hnRNPL results in early embryonic lethality. Animals carrying two hnRNPL-floxed alleles (hnRNPLfl/fl) can be deleted at adult stage by the pIpC inducible MxCre transgene or by the VavCre transgene, which is expressed in all hematopoietic cells starting at embryonic stage E14. VavCre+hnRNPLfl/fl mice were not viable and did not progress further in their development than embryonic stage E17.5 and ablation of hnRNPL by pIpC injection caused a high rate of mortality in adult MxCre+hnRNPLfl/fl mice compared to control animals. Both the fetal liver (FL) of VavCre+hnRNPLfl/fl mice and the bone marrow (BM) of adult MxCre+hnRNPLfl/fl mice had a significantly reduced cellularity. Furthermore, flow cytometric analysis revealed in both FL and BM a significant reduction in frequency and absolute numbers of all mature blood cells, the lymphoid and myeloid precursors, CLPS, CMPs and GMPs and to a lesser extent the erythroid/megakaryocytic precursors (MEPs). Methylcellulose and both competitive and non-competitive transplantation assays demonstrated that HSCs lacking hnRNPL cannot generate lineage-committed progenitors and have lost their self-renewal capacity and reconstitution potential. A genome-wide analysis of mRNA expression and splicing through next-generation RNA sequencing of wild-type (WT)