Aberrant Alternative Splicing in U2af1/Tet2 Double Mutant Mice Contributes to Major Hematological Phenotypes.

Cristina Martínez-Valiente,Elisa González-Romero,Alessandro Liquori,Alejandra Sanjuan-Pla,Cristian Garcia-Ruiz,Beatriz Rosón,Isabel Gómez-Redondo,Alfonso Gutiérrez-Adán,José Cervera,Raúl Fernández-González

doi:10.3390/ijms22136963

Abstract

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3′ splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12–13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+ Tet2−/− mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+ Tet2−/− mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.

Highlights

Myelodysplastic syndromes (MDS) comprise clonal hematological diseases that appear more commonly in the elderly and that can adversely evolve into secondary acute myeloid leukemia in up to 40% of patients
We analyze the impact of mutant U2af1 on the hematopoietic system in a novel CRISPR/Cas9 mouse model as well as its effect on myelodysplastic syndromes (MDS) disease development in combination with Tet2 deficiency
Our heterozygous mutant U2af1 per se is not sufficient to cause MDS disease, even at an advanced age. This agrees with transgenic mice overexpressing U2AF1S34 [16] or U2AF1Q157 [17] and conditional knock-in U2af1S34F mice [20] since these animals do not develop full-blown malignancy albeit presenting mild phenotypes with some MDS-like features

Summary

Introduction

Myelodysplastic syndromes (MDS) comprise clonal hematological diseases that appear more commonly in the elderly and that can adversely evolve into secondary acute myeloid leukemia (sAML) in up to 40% of patients. U2AF1 mutations are heterozygous and are associated with adverse prognosis, increased risk of progression to AML, and poor overall survival (OS) in MDS and MPN patients [5], with U2AF1Q157 having a worse OS than U2AF1S34 [6]. It is unclear whether U2AF1 mutations result in a gain-of [7,8] or loss-of-function of the protein [5,9]. Mutated U2AF1 requires the presence of a wild-type allele for surviving and counteracting aberrant splicing [8,10]

Methods

Results

Conclusion