Single-cell multi-omics defines the cell-type-specific impact of splicing aberrations in human hematopoietic clonal outgrowths

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 30 splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 30 splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.

Automated summary

In this study, the impact of mutated splicing factors on RNA splicing during hematopoiesis was investigated using a combination of genotyping of transcriptomes, long-read single-cell transcriptomics, and proteogenomics. The study found that mutations in the core splicing factor SF3B1 led to expansion of erythroid progenitor cells, as well as stage-specific aberrant splicing during erythroid differentiation. Additionally, the study revealed specific cryptic 30 splice site usage in SF3B1-mutated cells and an erythroid bias in clonal hematopoiesis samples before overt myelodysplastic syndrome.