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

Abstract

RNA splicing factors are recurrently affected by alteration-of-function mutations in clonal blood disorders, highlighting the importance of splicing regulation in hematopoiesis. However, our understanding of the impact of dysregulated RNA splicing has been hampered by the inability to distinguish mutant and wildtype cells in primary patient samples, the cell-type complexity of the hematopoietic system, and the sparse and biased coverage of splice junctions by short-read sequencing typically used in single-cell RNA sequencing. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT) with enhanced efficiency long-read single-cell transcriptome profiling, as well as proteogenomics (with CITE-seq). This allowed for the simultaneous single-cell profiling of gene expression, cell surface protein markers, somatic mutation status, and RNA splicing. We applied GoT-Splice to bone marrow progenitors from patients with myelodysplastic syndrome (MDS) affected by mutations in the most prevalent mutated RNA splicing factor - the core RNA splicing factor SF3B1. High-resolution mapping of SF3B1mut vs. SF3B1wt hematopoietic progenitors revealed a fitness advantage of SF3B1mut cells in the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1mut erythroid progenitor (EP) cells. SF3B1mut EP cells exhibited upregulation of genes involved in regulation of cell cycle and mRNA translation. Long-read single-cell transcriptomes revealed the previously reported increase of aberrant 3' splicing site usage in SF3B1mut cells. However, the ability to profile splicing within individual cell populations uncovered distinct cryptic 3' splice site usage across different progenitor populations, as well as stage-specific aberrant splicing during erythroid maturation. Lastly, as splice factor mutations occur in clonal hematopoiesis (CH) with increased risk of neoplastic transformation, we applied GoT-Splice to CH samples. These data revealed that the erythroid lineage bias, as well as cell-type specific cryptic 3' splice site usage in SF3B1mut cells, precede overt MDS. Collectively, we present an expanded multi-omics single-cell toolkit to define the cell-type specific impact of somatic mutations on RNA splicing, from the earliest phases of clonal outgrowths to overt neoplasia, directly in human samples.

Unraveling Heterogeneity in the Aging Hematopoietic Stem Cell Compartment: An Insight From Single-cell Approaches

“…115 The simultaneous single-cell profiling of gene expression, cell surface protein markers, and somatic mutation status is now possible and has been used to investigate the impact of splicing aberrations in human age-related clonal hematopoiesis by combing Genotyping of Transcriptomes (GoT), utilizing long-read single-cell transcriptome profiling in addition to proteo-genomics using CITE-Seq. 116 Assessment of bone marrow progenitor cells derived from patients with defective RNA splicing, due to mutations of the core RNA splicing factor 3b subunit 1 (SF3B1), was shown to have a fitness advantage over WT SF3B1 cells, resulting in the expansion of a mutant population of erythroid progenitor cells, which displayed an upregulation of genes involved in the cell cycle and mRNA translation. 116 Moreover, another study using a multiomics approach to interrogate human clonal hematopoiesis combined scBS-Seq with scRNA-Seq and targeted DNMT3A genotyping.…”

“…116 Assessment of bone marrow progenitor cells derived from patients with defective RNA splicing, due to mutations of the core RNA splicing factor 3b subunit 1 (SF3B1), was shown to have a fitness advantage over WT SF3B1 cells, resulting in the expansion of a mutant population of erythroid progenitor cells, which displayed an upregulation of genes involved in the cell cycle and mRNA translation. 116 Moreover, another study using a multiomics approach to interrogate human clonal hematopoiesis combined scBS-Seq with scRNA-Seq and targeted DNMT3A genotyping. This study showed that mutations within DNMT3A resulted in a myeloid over lymphoid bias and that these DNMT3A mutations revealed patterns of selective hypomethylation of key hematopoietic transcription factors that results in the disruption of early progenitor states.…”

Application of single cell transcriptomics to characterize the progression of hematopoietic cells to myeloid malignancies

“…Pellagatti et al (2018) [174] explored the impact of mutations in splicing factors applying bulk RNA-seq to the study of differentially expressed isoforms. Finally, an interesting work by Gaiti et al [175], still unpublished, combines scRNA-seq with single cell genomics to shed light on the effects of an specific spliceosome mutation: SF3B1, on gene and isoform expression. With these methods, they are able to detect aberrant splicing in mutated cells occurring in a cell type-specific manner.…”