Oncogenic DEAD-box ATPase DDX41 establishes transcript ensembles via CLK3-dependent and -independent mechanisms

Post-transcriptional diversification of RNA transcripts mediated by complex processing machinery, including DEAD-box ATPases, establishes and maintains cellular phenotypes. For example, DDX41 controls RNA splicing, innate immune signaling, and genome stability. Although heterozygous DDX41 germline genetic variation occurs in familial myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), the DDX41 contribution to splicing globally, biological processes, and pathogenic mechanisms are incompletely defined. Using a genetic rescue system with Ddx41+/- myeloid progenitors, we established global wildtype DDX41 and pathogenic variant mechanisms. Differing from pathogenic variants of other RNA splicing regulators, DDX41 deficiency compromised multiple splicing steps. DDX41-regulated transcripts encoded factors controlling RNA splicing, including Cdc2-like kinase 3 (CLK3). DDX41 regulated Clk3 transcripts, and elevated CLK3 during myeloid differentiation. Loss-of-function analysis revealed that the majority of transcript regulation mediated by DDX41 requires CLK3, but splicing-related transcripts remain regulated upon CLK3 depletion. Thus, through a dual mechanism, with or without a splicing factor kinase that itself is DDX41-regulated, DDX41 establishes transcript ensembles in myeloid progenitors.