Ribosome profiling analysis reveals the roles of DDX41 in translational regulation.

DDX41 mutation has been observed in myeloid malignancies including myelodysplastic syndromes and acute myeloid leukemia, but the underlying causative mechanisms of these diseases have not been fully elucidated. The DDX41 protein is an ATP-dependent RNA helicase with roles in RNA metabolism. We previously showed that DDX41 is involved in ribosome biogenesis by promoting the processing of newly transcribed pre-ribosomal RNA. To build on this finding, in this study, we leveraged ribosome profiling technology to investigate the involvement of DDX41 in translation. We found that DDX41 knockdown resulted in both translationally increased and decreased transcripts. Both gene set enrichment analysis and gene ontology analysis indicated that ribosome-associated genes were translationally promoted after DDX41 knockdown, in part because these transcripts had significantly shorter transcript length and higher transcriptional and translational levels. In addition, we found that transcripts with 5'-terminal oligopyrimidine motifs tended to be translationally upregulated when the DDX41 level was low. Our data suggest that a translationally regulated feedback mechanism involving DDX41 may exist for ribosome biogenesis.

DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.

Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5' splice site (5'SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5'SS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.