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

Abstract

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 5ʹSS, 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.

DDX41: exploring the roles of a versatile helicase

“…Qin et al [ 60 ] overexpressed DDX41 in HeLa cells, and RNA-seq revealed that DDX41 regulates transcription and alternative splicing of genes involved in tumorigenesis and immune response. Recently, Shinriki et al [ 61 ] discovered that DDX41 mainly binds 5′ splice sites of coding RNA, indicating its role in forming activated spliceosomes ( Figure 3 ). Nevertheless, further studies are warranted to address how DDX41 executes its role in pre-mRNA splicing, especially related to its helicase activity.…”

“…Using a zebrafish Ddx41 loss-of-function mutant, Dr. Teresa Bowman's group recently found that R-loop levels increase nearly two-fold in Ddx41 mutants [ 74 ]. Increased R-loops are also observed in DDX41-KD or by DDX41 inhibitor in K562 cells [ 61 ]. Recently, Dr. Karlene Cimprich's lab found that R-loops bind and activate cGAS in the cytoplasm [ 77 ], while our laboratory [ 55 ] and another group [ 68 ] found that DDX41 functions in the upstream of cGAS–STING.…”

“…Furthermore, they found that DDX41 proteins can unwind RNA:DNA hybrids in vitro , DDX41 localizes in the promoter regions, and DDX41 depletion leads to increased DSBs [ 75 ]. Increased R-loops and DSBs are also observed in DDX41 KD or by DDX41 inhibitors in K562 cells [ 61 ]. In addition, increased R-loops have been found in DDX41 KO human cardiomyocyte cell line AC16 cells [ 68 ].…”

“…R-loops have been implicated in pre-mRNA splicing [ 79 ], ribosome biogenesis [ 80 ], and translation [ 81 ]. In parallel, DDX41 is involved in pre-mRNA splicing [ 23 , 59 , 61 ], ribosome biogenesis [ 67 , 69 , 70 ], and translation [ 71 ]. However, the precise role of R-loops and DDX41 in these processes remains to be explored.…”

Spliceosomal helicases DDX41/SACY-1 and PRP22/MOG-5 both contribute to proofreading against proximal 3′ splice site usage

“…The pleiotropic effects of disrupting spliceosomal components often make it difficult to untangle the different pathogenic mechanisms; for example, SF3b1 also contributes to malignancy through R-loop formation and DNA damage ( Singh et al 2020 ). DEAD-box helicase DDX41 is another spliceosome-associated protein linked to cancers, and its disruption causes changes to both splicing and R-loop formation, leading to replicative stress ( Shinriki et al 2022 ). sacy-1 , the Caenorhabditis elegans homolog of DDX41, also has multiple roles; it was initially identified in a screen for oocyte meiotic maturation factors (the name sacy-1 stands for “suppressor of acy-4 ”) ( Kim et al 2012 ), and was only later understood to be involved in splicing and to be associated with C complex proteins ( Tsukamoto et al 2020 ).…”