Insights into the Involvement of Spliceosomal Mutations in Myelodysplastic Disorders from Analysis of SACY-1/DDX41 inCaenorhabditis elegans

Abstract

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia (AML). DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase that is recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of AML. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here, we conduct a comprehensive analysis of the Caenorhabditis elegans DDX41 ortholog, SACY-1. Biochemical analyses defined SACY-1 as a component of the C. elegans spliceosome, and genetic analyses revealed synthetic lethal interactions with spliceosomal components. We used the auxin-inducible degradation system to analyze the consequence of SACY-1 depletion on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-dependent and splicing-independent mechanisms. Altered 3′ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 in the second step of splicing. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. The transcriptome changes observed after SACY-1 depletion suggest that disruption of the spliceosome induces a stress response, which could contribute to the cellular phenotypes conferred by sacy-1 mutant alleles. Multiple sacy-1/ddx41 missense mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that their incorporation into the spliceosome is detrimental. Antagonistic variants that perturb the function of the spliceosome may be relevant to the disease-causing mutations, including DDX41, affecting highly conserved components of the spliceosome in humans.

DDX41 is needed for pre-and post-natal hematopoietic stem cell differentiation in mice

“…While our RNASeq data showed that DDX41 had the greatest effects on transcript levels in LSK+ cells, the data also showed that DDX41 loss altered exon skipping and intron retention. In contrast to our results, in HeLa cells A3SS, A5SS and SE were all regulated by DDX41, while in C. elegans upon gene deletion, A3SS represented the predominant splicing defect (26,27). Whether these differences are species-or cell type-specific remains to be determined.…”

Decreased mechanotransduction prevents nuclear collapse in a Caenorhabditis elegans laminopathy

“…OOC-5::GFP::TEV::3×FLAG and interacting proteins were purified from soluble protein extracts and membrane protein fractions prepared from the DG4377 strain using modifications of methods we previously described ( 47 – 49 ) as detailed in SI Appendix . Purifications from the wild-type strain (N2) were run in parallel as a control.…”

“…Lanes were subdivided into six slices and mass spectrometry was performed at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School) using an LTQ ion-trap mass spectrometer. In addition to control immunopurifications from the N2 strain, to account for abundant contaminant proteins that bound nonspecifically to the immunopurification matrix, we utilized data from multiple purifications of GFP fusion proteins, which we previously reported ( 47 – 49 ; see SI Appendix , Materials and Methods for the details of the filtering criteria used).…”

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

“…Dead box helicase (DDX41) is a tumor suppressor that is conserved in D.melanogaster, C.elegans, D.rerio and plants, and is considered essential to cell growth and viability [32][33][34][35] .…”

“…DDX41 has been reported to interact with components of the spliceosome, and DDX41 deletion or mutations led to splicing defects and faulty RNA processing 38 . The role of DDX41 in RNA processing appears to be conserved: The C.elegans orthologue SACY-1 was recently shown to associate with the spliceosome, and impacts the transcriptome through splicing-dependent and -independent mechanisms 33 . Despite the relevance of DDX41 in cancer, the cellular and molecular functions of DDX41 remain poorly understood.…”