The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3′ splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3′ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

MRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.

Most short sequences can be precisely written into a selected genomic target using prime editing; however, it remains unclear what factors govern insertion. We design a library of 3,604 sequences of various lengths and measure the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems in varying DNA repair contexts. We find that length, nucleotide composition and secondary structure of the insertion sequence all affect insertion rates. We also discover that the 3′ flap nucleases TREX1 and TREX2 suppress the insertion of longer sequences. Combining the sequence and repair features into a machine learning model, we can predict relative frequency of insertions into a site with R = 0.70. Finally, we demonstrate how our accurate prediction and user-friendly software help choose codon variants of common fusion tags that insert at high efficiency, and provide a catalog of empirically determined insertion rates for over a hundred useful sequences.

Nearly 50% of breast cancer patients suffer from depression or anxiety. Selective serotonin reuptake inhibitors (SSRIs), the first-line pharmacological treatment for depression, have been implicated in breast cancer development through increased prolactin levels and tamoxifen metabolism inhibition. Previous studies of breast cancer progression have focused on tamoxifen users, or have been limited by their small sample size and methodology. Therefore, we used UK population-based data to more robustly investigate the association between SSRI use and cancer-specific mortality. A cohort of patients with newly-diagnosed breast cancer between 1998 and 2012 was selected from English cancer registries and linked to prescription records from the Clinical Practice Research Datalink, and to death records from the Office for National Statistics. We used Cox regression models to calculate hazard ratios (HRs) comparing mortality between post-diagnostic SSRI users and non-users (using time-dependant covariates), after adjusting for demographics, comorbidities and pre-diagnosis use of hormone replacement therapy or oral contraceptives. We conducted several additional analyses to assess causality. Our cohort included 23,669 breast cancer patients, of which 2672 used SSRIs and 3053 died due to their breast cancer during follow-up. After adjustment, SSRI users had higher breast cancer-specific mortality than non-users (HR = 1.27; 95% confidence interval (CI) 1.16, 1.40). However, this association was attenuated when restricting to patients with a prior history of depression (HR = 1.14; 95% CI 0.98, 1.33), and when comparing to users of other antidepressant medications (HR = 1.06; 95% CI 0.93, 1.20). There was some evidence of higher mortality among long-term SSRI users, even when restricting to patients with prior depression (HR = 1.54; 95% CI 1.03, 2.29). In this large breast cancer cohort, SSRI use was associated with a 27% increase in breast cancer mortality. The cause of this is unknown; however, confounding by indication seems likely as it was largely attenuated when restricting to patients with prior depression, or when comparing SSRIs to other antidepressant medications. Clinicians should not be unduly concerned when prescribing SSRIs to breast cancer patients, but the increase in mortality among long-term SSRI users warrants further investigation.

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically. Significance: The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.