Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.

TREX1 acts in concert with the SET complex in granzyme A-mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3' UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 x 10(-7)). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE.

The TREX1 enzyme processes DNA ends as the major 3 3 5 exonuclease activity in human cells. Mutations in the TREX1 gene are an underlying cause of the neurological brain disease Aicardi-Goutières syndrome implicating TREX1 dysfunction in an aberrant immune response. TREX1 action during apoptosis likely prevents autoimmune reaction to DNA that would otherwise persist. To understand the impact of TREX1 mutations identified in patients with Aicardi-Goutières syndrome on structure and activity we determined the x-ray crystal structure of the dimeric mouse TREX1 protein in substrate and product complexes containing single-stranded DNA and deoxyadenosine monophosphate, respectively. The structures show the specific interactions between the bound nucleotides and the residues lining the binding pocket of the 3 terminal nucleotide within the enzyme active site that account for specificity, and provide the molecular basis for understanding mutations that lead to disease. Three mutant forms of TREX1 protein identified in patients with AicardiGoutières syndrome were prepared and the measured activities show that these specific mutations reduce enzyme activity by 4 -35,000-fold. The structure also reveals an 8-amino acid polyproline II helix within the TREX1 enzyme that suggests a mechanism for interactions of this exonuclease with other protein complexes.Processing of DNA ends is an important step in many DNA metabolic pathways such as replication, repair, and recombination. The 3Ј 3 5Ј exonucleases play a critical role in correcting fragmented, modified, mispaired, or even normal nucleotides to generate 3Ј termini suitable for downstream events. The drastic consequences that result from impaired 3Ј exonuclease activities underscore the importance of these enzymes for cell survival. Proofreading of DNA synthesis by 3Ј exonucleases is one of the major determinants of mutagenesis and genome stability and cells lacking this ability show a high incidence of cancers (1-3) (for review, see Ref. 4). Cells with defects in proteins containing 3Ј exonuclease activity, such as the Werner syndrome protein, MRE11, APE1, and p53 proteins display chromosomal instability, cell cycle checkpoint defects, and sensitivity to ionizing radiation (5-9).The major 3Ј 3 5Ј exonuclease activity detected in human cell extracts is catalyzed by the TREX1 enzyme. The genes encoding the TREX1 and closely related TREX2 proteins have been identified and cloned (10, 11), and the recombinant proteins confirm the robust catalytic nature of these enzymes (12, 13). Amino acid sequence analysis reveals the TREX proteins belong to the DnaQ family of 3Ј 3 5Ј exonucleases; a structurally conserved group of exonucleases that span Archaea and bacteria to humans and includes such proteins as the exonuclease domains of Werner syndrome protein, the bacterial ⑀ subunit of DNA polymerase III (⑀ subunit), and exonuclease I (Exo I) 2 (14 -17). A hallmark of the DnaQ family exonucleases is three conserved sequence motifs known as Exo I, II, and III. These motifs contain four ...

Summary Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pulldown assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17β-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an ‘activated’ state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a ‘natural’ ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1-3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. .