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.

Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.

The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylationdependent mechanism. We found a phosphorylation stoichiometry of 3.0 ؎ 0.4 phosphorylated residues/ NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr 184 , Thr 189 , and Thr 202 ) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. The secretory Na-K-Cl cotransporter NKCC1 is a major pathway for net influx of Cl Ϫ in many cells (1, 2). Along with the absorptive Na-K-Cl cotransporter NKCC2 and the thiazidesensitive Na-Cl transporter NCC (or TSC), NKCC1 belongs to the sodium-coupled branch of the cation chloride cotransporter family; this family also includes the K-Cl transporters (KCCs) (3) and two other major branches of putative transporters, including human CIP (4) and SLC12-8 (GenBank TM /EBI accession number AAK94307), whose transport functions are unknown. These proteins are all predicted to have 12 transmembrane-spanning domains that are responsible for ion transport properties (5) and large cytoplasmic N and C termini that are candidates for regulatory domains. NKCC1 is expressed in many cell types and is involved in the regulation of both cell volume and intracellular Cl Ϫ concentration (2, 6). This isoform is also a major component of the basolateral membrane of secretory cells, mediating Cl Ϫ influx, the first step in the transepithelial movement of Cl Ϫ . In contrast, the NKCC2 isoform is limited to the apical membranes of absorptive epithelia such as the thick ascending limb of Henle's loop, where it is a major determinant of NaCl reabsorption from the tubular fluid.In order that the processes of regulatory volume increase, secretion, and absorption may be tightly controlled, the Na-K-Cl cotransporter is subject to strict regulation. The cotransporter is inactive in the basolateral membranes of secretory cells until the application of secretagogues or cell shrinkage causes a phosphorylation-dependent activation of the cotransporter (7-10). Studies on shark rectal gland secretory epithelia have shown an increase in phosphorylation at serines and threonines in response to forskolin or hypertonic stress, and the N terminus of NKCC1 has been shown to be the locus of at least some of the phosphoregulatory sites (7, 11). Although, in many cells, PKA 1 agonists effect an increase in Na-K-Cl co...

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared.