Alternative splicing of mRNA precursors enables one gene to produce multiple protein isoforms with differing functions. Under normal conditions, this mechanism is tightly regulated in order for the human genome to generate proteomic diversity sufficient for the functional requirements of complex tissues. When deregulated, however, cancer cells take advantage of this mechanism to produce aberrant proteins with added, deleted, or altered functional domains that contribute to tumorigenesis. Here we discuss aspects of alternative splicing misregulation in cancer, focusing on splicing events affected by deregulation of regulatory splicing factors and also recent studies identifying mutated components of the splicing machinery.

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the moreexcluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.spliceosome | pre-mRNA splicing | serine/arginine-rich proteins | myelodysplastic syndromes | leukemia M yelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders characterized by ineffective production of myeloid blood cells, which have various risks of progression into acute myeloid leukemia (AML) (1, 2). The most frequently occurring mutations found in patients with MDS involve genes encoding pre-mRNA splicing factors, including Splicing factor 3B, subunit 1 (SF3B1), Serine/argininerich splicing factor 2 (SRSF2), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit-related protein 2 (ZRSR2) (3-6), suggesting that altered RNA splicing may play a critical role in the pathogenesis of MDS. Despite some recent advances (e.g., ref. 7; see Discussion), the molecular mechanisms by which the mutated splicing factors misregulate pre-mRNA splicing have not been studied thoroughly. However, it is now well established that splicing deregulation contributes to multiple diseases, especially cancer (8, 9).SRSF2 is a well-studied serine/arginine-rich splicing factor (SR protein). SR proteins play important roles in the regulation of both constitutive and alternative pre-mRNA splicing, functioning, for ...

Unlike normal cells, which metabolize glucose by oxidative phosphorylation for efficient energy production, tumor cells preferentially metabolize glucose by aerobic glycolysis, which produces less energy but facilitates the incorporation of more glycolytic metabolites into the biomass needed for rapid proliferation. The metabolic shift from oxidative phosphorylation to aerobic glycolysis is partly achieved by a switch in the splice isoforms of the glycolytic enzyme pyruvate kinase. Although normal cells express the pyruvate kinase M1 isoform (PKM1), tumor cells predominantly express the M2 isoform (PKM2). Switching from PKM1 to PKM2 promotes aerobic glycolysis and provides a selective advantage for tumor formation. The PKM1/M2 isoforms are generated through alternative splicing of two mutually exclusive exons. A recent study shows that the alternative splicing event is controlled by heterogeneous nuclear ribonucleoprotein (hnRNP) family members hnRNPA1, hnRNPA2, and polypyrimidine tract binding protein (PTB; also known as hnRNPI). These findings not only provide additional evidence that alternative splicing plays an important role in tumorigenesis, but also shed light on the molecular mechanism by which hnRNP proteins regulate cell proliferation in cancer.Cancer Res; 70(22); 8977-80. ©2010 AACR.

SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3 0 splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.(A) Distribution of cryptic 3 0 ss around the associated canonical 3 0 ss. Red and black histograms indicate 1,145 cryptic 3 0 ss more frequently used (q value < 0.05) in six mutant SF3B1 MDS patient samples and 186 cryptic 3 0 ss in nine WT SF3B1 samples, respectively. (B) Hierarchical clustering and heatmap analysis of the 627 cryptic 3 0 ss differentially used in six mutant versus nine WT SF3B1 samples (q value < 0.05 and cryptic 3 0 ss closer than 100 nt upstream of the associated canonical 3 0 ss). Each row represents one cryptic 3 0 ss and each column one MDS patient sample. Z scores in the matrix represent normalized percent-spliced-in values. The color bars above the heatmap indicate the SF3B1 mutations. (C) Volcano plot representation of genes associated with the 169 cryptic 3 0 ss differentially used in mutant versus WT SF3B1 samples (q value < 0.05, closer than 50 nt upstream of the associated canonical 3 0 ss and more than 15 supporting reads averaged over mutant SF3B1 samples). The horizontal axis shows the PSI difference between mutant and WT SF3B1 samples, and the vertical axis shows the significance. Genes selected for further experimental validation are highlighted in red.

RecA-and RecBC-catalyzed repair in eubacteria assembles chromosomes fragmented by double-strand breaks. We propose that recA mutants, being unable to repair fragmented chromosomes, depend on various strategies designed to avoid chromosomal fragmentation. To identify chromosomal fragmentation-avoidance strategies, we screened for Escherichia coli mutants synthetically inhibited in combination with recA inactivation by identifying clones unable to lose a plasmid carrying the recA ؉ gene. Using this screen, we have isolated several RecA-dependent mutants and assigned them to three distinct areas of metabolism. The tdk and rdgB mutants affect synthesis of DNA precursors. The fur, ubiE, and ubiH mutants are likely to have increased levels of reactive oxygen species. The seqA, topA mutants and an insertion in smtA perturbing the downstream mukFEB genes affect nucleoid administration. All isolated mutants show varying degree of SOS induction, indicating elevated levels of chromosomal lesions. As predicted, mutants in rdgB, seqA, smtA, topA, and fur show increased levels of chromosomal fragmentation in recBC mutant conditions. Future characterization of these RecA-dependent mutants will define mechanisms of chromosomal fragmentation avoidance. C hromosomal fragmentation due to double-strand DNA breaks and disintegrated replication forks is a major contributor to genome instability in all organisms (1, 2). The two major pathways used by eukaryotic cells to repair fragmented chromosomes are nonhomologous end joining and homologous recombination (3). Nonhomologous end joining is an imprecise repair, frequently involving loss of genetic information, but it is nevertheless an efficient way to repair double-strand breaks in cells of higher eukaryotes (4). However, disintegrated replication forks, having a single double-strand end, cannot be reassembled by nonhomologous end joining and require error-free recombinational repair (5, 6). The importance of recombinational repair in higher eukaryotes is illustrated by the inviability of recombinational repair mutants in mice (7) and by the rapid death of recombinational repair-defective vertebrate cells due to chromosomal fragmentation (8). In the model eubacterium Escherichia coli, recombinational repair of fragmented chromosomes is catalyzed by the RecA and RecBCD enzymes (9). RecBCD is an exonuclease͞helicase that prepares the doublestrand DNA ends of the break for RecA polymerization (10). RecA filament catalyzes homologous strand exchange of the broken DNA duplex with an intact sister duplex, thus creating an opportunity for double-strand break repair (11).In recA or recBC mutants of E. coli, double-strand DNA breaks are not repaired (12) and cause chromosomal loss and cell death (13,14). However, recA mutant E. coli strains are still 50% viable (15, 16), which indicates that the chromosomal fragmentation is not a frequent event in E. coli and suggests the existence of strategies designed to avoid chromosomal fragmentation. Inactivation of one of these hypothetical avoidance a...