When oxygen is abundant, quiescent cells efficiently extract energy from glucose primarily by oxidative phosphorylation, whereas under the same conditions tumour cells consume glucose more avidly, converting it to lactate. This long-observed phenomenon is known as aerobic glycolysis 1 , and is important for cell growth 2, 3. Because aerobic glycolysis is only useful to growing cells, it is tightly regulated in a proliferation-linked manner4. Inmammals, this is partly achieved through control of pyruvate kinase isoform expression. The embryonic pyruvate kinase isoform, PKM2, is almost universally re-expressed in cancer2, and promotes aerobic glycolysis, whereas the adult isoform, PKM1, promotes oxidative phosphorylation 2 . These two isoforms result from mutually exclusive alternative splicing of the PKM pre-mRNA, reflecting inclusion of either exon 9 (PKM1) or exon 10 (PKM2). Here we show that three heterogeneous nuclear ribonucleoprotein (hnRNP) proteins, polypyrimidine tract binding protein (PTB, also known as hnRNPI), hnRNPA1 and hnRNPA2, bind repressively to sequences flanking exon 9, resulting in exon 10 inclusion. We also demonstrate that the oncogenic transcription factor c-Myc upregulates transcription of PTB, hnRNPA1 and hnRNPA2, ensuring a high PKM2/PKM1 ratio. Establishing a relevance to cancer, we show that human gliomas overexpress c-Myc, PTB, hnRNPA1 and hnRNPA2 in a manner that correlates with PKM2 expression. Our results thus define a pathway that regulates an alternative splicing event required for tumour cell proliferation.Alternative splicing of PKM has an important role in determining the metabolic phenotype of mammalian cells. The single exon difference imparts the enzymes produced with important functional distinctions. For example, PKM2, but not PKM1, is regulated by the binding of tyrosine phosphorylated peptides, which results in release of the allosteric activator fructose-1-6-bisphosphate and inhibition of pyruvate kinase activity 5 , a property that might allow growth-factor-initiated signalling cascades to channel glycolytic intermediates into biosynthetic processes. The importance of tumour reversion to PKM2 was underscored by experiments in which replacement of PKM2 with PKM1 in tumour cells resulted in markedly reduced growth 2 . Consistent with a critical role in proliferation, re-expression of PKM2 in tumours is robust 2 , although little is known about the regulation of this process. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 October 5. We set out to identify RNA binding proteins that might regulate PKM alternative splicing. To this end, we prepared an [α-32 P]UTP-labelled 250-nucleotide RNA spanning the exon 9 (E9) 5′ splice site (EI9), previously identified as inhibitory to E9 inclusion 6 , as well as a labelled RNA from a corresponding region of E10 (EI10) (Fig. 1b), and performed ultraviolet crosslinking assays with HeLa nuclear extracts 7 . After separation by SDS-polyacrylamide gel electrophoresis (PAGE), multiple proteins...

Alternative splicing of mRNA precursors provides an important means of genetic control and is a crucial step in the expression of most genes. Alternative splicing markedly affects human development, and its misregulation underlies many human diseases. Although the mechanisms of alternative splicing have been studied extensively, until the past few years we had not begun to realize fully the diversity and complexity of alternative splicing regulation by an intricate protein-RNA network. Great progress has been made by studying individual transcripts and through genome-wide approaches, which together provide a better picture of the mechanistic regulation of alternative premRNA splicing.Alternative splicing is a crucial mechanism for gene regulation and for generating proteomic diversity. Recent estimates indicate that the expression of nearly 95% of human multi-exon genes involves alternative splicing 1,2 . In metazoans, alternative splicing plays an important part in generating different protein products that function in diverse cellular processes, including cell growth, differentiation and death.Splicing is carried out by the spliceosome, a massive structure in which five small nuclear ribonucleoprotein particles (snRNPs) and a large number of auxiliary proteins cooperate to accurately recognize the splice sites and catalyse the two steps of the splicing reaction 1,2 (BOX 1). Spliceosome assembly (BOX 1) begins with the recognition of the 5′ splice site by the snRNP U1 and the binding of splicing factor 1 (SF1) to the branch point 3 and of the U2 auxiliary factor (U2AF) heterodimer to the polypyrimidine tract and 3′ terminal AG 4,5 . This assembly is ATP independent and results in the formation of the E complex, which is converted into the ATP-dependent, pre-spliceosomal A complex after the replacement of SF1 by the U2 snRNP at the branch point. Further recruitment of the U4/U6-U5 tri-snRNP complex leads to the formation of the B complex, which is converted into to the catalytically active C complex after extensive conformational changes and remodelling. Splicing and spliceosome assemblyPre-mRNA splicing is a process in which intervening sequences (introns) are removed from an mRNA precursor. Splicing consists of two transesterification steps, each involving a nucleophilic attack on terminal phosphodiester bonds of the intron. In the first step this is carried out by the 2′ hydroxyl of the branch point (usually adenosine) and in the second step by the 3′ hydroxyl of the upstream (5′) exon 1,2 . This process is carried out in the spliceosome, a dynamic molecular machine the assembly of which involves sequential binding and release of small nuclear ribonucleoprotein particles (snRNPs) and numerous protein factors as well as the formation and disruption of RNA-RNA, protein-RNA and protein-protein interactions.The basic mechanics of spliceosome assembly are well known. Briefly, the process begins with the base pairing of U1 snRNA to the 5′ splice site (ss) and the binding of splicing factor 1 (SF1) to the b...

TGF-β signaling can be pro-tumorigenic or tumor suppressive. We investigated this duality in pancreatic ductal adenocarcinoma (PDA), which, with other gastrointestinal cancers, exhibits frequent inactivation of the TGF-β mediator Smad4. We show that TGF-β induces an epithelial-mesenchymal transition (EMT), generally considered a pro-tumorigenic event. However, in TGF-β sensitive PDA cells, EMT becomes lethal by converting TGF-β-induced Sox4 from an enforcer of tumorigenesis into a promoter of apoptosis. This is the result of an EMT-linked remodeling of the cellular transcription factor landscape, including the repression of the gastrointestinal lineage-master regulator Klf5. Klf5 cooperates with Sox4 in oncogenesis and prevents Sox4-induced apoptosis. Smad4 is required for EMT but dispensable for Sox4 induction by TGF-β. TGF-β-induced Sox4 is thus geared to bolster progenitor identity, while simultaneous Smad4-dependent EMT strips Sox4 of an essential partner in oncogenesis. Our work demonstrates that TGF-β tumor suppression functions through an EMT-mediated disruption of a lineage-specific transcriptional network.

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.

SummaryPostnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocytespecific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER T2 ) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER T2 mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER T2 transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach. Keywordschondrocyte; Cre-mediated recombination; conditional knockout; tamoxifen; X-Gal staining Chondrocyte maturation and cartilage formation involves multiple steps that are regulated by numerous growth factors, their downstream signaling molecules and transcription factors. Conventional and tissue-specific gene deletions provide powerful tools to investigate roles of specific genes in chondrocyte maturation. However, embryonic lethality is often encountered because of the essential role of various genes in early embryonic development (Akiyama et al., 2004;Karaplis et al., 1994;Lanske et al., 1996Lanske et al., , 1999Razzaque et al., 2005;Sakamoto et al., 2005;St-Jacques et al., 1999). Furthermore, some gene deletions result in such severe skeletal malformation that interpretation of the phenotype is challenging even if the animal survive birth. An inducible conditional gene deletion approach is an exquisite method to determine the function of a gene that is either embryonic lethal or associated with marked abnormalities of morphology and tissue architectures. In this report, we present a chondrocytespecific and tamoxifen-inducible Cre transgenic mouse model. Type II collagen is a chondrocyte-specific protein and its expression is detected in growth plate and articular chondrocytes in long bones and other cartilage tissues in the body. Type II collagen promoter (col2a1) has been used in a variety of animal models to achieve tissuespecific gene expression in chondrocytes (Schipani et al., 1997;Stricker et al., 2002;Takeda et al., 2001;Ueta et al., 2001;Weir et al., 1996). In the present studies, we generated transgenic mouse lines in which the Cre recombinase was fused to a mutated ligand-binding domain of human estrogen receptor (ER) driven by the col2a1 promoter (Col2a1-CreER T2 ; Metzger et al., 2005). The fusion protein has been reported to be translocated into the nuclei in response to estrogen antagonist tamoxifen or 4-hydroxy tamoxifen (4-OH tamoxifen), an active metabolite of tamoxifen in vivo ...

Expression of the mammalian pyruvate kinase M (PKM) gene provides an important example of mutually exclusive splicing. We showed previously that the hnRNP proteins A1, A2 and PTB play a critical role in this process. Here we provide evidence that concentration-dependent interactions involving a network of these proteins are sufficient to determine the outcome of PKM splicing. At high concentrations, such as found in most cancer cells, hnRNP A1 binding to two sites in the upstream regulated exon (exon 9) orchestrates cooperative interactions leading to exon 9 exclusion. At lower concentrations, binding shifts to downstream intronic sites such that exon 9 is included and exon 10 largely excluded, with any mRNA including both exons degraded by nonsense-mediated decay. Together our results provide a mechanism by which a small number of general factors control alternative splicing of a widely expressed transcript.

hypertrophic zones, delayed formation of the secondary ossification center, and reduced skeletal growth. Isolated primary Col2a1-ICAT transgenic chondrocytes showed reduced expression of chondrocyte genes associated with maturation, and demonstrated that VEGF gene expression requires cooperative interactions between BMP2 and β-catenin signaling. Altogether the findings confirm a crucial role for Wnt/β-catenin in postnatal growth.

SRp38 is an atypical SR protein that functions as a general splicing repressor when dephosphorylated. We now show that phosphorylated SRp38 functions as a sequence-specific splicing activator. Unlike characterized splicing activators, SRp38 functions in the absence of other SR proteins but requires a cofactor for activity. SRp38 was able to induce formation of splicing complex A in the absence of the cofactor, but this factor was necessary for progression to complexes B and C. Mechanistically, SRp38 strengthens the ability of the U1 and U2 small nuclear ribonucleoproteins to stably recognize the pre-mRNA. Extending these findings, analysis of alternative splicing of pre-mRNA encoding the glutamate receptor B revealed that SRp38 alters its splicing pattern in a sequence-specific manner. Together, our data demonstrate that SRp38, in addition to its role as a splicing repressor, can function as an unusual sequence-specific splicing activator.