The insulin receptor (IR) exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. This alternative splicing is cell specific, and the relative proportions of exon 11 isoforms also vary during development, aging, and different disease states. We have previously demonstrated that both intron 10 and exon 11 contain regulatory sequences that affect IR splicing both positively and negatively. In this study, we sought to define the precise sequence elements within exon 11 that control exon recognition and cellular factors that recognize these elements. Using minigenes carrying linker-scanning mutations within exon 11, we detected both exonic splicing enhancer and exonic splicing silencer elements. We identified binding of SRp20 and SF2/ASF to the exonic enhancers and CUG-BP1 to the exonic silencer by RNA affinity chromatography. Overexpression and knockdown studies with hepatoma and embryonic kidney cells demonstrated that SRp20 and SF2/ASF increase exon inclusion but that CUG-BP1 causes exon skipping. We found that CUG-BP1 also binds to an additional intronic splicing silencer, located at the 3 end of intron 10, to promote exon 11 skipping. Thus, we propose that SRp20, SF2/ASF, and CUG-BP1 act antagonistically to regulate IR alternative splicing in vivo and that the relative ratios of SRp20 and SF2/ASF to CUG-BP1 in different cells determine the degree of exon inclusion.In mammals, alternative splicing is a common strategy for creating functional diversities of proteins that have cell and developmentally specific functions. Given the important role for splicing, it is not surprising that a recent estimate has proposed that 50 to 60% of mutations linked to disease affect splicing (21, 43). The majority of human genes undergo alternative pre-mRNA splicing through the use of competing 5Ј or 3Ј splice sites or through alternative inclusion/exclusion of exons in the pre-mRNA. These alternative exons often contain splice sites that diverge from the consensus site, and the presence of cis regulatory elements within the exon and/or the flanking introns determines whether these exons are recognized (18,20,31). These cis elements can have either a positive (enhancer) or a negative (silencer) effect on splicing. Both enhancers and silencers are thought to function through binding to specific trans-acting protein factors (1). Differences in the expression or activities of these trans-acting factors may modulate the recognition of the alternative exon and lead to developmental or tissue-specific differences in splicing. Proteins that bind to specific sequence elements to affect splice site selection include SR proteins, hnRNPs, and other related RNA binding proteins, such as the CELF family, TIA-1, and Raver-1 (11,12,14,25,32). Adding a further layer of regulation, local context, such as RNA secondary structure, may influence the way that binding motifs are recognized by their cognate factors (3, 10, 13).The human insulin receptor (IR) is encoded by a single INSR gene th...

The hypothalamic-pituitary-gonadal endocrine axis regulates reproduction through estrous phase-dependent release of the heterodimeric gonadotropic glycoprotein hormones, LH and FSH, from the gonadotropes of the anterior pituitary. Gonadotropin synthesis and release is dependent upon pulsatile stimulation by the hypothalamic neuropeptide GnRH. Alterations in pulse frequency and amplitude alter the relative levels of gonadotropin synthesis and release. The mechanism of interpretation of GnRH pulse frequency and amplitude by gonadotropes is not understood. We have examined gene expression in LbetaT2 gonadotropes under various pulse regimes in a cell perifusion system by microarray and identified 1127 genes activated by tonic or pulsatile GnRH. Distinct patterns of expression are associated with each pulse frequency, but the greatest changes occur at a 60-min or less interpulse interval. The immediate early gene mRNAs encoding early growth response (Egr)1 and Egr2, which activate the gonadotropin LH beta-subunit gene promoter, are stably induced at high pulse frequency. In contrast, mRNAs for the Egr corepressor genes Ngfi-A binding protein Nab1 and Nab2 are stably induced at low pulse frequency. We show that Ngfi-A binding protein members inhibit Egr-mediated frequency-dependent induction of the LH beta-subunit promoter. This pattern of expression suggests a model of pulse frequency detection that acts by suppressing activation by Egr family members at low frequency and allowing activation at sustained high-frequency pulses.

SUMMARY The creation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of transcription factors has galvanized the fields of regenerative medicine and developmental biology. Here, we report a kinome-wide RNAi-based analysis to identify kinases that regulate somatic cell reprogramming to iPSCs. We prepared 3,686 shRNA lentiviruses targeting 734 kinase genes covering the entire mouse kinome and individually examined their effects on iPSC generation. We identified 59 kinases as barriers to iPSC generation and characterized seven of them further. We found that shRNA-mediated knockdown of the serine/threonine kinases TESK1 or LIMK2 promoted mesenchymal-to-epithelial transition, decreased COFILIN phosphorylation, and disrupted Actin filament structures during reprogramming of mouse embryonic fibroblasts. Similarly, knockdown of TESK1 in human fibroblasts also promoted reprogramming to iPSCs. Our study reveals the breadth of kinase networks regulating pluripotency and identifies a role for cytoskeletal remodeling in modulating the somatic cell reprogramming process.

Exon 11 of the insulin receptor gene (INSR) is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5′ GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3′ end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5′ splice site of intron 11. The SR protein SRSF1 (SF2/ASF) co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.

The insulin receptor exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. These two isoforms show a cell-specific distribution, and their relative proportions also vary during development, aging, and in different disease states. We have previously demonstrated that both intron 10 and the alternatively spliced exon 11 contain regulatory sequences that affect insulin receptor splicing both positively and negatively and that these sequences bind the serine/arginine-rich (SR) proteins SRp20 and SF2/ASF and the CELF protein CUG-BP1. In this study, we describe a new intronic splicing element within intron 11 that is highly conserved across species. Using minigenes carrying deletion mutations within intron 11, we demonstrated that this sequence functions as an intronic splicing enhancer. We subsequently used RNA affinity chromatography to identify Mbnl1 as a splicing factor that recognizes this enhancer. By ribonucleoprotein immunoprecipitation, we also established that Mbnl1 binds specifically to the INSR (insulin receptor gene) RNA. Overexpression or knockdown of Mbnl1 in hepatoma and embryonic kidney cells altered the levels of exon 11 inclusion. Finally, we showed that deletion of the intronic enhancer eliminates the ability of Mbnl1 to promote exon inclusion. Collectively, these findings demonstrate a role for Mbnl1 in controlling insulin receptor exon 11 inclusion via binding to a downstream intronic enhancer element.In mammals, alternative splicing is a common strategy for creating functional diversity in proteins to confer cell and developmentally specific functions. Given its important role, it is not surprising that a recent estimate has proposed that 50 -60% of mutations linked to disease affect RNA splicing (1, 2). The majority of human genes undergo alternative pre-mRNA splicing through the use of competing 5Ј or 3Ј splice sites or through alternative inclusion/exclusion of exons in the pre-mRNA. These alternative exons often contain splice sites that diverge from the consensus, and the presence of cis regulatory elements within the exon and/or the flanking introns determines whether these exons are recognized (3-5). These cis-elements can either have a positive (enhancer) or negative (silencer) effect on splicing. Both enhancers and silencers are thought to function through binding to specific trans-acting protein factors (6). Differences in the expression or activity of these trans-acting factors may modulate the recognition of the alternative exon and lead to developmental or tissue-specific differences in splicing. Proteins that bind to specific sequence elements to affect splice site selection include SR proteins, hnRNPs, 2 and other related RNA-binding proteins such as the CELF family, TIA-1, NOVA1, and A2BP1 (also known as Fox-1) (7-13). Adding a further layer of regulation, local context, such as RNA secondary structure, may influence the way that binding motifs are recognized by their cognate factors (14 -16).The human insulin receptor...

Insulin is the central hormone required for the activation of lipogenic genes in the liver. Feeding animals a high carbohydrate diet enhances the expression of the lipogenic genes. This effect involves the stimulatory actions of both dietary glucose and insulin (1, 2). In contrast, dietary polyunsaturated fats attenuate the stimulatory effect of feeding a high carbohydrate diet (3, 4). We have used glucose-6-phosphate dehydrogenase (G6PD), 2 a member of the lipogenic gene family, as a model system for studying the mechanism of action of fatty acids. The advantage of this model is that insulin is the primary inducer of G6PD expression, and fatty acids such as arachidonic acid are the primary inhibitors of G6PD expression; this regulation is independent of other hormonal requirements (5, 6). The intracellular mechanisms by which polyunsaturated fats inhibit G6PD or other lipogenic genes are not completely understood. Inhibition by polyunsaturated fatty acid may represent a direct action of fatty acids on factors involved in gene expression. Alternatively, fatty acids may act indirectly via the inhibition of stimulatory signal transduction pathways of glucose or insulin. We hypothesized that fatty acids inhibit G6PD expression by inhibition of the insulin induction.Insulin transduces its signal upon binding to the insulin receptor. Transduction of this signal in liver involves phosphorylation of two intracellular substrates, insulin receptor substrate (IRS)-1 and IRS-2 (7). These proteins play complementary roles in insulin signaling (8). Activation of phosphoinositide (PI) 3-kinase is associated with the stimulatory effects of insulin on metabolic pathways, including lipogenesis (9 -11).The IRS proteins can be phosphorylated on both tyrosines and serines. A known mechanism for the inhibition of IRS-1 activation is by phosphorylation at serines 307, 612, and 632 (12). These serine residues, when phosphorylated might interfere with the interaction between IRS-1 and the insulin receptor or PI 3-kinase (13,14). Among the factors known to cause serine phosphorylation of IRS-1 are the mitogen-activated protein (MAP) kinases. Activation of the MAP kinases extracellular regulated kinase (ERK) (15, 16), c-Jun NH 2 -terminal kinase (JNK) (17-19), or p38 MAP kinase (MAPK) (17,20) is associated with the development of insulin resistance in muscle and adipose tissue.Known activators of MAP kinases include tumor necrosis factor ␣ (TNF␣) and very high fat diets. TNF␣, a potent mediator of insulin resistance, activates all three of the MAP kinases. Phosphorylation and activation of p38 MAPK by TNF␣ correlates with IRS-1 serine phosphorylation and a decrease in PI 3-kinase activity (17,20). In muscle and adipose tissue, this results in the decrease in glucose uptake associated with insulin resistance. Likewise, diets containing 40% or more of the energy content as fat also decrease PI 3-kinase activation and result in an insulin-resistant phenotype in intact animals (21-23). This may involve activation of MAP kinases (18,19). In c...

The inhibition of glucose-6-phosphate dehydrogenase (G6PD) expression by arachidonic acid occurs by changes in the rate of pre-mRNA splicing. Here, we have identified a cis-acting RNA element required for regulated splicing of G6PD mRNA. Using transfection of G6PD RNA reporter constructs into rat hepatocytes, the cis-acting RNA element involved in this regulation was localized to nucleotides 43-72 of exon 12 in the G6PD mRNA. In in vitro splicing assays, RNA substrates containing exon 12 were not spliced. In contrast, RNA substrates containing other regions (exons 8 and 9 or exons 10 and 11) of the G6PD mRNA were efficiently spliced. Furthermore, exon 12 can inhibit splicing when substituted for other exons in RNA substrates that are readily spliced. This activity of the exon 12 regulatory element suggests that it is an exonic splicing silencer. Consistent with its activity as a splicing silencer, spliceosome assembly was inhibited on RNA substrates containing exon 12 compared with RNAs representing other regions of the G6PD transcript. Elimination of nucleotides 43-72 of exon 12 did not restore splicing of exon 12-containing RNA; thus, the 30-nucleotide element may not be exclusively a silencer. The binding of heterogeneous nuclear ribonucleoproteins K, L, and A2/B1 from both HeLa and hepatocyte nuclear extracts to the element further supports its activity as a silencer. In addition, SR proteins bind to the element, consistent with the presence of enhancer activity within this sequence. Thus, an exonic splicing silencer is involved in the inhibition of splicing of a constitutively spliced exon in the G6PD mRNA.Glucose-6-phosphate dehydrogenase (G6PD) 2 is a member of a family of enzymes that catalyze the de novo synthesis of fatty acids. In liver, the lipogenic pathway plays an essential role in converting excess dietary energy into a storage form. Consistent with this role in energy homeostasis, the capacity of this pathway is regulated by dietary changes, such as fasting, feeding, and the amount and type of carbohydrate and polyunsaturated fat in the diet (1). For many of the lipogenic enzymes, regulation of enzyme amount occurs primarily by changes in the transcription rate of the gene, but posttranscriptional regulation via mRNA stability has also been implicated (1). G6PD differs from the other family members in that dietary regulation occurs exclusively at a posttranscriptional step (2-4).G6PD expression is inhibited by polyunsaturated fatty acids, such as arachidonic acid; this occurs at a unique posttranscriptional step involving a decrease in the rate of splicing of the nascent G6PD transcript. Several lines of evidence indicate that changes in mature mRNA accumulation are caused by changes in the efficiency of splicing of the G6PD transcript. First, changes in the cytoplasmic accumulation of G6PD mRNA are preceded by changes in the accumulation of mRNA in the nucleus in the absence of changes in transcriptional activity of the gene (3-5). Second, stimulatory treatments, such as refeeding, enhance the ...

Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as β-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser 307 phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.