Pharmacological modulation of LMNA SRSF1-dependent splicing abrogates diet-induced obesity in mice.

Bakground/Objectives:Intense drug discovery efforts in the metabolic field highlight the need for novel strategies for the treatment of obesity. Alternative splicing (AS) and/or polyadenylation enable the LMNA gene to express distinct protein isoforms that exert opposing effects on energy metabolism and lifespan. Here we aimed to use the splicing factor SRSF1 that contribute to the production of these different isoforms as a target to uncover new anti-obesity drug. SUBJECTS/METHODS: Small molecules modulating SR protein activity and splicing were tested for their abilities to interact with SRSF1 and to modulate LMNA (AS). Using an LMNA luciferase reporter we selected molecules that were tested in diet-induced obese (DIO) mice. Transcriptomic analyses were performed in the white adipose tissues from untreated and treated DIO mice and mice fed a chow diet. RESULTS: We identified a small molecule that specifically interacted with the RS domain of SRSF1. ABX300 abolished DIO in mice, leading to restoration of adipose tissue homeostasis. In contrast, ABX300 had no effect on mice fed a standard chow diet. A global transcriptomic analysis revealed similar profiles of white adipose tissue from DIO mice treated with ABX300 and from untreated mice fed a chow diet. Mice treated with ABX300 exhibited an increase in O2 consumption and a switch in fuel preference toward lipids. CONCLUSIONS: Targeting SRSF1 with ABX300 compensates for changes in RNA biogenesis induced by fat accumulation and consequently represents a novel unexplored approach for the treatment of obesity.

RasGAP-associated endoribonuclease G3Bp: selective RNA degradation and phosphorylation-dependent localization.

Mitogen activation of mRNA decay pathways likely involves specific endoribonucleases, such as G3BP, a phosphorylation-dependent endoribonuclease that associates with RasGAP in dividing but not quiescent cells. G3BP exclusively cleaves between cytosine and adenine (CA) after a specific interaction with RNA through the carboxyl-terminal RRM-type RNA binding motif. Accordingly, G3BP is tightly associated with a subset of poly(A)(+) mRNAs containing its high-affinity binding sequence, such as the c-myc mRNA in mouse embryonic fibroblasts. Interestingly, c-myc mRNA decay is delayed in RasGAP-deficient fibroblasts, which contain a defective isoform of G3BP that is not phosphorylated at serine 149. A G3BP mutant in which this serine is changed to alanine remains exclusively cytoplasmic, whereas a glutamate for serine substitution that mimics the charge of a phosphorylated serine is translocated to the nucleus. Thus, a growth factor-induced change in mRNA decay may be modulated by the nuclear localization of a site-specific endoribonuclease such as G3BP.

Specific inhibition of serine- and arginine-rich splicing factors phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506.

Specific phosphorylation of serine- and arginine-rich pre-mRNA splicing factors (SR proteins) is one of the key determinants regulating splicing events. Several kinases involved in SR protein phosphorylation have been identified and characterized, among which human DNA topoisomerase I is known to have DNA-relaxing activity. In this study, we have investigated the mechanism of splicing inhibition by a glycosylated indolocarbazole derivative (NB-506), a potent inhibitor of both kinase and relaxing activities of topoisomerase I. NB-506 completely inhibits the capacity of topoisomerase I to phosphorylate, in vitro, the human splicing factor 2/alternative splicing factor (SF2/ASF). This inhibition is specific, because NB-506 does not demonstrate activity against other kinases known to phosphorylate SF2/ASF such as SR protein kinase 1 and cdc2 kinase. Importantly, HeLa nuclear extracts competent in splicing but not splicing-deficient cytoplasmic S100 extracts treated with the drug fail to phosphorylate SF2/ASF and to support splicing of pre-mRNA substrates containing SF2/ASF-target sequences. Native gel analysis of splicing complexes revealed that the drug affects the formation of the spliceosome, a dynamic ribonucleoprotein structure where splicing takes place. In the presence of the drug, neither pre-spliceosome nor spliceosome is formed, demonstrating that splicing inhibition occurs at early steps of spliceosome assembly. Splicing inhibition can be relieved by adding phosphorylated SF2/ASF, showing that extracts treated with NB-506 lack a phosphorylating activity required for splicing. Moreover, NB-506 has a cytotoxic effect on murine P388 leukemia cells but not on P388CPT5 camptothecin-resistant cells that carry two point mutations in conserved regions of topoisomerase I gene (Gly361Val and Asp709Tyr). After drug treatment, P388 cells accumulated hypophosphorylated forms of SR proteins and polyadenylated RNA in the nucleus. In contrast, neither SR protein phosphorylation nor polyadenylated mRNA distribution was affected in P388 CPT5-treated cells. Consistently, NB506 treatment altered the mRNA levels and/or splicing pattern of several tested genes (Bcl-X, CD 44, SC35, and Sty) in P388 cells but not in P388 CPT5 cells. The study shows for the first time that indolocarbazole drugs targeting topoisomerase I can affect gene expression by modulating pre-mRNA splicing through inhibition of SR proteins phosphorylation.

Distinctive features of Drosophila alternative splicing factor RS domain: implication for specific phosphorylation, shuttling, and splicing activation.

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5' alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro.

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)(n)repeat] with high affinity and bind double-stranded telomeric DNA with a 100xreduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66-64, 45 and 35 kDa, respectively. To identify these polypeptides we screened alambdagt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.

Interaction of human DNA topoisomerase I with G-quartet structures.

Because of their role in the control of the topological state of DNA, topoisomerases are ubiquitous and vital enzymes, which participate in nearly all events related to DNA metabolism including replication and transcription. We show here that human topoisomerase I (Topo I) plays an unexpected role of 'molecular matchmaker' for G-quartet formation. G-quadruplexes are multi-stranded structures held together by square planes of four guanines ('G-quartets') interacting by forming Hoogsteen hydrogen bonds. Topo I is able to promote the formation of four-stranded intermolecular DNA structures when added to single-stranded DNA containing a stretch of at least five guanines. We provide evidence that these complexes are parallel G-quartet structures, mediated by tetrads of hydrogen-bonded guanine. In addition, Topo I binds specifically to pre-formed parallel and anti-parallel G4-DNA.

Recognition of exonic splicing enhancer sequences by the Drosophila splicing repressor RSF1.

The Drosophila repressor splicing factor 1 (RSF1) comprises an N-terminal RNA-binding region and a C-terminal domain rich in glycine, arginine and serine residues, termed the GRS domain. Recently, RSF1 has been shown to antagonize splicing factors of the serine/arginine-rich (SR) family and it is, therefore, expected to play a role in processing of a subset of Drosophila pre-mRNAs through specific interactions with RNA. To investigate the RNA-binding specificity of RSF1, we isolated RSF1-binding RNAs using an in vitro selection approach. We have identified two RNA target motifs recognized by RSF1, designated A (CAACGACGA)- and B (AAACGCGCG)-type sequences. We show here that the A-type cognate sequence behaves as an SR protein-dependent exonic splicing enhancer. Namely, three copies of the A-type ligand bind SR proteins, stimulate the efficiency of splicing of reporter pre-mRNAs several fold and lead to inclusion of a short internal exon both in vitro and in vivo. However, three copies of a B-type ligand were much less active. The finding that RSF1 acts as a potent repressor of pre-mRNA splicing in vitro led us to propose that the equilibrium between a limited number of structurally-related general splicing activators or repressors, competing for common or promiscuous binding sites, may be a major determinant of the underlying mechanisms controlling many alternative pre-mRNA process-ing events.

Antagonism between RSF1 and SR proteins for both splice-site recognition in vitro and Drosophila development.

Specific recognition of splice sites within metazoan mRNA precursors (pre-mRNAs) is a potential stage for gene regulation by alternative splicing. Splicing factors of the SR protein family play a major role in this regulation, as they are required for early recognition of splice sites during spliceosome assembly. Here, we describe the characterization of RSF1, a splicing repressor isolated from Drosophila, that functionally antagonizes SR proteins. Like the latter, RSF1 comprises an amino-terminal RRM-type RNA-binding domain, whereas its carboxy-terminal part is enriched in glycine (G), arginine (R), and serine (S) residues (GRS domain). RSF1 induces a dose-sensitive inhibition of splicing for several reporter pre-mRNAs, an inhibition that occurs at the level of early splicing complexes formation. RSF1 interacts, through its GRS domain, with the RS domain of the SR protein SF2/ASF and prevents the latter from cooperating with the U1 small nuclear ribonucleoprotein particle (U1 snRNP) in binding pre-mRNA. Furthermore, overproduction of RSF 1 in the fly rescues several developmental defects caused by overexpression of the splicing activator SR protein B52/ SRp55. Therefore, RSF1 may correspond to the prototypical member of a novel family of general splicing repressors that selectively antagonize the effect of SR proteins on 5' splice-site recognition.

Poisoning of topoisomerase I by an antitumor indolocarbazole drug: stabilization of topoisomerase I-DNA covalent complexes and specific inhibition of the protein kinase activity.

We have investigated the mechanism of topoisomerase I inhibition by an indolocarbazole derivative, R-3. The compound is cytotoxic to P388 leukemia cells, but not to P388CPT5 camptothecin-resistant cells having a deficient topoisomerase I. R-3 can behave both as a specific topoisomerase I inhibitor trapping the cleavable complexes and as a nonspecific inhibitor of a DNA-processing enzyme acting via DNA binding. In addition, the drug is a potent inhibitor of the kinase activity of topoisomerase I. Unlike camptothecin, R-3 completely inhibits the phosphorylation of SF2/ASF, a member of the SR protein family, in the absence of DNA. The inhibitory effect is also observed using mutant enzyme Y723F that lacks DNA cleavage/religation activity but does not affect phosphotransferase activity, indicating, therefore, that R-3 acts independently at both DNA cleavage and protein kinase sites. R-3 is the only compound known thus far that interferes specifically with the kinase activity of topoisomerase I and not with other kinases, such as protein kinase C and the cdc2 kinase. The study reinforces the view that topoisomerase I is a dual enzyme with a DNA cleavage site juxtaposed to a functionally independent kinase site and shows for the first time that indolocarbazole drugs can inhibit both the DNA cleavage/religation and kinase activities of the enzyme.

The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity.

Human DNA topoisomerase I not only has DNA relaxing activity, but also splicing factors phosphorylating activity. Topo I shows strong preference for ATP as the phosphate donor. We used photoaffinity labeling with the ATP analogue [alpha-32P] 8-azidoadenosine-5'-triphosphate combined with limited proteolysis to characterize Topo I domains involved in ATP binding. The majority of incorporated analogue was associated with two fragments derived from N-terminal and C-terminal regions of Topo I, respectively. However, mutational analysis showed that deletion of the first 138 N-terminal residues, known to be dispensable for topoisomerase activity, did not change the binding of ATP or the kinase activity. In contrast, deletion of 162 residues from the C-terminal domain was deleterious for ATP binding, kinase and topoisomerase activities. Furthermore, a C-terminal tyrosine 723 mutant lacking topoisomerase activity is still able to bind ATP and to phosphorylate SF2/ASF, suggesting that the two functions of Topo I can be separated. These findings argue in favor of the fact that Topo I is a complex enzyme with a number of potential intra-cellular functions.

Interaction between the N-terminal domain of human DNA topoisomerase I and the arginine-serine domain of its substrate determines phosphorylation of SF2/ASF splicing factor.

Human DNA topoisomerase I, known for its DNA-relaxing activity, is possibly one of the kinases phosphorylating members of the SR protein family of splicing factors, in vivo. Little is known about the mechanism of action of this novel kinase. Using the prototypical SR protein SF2/ASF (SRp30a) as model substrate, we demonstrate that serine residues phosphorylated by topo I/kinase exclusively located within the most extended arginine-serine repeats of the SF2/ASF RS domain. Unlike other kinases such as cdc2 and SRPK1, which also phosphorylated serines at the RS domain, topo I/kinase required several SR dipeptide repeats. These repeats possibly contribute to a versatile structure in the RS domain thereby facilitating phosphorylation. Furthermore, far-western, fluorescence spectroscopy and kinase assays using the SF2/ASF mutants, demonstrated that kinase activity and binding were tightly coupled. Since the deletion of N-terminal 174 amino acids of Topo I destroys SF2/ASF binding and kinase activity but not ATP binding, we conclude that at least two distinct domains of Topo I are necessary for kinase activity: one in the C-terminal region contributing to the ATP binding site and the other one in the N-terminal region that allows binding of SF2/ASF.

A novel phosphorylation-dependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability.

A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3'-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.

DNA topoisomerase I: customs officer at the border between DNA and RNA worlds?

DNA topoisomerase I is required for the normal development of multicellular organisms, probably because it plays a role in controlling gene activity, in addition to its function in relieving tortional stress during DNA replication and transcription. The discovery of DNA topoisomerase I as a specific kinase that phosphorylates serine-arginine rich (SR) splicing factors may provide new insights into their precise function in regulating gene expression. It is clear that the splicing factors phosphorylated by DNA topoisomerase I can modulate gene expression by changing the splicing pattern of structural genes. Studies of the splicing mechanism suggest that the phosphorylation of serine residues of SR proteins contribute to their activity. As this phosphorylation can be accomplished by several kinases, it remains to be determined whether phosphorylation by DNA topoisomerase I protein kinase is the limiting step in regulating this process. The availability of specific inhibitors of DNA topoisomerase I, structurally related to the alkaloid camptothecin, have made it possible to address this question experimentally. These inhibitors, which hold great promise as antineoplastic drugs, lead to specific inhibition of SR protein phosphorylation in cultured cells. This observation will hopefully lead to improved understanding of the mechanism by which these drugs act at cellular level.

Involvement of U1 small nuclear ribonucleoproteins (snRNP) in 5' splice site-U1 snRNP interaction.

U1 small nuclear ribonucleoprotein (snRNP) is an important ribonucleoprotein involved early in the spliceosome formation to commit pre-mRNAs to the splicing pathway. We have determined the association and dissociation kinetics of the 5' splice site-U1 snRNP interaction using purified U1 snRNP and a short RNA oligonucleotide comprising the 5' splice site (5'-SS) consensus sequence of pre-mRNAs (5'-SS RNA oligo). The association is rapid, does not require ATP, and is almost irreversible. Surprisingly, oligonucleotide-directed cleavage of the U1 small nuclear RNA (snRNA) 5' end sequence with RNase H has no significant effect on the rate of association of the 5'-SS RNA oligo, but it does lead to rapid dissociation. This provides evidence that U1-specific snRNP proteins are critical for the 5' splice site recognition while base pairing ensures the stability of the interaction. The recognition of the 5' splice site by U1 snRNP does not result from the individual action of one or more proteins but rather from their organization around U1 snRNA. A consequence of this organization is that the U1-C protein makes direct contacts with the site, as it becomes cross-linked to the RNA oligo upon exposition of the reactions to shortwave UV light.

Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I.

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.

Modulation of DNA topoisomerase I activity by p53.

The tumor suppressor protein p53 plays a central role in the cellular response to genotoxic lesions and has been shown to be activated by most anticancer agents such as mitomycin C. We here show that mitomycin C treatment of human MCF7 breast adenocarcinoma cells results in increased topoisomerase I activity as measured by relaxation of supercoiled DNA and by phosphorylation of SR protein splicing factor. The increase in catalytic activity occurs in parallel with the nuclear accumulation of p53, resulting in detectable activation of topoisomerase I within less than 1 h of drug treatment. Furthermore, topoisomerase I co-immunoprecipitates with nuclear p53, suggesting that the activation of topoisomerase I may be a result of a direct interaction between the two proteins. In vitro experiments with purified recombinant proteins show that p53 increases the catalytic activities of topoisomerase I as measured by relaxation of supercoiled DNA, stabilization of the covalent topoisomerase I-DNA complex (in the presence of camptothecin), and phosphorylation of SR protein splicing factor ASF/SF2. Furthermore, topoisomerase I sediments at a higher molecular weight in the presence of p53 as revealed by sucrose density gradient analysis in the absence of DNA. Finally, p53 modifies the thermal stability of topoisomerase I, protecting it from heat denaturation. Taken together, our results show that topoisomerase I and p53 form molecular complexes in vitro as in vivo, and we suggest that the p53-mediated response to DNA damage may, at least in part, involve activation of topoisomerase I.

Structural features of U6 snRNA and dynamic interactions with other spliceosomal components leading to pre-mRNA splicing.

In the spliceosome, the pre-mRNA, U2 and U6 snRNAs fold into a catalytic structure exhibiting striking similarities with domain V and VI of group II introns. Building of this tripartite structure implies that an evolutionary conserved base pairing between U4 and U6 snRNAs should be disrupted to allow potentially U6 catalytic residue to interact with U2 snRNAs and the pre-mRNA. The steps leading to U4/U6 disruption have been recently discovered and have been shown to involve a modification of the 3' end of U6 snRNA and the hnRNP C protein.

Disruption of base-paired U4.U6 small nuclear RNAs induced by mammalian heterogeneous nuclear ribonucleoprotein C protein.

Due to 3' end modifications, mammalian U6 small nuclear RNA (snRNA) is heterogeneous in size. The major form terminates with five U residues and a 2',3'-cyclic phosphate, but multiple RNAs containing up to 12 U residues have a 3'-OH end. They are labeled in the presence of [alpha-32P]UTP by the terminal uridylyl transferase activity present in HeLa cell nuclear extracts. That these forms all enter the U6 snRNA-containing particles, U4.U6, U4.U5.U6, and the spliceosome, has been demonstrated previously. Here, we report an interaction between the heterogeneous nuclear ribonucleoprotein (hnRNP) C protein, an abundant nuclear pre-mRNA binding protein, and the U6 snRNAs that have the longest uridylate stretches. This U6 snRNA subset is free of any one of the other snRNPs, since anti-Sm antibodies failed to immunoprecipitate hnRNP C protein. Furthermore, isolated U4.U6 snRNPs containing U6 snRNAs with long oligouridylate stretches are disrupted upon binding of hnRNP C protein either purified from HeLa cells or produced as recombinant protein from Escherichia coli. In view of these data and our previous proposal that the U6 snRNA active in splicing has 3'-OH end, we discuss a model where the hnRNP C protein has a decisive function in the catalytic activation of the spliceosome by allowing the release of U4 snRNP.

Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing.

The U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA. Mammalian U1 snRNP contains a 165-nucleotide long RNA molecule and at least 11 proteins: the U1-specific 70K proteins A and C, and the common U snRNP proteins (B', B, D1, D2, D3, E, F and G). One of the functions of U1 snRNP is recognition of the 5' splice site, an event that requires both U1 RNA and U1 proteins. The 70K protein is the only heavily phosphorylated U1 protein in the cell. Isolated U1 snRNPs are associated with a kinase activity that selectively phosphorylates the 70K protein in vitro in a reaction requiring ATP. Here we investigate the role of phosphorylation of the 70K protein in the splicing of pre-mRNA. The 70K protein on U1 snRNPs was phosphorylated in vitro with either ATP, or with ATP-gamma S, which gave a thiophosphorylated product that was resistant to dephosphorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous U1 snRNPs were complemented with U1 snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated U1 snRNPs were used instead, splicing was completely inhibited, although formation of the mature spliceosome was unaffected. Our data show that the state of phosphorylation of the U1-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.