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.

Mammalian U6 small nuclear RNA undergoes 3' end modifications within the spliceosome.

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.

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.

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.

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.

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.

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.

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.

B-B' proteins from small nuclear ribonucleoproteins have an endoribonuclease catalytic domain inactive in native particles.

Native small nuclear ribonucleoproteins (snRNPs) purified by several conventional procedures or reconstituted in vitro have no ribonuclease activity. However, when these same snRNPs are centrifuged in cesium chloride gradients at low [Mg2+] and in the presence of sarkosyl, an endoribonuclease is unmasked at the density of core particles (i.e. containing only the set of low molecular weight proteins common to all snRNPs), while an inhibitory component is released in soluble form. The nature of this inhibitor was not further investigated and the molecular events underlying this inhibition/activation process remained only a matter of speculation. On the other hand, evidence was obtained that the nuclease activity is carried by B-B' on the basis of its comigration with B-B' as well as with two of their cleavage products after SDS/polyacrylamide gel electrophoresis of snRNP proteins. One was identified by a B-B'-specific monoclonal antibody. Another one, especially prominent and migrating between D and E core proteins, was identified as the N-terminal half of B-B' by microsequence analysis. Although tightly associated with core snRNPs, the activity is not dependent upon the presence of an snRNA. For the time being, the functional significance of this nuclease remains entirely elusive.

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.

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.

Alternative chromatin structure at CpG islands.

Actively transcribed chromatin is structurally different from bulk inactive chromatin. It has been difficult to define the molecular basis of the difference, however, because purified fractions of active chromatin were not available. We have overcome this problem by releasing oligonucleosomes from the nonmethylated CpG-rich islands (CpG islands) of HeLa cell nuclei using restriction endonucleases. Since CpG islands very often include the promoters and 5' transcribed regions of genes, they represent a model for the "active" chromatin structure. CpG island chromatin differs in three respects from bulk chromatin prepared in the same way: histone H1 is present in very low amounts; histones H3 and H4 are highly acetylated; and nucleosome-free regions are present. Except for the latter regions, the average nucleosomal spacing is similar to that of bulk chromatin.

Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing.

We have investigated the function of ATP hydrolysis in mammalian pre-mRNA in vitro splicing using adenosine phosphorothioates (ATP alpha S and ATP tau S) known to affect the activity of a number of ATP-requiring enzymes. Spliceosome assembly, but neither one of the two transesterification reactions involved in splicing, occurs with ATP alpha S suggesting that at least two types of ATP-requiring factors are brought into play. ATP alpha S has no effect in the presence of normal ATP and, therefore, spliceosomes assembled in the presence of ATP alpha S remain competent for splicing when supplied with normal ATP. ATP tau S noticeably and irreversibly inhibits the second transesterification reaction, i.e. at a time when most of the analog has been hydrolyzed and regenerated to normal ATP by creatine phosphate. This indicates that the inhibition results from an earlier event, most likely the thiophosphorylation of spliceosomal proteins. Under this assumption, the inhibition could be due to the failure of the thiophosphorylated proteins to be dephosphorylated. Indeed, okadaic acid, a potent inhibitor of protein phosphatases, inhibits the second step of a reaction in the presence of normal ATP. We propose that some splicing factors undergo phosphorylation-dephosphorylation cycles during spliceosome assembly and splicing, while others that could be the mammalian equivalents of the RNA helicase-like proteins recently discovered in yeast most likely bind and hydrolyze ATP.

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.

A protein that specifically recognizes the 3' splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein.

Using a protein blotting method for the detection of nucleic acid binding proteins, we have identified in HeLa cell nuclear extracts an intron binding protein (IBP) that selectively recognizes the 3' splice site region of mammalian pre-mRNAs. The binding site was accurately delineated using oligonucleotides complementary to human beta-globin pre-mRNA. It spans the 3' splice site AG dinucleotide and the crucial polypyrimidine stretch upstream, but includes neither the branchpoint nor the lariat structure. Although the technique used here shows that the binding specificity is an intrinsic property of IBP and does not depend on snRNA-pre-mRNA interactions, it comigrates with U5 snRNP and is immunoprecipitated by anti-Sm antibody. This strongly suggests that IBP belongs to U5 snRNP. We propose that it is involved in one of the earliest steps of the splicing reaction by mediating the interaction of U5 snRNP with the 3' splice site.

Interplay between U2 snRNP and 3' splice factor(s) for branch point selection on human beta-globin pre-mRNA.

We investigated the interaction of U2 snRNP with the branch-3' splice site region of three human beta-globin pre-mRNAs carrying nearly complete (BamHI RNA), 24 nt (Avall RNA) and 14 nt (Accl RNA) of exon 2. All supported splicing, but mRNAs yields were respectively 2 and 10 times lower for Avall and Accl RNAs than for BamHI. Analysis of RNase T1-resistant fragments immunoprecipitated by an anti-(U2)RNP antibody at early times of the splicing reaction showed that the protection encompasses both the branch point region and the end of the intron in BamHI and Avall, but essentially only the branch point in Accl RNAs. Later on, this protection becomes less detectable in BamHI, is reinforced in Avall and remains poorly detectable in Accl RNAs. Similar experiments performed at late times with an anti-Sm antibody recognizing all snRNPs showed that the end of the intron is protected in all but BamHI RNAs. These results support the conclusion that U2 snRNP binds to a fully efficient precursor (BamHI RNA) through another factor(s) recognizing the 3' splice site (U5 snRNP and the so-called U2AF protein are likely candidates). Either the absence of an initial contact between U2 snRNP and the factor(s) recognizing the end of the intron (Accl RNA) or the unability of this ternary complex to undergo a conformational change (Avall RNA) could render these severely truncated precursors poor substrates. These different situations have consequences on the branch point selection itself. BamHI and Avall RNAs use three functional branch points at early times, the usual A residue at -37 and two U residues at -17 and -22. Accl RNA uses only one branch point at -37. Later on, all three branch points are used at the same rate in Avall, while the usual one prevails in BamHI RNAs.

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.

U1-U2 snRNPs interaction induced by an RNA complementary to the 5' end sequence of U1 snRNA.

Several lines of evidences indicate that U1 and U2 snRNPs become interacting during pre-mRNA splicing. Here we present data showing that an U1-U2 snRNPs interaction can be mediated by an RNA only containing the consensus 5' splice site of all of the sequences characteristic of pre-mRNAs. Using monospecific antibodies (anti-(U1) RNP and anti-(U2) RNP), we have found that a tripartite complex comprising U1 and U2 snRNPs is immunoprecipitated in the presence of a consensus 5' splice site containing RNA, either from a crude extract or from an artificial mixture enriched in U1 and U2 snRNPs. This complex does not appear in the presence of an RNA lacking the sequence complementary to the 5' terminus of U1 snRNA. Moreover, RNAse T1 protection coupled to immunoprecipitation experiments have demonstrated that only the 5' end sequence of U1 snRNA contacts the consensus 5' splice site containing RNA, arguing that U2 snRNP binding in the tripartite complex is mediated by U1 snRNP.