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International audience; Splicing factor mutations are frequent in myeloid neoplasms, blood cancers,and solid tumors. Cancer cells harboring these mutations present aparticular vulnerability to drugs that target splicing factors such asSF3b155 or CAPERα. Still, the arsenal of chemical probes that targets thespliceosome is very limited. U2AF homology motifs (UHMs) are commonprotein interaction domains among splicing factors. They present ahydrophobic pocket ideally suited to anchor small molecules with the aimto inhibit protein–protein interaction. Here, we combined a virtual screeningof a small molecules database and an in vitro competition assay andidentified a small molecule, we named UHMCP1 that prevents theSF3b155/U2AF interaction. NMR analyses and molecular dynamics simulationsconfirmed the binding of this molecule in the hydrophobic pocketof the U2AF UHM domain. We further provide evidence that UHMCP1impacts splicing and cell viability and is therefore an interesting novel compoundtargeting an UHM domain with potential anticancer properties.

International audience; The essential splicing factor U2AF65 is known to help anchoring U2 snRNP at the branch site. Its C-terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF65 and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF65 drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF65 or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine-rich motifs. These results support a model in which liquid-like assemblies of U2AF65 and CAPERα on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF65 and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.

U2AF homology motifs (UHMs) mediate protein-protein interactions with U2AF ligand motifs (ULMs) of pre-mRNA splicing factors. The UHM-containing alternative splicing factor CAPERα regulates splicing of tumor-promoting VEGF isoforms, yet the molecular target of the CAPERα UHM is unknown. Here we present structures of the CAPERα UHM bound to a representative SF3b155 ULM at 1.7 Å resolution and, for comparison, in the absence of ligand at 2.2 Å resolution. The prototypical UHM/ULM interactions authenticate CAPERα as a bona fide member of the UHM family of proteins. We identify SF3b155 as the relevant ULM-containing partner of full-length CAPERα in human cell extracts. Isothermal titration calorimetry comparisons of the purified CAPERα UHM binding known ULM-containing proteins demonstrate that high affinity interactions depend on the presence of an intact, intrinsically unstructured SF3b155 domain containing seven ULM-like motifs. The interplay among bound CAPERα molecules gives rise to the appearance of two high affinity sites in the SF3b155 ULM-containing domain. In conjunction with the previously identified, UHM/ULM-mediated complexes of U2AF(65) and SPF45 with SF3b155, this work demonstrates the capacity of SF3b155 to offer a platform for coordinated recruitment of UHM-containing splicing factors.

SummaryThe essential splicing factors U2AF65 and SF1 cooperatively bind consensus sequences at the 3′ end of introns. Phosphorylation of SF1 on a highly conserved “SPSP” motif enhances its interaction with U2AF65 and the pre-mRNA. Here, we reveal that phosphorylation induces essential conformational changes in SF1 and in the SF1/U2AF65/3′ splice site complex. Crystal structures of the phosphorylated (P)SF1 domain bound to the C-terminal domain of U2AF65 at 2.29 Å resolution and of the unphosphorylated SF1 domain at 2.48 Å resolution demonstrate that phosphorylation induces a disorder-to-order transition within a previously unknown SF1/U2AF65 interface. We find by small-angle X-ray scattering that the local folding of the SPSP motif transduces into global conformational changes in the nearly full-length (P)SF1/U2AF65/3′ splice site assembly. We further determine that SPSP phosphorylation and the SF1/U2AF65 interface are essential in vivo. These results offer a structural prototype for phosphorylation-dependent control of pre-mRNA splicing factors.

International audience; Splicing factor 1 (SF1) recognizes 3' splice sites of the major class of introns as a ternary complex with U2AF65 and U2AF35 splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF65 protein partner. Conversely, quantitative fluorescence anisotropy RNA binding assays and isothermal titration calorimetry experiments establish that double-SPSP phosphorylation reduces phospho-SF1 and phospho-SF1-U2AF65 binding affinities for either optimal or suboptimal splice-site RNAs. Domain-substitution and mutagenesis experiments further demonstrate that arginines surrounding the phosphorylated SF1 loop are required for cooperative 3' splice site recognition by the SF1-U2AF65 complex (where cooperativity is defined as a nonadditive increase in RNA binding by the protein complex relative to the individual proteins). In the context of local, intracellular concentrations, the subtle effects of SF1 phosphorylation on its associations with U2AF65 and splice-site RNAs are likely to influence pre-mRNA splicing. However, considering roles for SF1 in pre-mRNA retention and transcriptional repression, as well as in splicing, future comprehensive investigations are needed to fully explain the requirement for SF1 SPSP phosphorylation in proliferating human cells.

The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF homology motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF65. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF65 and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF65, the UHM-containing C-terminal domain of KIS is required for binding to the splicing factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF65 in pull-down assays. These results further support the functional link of KIS with splicing factors. Interestingly, when compared to other UHM-containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains.

KIS is the only known protein kinase that possesses an RNA recognition motif. This original structure indicates a role for KIS in the maturation of RNAs possibly by phosphorylating and regulating the activities of RNA associated factors. Another function of KIS has recently been unravelled--it negatively regulates the cdk inhibitor p27Kip1 and thus promotes cell cycle progression through G1. In order to explore the functional expression of this kinase, we quantified its mRNA in a wide range of rat and human tissues, during development and in tumors. In both species, the highest level of KIS gene expression was in adult neural tissues. Interestingly, within the adult rat brain, KIS mRNA is enriched in several areas including the substantia nigra compacta and nuclei of the brain stem. Furthermore, KIS gene expression increases dramatically during brain development. Altogether our results point to a ubiquitous function for KIS together with a particular implication during neural differentiation or in the function of mature neural cells. No dysregulation of KIS gene expression was detected in human tumors from breast, bladder, prostate, liver and kidney origins. On the other hand, the KIS gene was overexpressed in NF1-associated plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) as compared to dermal neurofibroma which suggests a possible implication of KIS in the genesis of NF1-associated tumors.

Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and/or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and/or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological actions: it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3'/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.

Protein phosphorylation is involved at multiple steps of RNA processing and in the regulation of protein expression. We present here the first identification of a serine/threonine kinase that possesses an RNP-type RNA recognition motif: KIS. We originally isolated KIS in a two-hybrid screen through its interaction with stathmin, a small phosphoprotein proposed to play a general role in the relay and integration of diverse intracellular signaling pathways. Determination of the primary sequence of KIS shows that it is formed by the juxtaposition of a kinase core with little homology to known kinases and a C-terminal domain that contains a characteristic RNA recognition motif with an intriguing homology to the C-terminal motif of the splicing factor U2AF. KIS produced in bacteria has an autophosphorylating activity and phosphorylates stathmin on serine residues. It also phosphorylates in vitro other classical substrates such as myelin basic protein and synapsin but not histones that inhibit its autophosphorylating activity. Immunofluorescence and biochemical analyses indicate that KIS overexpressed in HEK293 fibroblastic cells is partly targetted to the nucleus. Altogether, these results suggest the implication of KIS in the control of trafficking and/or splicing of RNAs probably through phosphorylation of associated factors.

The CATS protein (also known as FAM64A and RCS1) was first identified as a novel CALM (PICALM) interactor that influences the subcellular localization of the leukemogenic fusion protein CALM/AF10. CATS is highly expressed in cancer cell lines in a cell cycle dependent manner and is induced by mitogens. CATS is considered a marker for proliferation, known to control the metaphase-to-anaphase transition during the cell division. Using CATS as a bait in a yeast two-hybrid screen we identified the Kinase Interacting Stathmin (KIS or UHMK1) protein as a CATS interacting partner. The interaction between CATS and KIS was confirmed by GST pull-down, co-immunopreciptation and co-localization experiments. Using kinase assay we showed that CATS is a substrate of KIS and mapped the phosphorylation site to CATS serine 131 (S131). Protein expression analysis revealed that KIS levels changed in a cell cycle-dependent manner and in the opposite direction to CATS levels. In a reporter gene assay KIS was able to enhance the transcriptional repressor activity of CATS, independent of CATS phophorylation at S131. Moreover, we showed that CATS and KIS antagonize the transactivation capacity of CALM/AF10.In summary, our results show that CATS interacts with and is a substrate for KIS, suggesting that KIS regulates CATS function.

The brain-enriched protein kinase KIS (product of the gene UHMK1) has been shown to phosphorylate the human splicing factor SF1 in vitro. This phosphorylation in turn favors the formation of a U2AF(65)-SF1-RNA complex which occurs at the 3' end of introns at an early stage of spliceosome assembly. Here, we analyzed the effects of KIS knockout on mouse SF1 phosphorylation, physiology, adult behavior, and gene expression in the neonate brain. We found SF1 isoforms are differently expressed in KIS-ko mouse brains and fibroblasts. Re-expression of KIS in fibroblasts restores a wild type distribution of SF1 isoforms, confirming the link between KIS and SF1. Microarray analysis of transcripts in the neonate brain revealed a subtle down-regulation of brain specific genes including cys-loop ligand-gated ion channels and metabolic enzymes. Q-PCR analyses confirmed these defects and point to an increase of pre-mRNA over mRNA ratios, likely due to changes in splicing efficiency. While performing similarly in prepulse inhibition and most other behavioral tests, KIS-ko mice differ in spontaneous activity and contextual fear conditioning. This difference suggests that disregulation of gene expression due to KIS inactivation affects specific brain functions.

Stathmin, a probable relay protein possibly integrating multiple intracellular regulatory signals [reviewed in Sobel (1991) Trends Biochem. Sci. 16, 301-305], was expressed in Escherichia coli at levels as high as 20% of total bacterial protein. Characterization of the purified recombinant protein revealed that it had biochemical properties very similar to those of the native protein. It is a good substrate for both cyclic AMP-dependent protein kinase (PKA) and p34cdc2, on the same four sites as the native eukaryotic protein. As shown by m.s., the difference in isoelectric points from the native protein is probably due to the absence of acetylation of the protein produced in bacteria. C.d. studies indicate that stathmin probably contains about 45% of its sequence in an alpha-helical conformation, as also predicted for the sequence between residues 47 and 124 by computer analysis. Replacement of Ser-63 by alanine by in vitro mutagenesis resulted in a ten times less efficient phosphorylation of stathmin by PKA which occurred solely on Ser-16, confirming that Ser-63 is the major target of this kinase. Replacement of Ser-25, the major site phosphorylated by mitogen-activated protein kinase in vitro and in vivo, by the charged amino acid glutamic acid reproduced, in conjunction with the phosphorylation of Ser-16 by PKA, the mobility shift on SDS/polyacrylamide gels induced by the phosphorylation of Ser-25. This result strongly suggests that glutamic acid in position 25 is able to mimic the putative interactions of phosphoserine-25 with phosphoserine-16, as well as the resulting conformational changes that are probably also related to the functional regulation of stathmin.

The ubiquitous cytoplasmic phosphoprotein stathmin was proposed to play a general role as an intracellular relay integrating diverse signals regulating the proliferation, differentiation, and functions of cells (Sobel, A. (1991) Trends Biol. Sci. 16, 301-305). It was originally identified in mammalian cells and tissues, but antibodies directed against the mammalian protein also recognized a stathmin-like 19-kDa protein in all vertebrate classes. The immunoreactive protein in Xenopus laevis displayed, like mammalian stathmin, several nonphosphorylated and phosphorylated heat-soluble forms with distinct migration on two-dimensional polyacrylamide gel electrophoresis. Screening of Xenopus oocyte and brain cDNA libraries with a rat stathmin cDNA probe allowed us to isolate several stathmin-related cDNA clones, among which clone XO35 encodes the Xenopus homologue of stathmin whose deduced amino acid sequence is 79% identical to and displays most of the characteristic structural features of the mammalian protein. In particular, one of the cAMP-dependent protein kinase and the two "proline-directed" kinase-specific sites known to be phosphorylated in rat stathmin are also present in the Xenopus protein. Furthermore, two other sets of clones coding for related proteins belonging to the stathmin gene family were also isolated; clone SC15 encodes the Xenopus homologue of SCG10, a rat protein specifically related to neuronal differentiation; clone XB3 encodes a protein which, as SCG10 or SC15, possesses a stathmin-like domain and an additional N-terminal extension but is more distant from SCG10 than SC15. Interestingly, the mRNA transcripts of Xenopus stathmin (XO35) appear ubiquitous, like stathmin in mammals, whereas the SC15 and XB3 mRNAs appeared as markers of the nervous tissue in Xenopus. During Xenopus oogenesis, stathmin accumulates and remains stable as a maternal product throughout early development. Concurrently, its phosphorylation is regulated from essentially unphosphorylated forms to highly phosphorylated ones in the mature egg, which are then progressively dephosphorylated again from the midblastula to the tailbud stage. Altogether, our results demonstrate the high evolutionary conservation of stathmin together with the members of its related gene family, not only at the level of their molecular structures, but also of their biochemical and biological regulation. These observations are thus further in favor of a very general and likely essential role of stathmin in the normal control of cells throughout development and in the adult.

Protein phosphorylation ensures the accurate and controlled expression of the genome, for instance by regulating the activities of pre-mRNA splicing factors. Here we report that splicing factor 1 (SF1), which is involved in an early step of intronic sequence recognition, is highly phosphorylated in mammalian cells on two serines within an SPSP motif at the junction between its U2AF65 and RNA binding domains. We show that SF1 interacts in vitro with the protein kinase KIS, which possesses a ‘U2AF homology motif’ (UHM) domain. The UHM domain of KIS is required for KIS and SF1 to interact, and for KIS to efficiently phosphorylate SF1 on the SPSP motif. Importantly, SPSP phosphorylation by KIS increases binding of SF1 to U2AF65, and enhances formation of the ternary SF1–U2AF65–RNA complex. These results further suggest that this phosphorylation event has an important role for the function of SF1, and possibly for the structural rearrangements associated with spliceosome assembly and function.

Stathmin is a ubiquitous phosphoprotein proposed to be a relay integrating various intracellular signaling pathways. Its high phylogenetic conservation and the identification of the related molecules, SCG10 in rat and XB3 in Xenopus, suggested the existence of a stathmin-related family. A systematic PCR-based approach allowed the identification of several novel mammalian sequences of which two coded for expressed members of the stathmin family; the translated RB3 sequence shares 88% amino-acid identity with that of XB3 and is thus its rat homologue, and RB3' corresponds to an alternatively spliced product of the same gene, encoding a truncated form. Within their stathmin-like domain, the alpha helix, probably responsible for coiled-coil protein-protein interactions, is conserved, as well as are two consensus phosphorylation sites; in their N-terminal extension domain, two cystein residues most likely responsible for membrane attachment through palmitoylation, are present in RB3/RB3' as in SCG10. The novel identification and characterization of the corresponding proteins showed that all three are associated with the particulate, membrane-containing fraction. They furthermore display several spots of decreasing pI on two-dimensional immunoblots, suggesting that they are phosphorylated in vivo. As for SCG10, RB3 mRNA is detectable only in the nervous system by in situ hybridization, but at similar levels in the newborn and the adult brain as revealed by Northern blots, whereas SCG10 expression decreases in the adult. Furthermore, RB3 mRNA is undetectable in PC12 cells, whereas SCG10 mRNA increases after treatment with nerve growth factor, inducing neuronal differentiation. In conclusion, we demonstrate here the existence of a highly conserved stathmin-related family in mammals, of which each member seems to play specific roles, related to the control of cell proliferation and activities for stathmin and to that of neuronal differentiation for SCG10, the novel RB3/RB3' proteins being rather related to the expression of differentiated neuronal functions.

Stathmin is a 19 kDa cytoplasmic phosphoprotein proposed to act as a relay for signals activating diverse intracellular regulatory pathways. After two-thirds partial hepatectomy, the concentration of stathmin reached a peak between 48 and 72 hours, comparable to the levels observed in neonatal liver, at about 10 times the basal adult level. Stathmin then decreased to basal levels within 7 days, more rapidly than during postnatal tissue development (7 weeks), with no detectable change in its phosphorylation state. Interestingly, the mRNA for stathmin reached a peak much earlier than the protein, at 24 hours posthepatectomy, and decreased to a still detectable level until 96 hours after hepatectomy. Altogether, the present results further support the generatility of the implication of stathmin in regulatory pathways of cell proliferation and differentation during normal tissue development and posttraumatic regeneration.

Stathmin is a ubiquitous phosphoprotein proposed to play a general role as an intracellular relay integrating diverse regulatory signals of the cell's environment. We used a rat stathmin probe to isolate two classes of cDNAs coding for the human protein and corresponding to the usage of different polyadenylation sites. Compared to the rat sequences, they displayed a very high conservation both at the nucleic acid and the deduced protein sequence levels, with a single conservative amino acid difference. Further analysis of the protein sequence revealed novel putative phosphorylation sites, as well as internal repeated sequences which might reflect structural features involved in the molecular mechanisms by which stathmin fulfills its biological functions. The extreme conservation of the entire stathmin sequence further stresses the essential and general role of stathmin in cell regulations.