Publications by authors named "Alexandre Maucuer"

14 Publications

  • Page 1 of 1

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain.

Commun Biol 2021 Mar 19;4(1):359. Epub 2021 Mar 19.

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025, Evry, France.

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved β-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.
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http://dx.doi.org/10.1038/s42003-021-01862-3DOI Listing
March 2021

U2AF assemblies drive sequence-specific splice site recognition.

EMBO Rep 2019 08 4;20(8):e47604. Epub 2019 Jul 4.

SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay, Evry, France.

The essential splicing factor U2AF 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 U2AF and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF 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 U2AF 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 U2AF and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.
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http://dx.doi.org/10.15252/embr.201847604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681011PMC
August 2019

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

Nucleic Acids Res 2019 04;47(6):3127-3141

SABNP, University of Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.
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http://dx.doi.org/10.1093/nar/gky1303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451097PMC
April 2019

Microtubules as platforms for probing liquid-liquid phase separation in cells - application to RNA-binding proteins.

J Cell Sci 2018 06 11;131(11). Epub 2018 Jun 11.

SABNP Lab, Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France

Liquid-liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid-liquid phase separation and their deregulation in human diseases.
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http://dx.doi.org/10.1242/jcs.214692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031325PMC
June 2018

SF1 Phosphorylation Enhances Specific Binding to U2AF and Reduces Binding to 3'-Splice-Site RNA.

Biophys J 2016 Dec;111(12):2570-2586

Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York. Electronic address:

Splicing factor 1 (SF1) recognizes 3' splice sites of the major class of introns as a ternary complex with U2AF and U2AF 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 U2AF 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-U2AF 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-U2AF 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 U2AF 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.
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http://dx.doi.org/10.1016/j.bpj.2016.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192697PMC
December 2016

Cancer-relevant splicing factor CAPERα engages the essential splicing factor SF3b155 in a specific ternary complex.

J Biol Chem 2014 Jun 2;289(25):17325-37. Epub 2014 May 2.

From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 and

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.
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http://dx.doi.org/10.1074/jbc.M114.558825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067167PMC
June 2014

The CATS (FAM64A) protein is a substrate of the Kinase Interacting Stathmin (KIS).

Biochim Biophys Acta 2013 May 16;1833(5):1269-79. Epub 2013 Feb 16.

Hematology and Hemotherapy Center, State University of Campinas, Campinas, Brazil.

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-immunoprecipitation 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.
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http://dx.doi.org/10.1016/j.bbamcr.2013.02.004DOI Listing
May 2013

Structure of phosphorylated SF1 bound to U2AF⁶⁵ in an essential splicing factor complex.

Structure 2013 Feb 27;21(2):197-208. Epub 2012 Dec 27.

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

The 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.
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http://dx.doi.org/10.1016/j.str.2012.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570649PMC
February 2013

The protein kinase KIS impacts gene expression during development and fear conditioning in adult mice.

PLoS One 2012 24;7(8):e43946. Epub 2012 Aug 24.

INSERM, UMR-S 839, Paris, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043946PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427225PMC
February 2013

Genetic and molecular exploration of UHMK1 in schizophrenic patients.

Psychiatr Genet 2011 Dec;21(6):315-8

Psychiatrie Génétique, Hôpital Henri Mondor, France.

In two recent papers, polymorphisms located in U2AF homology motif kinase 1 (UHMK1) gene have been associated to schizophrenia. This gene encodes the serine/threonine kinase, kinase interacting with Stathmin, and has been functionally related to RNA metabolism and neurite outgrowth. In this study, we explored the contribution of this gene in schizophrenia susceptibility, using a case-control association study, a mutation screening, a transcription level analysis, and by the investigation of the phosphorylation status of the splicing factor, SF1, in B-lymphoblastoid cell lines of patients and controls. No association was observed in our French cohort, and no amino acid substitution was predicted in the subsample studied for mutation screening. No difference was observed in expression level or in SF1 phosphorylation between patients and controls. Despite a slight difference persisting in the meta-analysis carried out using four European populations, these data suggest, altogether, that UHMK1 does not play a major role in susceptibility to schizophrenia.
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http://dx.doi.org/10.1097/YPG.0b013e3283458a37DOI Listing
December 2011

Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to splicing factors.

J Mol Biol 2008 Sep 17;381(3):748-62. Epub 2008 Jun 17.

Institut National de la Santé et de la Recherche Médicale, UMR839, 17, rue du Fer à Moulin, F-75005 Paris, France.

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, U2AF(65). KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF(65) 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 U2AF(65), 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 U2AF(65) 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.
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http://dx.doi.org/10.1016/j.jmb.2008.06.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632974PMC
September 2008

Major phosphorylation of SF1 on adjacent Ser-Pro motifs enhances interaction with U2AF65.

FEBS J 2006 Feb;273(3):577-87

INSERM, U706, UPMC, Institut du Fer à Moulin, Paris, France.

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.
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http://dx.doi.org/10.1111/j.1742-4658.2005.05091.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949809PMC
February 2006

Quantitative RT-PCR reveals a ubiquitous but preferentially neural expression of the KIS gene in rat and human.

Brain Res Mol Brain Res 2003 May;114(1):55-64

Laboratoire de Génétique Moléculaire-UPRES EA 3618, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes-Paris V, 75006 Paris, France.

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.
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http://dx.doi.org/10.1016/s0169-328x(03)00132-3DOI Listing
May 2003

Expression of stathmin family genes in human tissues: non-neural-restricted expression for SCLIP.

Genomics 2003 Apr;81(4):400-10

Laboratoire de Génétique Moléculaire, UPRES JE 2195, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes-Paris V, Paris, France.

The stathmin family consists of phosphoproteins highly conserved in vertebrates and thought to be implicated in the development and functional regulation of various organs, most notably the nervous system. This family includes stathmin, SCG10, SCLIP, and RB3, phosphoproteins that are related by structural and functional homologies. They all sequester tubulin and interfere with microtubule dynamics, a property due to their shared stathmin-like domain. Little is known about the expression of the stathmin gene family in humans. Herein, we describe for the first time, for a collection of human tissues, the expression of each member of this family, using real-time quantitative RT-PCR. We found that stathmin is ubiquitously expressed, whereas SCG10 and RB3 are neural enriched, expression patterns similar to those reported for other mammals. Surprisingly, SCLIP, whose expression is thought to be neural-specific, exhibits a broader tissue distribution. Analyses of the SCLIP gene (approved symbol STMN3) show that it contains several NRSE-like elements that display low or no affinity for the cognate binding protein NRSF. The substantial expression of SCLIP in most tissues points out a novel function for this protein outside the nervous system and raises the possibility that its coexpression with stathmin could provide some degree of functional redundancy.
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http://dx.doi.org/10.1016/s0888-7543(03)00031-4DOI Listing
April 2003