A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2–RNA complex

AbstractDysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates in cells and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges an active conformation of the U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing inactive checkpoints may offer a breakthrough approach for small molecule inhibition of multi-stage macromolecular assemblies.

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Phylogeny of the sex-determining gene Sex-lethal in insects

Genome ◽

10.1139/g05-107 ◽

2006 ◽

Vol 49 (3) ◽

pp. 254-262 ◽

Cited By ~ 34

Author(s):

Walther Traut ◽

Teruyuki Niimi ◽

Kazuho Ikeo ◽

Ken Sahara

Keyword(s):

Drosophila Melanogaster ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Splicing ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

The Family ◽

Mosquito Anopheles Gambiae ◽

Sex Lethal ◽

Recognition Motifs

The Sex-lethal (SXL) protein belongs to the family of RNA-binding proteins and is involved in the regulation of pre-mRNA splicing. SXL has undergone an obvious change of function during the evolution of the insect clade. The gene has acquired a pivotal role in the sex-determining pathway of Drosophila, although it does not act as a sex determiner in non-drosophilids. We collected SXL sequences of insect species ranging from the pea aphid (Acyrtho siphom pisum) to Drosophila melanogaster by searching published articles, sequencing cDNAs, and exploiting homology searches in public EST and whole-genome databases. The SXL protein has moderately conserved N- and C-terminal regions and a well-conserved central region including 2 RNA recognition motifs. Our phylogenetic analysis shows that a single orthologue of the Drosophila Sex-lethal (Sxl) gene is present in the genomes of the malaria mosquito Anopheles gambiae, the honeybee Apis mellifera, the silkworm Bombyx mori, and the red flour beetle Tribolium castaneum. The D. melanogaster, D. erecta, and D. pseudoobscura genomes, however, contain 2 paralogous genes, Sxl and CG3056, which are orthologous to the Anopheles, Apis, Bombyx, and Tribolium Sxl. Hence, a duplication in the fly clade generated Sxl and CG3056. Our hypothesis maintains that one of the genes, Sxl, adopted the new function of sex determiner in Drosophila, whereas the other, CG3056, continued to serve some or all of the yet-unknown ancestral functions.Key words: sex determination, Sxl, CG3056, Bombyx, Apis, Anopheles, Aedes, Acyrtosiphon, Megaselia, Lucilia, Musca, Drosophila, Tribolium, Sciarids, Drosophila melanogaster, Drosophila erecta, Drosophila pseudoobscura, gene duplication.

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Pre-mRNA Splicing Factor U2AF2 Recognizes Distinct Conformations of Nucleotide Variants at the Center of the pre-mRNA Splice Site Signal

10.1101/2021.09.27.462007 ◽

2021 ◽

Author(s):

Eliezra Glasser ◽

Debanjana Maji ◽

Guilia Biancon ◽

Anees Mohammed Keedakkatt Puthenpeedikakkal ◽

Chapin Cavender ◽

...

Keyword(s):

Splice Site ◽

Rna Binding ◽

Mrna Splicing ◽

Splicing Factor ◽

Central Position ◽

Nucleotide Substitutions ◽

Rna Recognition Motifs ◽

Recognition Motifs ◽

Polypeptide Backbone ◽

Dynamics Simulations

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

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Faculty Opinions recommendation of The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1025155.298750 ◽

2005 ◽

Author(s):

Reinhard Lührmann

Keyword(s):

Rna Binding ◽

Splicing Factor ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Recognition Motifs ◽

U6 Rna

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An RNA-binding protein gene (RBP1) of Saccharomyces cerevisiae encodes a putative glucose-repressible protein containing two RNA recognition motifs

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)82440-1 ◽

1993 ◽

Vol 268 (20) ◽

pp. 15080-15087

Author(s):

F.J. Lee ◽

J. Moss

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Binding ◽

Protein Gene ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Binding Protein Gene ◽

Recognition Motifs

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Sla1, a Schizosaccharomyces pombe Homolog of the Human La Protein, Induces Ectopic Meiosis when Its C Terminus Is Truncated

Eukaryotic Cell ◽

10.1128/ec.2.6.1274-1287.2003 ◽

2003 ◽

Vol 2 (6) ◽

pp. 1274-1287 ◽

Cited By ~ 9

Author(s):

Kaori Tanabe ◽

Noriko Ito ◽

Tomomi Wakuri ◽

Fumiyo Ozoe ◽

Makoto Umeda ◽

...

Keyword(s):

Schizosaccharomyces Pombe ◽

Rna Binding ◽

Rna Binding Proteins ◽

Small Region ◽

Full Length ◽

Rna Recognition Motifs ◽

La Protein ◽

C Terminus ◽

Localization Signal ◽

Recognition Motifs

ABSTRACT Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1ΔC) induced ectopic sporulation in the ras1Δ strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1ΔC is found throughout the cell, suggesting the cytoplasmic localization of Sla1ΔC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1ΔC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1ΔC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1ΔC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1ΔC but not full-length Sla1. Thus, Sla1ΔC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.

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Specificity of RNA Binding by CPEB: Requirement for RNA Recognition Motifs and a Novel Zinc Finger

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.685 ◽

1998 ◽

Vol 18 (2) ◽

pp. 685-693 ◽

Cited By ~ 108

Author(s):

Laura E. Hake ◽

Raul Mendez ◽

Joel D. Richter

Keyword(s):

Zinc Finger ◽

Rna Binding ◽

Rna Recognition ◽

Cytoplasmic Polyadenylation ◽

Functional Motif ◽

Rna Recognition Motifs ◽

Amino Terminal ◽

Terminal Amino ◽

Maternal Mrnas ◽

Recognition Motifs

ABSTRACT CPEB is an RNA binding protein that interacts with the maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif, two RNA recognition motifs (RRMs), and a cysteine-histidine region that is reminiscent of a zinc finger. In this study, we investigated the physical properties of CPEB required for RNA binding. CPEB can interact with RNA as a monomer, and phosphorylation, which modifies the protein during oocyte maturation, has little effect on RNA binding. Deletion mutations of CPEB have been overexpressed inEscherichia coli and used in a series of RNA gel shift experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids bind CPE-containing RNA avidly, proteins that have had either RRM deleted bind RNA much less efficiently. CPEB that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA binding, pointing to the importance of this highly conserved domain of the protein. Chelation of metal ions by 1,10-phenanthroline inhibits the ability of CPEB to bind RNA; however, RNA binding is restored if the reaction is supplemented with zinc. CPEB also binds other metals such as cobalt and cadmium, but these destroy RNA binding. These data indicate that the RRMs and a zinc finger region of CPEB are essential for RNA binding.

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Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1

Gene ◽

10.1016/s0378-1119(96)00673-7 ◽

1997 ◽

Vol 186 (1) ◽

pp. 21-27 ◽

Cited By ~ 14

Author(s):

Yasuyuki Kurihara ◽

Takashi Nagata ◽

Takao Imai ◽

Ado Hiwatashi ◽

Masataka Horiuchi ◽

...

Keyword(s):

Structural Properties ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Recognition Motifs

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Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV

Journal of Cell Science ◽

10.1242/jcs.112.24.4501 ◽

1999 ◽

Vol 112 (24) ◽

pp. 4501-4512 ◽

Cited By ~ 1

Author(s):

Y.M. Yannoni ◽

K. White

Keyword(s):

Nuclear Localization ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Localization Sequence ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Mutant Proteins ◽

Localization Sequence ◽

Recognition Motifs

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333–374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

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