title: Inhibition of pre-mRNA splicing by a synthetic Blom7α-interacting small RNA.
abstract: Originally the novel protein Blom7α was identified as novel pre-mRNA splicing factor that interacts with SNEV(Prp19/Pso4), an essential protein involved in extension of human endothelial cell life span, DNA damage repair, the ubiquitin-proteasome system, and pre-mRNA splicing. Blom7α belongs to the heteronuclear ribonucleoprotein K homology (KH) protein family, displaying 2 KH domains, a well conserved and widespread RNA-binding motif. In order to identify specific sequence binding motifs, we here used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with a synthetic RNA library. Besides sequence motifs like (U/A)(1-4) C(2-6) (U/A)(1-5), we identified an AC-rich RNA-aptamer that we termed AK48 (Aptamer KH-binding 48), binding to Blom7α with high affinity. Addition of AK48 to pre-mRNA splicing reactions in vitro inhibited the formation of mature spliced mRNA and led to a slight accumulation of the H complex of the spliceosome. These results suggest that the RNA binding activity of Blom7α might be required for pre-mRNA splicing catalysis. The inhibition of in-vitro splicing by the small RNA AK48 indicates the potential use of small RNA molecules in targeting the spliceosome complex as a novel target for drug development.
authors.affiliation: Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Marlies
authors.initials: M
authors.lastname: Löscher
authors.forename: Markus
authors.initials: M
authors.lastname: Schosserer
authors.forename: Eric
authors.initials: E
authors.lastname: Dausse
authors.forename: Kiseok
authors.initials: K
authors.lastname: Lee
authors.forename: Paul
authors.initials: P
authors.lastname: Ajuh
authors.forename: Regina
authors.initials: R
authors.lastname: Grillari-Voglauer
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Toulmé
authors.forename: Johannes
authors.initials: J
authors.lastname: Grillari
mesh_terms: D020856:AT Rich Sequence / Q000235:genetics; D000595:Amino Acid Sequence; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000235:genetics / Q000378:metabolism*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D052156:SELEX Aptamer Technique / Q000379:methods; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C000590914:KHDC4 protein, human; D012322:RNA Precursors