title: NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins.
abstract: NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS-NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing.
authors.affiliation: Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine Cologne (CMMC), 50931 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany, Institute of Genetics, University of Cologne, 50931 Cologne, Germany and Leibniz-Institute of Freshwater Ecology and Inland Fisheries, IGB, Müggelseedamm 301, 12587 Berlin, Germany.
authors.forename: Bhagyashri D
authors.initials: BD
authors.lastname: Burgute
authors.forename: Vivek S
authors.initials: VS
authors.lastname: Peche
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Steckelberg
authors.forename: Gernot
authors.initials: G
authors.lastname: Glöckner
authors.forename: Berthold
authors.initials: B
authors.lastname: Gaßen
authors.forename: Niels H
authors.initials: NH
authors.lastname: Gehring
authors.forename: Angelika A
authors.initials: AA
authors.lastname: Noegel
mesh_terms: D000818:Animals; D002467:Cell Nucleus; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000032:analysis; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism; D034702:RNA-Binding Protein FUS / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: C560431:NKAP protein, mouse; D009687:Nuclear Proteins; D012342:RNA, Small Nuclear; D034702:RNA-Binding Protein FUS; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D012313:RNA; D020365:RNA Helicases