title: The transcription-splicing protein NonO/p54nrb and three NonO-interacting proteins bind to distal enhancer region and augment rhodopsin expression.
abstract: Phototransduction machinery in vertebrate photoreceptors is contained within the membrane discs of outer segments. Daily renewal of 10% of photoreceptor outer segments requires stringent control of gene expression. Rhodopsin constitutes over 90% of the protein in rod discs, and its altered expression or transport is associated with photoreceptor dysfunction and/or death. Two cis-regulatory sequences have been identified upstream of the rhodopsin transcription start site. While the proximal promoter binds to specific transcription factors, including NRL and CRX, the rhodopsin enhancer region (RER) reportedly contributes to precise and high-level expression of rhodopsin in vivo. Here, we report the identification of RER-bound proteins by mass spectrometry. We validate the binding of NonO (p54(nrb)), a protein implicated in coupling transcription to splicing, and three NonO-interacting proteins-hnRNP M, Ywhaz and Ppp1ca. NonO and its interactors can activate rhodopsin promoter in HEK293 cells and function synergistically with NRL and CRX. DNA-binding domain of NonO is critical for rhodopsin promoter activation. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analysis demonstrates high occupancy of NonO at rhodopsin and a subset of phototransduction genes. Furthermore, shRNA knockdown of NonO in mouse retina leads to loss of rhodopsin expression and rod cell death, which can be partially rescued by a C-terminal NonO construct. RNA-seq analysis of the NonO shRNA-treated retina revealed splicing defects and altered expression of genes, specifically those associated with phototransduction. Our studies identify an important contribution of NonO and its interacting modulator proteins in enhancing rod-specific gene expression and controlling rod homeostasis.
authors.affiliation: Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Sharda P
authors.initials: SP
authors.lastname: Yadav
authors.forename: Hong
authors.initials: H
authors.lastname: Hao
authors.forename: Hyun-Jin
authors.initials: HJ
authors.lastname: Yang
authors.forename: Marie-Audrey I
authors.initials: MA
authors.lastname: Kautzmann
authors.forename: Matthew
authors.initials: M
authors.lastname: Brooks
authors.forename: Jacob
authors.initials: J
authors.lastname: Nellissery
authors.forename: Bernward
authors.initials: B
authors.lastname: Klocke
authors.forename: Martin
authors.initials: M
authors.lastname: Seifert
authors.forename: Anand
authors.initials: A
authors.lastname: Swaroop
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M / Q000235:genetics / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012160:Retina / Q000166:cytology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D048948:14-3-3 Proteins; D015415:Biomarkers; D004268:DNA-Binding Proteins; D034761:Heterogeneous-Nuclear Ribonucleoprotein Group M; D034521:Nuclear Matrix-Associated Proteins; D054645:Protein Phosphatase 1; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012243:Rhodopsin; C000590975:Ywhaz protein, mouse; C500331:Nono protein, mouse; C527795:PPP1CA protein, mouse