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.

Pro-oncogenic Roles of HLXB9 Protein in Insulinoma Cells through Interaction with Nono Protein and Down-regulation of the c-Met Inhibitor Cblb (Casitas B-lineage Lymphoma b)

“…Plasmids, shRNA, siRNA, and Antibodies-The following mammalian expression plasmids were used: pcDNA3.1-Myc-His vector (pcDNA3.1-mh) (Invitrogen), mouse-HLXB9 (pcDNA3.1-mh-HB9-WT, and pcDNA3.1-mh-HB9-AA (phospho-dead mutant of HLXB9 with alanine substitution at serine 78 and serine 80)) (11), pcDNA3.1-mh-menin (27), pCMV6-XL4-CBLB (Origene, SC107022), pFLAG-p54 (Nono) (Addgene, plasmid 35379), control and MEN1 shRNA (28), and control and Nono shRNA (29). The following siRNAs were used: negative control (Qiagen, 1027280) and mouse HLXB9 (Dharmacon, L-049859-01).…”

SRSF6-regulated alternative splicing that promotes tumour progression offers a therapy target for colorectal cancer

“…As a new hallmark of cancer, AS has been involved in multiple oncological processes, including angiogenesis,1 invasion and metastasis,2 immune destruction3 and cellular energetics 45.…”

A conserved charged single α-helix with a putative steric role in paraspeckle formation

“…In summary, the most likely explanation for the apparent inconsistencies between observations on DBHS protein assembly is that the extended coiled-coil/CSAH segment of DBHS proteins is highly versatile and can mediate a number of different interactions and multimerization modes in the different proteins in a context-dependent manner, as exemplified by the behavior of NONO/SFPQ tetramers and multimers depending on the ionic strength applied (Snijders et al 2015). Moreover, NONO can bind to specific DNA segments and is involved in transcriptional activation (Park et al 2013;Yadav et al 2014). NONO was shown to bind double-stranded DNA independently of RNA and single-stranded DNA (Yang et al 1993).…”