p54^nrb/NONO Regulates Cyclic AMP-Dependent Glucocorticoid Production by Modulating Phosphodiesterase mRNA Splicing and Degradation

Abstract

Glucocorticoid production in the adrenal cortex is activated in response to an increase in cyclic AMP (cAMP) signaling. The nuclear protein p54 nrb /NONO belongs to the Drosophila behavior/human splicing (DBHS) family and has been implicated in several nuclear processes, including transcription, splicing, and RNA export. We previously identified p54 nrb /NONO as a component of a protein complex that regulates the transcription of CYP17A1, a gene required for glucocorticoid production. Based on the multiple mechanisms by which p54 nrb /NONO has been shown to control gene expression and the ability of the protein to be recruited to the CYP17A1 promoter, we sought to further define the molecular mechanism by which p54 nrb /NONO confers optimal cortisol production. We show here that silencing p54 nrb /NONO expression in H295R human adrenocortical cells decreases the ability of the cells to increase intracellular cAMP production and subsequent cortisol biosynthesis in response to adrenocorticotropin hormone (ACTH) stimulation. Interestingly, the expression of multiple phosphodiesterase (PDE) isoforms, including PDE2A, PDE3A, PDE3B, PDE4A, PDE4D, and PDE11A, was induced in p54 nrb /NONO knockdown cells. Investigation of the mechanism by which silencing of p54 nrb /NONO led to increased expression of select PDE isoforms revealed that p54 nrb /NONO regulates the splicing of a subset of PDE isoforms. Importantly, we also identify a role for p54 nrb /NONO in regulating the stability of PDE transcripts by facilitating the interaction between the exoribonuclease XRN2 and select PDE transcripts. In summary, we report that p54 nrb /NONO modulates cAMP-dependent signaling, and ultimately cAMP-stimulated glucocorticoid biosynthesis by regulating the splicing and degradation of PDE transcripts. T he non-POU domain-containing octamer-binding protein p54nrb /NONO is a member of the Drosophila behavior/human splicing (DBHS) family of RNA-binding proteins and is comprised of two tandem RNA recognition motif (RRM) domains followed by a charged protein-protein interaction module (1-3). DBHS proteins have been described as multifunctional nuclear proteins, with p54 nrb /NONO being implicated in numerous nuclear processes, including transcription regulation, mRNA splicing, nuclear retention, and subnuclear body formation (4-7). In addition to binding to DNA elements such as intracisternal A particles (8), p54 nrb /NONO is also known to promote the binding of other transcription factors to their response elements (9). The polypyrimidine tract-binding protein-associated splicing factor (PSF)/p54 nrb /NONO heterodimer regulates the transcriptional activity of several nuclear receptors (NRs), including the thyroid hormone receptor (10) and the androgen receptor (11). This heterodimeric complex also promotes nuclear retention of hyperedited RNAs by binding to the inner nuclear matrix structural protein matrin 3 (12, 13) and stimulates topoisomerase I activity (14). A heterodimer of p54 nrb /NONO and paraspeckle protein 1 has been shown to be...

Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb

“…B‐C). These findings were consistent to the previous report that knockdown of p54nrb resulted in an increased expression of not only the splice variant PDE4D5 transcripts, but also total PDE4D gene transcripts (Lu and Sewer, ). These results confirm the importance of the Tyr residues on p54nrb function in transcription and RNA splicing of the PED4D gene.…”

The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold

“…Specifically, SFPQ has been identified as a regulator of splicing for CD45 ( 90 ), neuronal cell-specific genes ( 91 ), the preprotachykini ( PPT ) minigene ( 22 ), the microtubule-binding protein Tau ( 45 ) and spinal muscular atrophy genes SMN1 / SMN2 ( 92 ). Similarly, NONO is cited in rod-specific gene expression ( 71 ), phosphodiesterase splicing ( 93 ) and together SFPQ and NONO bind to specific A-U rich elements in pre-mRNA such as TNF-α ( 94 ). SFPQ/NONO also facilitates pre-mRNA 3′-end processing by promoting polyadenylation and pre-mRNA cleavage ( 84 , 95 , 96 ).…”

A crystallographic study of human NONO (p54nrb): overcoming pathological problems with purification, data collection and noncrystallographic symmetry

“…DBHS proteins are well established as obligatory ISSN 2059-7983 # 2016 International Union of Crystallography dimers, and X-ray crystallography has illustrated the intimacy of dimerization with the structures of the PSPC1-NONO heterodimer, SFPQ homodimer and NONO-1 homodimer (Passon et al, 2011(Passon et al, , 2012Lee et al, 2015;Knott et al, 2015). NONO can be found as a heterodimer with SFPQ or PSPC1 (Straub et al, 1998;Fox et al, 2005;Passon et al, 2012) and has been reported to function in transcriptional co-activation and co-repression (Ishitani et al, 2003;Dong et al, 2005;Yadav et al, 2014), post-transcriptional processing (Peng et al, 2002;Kameoka et al, 2004;Liang & Lutz, 2006;Hall-Pogar et al, 2007;Kaneko et al, 2007;Izumi et al, 2014;Yadav et al, 2014;Lu & Sewer, 2015), paraspeckle assembly and function (Fox et al, 2002(Fox et al, , 2005Chen & Carmichael, 2009), DNA repair (Udayakumar & Dynan, 2015) and circadian rhythm (Kowalska et al, 2012(Kowalska et al, , 2013. However, emerging evidence indicates that NONO itself is a key regulator of the cell-cycle G1-S checkpoint (Kowalska et al, 2013), viral infection (Cao et al, 2015) and cancer (Zhu et al, 2016).…”