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The RNA-binding protein [p54.sup.nrb] is involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, [p54.sup.nrb] localizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, [p54.sup.nrb] becomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that [p54.sup.nrb] phosphorylation does not affect the interactions with its protein partners but rather diminishes its general RNA-binding ability. Biochemical assays indicate that in vitro phosphorylation of a GST-[p54.sup.nrb] construct by CDK1 abolishes the interaction with 5' splice site RNA sequence. Site-directed mutagenesis shows that the threonine 15 residue, located N-terminal to the RRM tandem domains of [p54.sup.nrb], is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with [p54.sup.nrb] in either interphase or mitotic cells, suggesting that [p54.sup.nrb]-Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-[p54.sup.nrb] still interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with [p54.sup.nrb]. In vitro RNA binding assays show that CDK1-phosphorylation of a GST-[p54.sup.nrb] construct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that [p54.sup.nrb] interaction with RNA could be selectively modulated by phosphorylation during mitosis. Key words: [p54.sup.nrb], NonO, RNA-binding proteins, mitosis, paraspeckles, Neat1. La proteine de liaison a l'ARN [p54.sup.nrb] est impliquee dans divers processus nucleaires comme la transcription, la maturation des ARNs et la retention des ARNs hyperedites. En interphase, [p54.sup.nrb] est presente dans le nucleoplasme et est aussi concentree avec ses partenaires proteiques dans les paraspeckles via une interaction avec l'ARN non codant Neat1. En mitose, [p54.sup.nrb] devient multiphosphorylee et les effets de cette modification sont inconnus. Dans la presente etude, nous montrons que la phosphorylation de [p54.sup.nrb] n'affecte pas les interactions avec ses partenaires proteiques mais diminue sa capacite de lier l'ARN. Des tests biochimiques indiquent que la phosphorylation in vitro d' une construction GST-[p54.sup.nrb] par CDK1 abolit son interaction avec des sequences d'ARN du site 5' d'epissage. Par mutagenese dirigee, il est montre que cette inhibition depend de la phosphorylation de la threonine 15 situee dans la partie N-terminale en amont des deux domaines RRM de liaison a l'ARN. Des analyses in vivo revelent que l'ARN non codant Neat1 est co-immunoprecipite avec [p54.sup.nrb] aussi bien dans les conditions interphasiques que mitotiques, suggerant que l'interaction entre [p54.sup.nrb] et Neat1 n'est pas affectee par la phosphorylation mitotique. De fait, GST-[p54.sup.nrb] phosphorylee in vitro se lie a l'ARN PIR-1, une sequence riche en G de Neat1 connue pour interagir avec [p54.sup.nrb]. Des analyses de liaison a l'ARN in vitro montre que la phosphorylation de GST-[p54.sup.nrb] par CDK1 affecte son interaction avec des homoribopolymeres poly(A), poly(C) et poly(U) mais pas les poly (G). Ces resultats suggerent que l'interaction de [p54.sup.nrb] avec l'ARN peut etre modulee selectivement par phosphorylation durant la mitose., Introduction The protein [p54.sup.nrb] (NonO in mouse) is a highly conserved nuclear matrix-associated factor involved in numerous nuclear processes (Shav-Tal and Zipori 2002). This multifunctional protein, characterized by the presence [...]

The NO-cGMP pathway plays a key role in the male and female genital sexual arousal response. Nitric oxide synthase (NQS) utilizes L-arginine and oxygen as substrates to produce nitric oxide (NO) and citrulline. Arginase is a metalloenzyme that catalyzes the hydrolysis of L-arginine to produce L-ornithine and urea. It is proposed that arginase competes for L-arginine and reduces NOS activity in genital tissues, thus modulating sexual function. Using 2 transition state analogue inhibitors of arginase, 2(S)-Amino-6-boronohexanoic acid (ABH) and S-(2-boronoethyl)-L-cysteine (BEC), we have characterized arginase activity in penile and vaginal tissue. Neither of these inhibitors has activity against NOS. Thus, ABH and BEC are useful compounds for examining the role of arginase in genital tissue physiology, without directly influencing NOS activity. We present data to suggest that arginase may regulate NO production by competing for endogenous pools of L-arginine. In this fashion, arginase is an indirect regulator of penile and vaginal blood flow and specific arginase inhibitors may improve genital blood flow during sexual arousal. As evidenced by the upregulation of arginase in specific disease states, its distribution in the vagina, and its modulation by sex steroid hormones, this enzyme may also participate in numerous other physiological and pathophysiological processes, such as tissue growth, fibrosis, and immune function. KEY WORDS: * arginase * arginine * nitric oxide synthase * erectile function * genital hemodynamics