NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss.

Abstract

We have cloned the ubiquitous form of an octamer-binding, 60-kDa protein (NonO) that appears to be the mammalian equivalent of the Drosophila visual and courtship song behavior protein, no-on-transient A/dissonance (nonAdiss). A region unprecedently rich in aromatic amino acids containing two ribonuclear protein binding motifs is highly conserved between the two proteins. A ubiquitous form of NonO is present in all adult tissues, whereas lymphocytes and retina express unique forms of NonO mRNA. The ubiquitous form contains a potential helix-turn-helix motif followed by a highly charged region but differs from prototypic octamer-binding factors by lacking the POU DNA-binding domain. In addition to its conventional octamer duplex-binding, NonO binds single-stranded DNA and RNA at a site independent of the duplex site.

Fusion of splicing factor genes PSF and NonO (p54nrb) to the TFE3 gene in papillary renal cell carcinoma

“…For both 5' and 3'-RACE, sequences were obtained that diverged from known TFE3 sequences at the exon 3-exon 4 junction (eg, the 5'-RACE product is shown in Figure 2b). Database searches of the new sequences present in the 5'-RACE and 3'-RACE products revealed that they exactly matched adjacent regions of a gene designated NonO (p54 nrb ) that is closely related to PSF (Dong et al, 1993;Yang et al, 1993). The 471 amino acid NonO (p54 nrb ) protein contains several distinct domains ( Figure 3) including (i) a short N-terminal sequence composed entirely of histidine, glutamine and proline residues, (ii) a helix ± turn ± helix domain¯anked by charged amino acids that is responsible for binding to the octamer sequence in double stranded DNA and (iii) a short C-terminal proline-rich region.…”

“…The 471 amino acid NonO (p54 nrb ) protein contains several distinct domains ( Figure 3) including (i) a short N-terminal sequence composed entirely of histidine, glutamine and proline residues, (ii) a helix ± turn ± helix domain¯anked by charged amino acids that is responsible for binding to the octamer sequence in double stranded DNA and (iii) a short C-terminal proline-rich region. The NonO (p54 nrb ) protein has a region of 320 amino acids with 71% identity and 7% similarity to a 320aa region of PSF (Patton et al, 1993;Dong et al, 1993;Yang et al, 1993). This same region of NonO (p54 nrb ) exhibits 42% identity and 7% similarity to a 321aa region of the 700aa Drosophila NonA diss protein.…”

PSF and p54nrb bind a conserved stem in U5 snRNA

“…To further define the functional role of PSF, yeast twohybrid protein-protein interaction screens (Fields & Song, 1989) were performed to identify factors that interact with PSF+ A cDNA clone encoding PSF was fused to the LexA DNA-binding domain and used to screen a mouse 7-day embryo cDNA two-hybrid library fused to the VP16 transactivation domain+ Sixty-three positive clones were sequenced and 29 were found to encode the mouse protein NonO (Yang et al+, 1993)+ NonO is the mouse homolog of human p54 nrb (also referred to as nmt 55; Dong et al+, 1993;Traish et al+, 1997), which is very similar in sequence to PSF (Fig+ 1A)+ For the rest of the clones, only the mouse RING Finger protein 4 (RNF4, Accession No+ AF169300) and an as yet unidentified protein were identified five and six times, respectively, whereas none of the remaining clones were detected more than twice+ Interestingly, separate two-hybrid screens have shown that zinc finger proteins, such as RNF4, can interact with the second RRM in PSF and may be involved in nuclear localization of PSF (Dye & Patton, 2001)+…”

“…The interaction between PSF and NonO/p54 nrb is especially interesting because the two proteins are so similar+ Originally identified as a protein that crossreacts with an antibody raised against the yeast U5 snRNP-associated second-step splicing factor Prp18, p54 nrb is 71% identical to PSF over a region of 320 amino acids that encompasses their RRMs (Fig+ 1A;Dong et al+, 1993)+ Other homologs of PSF and p54 nrb include NonA/BJ6 from Drosophila, which has been shown to be important in Drosophila visual acuity and male courtship song (Besser et al+, 1990;Jones & Rubin, 1990), hrp65 from Chironomus tentans, a component of nuclear fibers associated with specific pre-mRNPs (Wurtz et al+, 1996;Miralles et al+, 2000), and PSP1 from humans, a paraspeckle protein of unknown function (Andersen et al+, 2002;Fox et al+, 2002)+ To verify that p54 nrb interacts with PSF and to identify the regions of p54 nrb that are required for binding, coimmunoprecipitation experiments and in vitro interaction assays were performed+ As shown in Figure 1B, polyclonal anti-PSF antibodies were capable of coimmunoprecipitating p54 nrb from HeLa nuclear extract, whereas p54 nrb was not precipitated in control reactions using nonimmune serum or protein G beads alone+ The converse experiment using antibodies against p54 nrb also resulted in coimmunoprecipitation (data not shown)+ To identify which sequences of p54 nrb are re-quired for binding PSF, a series of glutathione-Stransferase-p54 nrb fusion proteins (GST-p54 nrb ) were assayed for their ability to precipitate PSF using glutathione-agarose pull-down assays (Fig+ 1C)+ Fulllength GST-p54 nrb and two deletion mutants (GSTp54 nrb ⌬17-220 and GST-p54 nrb ⌬71-220), both of which contain the putative helix-turn-helix motif and basic/ acidic region (Yang et al+, 1993), were capable of binding PSF+ In contrast, GST fusions that lack either or both of these regions (GST-p54 nrb ⌬226-464, GSTp54 nrb ⌬71-464, and GST-p54 nrb ⌬17-369) could not precipitate PSF+ Thus, it appears that the C-terminus of p54 nrb is required for its interaction with PSF+ These results were corroborated by yeast two-hybrid assays, which showed that both p54 nrb ⌬17-220 and p54 nrb ⌬71-220 interacted with PSF in the two-hybrid system, whereas p54 nrb ⌬226-464, p54 nrb ⌬71-464, and p54 nrb ⌬17-369, did not (data not shown)+ When the same mapping experiments were performed with a series of PSF deletion constructs, only full-length PSF was capable of interacting with p54 nrb (data not shown)+ It appears that multiple contacts, or a precise tertiary structure, are needed for PSF to interact with p54 nrb + Determination of the optimal RNA-binding sites of PSF and p54 nrb Iterative selection assays (Tuerk & Gold, 1990;Szostak, 1992) were used to determine the optimal RNA binding sequence for PSF and p54 nrb + A pool of in vitrotranscribed RNAs representing over 10 12 different sequences was incubated with recombinant, hexahistidine-tagged (his-tagged) proteins, and bound RNAs were recovered by copurification over Ni-NTA agarose+ Sequencing of 20 independent clones from the initial pool showed that the randomized region contained roughly equal amounts of each nucleotide (data not shown)+ Prior to incubation wi...…”

The Splicing Factor PSF is Part of a Large Complex That Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs

“…14,[16][17][18][19][20][21][22][23] It is comprised of an N-terminal glycine-rich domain, a proline/glutamine (P/Q) rich domain, two RNA recognition motifs (RRMs), and a C-terminal region with two nuclear localization signals. A closely related protein with a similar domain structure, p54 nrb (and its mouse homolog nonO) 24,25 has been found to associate with PSF. 23,[26][27][28] Together, these proteins interact with the C-terminal domain (CTD) of RNA Pol II 29 and contact 5' splice sites within large transcription/splicing complexes, 30 consistent with coupling between transcription and splicing.…”