Abstract

Background

   Neuroblastoma (NB), the most prevalent solid tumor in children, arises
   from sympathetic nervous system and accounts for 15% of pediatric
   cancer mortality. This malignancy exhibits substantial genetic and
   clinical heterogeneity, thus complicating treatment strategies.
   Poly(ADP-ribose) polymerase 1 (PARP1), a key enzyme catalyzing
   polyADP-ribosylation (PARylation), plays critical roles in various
   cellular processes, and contributes to tumorigenesis and
   aggressiveness. However, the functions and regulatory mechanisms of
   PARP1 in NB progression still remain to be determined.

Methods

   The association of PARP1 expression with NB patients’ survival was
   analyzed by mining of R2 database. Western blotting, reverse
   transcription-polymerase chain reaction, MTT colorimetric, soft agar,
   and matrigel invasion assays were utilized to assess PARP1 expression
   and its effects on aggressiveness of NB cell lines. Chromatin
   immunoprecipitation (ChIP) sequencing and ChIP assays were employed to
   investigate the binding of Yin Yang 1 (YY1) to PARP1 promoter. Protein
   interactions were explored by BioGRID database analysis, molecular
   docking, and co-immunoprecipitation assay. RNA sequencing and
   crosslinking-immunoprecipitation high throughput sequencing datasets
   were used to identify precursor mRNA splicing targets of non-POU domain
   containing octamer binding protein (NONO).

Results

   High PARP1 expression was associated with poor survival of NB patients.
   PARP1 over-expression enhanced the proliferation and invasion of NB
   cell lines, confirming its oncogenic roles. YY1 was identified as a key
   transcriptional regulator facilitating PARP1 expression. Additionally,
   PARP1 interacted with NONO to induce its PARylation, resulting in
   stabilization of NONO protein via preventing ubiquitin-mediated
   degradation. NONO facilitated the splicing and mRNA maturation of
   target genes a disintegrin and metalloproteinase domain 8 (ADAM8) and
   testis-expressed gene 14 (TEX14) in a PARylation-dependent manner.
   Rescue experiments indicated that YY1 facilitated PARP1-mediated
   PARylation of NONO and subsequent mRNA maturation of ADAM8 and TEX14 in
   NB cells. In clinical NB cases, high expression of YY1, PARP1, NONO,
   ADAM8, or TEX14 was associated with poor survival of patients.

Conclusions

   These findings indicate that YY1 drives PARP1 expression essential for
   PARylation of NONO in mRNA maturation during NB progression.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12967-024-05956-4.

   Keywords: Neuroblastoma, Poly(ADP-ribose) polymerase 1, Yin Yang 1,
   Non-POU domain containing octamer binding protein, Transcriptional
   regulation, Protein PARylation, MRNA maturation

Introduction

   Neuroblastoma (NB), a malignancy arising from developing sympathetic
   nervous system, is the most common solid tumor in children, accounting
   for approximately 15% of pediatric cancer-related mortality [[46]1,
   [47]2]. This malignancy is characterized by considerable genetic,
   morphological, or clinical heterogeneity, which challenges the
   effectiveness of current therapeutic strategies [[48]3–[49]5]. However,
   comprehension of molecular features and genetic alterations underlying
   its pathogenesis remains incomplete. As an adenosine diphosphate (ADP)
   ribosyltransferase, poly(ADP-ribose) polymerase 1 (PARP1) directly
   transfers multiple ADP-ribose units onto protein substrates in an
   iterative manner, a process known as polyADP-ribosylation (PARylation)
   [[50]6, [51]7]. PARylation plays a critical role in various biological
   processes, such as DNA repair, genome integrity maintenance, gene
   expression, or cell death [[52]8–[53]10]. For instance, PARP1 inhibits
   the transcription of programmed cell death ligand 1 (PD-L1) via
   interaction with nucleophosmin 1 [[54]11]. Emerging evidence indicates
   that PARP1 can influence RNA modification. For instance, PARP1
   facilitates the transcription of methyltransferase 3 (METTL3),
   resulting in N6-methyladenosine modification of target mRNAs [[55]12].
   It has been established that PARP1 is a promising target for cancer
   therapy, while its inhibitors function as chemosensitizers or efficient
   agents for tumors deficient in homologous recombination [[56]13].
   However, current research mainly focuses on the functions of PARP1,
   while regulatory mechanisms governing PARP1 expression in NB remain to
   be determined.

   Yin Yang 1 (YY1), a highly conserved C2H2 zinc finger nuclear
   transcription factor [[57]14], plays a critical role in essential
   biological processes, such as embryogenesis, differentiation,
   replication, or cellular proliferation [[58]15, [59]16]. In recent
   years, emerging studies have shown the oncogenic or suppressive roles
   of YY1 in tumor progression [[60]17]. For instance, YY1 enhances the
   expression of several oncogenic transcription factors, including MYC
   and snail family transcriptional repressor 1 (SNAIL1) [[61]18]. YY1
   promotes phosphoinositide 3 kinase-serine/threonine kinase 1 signaling,
   thereby increasing proliferation and metastasis of bile duct carcinoma
   [[62]19]. In addition, YY1-induced zinc finger protein FOG family
   member 2 antisense RNA 1 (ZFPM2-AS1), a long non-coding RNA (lncRNA),
   enhances cellular proliferation and invasion of small cell lung cancer
   [[63]20]. In contrast, YY1 inhibits the migration and invasion of
   pancreatic ductal adenocarcinoma [[64]21]. However, the functions and
   transcriptional targets of YY1 in NB progression warrant investigation.

   In this study, by employing an integrative screening approach, we
   identify that PARP1 is transcriptionally regulated by YY1, which
   interacts with non-POU domain containing octamer binding protein
   (NONO). As a multifunctional splicing factor, NONO also seems to plays
   critical roles in maintaining genome integrity and regulating gene
   expression at multiple levels, including transcription initiation,
   elongation, termination, precursor mRNA (pre-mRNA) processing and
   splicing, or nuclear RNA retention [[65]22–[66]25]. Previous evidences
   show that NONO expression is associated with poor survival outcomes of
   NB patients [[67]22], and interacts with specific lncRNAs, such as
   LncMycnUS [[68]26], while its functions and acting mechanisms are still
   largely unknown. In this study, we found that PAPR1 was essential for
   PARylation and stabilization of NONO, thereby impacting the pre-mRNA
   splicing and mRNA maturation, indicating the critical roles of
   YY1/PARP1/NONO axis in NB progression.

Materials and methods

Cell culture

   Human NB cell lines SK-N-AS (CRL-2137), IMR32 (CCL-127), SH-SY5Y
   (CRL-2266), SK-N-BE(2) (CRL-2271), cervical cancer HeLa cells (CCL-2),
   gastric cancer AGS cells (CRL-1739), and embryonic kidney HEK293
   (CRL-1573) cells were obtained from American Type Culture Collection
   (Rockville, MD). Cells were authenticated by short tandem repeat
   profiling, and used within 6 months after resuscitation of frozen
   aliquots. Mycoplasma contamination was regularly examined using Lookout
   Mycoplasma PCR Detection Kit (MP0035, Sigma, St. Louis, MO). Cells were
   maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented
   with 10% fetal bovine serum (Gibco, Grand Island, NY) at 37 °C in a
   humidified atmosphere of 5% CO[2], and treated with cycloheximide
   (CHX), MG-132, Z-VAD-FMK, 3-methyladenine (3-MA), Olaparib, or PJ34
   (MedChemExpress, Shanghai, China).

Real-time quantitative reverse transcription-polymerase chain reaction
(RT-qPCR)

   Total RNA was isolated with RNeasy Mini Kit (Qiagen Inc., Valencia,
   CA). Reverse transcription reactions were conducted with Transcriptor
   First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). Real-time
   PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems,
   Foster City, CA) and primers (Supplementary Table S1) [[69]7, [70]22,
   [71]27].

Western blot

   Tissue or cellular protein was extracted using a 1 × cell lysis buffer
   (Promega, Madison, WI). Western blotting was performed as previously
   described [[72]28–[73]31]. The membranes were blocked for 1 h in
   10 mmol/L Tris-buffered saline (TBS) containing 5% skimmed milk powder
   (Beyotime Biotechnology, Shanghai, China), and subsequently incubated
   overnight at 4 °C with primary antibodies specific for YY1 (D3D4Q, Cell
   Signaling Technology, Danvers, MA), maltose binding protein (MBP;
   #2396, Cell Signaling Technology), PARylation (4335-MC-100, Bio-Techne,
   Minneapolis, MN), PARP1 (ab227244), NONO (ab70335), Ubiquitin (ab7254),
   glutathione S-transferase (GST)-tag (ab36415), or β-actin (ab6276,
   Abcam Inc., Cambridge, MA). The membranes were washed three times in
   TBS containing 0.1% Tween 20, and incubated for 1 h at room temperature
   with anti-rabbit (40 ng/mL; Sigma) or anti-mouse (1:10,000; Sigma)
   horseradish peroxidase-conjugated secondary antibody. After several
   washing with TBS-Tween 20, the membranes were treated with an ECL
   chemiluminescence substrate (Keygen BioTECH, Jiangsu, China), while
   protein bands were visualized and quantified by using an imaging system
   (Thermo Fisher Scientific, Inc., Waltham, MA) and ImageJ software
   ([74]https://imagej.nih.gov/ij) [[75]28–[76]31].

Gene over-expression and knockdown

   Human PARP1 cDNA (3045 bp), NONO cDNA (1416 bp), and their
   corresponding truncations were obtained from NB tissues by PCR primers
   (Supplementary Table S2), and inserted into pGEX-6P-1, pMAL-c4X, or
   lentiviral expression vector CV186 (Genechem Co., Ltd, Shanghai,
   China), respectively. For preparing recombinant proteins, GST-tagged
   PARP1 or MBP-tagged NONO construct was transferred into E. coli BL21
   strain (Thermo Fisher Scientific, Inc.) [[77]7, [78]24], while proteins
   were purified by GST-Tag Protein Purification Kit (Beyotime
   Biotechnology) or PurKine^™ MBP-Tag Protein Purification Kit (Abbkine,
   Wuhan, China). Oligonucleotides encoding short hairpin RNAs (shRNAs,
   Supplementary Table S3) specific for YY1, PARP1 and NONO were subcloned
   into GV298 (Genechem Co., Ltd). Stable cell lines were screened by
   administration of puromycin (Invitrogen).

Co-immunoprecipitation (co-IP)

   Co-IP was performed as previously described [[79]27, [80]29, [81]30],
   with antibodies for MBP (#2396, Cell Signaling Technology), GST-tag
   (ab36415), NONO (ab70335), PARP1 (ab227244, Abcam Inc.). For co-IP
   assay, cell lysates were prepared and subsequently incubated with
   specific magnetic beads conjugated with antibodies against target
   proteins. The immunocomplexes were captured by beads, washed
   extensively to remove non-specifically bound proteins, and eluted for
   further analysis. The immunoprecipitated proteins were analyzed by
   western blot assay [[82]27, [83]29, [84]30].

Immunofluorescence assay

   Cells were cultured on coverslips, fixed with 4% paraformaldehyde,
   blocked with 5% milk for 1 h, and incubated overnight at 4 °C with
   antibodies specific for PARP1 (ab227244; 1:100 dilution) or NONO
   (ab70335, Abcam Inc.; 1:200 dilution). Then, coverslips were treated
   with Alexa Fluor 488-conjugated goat anti-rabbit IgG (ab150081; 1:1000
   dilution) or Alexa Fluor 594-conjugated goat anti-rabbit IgG (ab150160,
   Abcam Inc.; 1:1000 dilution), followed by staining with 4’,6-diamidino-
   2-phenylindole (DAPI, 300 nmol·L^−1; D9542, Sigma). Images were
   captured using a Nikon A1Si Laser Scanning Confocal Microscope (Nikon
   Instruments Inc., Japan).

Chromatin immunoprecipitation (ChIP)

   ChIP assay was conducted following the manual of EZ-ChIP kit (Upstate
   Biotechnology, Temecula, CA). Briefly, cells were cross-linked with 1%
   formaldehyde for 10 min at 37 °C. The chromatin was sonicated to yield
   DNA fragments ranging from 200 to 500 bp in length. The YY1-specific
   antibody, with IgG serving as a negative control, was employed to
   immunoprecipitate the cross-linked protein-DNA complexes. Following
   immunoprecipitation, chromatin DNA was purified using a DNA
   Purification Kit. Real-time quantitative PCR (qPCR) was subsequently
   performed using SYBR Green PCR Master Mix (Applied Biosystems) with
   specific primers (Supplementary Table S1) [[85]29–[86]31].

Bimolecular fluorescence complementation (BiFC) assay

   Human NONO cDNA (1416 bp) and PARP1 cDNA (3045 bp) were cloned into
   BiFC vectors pBiFC-VC155 and pBiFC-VN173 (Addgene, Cambridge, MA),
   respectively (Supplementary Table S2). The recombinant plasmids were
   co-transfected into cancer cells. Twenty-four hours post-transfection,
   cells were fixed in 4% paraformaldehyde, stained with DAPI for 5 min,
   and then examined using a confocal microscope (Nikon Instruments Inc.,
   Japan), with excitation at 488 nm and emission at 500 nm, respectively.
   Images were captured to observe fluorescence complementation indicating
   protein–protein interactions [[87]27, [88]28].

Cross-linking RNA immunoprecipitation (RIP)

   Tumor cells were rinsed twice with ice-cold phosphate-buffered saline
   (PBS), and subjected to ultra-violet cross-linking at 400 mJ/cm^2 on
   ice for 10 min. Subsequently, cells were resuspended in 100 μl of RIPA
   buffer [25 mmol/L Tris–HCl, pH 7.5; 150 mmol/L NaCl; 1%
   octylphenyl-polyethylene glycol; 1% sodium deoxycholate; 0.1% sodium
   dodecyl sulfate (SDS); 1 × protease inhibitor cocktail (04693132001,
   Merck Millipore, Burlington, MA); and 2 U/μl SUPERase-In RNase
   inhibitor (AM2696, Thermo Fisher Scientific, Inc.)]. Then, cells were
   subjected to sonication using an ultrasonicator. The lysates were
   clarified by centrifugation at 20,000 × g for 15 min at 4 °C. The
   resulting supernatants were diluted in 300 μl of Tris-Nacl (TN) buffer
   (25 mmol/L Tris–HCl, pH 7.5; 150 mmol/L NaCl; 0.5%
   octylphenyl-polyethylene glycol) containing RNase inhibitor, and
   pre-cleared with 15 μl of Dynabeads Protein G (10004D, Thermo Fisher
   Scientific, Inc.) for 3 h at 4 °C. Post pre-clearance, aliquots were
   taken for pre-RIP analysis. The remaining supernatants were split into
   two equal fractions and incubated overnight at 4 °C with 15 μl of
   Dynabeads pre-bound with either 1.5 μg of anti-NONO antibody or an
   isotype IgG (30000-0-AP, Proteintech, Wuhan, China). Following
   incubation, bead complexes were washed twice with RIPA buffer and twice
   with TN buffer. The bead-bound samples were then subjected to treatment
   with TN buffer containing 0.5% SDS and proteinase K (EO0491, Thermo
   Fisher Scientific, Inc.) at 55 °C for 30 min to release the
   immunoprecipitated RNA. RNA was subsequently extracted,
   reverse-transcribed, and quantified by RT-qPCR [[89]32, [90]33].

In vitro cell viability, growth, and invasion assays

   The 3-(4,5-dimethyltriazol-2-yl)−2,5-diphenyl tetrazolium bromide (MTT,
   Sigma) colorimetric, soft agar, and matrigel (BD Matrigel^™ Matrix, BD
   Biosciences, Franklin Lakes, NJ) invasion assays for measuring the
   viability, growth, and invasion capability of tumor cells were
   conducted as previously described [[91]28–[92]31, [93]34].

Statistical analysis

   All data were shown as mean ± standard error of the mean (SEM). Cutoff
   values were determined by average gene expression levels. Student's t
   test, one-way analysis of variance (ANOVA), and χ^2 analysis were
   applied to compare the difference of tumor cells or tissues. Fisher's
   exact test was applied to analyze the statistical significance of
   overlap between two gene lists. Pearson's correlation coefficient was
   applied for analyzing the relationship among gene expression. Log-rank
   test was used to assess survival difference and hazard ratio. All
   statistical tests were two-sided and considered statistically
   significant when false discovery rate-corrected P values were less than
   0.05 [[94]7, [95]27, [96]28].

Results

PARP1 enhances the proliferation, migration, and invasion of NB cells

   To explore the prognostic values of PARP1 expression in NB, mining of
   R2 database ([97]https://hgserver2.amc.nl/cgi-bin/r2/main.cgi) was
   performed. Kaplan–Meier plots revealed that NB patients with high PARP1
   expression had significantly poorer overall survival (OS) compared to
   those with low PARP1 expression ([98]GSE62564, P < 0.001; [99]GSE45547,
   P < 0.01, Fig. [100]1A). The protein levels of PARP1 were detected in
   various human cell lines, including HEK293 (embryonic kidney cells),
   SK-N-SH, SK-N-AS, SK-N-BE(2), IMR32, SH-SY5Y (NB cells), HeLa (cervical
   cancer cells), and AGS (gastric cancer cells; Fig. [101]1B). Western
   blot and quantification assays revealed that PARP1 expression was
   significantly elevated in tumor cells compared to normal cells
   (Fig. [102]1B). From five NB cell lines, SK-N-AS and SK-N-BE(2)
   (displaying medium PARP1 levels) were chosen as models for subsequent
   investigations. Furthermore, SK-N-AS and SK-N-BE(2) cell lines with
   stable PARP1 over-expression or knockdown were established, and
   validated by western blot and RT-qPCR assays (Fig. [103]1C, D). In MTT
   colorimetric, soft agar, and matrigel invasion assays, the increase or
   decrease of viability, growth, and invasiveness of NB cells were noted
   following stable ectopic expression or knockdown of PARP1, respectively
   (Fig. [104]1E–G). These findings revealed that PARP1 promoted
   proliferation, invasion, and migration in NB cells in vitro.

Fig. 1.

   [105]Fig. 1
   [106]Open in a new tab

   PARP1 enhances the proliferation, migration, and invasion of NB cells.
   A Kaplan–Meier curve showing overall survival of NB patients (left
   panel: [107]GSE62564, cutoff value = 7.05; right panel: [108]GSE45547,
   cutoff value = 14.29) stratified by high or low PARP1 expression. B
   Western blot assay (left panel) and quantification analysis via ImageJ
   software (right panel) revealing the expression of PARP1 in HEK293,
   HeLa, AGS, SK-N-SH, SK-N-AS, SK-N-BE(2), IMR32, and SH-SY5Y cells. C
   and D Western blot and RT-qPCR (normalized to β-actin) assays showing
   the levels of PARP1 in SK-N-AS and SK-N-BE(2) cells stably transfected
   with empty vector (mock), PARP1, scramble shRNA (sh-scb), sh-PARP1 #1,
   or sh-PARP1 #2 (n = 3). E MTT colorimetric assay depicting changes in
   viability of SK-N-AS and SK-N-BE(2) cells stably transfected with mock,
   PARP1, sh-scb, sh-PARP1 #1, or sh-PARP1 #2 (n = 5). F and G
   Representative images (left panels) and quantification (right panels)
   of soft agar (F) and matrigel invasion (G) assays demonstrating
   anchorage-independent growth and invasion of SK-N-AS and SK-N-BE(2)
   cells stably transfected with mock, PARP1, sh-scb, sh-PARP1 #1, or
   sh-PARP1 #2 (n = 3). Log-rank test for survival comparison in (A).
   Student’s t test or ANOVA compared the difference in (D–G). **P < 0.01,
   ***P < 0.001, ****P < 0.0001 vs. mock or sh-Scb. Data are shown as
   mean ± s.e.m. (error bars) or representative of three independent
   experiments in (B–G)

Identification of YY1 as a transcription factor activating PARP1 expression
in NB

   To investigate key transcription factors (TFs) regulating PARP1
   expression in NB, we performed a comprehensive analysis of hTFtarget
   ([109]http://bioinfo.life.hust.edu.cn/hTFtarget) and PROMO
   ([110]http://acgt.cs.tau.ac.il/promo/) databases, which identified nine
   TFs regulating PARP1 expression (Fig. [111]2A). Among them, by
   analyzing datasets from R2 database ([112]https://hgserver2.amc.nl/),
   YY1 was identified as top-ranked TF with oncogenic features [[113]15,
   [114]16, [115]35], which was associated with poor survival and PARP1
   expression in 498 NB cases (Fig. [116]2B, C). Through analysis of a
   public ChIP-seq dataset of YY1 ([117]GSE155839), significant enrichment
   of YY1 was noted at promoter region of PARP1 (Fig. [118]2D). Validating
   ChIP-qPCR assay demonstrated that stable ectopic expression of YY1
   enhanced its enrichment on PARP1 promoter regions in NB cells, whereas
   YY1 knockdown accordingly diminished its binding (Fig. [119]2E).
   Furthermore, stable over-expression or knockdown of YY1 resulted in an
   increase or decrease in the transcript and protein levels of PARP1,
   respectively (Fig. [120]2F, G). The increase in PARP1 expression,
   anchorage-independent growth, and invasion capabilities of SK-N-AS and
   SK-N-BE(2) cells with stable over-expression of YY1 were abolished by
   knockdown of PARP1 (Fig. [121]2H–J). These findings indicted that YY1
   up-regulated the transcription of PARP1, thereby promoting the
   proliferation, migration, and invasion of NB cells.

Fig. 2.

   [122]Fig. 2
   [123]Open in a new tab

   Identification of YY1 as a transcription factor activating PARP1
   expression in NB. A Prediction of transcription factors regulating
   PARP1 expression via analyzing hTFtarget and PROMO databases. B
   Kaplan–Meier curve showing overall survival of NB patients (left panel:
   [124]GSE62564, cutoff value = 5.53; right panel: [125]GSE45547, cutoff
   value = 12.35) stratified by high or low YY1 expression. C Pearson's
   correlation coefficient for analyzing the relationship between gene
   expression of PARP1 and YY1 in 498 NB cases ([126]GSE62564). D ChIP-seq
   analysis of YY1 binding to PARP1 promoter in glioblastoma stem cells
   ([127]GSE155839). E ChIP-qPCR assay showing YY1 binding to PARP1
   promoter in SK-N-AS cells stably transfected with empty vector (mock),
   PARP1, scramble shRNA (sh-scb), sh-PARP1 #1, or sh-PARP1 #2 (n = 3). F
   and G Real-time RT-qPCR (F, normalized to β-actin, n = 3) and western
   blot (G) assays indicating the transcript and protein levels of PARP1
   in SK-N-AS and SK-N-BE(2) cells stably transfected with mock, YY1,
   sh-scb, sh-YY1 #1, or sh-YY1 #2. H Western blot assay indicating the
   protein levels of YY1 and PARP1 in SK-N-AS and SK-N-BE(2) cells stably
   transfected with mock, YY1, sh-scb, or sh-PARP1 #1. I and J
   Representative images (left panels) and quantification (right panels)
   of soft agar (I) and matrigel invasion (J) assays showing
   anchorage-independent growth and invasion of SK-N-AS and SK-N-BE(2)
   cells stably transfected with mock, YY1, sh-scb, or sh-PARP1 #1
   (n = 3). Fisher's exact test for overlapping analysis in (A). Log-rank
   test for survival comparison in (B). Pearson’s correlation coefficient
   analysis for gene expression in (C). Student’s t test or ANOVA compared
   the difference in (E, F, I and J). **P < 0.01, ***P < 0.001,
   ****P < 0.0001 vs. mock, sh-Scb, or mock + sh-Scb. Data are shown as
   mean ± s.e.m. (error bars) or representative of three independent
   experiments in (E–J)

PARP1 interacts with NONO in NB cells

   To explore protein substrates of PARP1 in NB, we conducted a search of
   BioGRID database ([128]https://thebiogrid.org/) [[129]36]. The results
   indicated 1,320 potential protein partners of PARP1, and pathway
   enrichment analysis using Metascape program
   ([130]https://metascape.org/gp/index.html#/ main/step1) revealed that
   they were related to regulation of RNA metabolism [[131]12, [132]37] or
   DNA damage repair [[133]38, [134]39] (Fig. [135]3A). To further screen
   essential RNA-binding proteins (RBPs) involved in mRNA metabolism,
   integrated analysis of POSTAR3 ([136]http://111.198.139.65/), AmiGO2
   ([137]https://amigo.geneontology.org/amigo), and R2 databases was
   undertaken, which indicated twelve RBP candidates (Fig. [138]3B). Of
   them, only NONO protein was consistently associated with poor overall
   survival (OS) of three independent cohorts consisting of 498
   ([139]GSE62564, P < 0.05), 649 ([140]GSE45547, P < 0.0001), and 101
   ([141]GSE3960, P < 0.0001) NB patients (Fig. [142]3B, C). Based on
   three-dimensional protein structures of NONO and PARP1 from UniProt
   database ([143]https://www.uniprot.org/), molecular docking via HDOCK
   ([144]http://hdock.phys.hust.edu.cn/) and PyMOL
   ([145]https://www.pymol.org/) programs further implicated their
   physical interaction (Fig. [146]3D). Co-IP and western blot assays
   indicated that PARP1 protein was able to interact with NONO in SK-N-AS
   and SK-N-BE(2) cells (Fig. [147]3E). In immunofluorescence assay,
   co-localization of PARP1 and NONO was observed in the nucleus of NB
   cells (Fig. [148]3F). Physical interaction of PARP1 with NONO was
   observed in SK-N-AS cells transfected with VN173-PARP1 and VC155-NONO
   constructs (Fig. [149]3G). These findings suggested that PARP1
   interacted with NONO in the nucleus and might influence mRNA
   metabolism.

Fig. 3.

   [150]Fig. 3
   [151]Open in a new tab

   PARP1 interacts with NONO in NB. A Functional enrichment analysis of
   proteins interacting with PARP1 using Metascape program
   ([152]https://metascape.org/gp/index.html#/main/step1). B
   Identification of NONO as a substrate of PARP1 via integration analysis
   of AmiGO2, POSTAR3, and R2 databases. C Kaplan–Meier curve showing
   overall survival of NB patients (left panel: [153]GSE62564, cutoff
   value = 7.58; middle panel: [154]GSE45547, cutoff value = 13.65; right
   panel: [155]GSE3960, cutoff value = 9.59) stratified by high or low
   NONO expression. D Top four docking conformations of 3D protein
   structures of PARP1 and NONO proteins visualized by using PyMOL
   program. E Co-IP and western blot assays indicating endogenous
   interaction between PARP1 and NONO in SK-N-AS and SK-N-BE(2) cells. F
   Immunofluorescence showing co-localization of PARP1 and NONO in SK-N-AS
   and SK-N-BE(2) cells. Scale bars: 10 μm. G BiFC assay showing physical
   interaction of PARP1 with NONO in SK-N-AS cells co-transfected with
   VC155-NONO and VN173-PARP1 constructs. Scale bars: 10 μm. Fisher's
   exact test for overlapping analysis in (B). Log-rank test for survival
   comparison in (C). Data are shown as mean ± s.e.m. (error bars) or
   representative of three independent experiments in (E–G)

YY1 facilitated PARP1-mediated PARylation and stabilization of NONO

   To investigate interaction domains of PARP1 and NONO, recombinant
   GST-tagged PARP1 truncation (Fig. [156]4A) and MBP-tagged NONO proteins
   were applied. Co-IP and western blot assays confirmed that full-length
   and catalytic (CAT) domain (amino acids 662–1014) [[157]40, [158]41] of
   PARP1, but not zinc finger (ZnF) or BRCA1 C terminus (BRCT) domain,
   were able to interact with NONO protein, suggesting that CAT domain was
   required for its binding to NONO (Fig. [159]4B). Since previous studies
   have identified CAT domain as an indicator of PARylation capability in
   PARP protein family [[160]6, [161]40, [162]42], we hypothesized that
   PARP1 might induce the PARylation of NONO protein. Treatment with
   Olaparib and PJ34, established PARP1 activity inhibitors [[163]43,
   [164]44], resulted in a dose-dependent decrease in both PARylation and
   protein levels of NONO in SK-N-AS cells (Fig. [165]4C, D). Notably,
   incubation with Olaparib also markedly decreased the half-life of NONO
   protein in NB cells (Fig. [166]4E). Conversely, treatment with
   proteasome inhibitor MG132, but not with autophagy inhibitor (3-MA) or
   Z-VAD-FMK (apoptosis inhibitor), reversed the Olaparib-induced
   degradation of NONO protein (Fig. [167]4F). Ubiquitination assay
   demonstrated a significant increase in ubiquitin levels of NONO protein
   following Olaparib treatment in SK-N-AS cells (Fig. [168]4G).
   Over-expression of YY1 led to an increase in both PARylation and
   protein levels of NONO in SK-N-AS cells, which were reversed by
   knockdown of PARP1 (Fig. [169]4H). These findings indicted that YY1
   facilitated PARP1-mediated PARylation and stabilization of NONO in NB.

Fig. 4.

   [170]Fig. 4
   [171]Open in a new tab

   YY1 facilitated PARP1-mediated PARylation and stabilization of NONO
   protein. A and B Schematic diagram, co-IP, and western blot assays
   indicating interaction between recombinant full-length or truncated
   GST-tagged PARP1 and MBP-tagged NONO proteins. C and D Co-IP and
   western blot assays showing the impact of Olaparib (5 nmol/L) and PJ34
   (110 nmol/L) on PARylation and protein levels of NONO in SK-N-AS cells.
   E Western blot and quantification analysis showing NONO protein levels
   in SK-N-AS cells treated with Olaparib (5 nmol/L) and CHX (20 μg/ml)
   for indicated durations. F Western blot analysis showing NONO protein
   levels in SK-N-AS cells treated with MG132 (20 μmol/L), Z-VAD-FMK
   (100 nmol/L), 3-MA (5 mmol/L), or Olaparib (5 nmol/L). G Co-IP and
   western blot assays revealing ubiquitination of NONO in SK-N-AS cells
   treated with Olaparib (5 nmol/L). H Co-IP and western blot assays
   showing the PARylation and protein levels of NONO in SK-N-AS cells
   stably transfected with empty vector (mock), YY1, scramble shRNA
   (sh-scb), or sh-PARP1 #1. Data are shown as mean ± s.e.m. (error bars)
   or representative of three independent experiments in (B–H)

NONO enhances splicing and mRNA maturation of ADAM8 and TEX14 in a
PARylation- dependent manner

   To further analyze target genes of NONO, 187 down-regulated genes upon
   NONO knockdown ([172]GSE114376, Fig. [173]5A) [[174]22] were
   over-lapped with mRNA targets of NONO derived from POSTAR3 database.
   The results indicated two potential target genes, including a
   disintegrin and metalloproteinase domain 8 (ADAM8) and testis-expressed
   gene 14 (TEX14) (Fig. [175]5B). Kaplan–Meier analysis revealed that NB
   patients with elevated levels of ADAM8 and TEX14 had significantly
   reduced OS rates (Fig. [176]5C). Based on protein-nucleic acid
   interaction analysis using crosslinking-immunoprecipitation and high
   throughput sequencing (CLIP-seq) dataset from ENCORI
   ([177]https://rnasysu.com/encori/) database and 3dRNA/DNA
   ([178]http://biophy.hust.edu.cn/new/), HDOCK, or PyMOL programs, the
   binding of NONO protein to introns of ADAM8 (chr10:133276313–133276352)
   and TEX14 (chr17:58659190–58659230) was noted (Fig. [179]5D). RIP assay
   demonstrated that over-expression or knockdown of NONO significantly
   increased or decreased its binding to ADAM8 and TEX14 intronic regions
   (Fig. [180]5E). We further hypothesized that NONO might be essential
   for maturation of ADAM8 and TEX14 pre-mRNAs. To investigate this,
   mature mRNAs and immature pre-mRNAs were quantified by using primer
   sets specific to intron–exon and exon-exon junctions [[181]23]
   (Fig. [182]5F). Stable knockdown of NONO resulted in a decrease in the
   levels of mature ADAM8 and TEX14 mRNAs, with pre-mRNAs remaining stable
   in SK-N-AS and SK-N-BE(2) cells (Fig. [183]5G, H). Notably, the
   relative pre-mRNA/mRNA ratio was significantly elevated upon NONO
   knockdown in both cell lines (Fig. [184]5I), indicating that diminished
   splicing efficiency in absence of NONO led to an accumulation of
   pre-mRNA. Conversely, overexpression of NONO enhanced the splicing
   efficiency of ADAM8 and TEX14 mRNAs (Fig. [185]5G–I). Furthermore,
   western blot assay showed that stable over-expression or knockdown of
   NONO resulted in an increase or decrease in the protein levels of ADAM8
   and TEX14, respectively (Supplementary Fig. S1A). Of note, Olaparib
   treatment abolished the increase in NONO enrichment (Supplementary Fig.
   S1B) or mature mRNA levels (Supplementary Fig. S1C), and decrease in
   pre-mRNA/mRNA ratio (Supplementary Fig. S1D, E) of ADAM8 and TEX14 in
   SK-N-AS and SK-N-BE(2) cells with stable over-expression of NONO. These
   data indicated that NONO enhanced splicing and mRNA maturation of ADAM8
   and TEX14 in a PARylation-dependent manner.

Fig. 5.

   [186]Fig. 5
   [187]Open in a new tab

   NONO enhances splicing and mRNA maturation of ADAM8 and TEX14. A
   RNA-seq analysis of a public dataset (GSE114376_KELLY_NONO-KD) showing
   differentially expressed gene in NB cells with NONO knockdown. B
   Identification of ADAM8 and TEX14 as downstream targets of NONO by
   integration analyses of RNA-seq (GSE114376_KELLY_NONO-KD) and CLIP-seq
   (POSTAR3) datasets. C Kaplan–Meier curve showing overall survival of
   649 NB patients ([188]GSE45547) stratified by high or low expression of
   ADAM8 (left panel, cutoff value = 12.63) or TEX14 (right panel, cutoff
   value = 8.82). D Docking conformations of NONO protein biniding to
   intronic regions of ADAM8 and TEX14 pre-mRNAs visualized by using PyMOL
   program. E RIP assay using NONO antibody showing the interaction of
   NONO and ADAM8/TEX14 pre-mRNA in SK-N-AS and SK-N-BE(2) cells stably
   transfected with empty vector (mock), NONO, sh-scb, sh-NONO #1, or
   sh-NONO #2. F Primer design (arrows) for identifying intron–exon
   (unspliced, pre-mRNA) and exon-exon (spliced, mature RNA) junctions.
   G–I Relative levels and ratios of unspliced and spliced ADAM8 and TEX14
   transcripts in SK-N-AS and SK-N-BE(2) cells stably transfected with
   mock, NONO, sh-scb, sh-NONO #1, or sh-NONO #2. Fisher's exact test for
   overlapping analysis in (B). Log-rank test for survival comparison in
   (C). Student’s t test or ANOVA compared the difference in (E, G–I).
   *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. mock or sh-scb.
   Data are shown as mean ± s.e.m. (error bars) or representative of three
   independent experiments in (C and E–I)

YY1/PARP1/NONO axis drives mRNA maturation of ADAM8 and TEX14 in NB

   To further investigate the roles of YY1/PARP1/NONO axis in mRNA
   maturation of ADAM8 and TEX14, rescue experiments were undertaken. The
   increase in protein expression of ADAM8 and TEX14 of SK-N-AS and
   SK-N-BE(2) cells with stable over-expression of YY1 were abolished by
   knockdown of PARP1 or NONO (Fig. [189]6A). Over-expression of YY1
   significantly increased NONO’s binding to ADAM8 and TEX14 intronic
   regions, which was abolished by knockdown of PARP1 or NONO
   (Fig. [190]6B). Stable over-expression of YY1 led to increased levels
   of mature ADAM8 and TEX14 mRNAs, and this effect was reversed by
   knockdown of PARP1 or NONO (Fig. [191]6C). Notably, the relative
   pre-mRNA/mRNA ratio was elevated upon NONO or PARP1 knockdown in both
   cell lines, which were abolished by over-expression of YY1
   (Fig. [192]6D, E). These data indicated that YY1/PARP1/NONO axis drove
   mRNA maturation of ADAM8 and TEX14 in NB.

Fig. 6.

   [193]Fig. 6
   [194]Open in a new tab

   YY1/PARP1/NONO axis drives mRNA maturation of ADAM8 and TEX14 in NB. A
   Western blot assay showing the protein levels of YY1, PARP1, NONO,
   ADAM8 and TEX14 in SK-N-AS and SK-N-BE(2) cells stably transfected with
   empty vector (mock), YY1, scramble shRNA (sh-scb), sh-PARP1 #1, or
   sh-NONO #1. B RIP assay using NONO antibody showing the interaction of
   NONO and ADAM8/TEX14 pre-mRNA in SK-N-AS and SK-N-BE(2) cells stably
   transfected with mock, YY1, sh-scb, sh-PARP1 #1, or sh-NONO #1 (n = 3).
   C–E Relative levels and ratios of unspliced and spliced ADAM8 and TEX14
   transcripts in SK-N-AS and SK-N-BE(2) cells stably transfected with
   mock, YY1, sh-scb, sh-PARP1 #1, or sh-NONO #1 (n = 3). ANOVA compared
   the difference in (B–E). **P < 0.01, ***P < 0.001, ****P < 0.0001 vs.
   mock + sh-Scb. Data are shown as mean ± s.e.m. (error bars) or
   representative of three independent experiments in (B–E)

Discussion

   Since the discovery of PARylation and its major enzyme PARP1 over
   50 years ago, significant advancements in understanding PARP1's roles
   in DNA repair, gene transcription, or cell death have highlighted its
   functions in tumorigenesis and spurred the investigation of therapeutic
   PARP1 inhibitors [[195]45]. Previous studies show that YY1 is able to
   interact with and facilitate the enzymatic activity of PARP1 during DNA
   repair in cervical cancer HeLa cells [[196]46]. In addition, RNA
   recognition motif 1 (RRM1) of NONO is responsible for PAR-binding,
   while recruitment of NONO to DNA damage sites completely depends on
   PARP1-mediated PAR in HeLa cells [[197]47]. However, the roles of YY1,
   PARP1, and NONO during NB progression remain to be determined. This
   study provides significant insights into the oncogenic role of PARP1 in
   NB, elucidating its regulatory mechanisms, interaction networks, and
   its impact on cellular behaviors (Fig. [198]7). Our findings indicate
   PARP1 as a critical oncogenic driver that directly enhances the
   proliferation, migration, and invasion of NB cells. In clinical NB
   cases, PARP1 expression is correlated with poorer survival. By mining
   public datasets, we identified YY1 as a key transcription factor
   up-regulating PARP1 expression. ChIP-seq and ChIP assays collectively
   verified YY1’s direct binding to PARP1 promoter region, thereby
   up-regulating its transcriptional levels. In addition, YY1 exerted
   oncogenic roles in proliferation, migration, and invasion in NB, at
   least in part, through up-regulating PARP1 expression.

Fig. 7.

   [199]Fig. 7
   [200]Open in a new tab

   Roles of YY1/PARP1/NONO axis in tumor progression. YY1 binds to PARP1
   promoter to enhance its expression. PARP1 stabilizes NONO through
   inducing PARylation and preventing it from ubiquitin-mediated
   degradation, while NONO is crucial for efficient splicing of pre-mRNAs
   such as ADAM8 and TEX14 in a PARylation-dependent manner, illustrating
   the roles of YY1/PARP1/NONO axis as a potential target for tumor
   therapeutics

   Previous studies show that PARylation regulates mitotic spindle, Cajal
   bodies, DNA damage repair, and microRNA-mediated translational
   repression in tumor progression [[201]48]. For instance, PARylation of
   DNA repair protein O^6-methylguanine DNA methyltransferase (MGMT) is
   essential for repairing temozolomide-induced O^6-methylguanine, while
   inhibitors of PARylation decrease MGMT function and sensitize tumor
   cells to temozolomide in glioblastoma [[202]49]. Moreover, PARylation
   is specifically concentrated in the regulation of RNA/DNA-binding
   proteins and their associated complexes [[203]48]. Previous studies
   have elucidated that ZnF domain is crucial for binding of PARP1 to DNA
   strand breaks, while its BRCT domain typically engages in
   protein–protein interactions and is essential for recognizing
   phosphorylated proteins involved in DNA repair processes [[204]40]. The
   CAT domain is responsible for PARP1's ADP-ribosylation activity,
   transferring ADP-ribose units to target proteins, a vital step in DNA
   repair and other cellular processes [[205]40, [206]41]. Our research
   uncovered the interaction between PARP1 and splicing factor NONO in NB
   cells, which was primarily mediated through CAT domain of PARP1,
   indicating the potential impact of PARP1 on PARylation of NONO protein.
   We further demonstrated that through PARylation, PARP1 stabilized NONO
   protein via preventing its ubiquitin-mediated degradation.
   Administration of Olaparib or PJ34 led to a dose-dependent decrease in
   PARylation and expression of NONO, further verifying PARP1's role in
   regulating NONO expression. In the realm of post-translational
   modifications (PTMs), competitive inhibition between different
   modifications on a single protein is pivotal in modulating its
   function, stability, or protein interaction [[207]50]. For example,
   phosphorylation may activate or deactivate a protein, while
   ubiquitination typically marks it for degradation [[208]51]. We
   analyzed the potential PARylation and ubiquitination sites of NONO
   protein by using ADPriboDB 2.0 ([209]https://adpribodb.adpribodb.org/)
   and GPS-Uber ([210]http://gpsuber.biocuckoo.cn/) programs. Notably, we
   found that several PARylation sites, specifically the 10th, 87th. and
   462th glutamic acid residues, were in close proximity to ubiquitination
   sites. Based on our findings that Olaparib was able to induce
   ubiquitination of NONO, and proteasome inhibitor was able to reverse
   Olaparib-induced degradation of NONO protein, we hypothesized that
   PARylation might stabilize NONO protein levels by competitively
   inhibiting ubiquitination, which warrant further investigation.

   Emerging evidence indicates that NONO is involved in DNA unwinding,
   transcriptional regulation, mRNA splicing, or nuclear RNA retention
   [[211]52]. Our experiments demonstrated that NONO knockdown
   significantly reduced the splicing and mRNA maturation of ADAM8 and
   TEX14, while their pre-mRNAs remained stable rather than accumulative
   in NB cells. Since previous research shows that pre-mRNA maturation
   frequently occurs simultaneously with transcription [[212]53], we
   believe that these findings may be attributed to reduced
   splicing-coupled transcription, suggesting a complex interplay of NONO
   during this process. Furthermore, ADAM8 and TEX14 exhibit
   tumor-promoting activities in various malignancies. ADAM8 has emerged
   as a critical factor in the progression of breast, pancreatic, and
   brain cancers [[213]54, [214]55]. High expression levels of ADAM8 are
   associated with increased invasiveness and poor outcome of patients
   [[215]54, [216]55]. TEX14 is integral to reproductive cell stability,
   particularly through its influence on intercellular bridge function,
   and its DNA methylation has been implicated in early-onset familial
   breast cancer [[217]56, [218]57]. Recent studies have also demonstrated
   that TEX14 mediates pro-proliferative or anti-apoptotic effects of
   transforming growth factor-beta in ovarian granulosa cells [[219]56,
   [220]57]. In this study, both ADAM8 and TEX14 are significantly
   associated with poor outcome of NB patients, with their functions
   requiring further investigation.

Conclusion

   In summary, our data indicate that high PARP1 expression is
   significantly correlated with poorer survival of NB patients,
   highlighting its potential as a prognostic marker. Functionally, PARP1
   acts as a pro-oncogene, enhancing proliferation, migration, and
   invasion in NB cell lines. The transcription factor YY1 directly
   regulates PARP1 expression by binding to its promoter. Furthermore,
   PARP1 interacted with NONO to induce its PARylation, preventing its
   ubiquitin-mediated degradation. NONO, in turn, is essential for
   efficient splicing and mRNA maturation of target genes such as ADAM8
   and TEX14 in a PARylation-dependent manner. These findings offer deeper
   molecular mechanistic insights and highlight the YY1/PARP1/NONO axis as
   a potential therapeutic target. Future research should explore how
   PAR-dependent modification impacts NONO's RNA-binding capability.
   Concurrent inhibition of PARP1 and YY1 might result in significant
   disruption of crucial pre-mRNA splicing process, and ultimately
   repressing tumorigenesis and aggressiveness, which warrant further in
   vivo studies using animal models.

Supplementary Information

   [221]Supplementary material 1.^ (402KB, pdf)

Acknowledgements