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RNA-binding proteomics reveals MATR3 interacting with lncRNA SNHG1 to enhance neuroblastoma progression Tz-Wen Yang, Divya Sahu, Yi-Wen Chang, Chia-Lang Hsu, Chiao-Hui Hsieh, Hsuan-Cheng Huang, and Hsueh-Fen Juan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00693 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Journal of Proteome Research

RNA-binding proteomics reveals MATR3 interacting with lncRNA SNHG1 to enhance neuroblastoma progression Tz-Wen Yang1#, Divya Sahu2#, Yi-Wen Chang3#, Chia-Lang Hsu3,4, Chiao-Hui Hsieh1, HsuanCheng Huang2*, Hsueh-Fen Juan1,3* 1

Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan

2

Institute of Biomedical Informatics, National Yang-Ming University, Taipei 112, Taiwan

3

Department of Life Science, National Taiwan University, Taipei 106, Taiwan

4

Department of Medical Research, National Taiwan University Hospital, Taipei 100, Taiwan

#These authors contributed equally to this work

Correspondence to: Hsuan-Cheng Huang, Ph.D. Institute of Biomedical Informatics, National Yang-Ming University, No. 155, Sec. 2, Linong St., 112 Taipei, Taiwan. Tel: +886-2-28267357; E-mail: [email protected]

Hsueh-Fen Juan, Ph.D. Department of Life Science, Institute of Molecular and Cellular Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., 106 Taipei, Taiwan. Tel: +886-2-3366-4536; Fax: +886-2-23673374; E-mail: [email protected]

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Abstract The interaction of long noncoding RNAs (lncRNAs) with one or more RNA-binding proteins (RBPs) are important to a plethora of cellular and physiological processes. The lncRNA SNHG1 was reported to be aberrantly expressed and associated with poor patient prognosis in several cancers, including neuroblastoma. However, the interacting RBPs and biological functions associated with SNHG1 in neuroblastoma remains unknown. In this study, we identified 283, 31, and 164 SNHG1-interacting proteins in SK-N-BE(2)C, SK-N-DZ and SK-N-AS neuroblastoma cells, respectively, using a RNA-protein pull-down assay coupled with liquid chromatographytandem mass spectrometry (LC-MS/MS). Twenty-four SNHG1-interacting RBPs were identified in common from these three neuroblastoma cell lines. RBPs MATR3, YBX1 and HNRNPL have the binding sites for SNHG1 predicted by DeepBind motif analysis. Furthermore, the direct binding of MATR3 with SNHG1 was validated by western blot and confirmed by RNA immunoprecipitation assay (RIP). Co-expression analysis revealed that the expression of SNHG1 is positively correlated with MATR3 (P = 3.402E-13). The high expression of MATR3 is associated with poor event-free survival (P = 0.00711) and overall survival (P = 0.00064). Biological functions such as ribonucleoprotein complex biogenesis, RNA processing, and RNA splicing are significantly enriched and in common between SNHG1 and MATR3. In conclusion, we identified MATR3 as binding to SNHG1 and the interaction might be involved in splicing events that enhance neuroblastoma progression. Keywords RNA-binding proteomics, Neuroblastoma, lncRNA-SNHG1, RNA-protein pull down assay, LCMS/MS, RNA immunoprecipitation, MATR3


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1. Introduction Neuroblastoma is the most common childhood malignant cancer of the developing sympathetic nervous system (1, 2). It accounts for approximately 6% of all childhood cancers (3) and more than 90% of the cases are diagnosed by five year of age (4). The tumor displays wide range of enigmatic behavior. It regresses spontaneously in infants but undergo persistent proliferation in children older than 1 year of age (1, 2). Amplification of v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) gene serves as potent oncogenic driver of neuroblastoma (5). It is located in short arm of chromosome 2 (2p24) (6, 7), contributes to approximately 25% of the neuroblastoma cases, and associated with poor patient outcome (4, 8). Risk factors including patient age at diagnosis, MYCN amplification status, tumor stage, chromosomal aberrations, and tumor histology are used in clinical practice for evaluation of biological behavior of the tumor, risk assessment of the disease and determination of appropriate treatments (9-11). Yet, five-year survival chance for neuroblastoma patient after the disease relapse remains disappointingly low (12). Long noncoding RNAs (lncRNAs) are pivotal regulatory RNAs of the mammalian noncoding transcriptome (13, 14). They are predominantly characterized by having length greater than 200 nucleotides, transcribed by RNA polymerase II, polyadenylated and typically located in either nucleus or cytoplasm (15, 16). LncRNAs are involved in a plethora of cellular and physiological processes. They are associated in posttranscriptional regulation, splicing of the multiple exons into mature transcripts, differentiation, chromatin modification, dosage compensation and proteins synthesis (17-21). Most of these biological functions require interaction with one or more protein partners known as RNA-binding proteins (RBPs) (22). They bind with target RNA in sequence specific manner through their unique RNA binding domain (23). 3 ACS Paragon Plus Environment


LncRNAs are significantly dysregulated in various types of cancers, including neuroblastoma (24-30). For instance, the widely studied lncRNA SNHG1 was significantly up-regulated in nonsmall cell lung cancer, both in cell lines and tissues (31). It promotes tumor cell proliferation via interaction with miR-101-3p regulating expression of SOX9 activating Wnt/β-catenin signaling pathway (31). SNHG1 was significantly up-regulated in intervertebral disk degeneration tissues leading to nucleus pulposus cell proliferation via inhibiting miR-326 expression and promoting CCND1 expression (32). SNHG1 was significantly up-regulated in neuroblastoma and associated with poor patient survival (33). However, biological functions of lncRNA SNHG1 in neuroblastoma remains unknown. Herein, using a RNA-protein pull down assay coupled with LCMS/MS, we identified 283, 31 and 164 SNHG1-interacting proteins in three neuroblastoma cell lines, SK-N-BE(2)C, SK-N-DZ and SK-N-AS, respectively. We further found the RBP Matrin 3 (MATR3) to be associated with SNHG1, confirmed by western blot and RNA immunoprecipitation (RIP). The subsequent gene set enrichment analysis of SNHG1 and MATR3 revealed ribonucleoprotein complex biogenesis, RNA processing, and RNA splicing to be significantly enriched in common as potential prospective biological functions.

2. Experimental Section 2.1. Cell lines and cell culture Human neuroblastoma cell lines SK-N-DZ and SK-N-BE(2)C were obtained from American Type Tissue Collection (ATCC, USA). SK-N-AS cell lines were kindly provided by Dr. Yung-Feng Liao (Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan). All neuroblastoma cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA)


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supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), and incubated at 37oC in an atmosphere with 5% CO2. 2.2. Construction and in vitro transcription assay The SP6 promoter region was first introduced onto 5’-end of SNHG1 by primers as follows: SNHG1-forward (cloning)-XhoI: 5’-CCG-CTC-GAG-ATT-TAG-GTG-ACA-CTA-TAG-AAGTTC-TCA-TTT-TTC-TAC-TGC-TCG-3’, SNHG1-reverse (cloning)-NotI: 5’-ATA-GTT-TAGCGG-CCG-CTT-TTT-TTT-TTT-TTT-TTA-TGT-AAT-CAA-TCA-TTT-TAT-3’.

Next,

the

amplified PCR product was further constructed into the pBlueScript SK (+) vector at XhoI and NotI sites. After resultant SNHG1 plasmid was linearized by NotI, in vitro transcription was then implemented using MEGAscript® SP6 Transcription Kit (Thermo Scientific, USA) following the manufacturer’s protocol. RNase inhibitors provided by MEGAscript® SP6 Transcription Kit were used through all procedures to avoid RNA degradation. Finally, DNA template was removed by TURBO DNaseI and synthetic SNHG1 was precipitated with LiCl and chilled on -20°C for 1 hour. RNA pellet was dissolved with RNase-free water and the concentration was determined by NanoDrop ND-1000 (NanoDrop Technologies, USA). 2.3. RNA-protein pull down assay Pull-down assay was carried out with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA). First, the 3’-end of in vitro transcripted SNHG1 was biotinylated using Pierce RNA 3’ End Desthiobiotinylation Kit (Thermo Fisher Scientific, USA) following manufacturer’s protocol and then incubated with Streptavidin magnetic beads at room temperature for 30 minutes. Meanwhile, cell lysate was harvested by Pierce IP Lysis Buffer (Pierce, USA) with protease inhibitor cocktail (Bioshop, Canada), tyrosine phosphatase inhibitor cocktail and serine/threonine phosphatase inhibitor cocktail (Bionovas, USA) at 4°C. Next, the biotin-labeled 5 ACS Paragon Plus Environment


SNHG1-beads complex were further mixed well with cell lysate at 4°C with rotation for 60 minutes. After beads collection and washing, eluted protein concentration was measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The precipitated SNHG1interacting proteins were then analyzed by LC-MS/MS or western blot. Streptavidin magnetic beads were served as negative controls. 2.4. Sample preparation for proteome Whole SNHG1-interacting proteins were dissolved with 50 mM triethylammonium bicarbonate (TEABC, Sigma-Aldrich) (pH 8.5). Then, proteins were reduced by 5 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) for 30 minutes at 37oC and were alkylated by using 2 mM SMethyl methanethiosulfonate (MMTS) for 30 minutes at room temperature in the dark. We performed gel-assisted protein digestion to obtain peptides. Acrylamide/bisacrylamide (40%, v/v, 37.5:1), 10% APS (w/v) and TEMED were mixed with the protein solution by vortex for few seconds and spin down soon (the ratio of protein solution: acrylamide/bisacrylamide: APS: TEMED = 14:5:0.3:0.3, v/v) until the solution polymerized into a gel. The gels were excised into small pieces and washed continuously with 25 mM TEABC and 25 mM TEABC/50% (v/v) acetonitrile (ACN, Thermo Fisher Scientific) until no bubbles were visible. The gel pieces were further dehydrated with 100% ACN dried completely with a centrifugal evaporator (miVac Duo Concentrator; UK; Eppendorf concentrator 5301). 25 mM TEABC was added to rehydrate the gel and proteins were further digested by trypsin (protein: trypsin = 10:1, w/w) (Thermo Fisher Scientific, USA) at 37oC overnight. After in-gel digestion, peptides were extracted from the gel with 0.1% (v/v) Trifluoroacetic acid (TFA), 50% (v/v) ACN /0.1% (v/v) TFA, and 100% ACN sequentially. The extracted peptide solution was dried with a sample concentrator (miVac Duo


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Concentrator; Genevac, Ipswich, UK; Eppendorf concentrator 5301) and then subjected to desalting by using ZipTip® Pipette Tips (Millipore, USA). 2.5. Peptide desalting by ZipTip pipette tips Dried peptides were resolved by 20 μl of 0.1% Pierce™ Trifluoroacetic Acid (TFA) (Thermo Fisher Scientific, Waltham, MA, USA) and added 10 % TFA to adjust the pH to pH 2-3. The ZipTip was wetted by 50% (v/v) acetonitrile (ACN) / 0.1% TFA for 5 cycles first and followed by equilibrated in 0.1% TFA with 10 cycles. Aspirated and dispensed the sample peptides with ZipTip for 20 cycles by pipetting slowly to let peptides bind to ZipTip. We washed the ZipTip with 0.1% TFA for 10 cycles. At the final stage, peptides were eluted with 20 μl of 50% (v/v) acetonitrile (ACN)/0.1% TFA for 10 cycles by aspirating and dispensing for 10 cycles to new tubes. The eluted solution was dried with a centrifugal evaporator (CVE-2000, Eyela, Tokyo, Japan). 2.6. NanoLC-MS/MS analysis NanoLC-MS/MS analysis was performed on a nanoACQUITY UPLC system (Waters, Milford, MA) connected to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nanospray interface (Proxeon, Odense, Denmark). Peptide samples were loaded onto a 2 cm × 180 µm capillary trap column and then separated in a 75 µm × 25 cm nanoACQUITY 1.7 µm BEH C18 column (Waters, Milford, MA) at a flow rate of 300 nL/minute. Mobile phase A consisted of 0.1% formic acid, and B consisted of 0.1% formic acid and 80% ACN. A linear gradient of 10% to 40% B in 90 minutes and 40% to 85% B in 10 minutes was employed throughout this study. Mass spectra from survey full scans were acquired on the Orbitrap (m/z 350–1500). The resolution was set to 60,000 at m/z 400 and the automatic gain control (AGC) was set to 1 × 106 ions. The m/z values triggering MS/MS were put on an exclusion list for 90



seconds. The top ten most-intense precursor ions were selected from the MS scan for subsequent collision-induced dissociation MS/MS scan by ion trap (AGC target at 7000). 2.7. Proteome data analysis Raw MS spectral data was processed for peak detection and identification by using MaxQuant software version 1.5.2.8 (34). Peptide identification was performed by using the Andromeda search engine (35) against the Reviewed Swiss-Prot human database version canonical (Published February, 2017) (36). Search criteria used in this study were trypsin specificity, fixed modification of carbamidomethyl (C), variable modifications of carbamidomethyl (C), oxidation (M), carbamyl (N-term) and deamidation (NQ), and allowed for up to two missed cleavages. The following parameters were chose: the minimal peptide length is 7; the precursor mass tolerance is 3 ppm; and the fragment ion tolerance is 0.5 Da. By using a decoy database strategy, peptide identification was accepted based on the posterior error probability with a false discovery rate of 1%. The leading proteins were selected for further bioinformatics analysis. 2.8. Bioinformatics analysis The comparison of proteins list among biological duplicates and negative control were performed by Venny (37). The annotations of cellular location of each protein were obtained from UniProt (36). We considered five major cellular compartments, including nucleus, cytoplasm, mitochondrion, endoplasmic reticulum and golgi apparatus. The proteins not located in these cellular compartment were assigned as “other”. The over-representation analysis was performed by ConsensusPathDB (38) with gene sets of GO molecular function. We only considered the GO molecular function terms at level 4 and 5 and q-value of 0.05 as the cutoff to determine the overrepresented GO terms. For motif analysis, DeepBind tool (39) were used to scan the whole SNHG1 sequence with the window size of 20 nucleotides. 8 ACS Paragon Plus Environment

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2.9. Western blot The protein samples were denatured at 95 °C for 10 minutes, and separated by 10% SDS-PAGE. The linear protein in SDS-PAGE gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) and blocked the membrane in 5% skim milk/TBST at room temperature for 1 hour. The membrane was incubated overnight with the primary antibodies of interest in 5% skim milk/TBST at 4°C. After washing three times in 10 minutes, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for two hours. Finally, the bands of membrane were detected by FluorChem M (proteinsimple, San Jose, California). All of the primary antibodies, anti-MATR3 (GTX115291), anti-DDX5 (GTX100234) and anti-YBX1 (GTX131630), were purchased from Genetex (GeneTex International Corporation, Hsinchu City, Taiwan). The secondary antibodies conjugated with HRP were purchase from Sigma-Aldrich (Taipei, Taiwan). 2.10. RNA immunoprecipitation The procedure was followed by the manuscript protocol of Magna RIPTM RNA-Binding Protein Immunoprecipitation kit (Millipore, Bedford, MA, USA). The antibody of MATR3 for immunoprecipitation was obtained from Abcam (Cambridge, MA, USA). Briefly, the precipitated RNAs were reverse transcribed using RevertAid H Minus First Strand cDNA Synthase Kit (Thermo Fisher Scientific, Waltham, MA, USA) and RT-PCR analyses were further carried out to detect SNHG1 by using primers as follows: SNHG1-F: 5’-ACAGCAGTTGAGGGTTTGCT-3’ and SNHG1-R: 5’-ACAGTGCCTGAGTTTGGGTT-3’. The PCR products were resolved by 1.7 % agarose gel electrophoresis for 110V/30 minutes. 2.11. Survival analysis



The Kaplan-Meier event-free survival (EFS) and overall survival (OS) analysis were performed for the neuroblastoma patients with Gene Expression Omnibus accession number GSE62564 (40) using the survival R package (41). The patients were divided into two groups, classified on the basis of median gene expression value. The significance of the survival curve was evaluated using log-rank (Mantel-Haenszel) test. 2.12. Gene Set Enrichment Analysis We calculated Spearman correlation coefficients (SCC) between the protein-coding gene (PCG) MATR3 and whole-genome protein-coding genes (19,199 PCGs) from the neuroblastoma RNAseq cohort. Gene Set Enrichment Analysis (GSEA v2.2.3, Broad Institute) (42) was performed by the stand-alone tool using MSigDB C5.bp.v6.1.symbols.gmt gene set collection, ranked list as MATR3-correlated PCGs and their corresponding SCC, maximum gene set size of 5000, minimum gene set size of 15, 1000 permutations, and weighted enrichment statistics. Overrepresented gene sets with false discovery rate (FDR) q value 0.05 were filtered. Similarly, GSEA was also performed for lncRNA SNHG1.

3. Results 3.1. Identification of the SNHG1 interacting proteins from human neuroblastoma cells LncRNAs play important regulatory functions via interacting with their protein partners. In order to specifically identify SNHG1-interacting proteins in neuroblastoma, we performed lncRNAprotein interactomic profiling (Figure 1A). First, we cloned SNHG1 sequence into pBlueScript SK+ vector containing SP6 RNA polymerase binding site (Figure 1B). Next, the circular plasmid was linearized using NotI restriction enzyme and followed by in vitro transcription. In vitro 10 ACS Paragon Plus Environment

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synthetic SNHG1 transcripts were further labeled by biotin at 3’-end and incubated with whole cell lysate freshly extracted from two MYCN-amplified (SK-N-BE(2)C and SK-N-DZ) and one MYCN-nonamplified (SK-N-AS) neuroblastoma cell lines. Biotin labeled SNHG1-protein complex were precipitated by using streptavidin agarose beads. After RNA pull-down assay, potential SNHG1-interacting proteins were identified by LC-MS/MS and subsequently analyzed by MaxQuant software. To avoid non-specific interacting proteins, beads alone were performed as negative control. 3.2. SNHG1 interacting proteins partners are localized in nucleus and cytoplasm To obtain the true positive SNHG1-interacting proteins, we only considered the proteins which were commonly identified in two biological replicates but not in negative control samples. A total of 283, 31 and 164 SNHG1-interacting proteins in SK-N-BE(2)C, SK-N-DZ and SK-N-AS respectively, were identified (Table S1-3 and Figure 2A). Most of SNHG1-interacting proteins are located in nucleus and cytoplasm (Figure 2B), consistent with the previous report that SNHG1 localizes not only nucleus but also cytoplasm (43). Moreover, GO enrichment analysis revealed that the terms related to nucleotide binding were statistically significantly enriched in all the neuroblastoma cell lines (Figure 2C). These evidence further confirm that the proteins identified by lncRNA pull down combined with proteomics are potential SNHG1-interacting partners. 3.3. SNHG1 involved in various biological pathways via interaction with diverse regulators Among the SNHG1-interacting proteins, 24 were shared in the three neuroblastoma cell lines, SKN-BE(2)C, SK-N-DZ and SK-N-AS (Figure 3A). These 24 SNHG1-interacting proteins were divided into four groups by their biological functions, including ribosome-related, hnRNP-related, splicing-related and others (Figure 3B). 60S ribosomal protein L22 (RPL22), 60S ribosomal



protein L6 (RPL6), 60S ribosomal protein L3 (RPL3) and 60S ribosomal protein L19 (RPL19) are involved in ribosome-related functions. Heterogeneous nuclear ribonucleoprotein M (HNRNPM), Heterogeneous nuclear ribonucleoprotein L (HNRNPL), heterogeneous nuclear ribonucleoprotein A0 (HNRNPA0), heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB), heterogeneous nuclear ribonucleoprotein D-like (HNRPNDL) and heterogeneous nuclear ribonucleoprotein Q (SYNCRIP) belong to hnRNP-related functions. Probable ATP-dependent RNA helicase DDX5 (DDX5), nuclease-sensitive element-binding protein 1 (YBX1), non-POU domain containing octamer binding protein (NONO), small nuclear ribonucleoprotein Sm D1 (SNRPD1), small nuclear ribonucleoprotein Sm D3 (SNRPD3) and Aly/REF export factor 2 (ALYREF) are important players in splicing. Matrin 3 (MATR3), Ras GTPase-activating protein-binding protein 1 (G3BP1), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), histone H1x (H1FX), leucine-rich PPR motif-containing protein (LRPPRC), interleukin enhancer-binding factor 3 (ILF3), ribonuclease inhibitor (RNH1) and KH domain-containing, RNA-binding, signal transduction-associated protein 1 (KHDRBS1) have the other functions not described above “ribosome-related, hnRNP-related, splicing-related”. 3.4. SNHG1 recognized by regulators through binding motif To elucidate the SNHG1 directly interacting proteins, we searched predicted binding motif from the DeepBind tool for all the 24 proteins. Three proteins, nuclease-sensitive element-binding protein 1 (YBX1), heterogeneous nuclear ribonucleoprotein L (HNRNPL) and Matrin 3 (MATR3) were recognized (Figure 4). These three proteins were recognized as the highest score of the twenty nucleotides on SNHG1. We also checked the MS/MS spectrum peaks and coverage percentage for each of these three proteins (Figure S1) and selected them for further experimental validation. 3.5. Direct binding of MATR3 with SNHG1 12 ACS Paragon Plus Environment

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To validate the binding association of YBX1, HNRNPL, and MATR3 with SNHG1, the SNHG1pull down proteins were further detected by western blot. The results revealed only MATR3 to be associated with SNHG1 in all the three neuroblastoma cell lines (Figure 5A). To cross validate this direct binding, we performed RNA immunoprecipitation assay (RIP). The results depicted a high affinity binding of MATR3 with SNHG1 in MYCN amplified neuroblastoma cell lines (Figure 5B). Moreover, we also found a statistical significant positive correlation between SNHG1 and MATR3 from a RNA-seq neuroblastoma cohort (n = 493) (Figure 5C). Interestingly, the protein level of MATR3 was upregulated in SK-N-AS, which expressed less SNHG1 transcript, compared to SK-N-DZ and SK-N-BE(2)C (Figure 5A, left panel). These results indicate that SNHG1 is able to directly interact with MATR3 and possibly regulates MATR3 protein stability. 3.6. MATR3 is up-regulated in high-risk neuroblastoma MATR3 is found to be significantly up-regulated in MYCN amplified (Figure 6A) and high-risk (Figure 6B) neuroblastoma patients. To address the possibility that expression of MATR3 is associated with patient survival status, we performed Kaplan-Meier survival analysis on the expression value of MATR3 from the RNA-seq cohort. First, we classified the patients into lowexpression (n = 247) and high-expression (n = 246) groups using the median expression of MATR3. We observed patients in high-expression group had poorer event-free survival (P = 0.00711) and overall survival (P = 0.00064) than those in the low-expression group (Figures 6C and 6D). Biological functions regulated by SNHG1 and MATR3 in neuroblastoma LncRNAs are devoid of protein-coding capacity. Thus, we applied guilt-by-association strategy (44) and performed gene set enrichment analysis (GSEA) to explore the biological functions of lncRNA SNHG1 (Table S4). Moreover, we also performed GSEA for MATR3 (Table S5). We



found biological functions such as ribonucleoprotein complex biogenesis, ncRNA processing, translation initiation, protein localization, and RNA splicing to be significantly enriched and in common between SNHG1 and MATR3 (Figure 7). Taken together, these results indicate that MATR3 might associate with SNHG1 and enhances neuroblastoma progression.

Discussion RBPs control the fate of various transcripts including lncRNAs at the posttranscriptional level in the cell (22, 45). By the use of SNHG1-pull down assay followed by LC-MS/MS spectrometry, we were able to identify 24 potential RBPs binding with SNHG1. Study reported that SNHG1 might associate with spliceosomes or ribosomal proteins that facilitate in its transcription or modification (46). As expected, we found the potential SNHG1-intereacting proteins were classified either into ribosome-related, spliceosome-related or hnRNP-related. Among these 24 proteins, Probable ATP-dependent RNA helicase DDX5 (DDX5), was reported to form complex with ROR t, a ligand receptor that controls the differentiation of T helper 17 lymphocytes. DDX5ROR t complex coordinates with lncRNA Rmrp and regulates tissue-specific transcription in T helper 17 lymphocytes (47). To validate the interaction of DDX5 and SNHG1, we analyzed the MS/MS spectrum, coverage percentage of DDX5 and performed western blot (Figure S1, Figure 1C and Figure 5A). The result depicted that DDX5 might interact indirectly or bind other proteins with SNHG1. DeepBind motif analysis identified only three proteins YBX1, HNRNPL, and MATR3 to have the predicted binding motifs for SNHG1. The protein, YBX1 is a highly conserved protein with cold-shock domain and involved in cellular processes such as DNA repair, proliferation,


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transcription (48). Studies indicated that YBX1 participated in drug-resistance in human cancer cells (49), and acted as a transcription factor that regulates embryogenesis (50). YBX1 has also four RNA recognized binding motifs in arginine-rich (51). These features suggest that YBX1 might interacts with RNA through RNA binding motifs. DeepBind motif analysis identified YBX1 recognized SNHG1 through binding motif. However, western blot analysis shows no association between YBX1 and SNHG1. Thus, we hypothesize that there might be some other proteins which indirectly helps in association of YBX1 with SNHG1. The heterogeneous nuclear ribonucleoproteins (hnRNPs) comprises of RNA binding proteins, which helps in processing of heterogeneous nuclear RNAs to mature mRNAs (52). The protein, hnRNPL interacts with linc1992, and regulates TNF α expression (53). Study shows that during fasting, hnRNPL interacted with lncRNA lncLGR and suppressed the expression of hepatic glucokinase (54). Moreover, hnRNPL interacts with Sam68 and forms a SLM/Sam nuclear bodies (55). Among all the 24 candidate proteins, only MATR3 found to be associated with SNHG1, confirmed by western blot and RIP. MATR3 is a RNA and DNA binding proteins with its mutation associated to amyotrophic lateral sclerosis (ALS) (56). It is primarily located in nucleus (57) and participates in RNA splicing (58). From the Kaplan-Meier survival analysis, MATR3 is found to be associated with neuroblastoma patient survival status. The patients with high MATR3 expression had poorer event-free and overall survival than those in the low expression group. This finding suggests that MATR3 could be as a prognostic biomarker for neuroblastoma progression. Molecular functions of lncRNAs are critically based on their subcellular localization (59). We found majority of the SNHG1-binding proteins to be located in nucleus suggesting ribonucleoprotein complex biogenesis, RNA processing, and RNA splicing as its potential prospective functions. 15 ACS Paragon Plus Environment


Conclusion In conclusion, our integrative analysis revealed RBP MATR3 has the binding association with lncRNA SNHG1, confirmed by RNA pull-down and RIP assay. MATR3 expression was significantly up-regulated in MYCN amplified and high-risk neuroblastoma patients. High expression of MATR3 was associated with poor patient survival. MATR3 after binding with SNHG1 might be involved in RNA splicing and cell cycle that enhances neuroblastoma progression.

SUPPORTING INFORMATION: The following supporting information is available free of charge at ACS website http://pubs.acs.org. Figure S1. The mass spectrum of candidate proteins. Table S1. The list of identified proteins interacting with SNHG1 in SK-N-BE(2)C cells. Protein identification was performed by MaxQuant Version 1.5.2.8. Table S2. The list of identified proteins interacting with SNHG1 in SK-N-DZ cells. Protein identification was performed by MaxQuant Version 1.5.2.8. Table S3. The list of identified proteins interacting with SNHG1 in SK-N-AS cells. Protein identification was performed by MaxQuant Version 1.5.2.8. Table S4. GSEA report for SNHG1 positive correlated genes. Table S5. GSEA report for MATR3 positive correlated genes. 16 ACS Paragon Plus Environment

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Acknowledgements This work was supported by the Ministry of Science and Technology (MOST 105-2320-B-002057-MY3 and MOST 106-2320-B-002-053-MY3) and the National Health Research Institutes (NHRI-EX107-10530PI and NHRI-EX107-10709BI) in Taiwan. We thank Dr. Yung-Feng Liao (Academia Sinica, Taiwan) for kindly providing the neuroblastoma cell lines, Dr. Jin-Der Wen (Institute of Molecular and Cellular Biology, National Taiwan University, Taiwan) for the help in SNHG1 RNA construction, and Technology Commons at the College of Life Science, National Taiwan University for technical assistance.

Author Contributions T.-W. Y., Y.-W. C. and C.-H. H. executed the experiments; D. S., Y.-W. C. and C.-L. H. analyzed the data; H.-C. H. and H.-F. J. designed and supervised all experiments; T.-W. Y., D. S., Y.-W. C., C.-L. H., H.-C. H. and H.-F. J. wrote the manuscript. All authors approved the final manuscript.

Declaration of Interests The authors declare no competing financial interests.

Abbreviations



EFS: Event-free survival; FDR: False discovery rate; GO: Gene Ontology; GSEA: Gene set enrichment analysis; HNRNPL: Heterogeneous Nuclear Ribonucleoprotein L; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LncRNAs: long noncoding RNAs; MATR3: Matrin 3; MYCN: v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog; OS: Overall survival; PCGs: Protein-coding genes; RBPs: RNA-binding proteins; RIP: RNA immunoprecipitation; RNA-seq: RNA sequencing; SNHG1: small nucleolar RNA host gene 1; YBX1: Y-box binding protein 1


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Figure Legends Figure 1: Identification of lncRNA SNHG1 interacting proteins by RNA-protein pull down assay and proteomics approaches. (A) Work flow of the SNHG1-protein interactomic profiling. (B) The map of constructed plasmid for in vitro transcription, which containing full length of SNHG1 sequence along with SP6 RNA polymerase binding site. (C) Schematic diagram of whole experiment procedures. First, the constructed SNHG1-plasmid was digested to linear form for in vitro transcription. Then, biotin was labeled onto SNHG1, and proteins interacting with this biotinlabeled-SNHG1 were precipitated by RNA pull-down assay. LC-MS/MS and MaxQuant were performed to identify SNHG1-interacting proteins. Experiments were performed in biological duplicates. Figure 2: SNHG1-interacting protein in neuroblastoma cell lines. (A) Venn-diagram shows the number of interacted proteins with SNHG1 in three neuroblastoma cell lines (MYCN amplified: SK-N-BE(2)C and SK-N-DZ and MYCN nonamplified: SK-N-AS). SNHG1 was RNA pull-down with biotin-labeled SNHG1 and Empty was beads alone severed as negative control. Rep1 and Rep2 represented the first and second biological replicates. (B) Pie chart shows cellular distribution of the SNHG1-interacting proteins in the neuroblastoma cell lines, respectively. (C) The point plot shows molecular functions of the SNHG1-interacting proteins in three neuroblastoma cell lines. The size and color of each of the circle denotes ratio of matched genes to the query gene set and P-value of the molecular function, respectively. Figure 3: Comparison of potential SNHG1-interacting proteins in three neuroblastoma cell lines. (A) Venn diagram shows overlapping of SNHG1-interacting proteins in three neuroblastoma cell lines, SK-N-BE(2)C and SK-N-DZ and SK-N-AS. (B) Classification of the 24 common


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SNHG1-interacting proteins into 4 groups based on their biological processes. The protein information is described in main text. Figure 4: The predicted binding motif of interacting proteins on SNHG1 sequence. (A-D) The highest peak of each candidate protein shows predicted binding motif on SNHG1 sequence. The highest score of each peak comprises twenty nucleotides of SNHG1. The IDs of the three proteins MATR3 (D00119.001), YBX1 (D00163.001 and D00163.002) and HNRNPL (D00110.001) were obtained from DeepBind tool. Figure 5: MATR3 interacts with SNHG1. (A) Interaction between MATR3 and SNHG1 is confirmed using SNHG1 pull-down combining western blotting. Input mean cell lysate without RNA precipitation process. Each cell line had beads only as negative control of RNA pull-down assay. Experiments were performed in biological triplicates. (B) The binding of MATR3 with SNHG1 is validated using RNA immunoprecipitation in two neuroblastoma cell lines. PC was RTPCR positive control which used pBlueScript SK (+)-SNHG1 plasmid as the template. Experiments were performed in biological duplicates. (C) Scatter plot shows the positive correlation between SNHG1 and MATR3 expression levels in neuroblastoma patients from RNAseq cohort (n = 493). Figure 6: MATR3 predicts poor clinical outcome in neuroblastoma patients. (A-B) Boxplot shows the normalized log2RPM expression value of MATR3 in risk status (A) and MYCN status (B) in a RNA-seq neuroblastoma cohort (n = 493). (C-D) Kaplan-Meier plots shows event-free survival (C) and overall survival (D) of low expression and high expression groups based on median expression value of MATR3 in neuroblastoma patient (n = 493). The P-values were obtained from log-rank (Mantel-Haenszel) test.



Figure 7: Gene set enrichment analysis of MATR3 and SNHG1. (A) Bar chart shows top overrepresented gene sets common between MATR3 and SNHG1 analyzed by GSEA. Colors represent normalized enrichment score (NES) of MATR3 and SNHG1. (B) Enrichment plot for the gene set of ribonucleoprotein complex biogenesis, ncRNA processing and RNA splicing. These biological processes enriched for MATR3 and SNHG1 correlated genes.


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Figure 1

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Figure 2 a

SK-N-BE(2)C

SK-N-DZ

SK-N-AS

b

SK-N-BE(2)C

SK-N-DZ

SK-N-AS

c


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Figure 3 a

b SK-N-AS

Ribosome-related

SK-N-BE(2)C RPL22 RPL6 RPL3 RPL19

MATR3 G3BP1 IFIT5 H1FX

SK-N-DZ

LRPPRC ILF3 RNH1 KHDRBS1

Others

hnRNP-related HNRNPM HNRNPL HNRNPA0 HNRNPAB HNRNPDL SYNCRIP

DDX5 YBX1 NONO

SNRPD1 SNRPD3 ALYREF

Splicing-related



Figure 4


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Figure 5



Figure 6


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Figure 7



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