The flightless I protein interacts with RNA-binding proteins and is involved in the genome-wide mRNA post-transcriptional regulation in lung carcinoma cells

  • Authors:
    • Cuihua Wang
    • Kezhi Chen
    • Shengyou Liao
    • Wei Gu
    • Xinlei Lian
    • Jing Zhang
    • Xuejuan Gao
    • Xiaohui Liu
    • Tong Wang
    • Qing-Yu He
    • Gong Zhang
    • Langxia Liu
  • View Affiliations

  • Published online on: May 11, 2017     https://doi.org/10.3892/ijo.2017.3995
  • Pages: 347-361
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Abstract

The flightless I protein (FLII) belongs to the gelsolin family. Its function has been associated with actin remodeling, embryonic development, wound repair, and more recently with cancer. The structure of FLII is characterized by the N-terminal leucine-rich repeats (LRR) and C-terminal gesolin related repeated units that are both protein-protein inter­action domains, suggesting that FLII may exert its function by interaction with other proteins. Therefore, systematic study of protein interactions of FLII in cells is important for the understanding of FLII functions. In this study, we found that FLII was downregulated in lung carcinoma cell lines H1299 and A549 as compared with normal HBE (human bronchial epithelial) cell line. The investigation of FLII interactome in H1299 cells revealed that 74 of the total 132 putative FLII interactors are involved in RNA post-transcriptional modification and trafficking. Furthermore, by using high-throughput transcriptome and translatome sequencing combined with cell fractionation, we showed that the overexpression or knockdown of FLII impacts on the overall nuclear export, and translation of mRNAs. IPA analysis revealed that the majority of these target mRNAs encode the proteins whose functions are reminiscent of those previously reported for FLII, suggesting that the post-transcriptional regulation of mRNA might be a major mechanism of action for FLII.

Introduction

Lung cancer has become the most common cause of cancer-related mortality and a serious public health concern worldwide. In China, the lung cancer-related mortality rate has been multiplied by 4.6 in the past 3 decades. It will reach the alarming 1 million per year by 2025, as estimated by WHO (1). Among all lung cancers, 85% are NSCLC (non-small cell lung cancer). At the moment of diagnosis, most of NSCLC patients present with locally advanced or metastatic disease, when the surgical intervention is no longer feasible despite its high success rate if applied earlier. For these patients, systemic chemotherapy with concurrent radiotherapy remains a necessary option for cure. For this reason, the identification of the proteins playing key roles in the physiopathology of NSCLC that could be potential targets for the chemotherapy reveals to be important and emergent.

The flightless I protein (FLII) belongs to the gelsolin family of actin severing proteins. At the time of its discovery, Drosophila melanogaster FLII was shown to play an important role in the embryonic development. The following studies on mammalian FLII demonstrated that this protein is also involved in the regulation of wound repair, skin barrier development, the recovery after blistering and regulation of immune response (29). Recently, studies have also demonstrated that FLII is involved in colorectal cancer, hepatocellular and prostate cancer (1013). Notably, a role of tumor suppressor in prostate cancer has been attributed to this protein (14).

Despite the previous attempts of identifying the FLII interactome in the cells using yeast-two hybrid system (15,16), the systemic picture of protein interaction network of FLII still remains elusive. In this study, by combining co-immunoprecipitation and mass spectrometry analysis, we have identified 132 putative FLII interactors in lung adenocarcinoma cancer H1299 cells, and found that more than a half of them are proteins involved in RNA post-transcriptional regulation and protein biosynthesis. By combining cell fractionation, mRNA-seq and translating mRNA sequencing (RNC-seq), we evaluated the function of FLII on the transcription, nuclear export and translation of mRNAs. We demonstrated that FLII affects the overall nuclear export and translation of mRNAs.

Materials and methods

Antibodies

Rabbit polyclonal anti-FLII (sc-30046) antibody, mouse polyclonal anti-HNRNPQ (sc-56703) antibody, goat polyclonal anti-TIAL1 (sc-1749) antibody mouse monoclonal anti-actin (sc-47778), mouse monoclonal anti-NuP88 (sc-136009) antibody, peroxidase-conjugated AffiniPure goat anti-Mouse IgG (H+L), peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), rabbit anti-goat IgG (H+L)-HRP (sc-2768) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell culture

Human H1299 and HBE cells (Cell Resource Center, Institute of Life Science Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Grand island, NY, USA) supplemented with 10% fetal bovine serum (PAA Laboratories, Weike Biochemical Reagent, Shanghai, China), 1% penicillin/streptomycin (Genom, Hangzhou, China) at 37°C in a humidified atmosphere containing 5% CO2.

Plasmid constructions

The pot-b7-FLII plasmid containing the cDNA of the human FLII was obtained from Yingrun Biotechnologies Inc. (Changsha, China). The cDNA fragment encoding FLII was PCR-amplified and inserted into pCMV-N-FLAg-vector using the EcoRV and SpeI restriction sites. A primer pair (upstream: 5′-ATTGATATCATGGAGGCCACCGGGGTGCTG-3′ and downstream: 5′-ATAACTAGTTTAGGCCAGGGCCTTGCAGAA-3′) was designed based on NCBI GenBank NP_002009.1. All the plasmids were accuratly confirmed by DNA sequencing.

Transfections and immunoprecipitation of FLII

Cells were transfected with either empty pCMV-N-Flag or pCMV-N-Flag-FLII vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 48 h after transfection, cells were lysed using EBC lysis buffer. Total soluble extract (1 mg) was incubated with anti-Flag antibody overnight at 4°C. Protein A/G-Sepharose was added and incubated for 4 h at 4°C. After washing, the precipitated proteins were eluted and separated using SDS-PAGE. The gel was stained with Silver nitrate after migration, and gel lanes were subsequently cut, destained, reduced, alkylated and digested with gold-trypsin at 37°C overnight. The tryptic peptides were extracted, and the peptide mixtures were concentrated by SpeedVac centrifuge to dryness and re-dissolved with 2% ACN in 0.1% formic acid before LC-MS/MS analysis. For the confirmation of the presence of Flag-FLII in the immune-complexes, the immunoprecipitates were washed with immunoprecipitation assay buffer three times, and subjected to western blot analysis using anti-Flag antibody.

MS analysis

The peptide mixtures were analyzed by reverse-phase liquid chromatography coupled with LTQ-Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) as previously described with minor modification (17). Briefly, the peptide mixtures were firstly loaded on a C18 reverse-phase column (100-mm i.d., 10-cm long, 5-mm resin from Michrom Bioresources, Auburn, CA, USA) using an auto sampler. The peptide mixtures were eluted with 0–40% gradient buffer solution (Buffer A, 0.1% formic acid, and 5% ACN; Buffer B, 0.1% formic acid and 95% ACN) over 180 min. The eluate was then analyzed online in the LTQ-Orbitrap mass spectrometer operated in a data-dependent mode with capillary temperature of 2001 C and spray voltage of 1.80 kV. A full MS scan with m/z 350–1800 was carried out in the Orbitrap at resolution r5100,000 at m/z 400, and followed by five MS2 scans in the LTQ with Dynamic Exclusion setting: 2 repeat counts with repeat duration of 30 sec and exclusion duration of 90 sec. MS3 was further performed if an ion had a neutral loss of −98.00, −58.00, −49.00, −38.67, −32.67 or −24.50 Da in the MS2 and the ion was one of the top five most intense ions in the MS2. Conditions with 35% normalized collision energy, activation q of 0.25 and activation time of 30 msec were applied for MS2 and MS3 acquisitions.

Stable FLII knockdown in HBE cells

Lentiviral shRNA vectors (GenePharma, Shanghai, China) targeting human FLII were utilized for stable knockdown in HBE cells. Procedures were conducted according to the manufacturer's protocol. The FLII siRNA sense sequence (5′-GGGCTAGACATCTACGTAT-3′) and Scramble siRNA sense sequence (5′-TTCTCCGAACGTGTCACGTTTC-3′) were used to design the shRNA that were inserted into PGLV3/H1/GFP+ pur Vector under H1 promoter. Cells resistant to puromycin (1.5 µg/ml) were selected and passaged for further study.

Cell fractionation

Six million human H1299 or HBE cells were resuspended in 500 µl ice cold Extraction Buffer (10 mM Tris-HCl, 10 mMKCl, 5 mM MgCl2, protease and phosphatase inhibitors, RNase inhibitor, VRC) and incubated on ice for 10 min, 500 µl 0.6% Triton/Extraction Buffer was then added, followed by 10 sec of vortex, and incubation on ice for further 10 min, then vortex for 10 sec again. Lysate was then subjected to vertical centrifugation for 5 min at 600 × g speed at RT. Supernatant corresponding to the cytoplasmic fraction was transferred into a new tube. The pellet corresponding to the cell nucleus was subsequently resuspended in 1 ml 0.6% Triton Extraction Buffer by pipetting 10 times, incubated on ice for 5 min, followed by vertical centrifugation for 5 min at 600 × g speed at RT, The supernatant corresponded to nuclear extract was then collected. This experiment was repeated thrice, and the cytoplasmic or nuclear extract resulted from the three experiments were pooled together, and served for the RNA extraction using Trizol reagent (Invitrogen).

Ribosome-nascent chain complex (RNC) extraction

The RNC extraction was performed as described by Esposito et al (18) with modifications. In brief, cells were pre-treated with 100 mg/ml cycloheximide for 15 min, followed by pre-chilled phosphate buffered saline washes and addition of 2 ml cell lysis buffer [1% Triton X-100 in ribosome buffer (RB buffer) 20 mM HEPES-KOH (pH 7.4), 15 mM MgCl2, 200 mMKCl, 100 mg/ml cycloheximide and 2 mM dithiothreitol]. After 30-min ice-bath, cell lysates were scraped and transferred to pre-chilled 1.5 ml tubes. Cell debris was removed by centrifuging at 13, 200 × g for 10 min at 4°C. Supernatants were transferred on the surface of 20 ml of sucrose buffer (30% sucrose in RB buffer). RNCs were pelleted after ultra-centrifugation at 185,000 × g for 5 h at 4°C. RNC-RNA was purified using TRIzol method.

Next-generation sequencing and sequence analysis

Total RNA and RNC-RNA of human H1299 and HBE cells were extracted as previously described (18). Equal amounts of total mRNA or RNC-mRNA isolated from three independent cultures were pooled and reverse-transcribed into cDNA. The yielded cDNA library was then constructed and subjected to RNA-seq analysis using Illumina HiSeq™ 2000. The sequencing data sets are deposited in Gene Expression Omnibus database under the accession number of GSE92979. Reads were mapped to human mRNA reference sequence (RefSeq) for GRCh37/hg19 in UCSC genome browser (downloaded from http://hgdownload.cse.ucsc.edu/downloads, accessed January 21, 2013) using FANSe2 mapping algorithm with the options -L78 -S8 -I0 -E9 -B1. The reads mapped to splice variants of one gene were summed. The mRNA abundance was normalized using both rpkM (reads per kilobase per million reads). Differential expression was evaluated using edgeR since it outperforms the other mainstream differential expression models (19). Genes with >10 mapped reads were considered as quantified genes. The cytoplasm/nucleus distribution of each given mRNA has been quantified in all experimental groups of cells, and the variation of such distribution between the FLII knocked down HBE or the FLII overexpressing H1299 cells and their respective control cells were calculated. Where Flag-FLII and Flag-Vector refer to H1299 cells transfected with Flag-FLII and Flag vector; shRNA-FLII and shRNA-Ctrl refer to HBE cells expressing the shRNA against FLII and the control shRNA. 'C' for cytoplasmic, and 'N' for nuclear.

Reverse transcription and PCR

Total RNA or RNC-RNA, isolated from both H1299 and HBE cells, were reverse transcribed to cDNA with poly-dT primer using Reverse Transcriptase XL (AMV) (Takara, Foster City, CA, USA), by following the manufacturer's instructions. The quantitative real-time PCR (qPCR) was then performed with gene-specific primers and the SsoFast™EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on a Bio-Rad MiniOpticon real-time PCR system (Bio-Rad) by following the manufacturer's instructions. The specificity of the primers was verified by both in Silico Computation (NCBI Primer-BLAST) and melting curve measurement after the qPCR amplification.

Computational analyses

Protein interaction network was built by using STRING-DB (string-db.org) with default settings (all interaction sources selected, 'medium confidence (0.400)' for minimum required interaction score, and 'query protein only' for max number of interactors to show). Ingenuity pathway analysis (IPA) was performed as described previously with minor modifications. Briefly, target proteins (DEPs) were uploaded into www.ingenuity.com (Ingenuity Systems, Inc., Redwood City, CA, USA). Core analyses were performed to identify top canonical pathways, bioprocesses and effects on functions.

Cell migration and invasion

In vitro migration and invasion assays were performed using Boyden chambers as previously described with minor modifications (20). In brief, for migration assays, cells transfected with pCMV-N-Flag-FLII or pCMV-N-Flag plasmid were resuspended in serum-free medium and seeded in the upper transwell chamber (8.0 mM pore size, Corning), and medium supplemented with 10% FCS was added to the bottom chamber, and cultured under regular conditions for 6 h, cells on the upper surface of filters were then removed and those on the under-surface were stained with 5% crystal violet. Images were captured from each membrane and the number of migrated cells was counted under a microscope. For invasion assays, similar transwell chambers coated with Matrigel (8.0 mM pore size, Chemicon) were used to analyze the invasive potential regulated by FLII.

Statistics

The Spearman correlation coefficients were calculated to determine bivariate relationships. The regression, data distribution and standard deviations were calculated by using MATLAB R2012a software package (MathWorks, Natick, MA, USA). Data are shown as mean ± standard deviation. Statistical difference was accepted at P<0.001.

Results

FLII is downregulated in lung carcinoma cells

In order to assess the possible role of FLII in the physiopathology of NSCLC, we firstly examined its expression pattern in two lung adenocarcinoma epithelial cell lines, namely H1299 and A549 cell lines. Of note, we observed that FLII was significantly downregulated in A549 and H1299 lung carcinoma cells, as compared with the human bronchial epithelial (HBE) cells (Fig. 1A and B). This result is consistent with the recent study reporting that FLII is a tumor suppressor (14).

Identification of FLII interactome in H1299 lung carcinoma cells

We then tried to identify the possible interactors of FLII in H1299 cells. Immunoprecipitation experiment was carried out using anti-Flag antibody in Flag-FLII transfected H1299 cells as described in Materials and methods. The immunoprecipitation of Flag-FLII was then confirmed by western blotting using an anti-Flag antibody with one tenth of the immunoprecipitates from the Flag-FLII transfected and the empty Flag vector transfected cells (Fig. 1C). The remaining immunoprecipitates were run on an SDS-PAGE gel that was subsequently silver-stained (Fig. 1D). The protein bands of both Flag-FLII transfected and the negative control lanes were extracted for tryptic in-gel digestion. Digested peptides were then subjected to LC-MS/MS analysis for protein identification. Proteins (263) were identified in Flag-FLII transfected lane. One hundred and thirty of them were also found in the negative control lane, thus considered to be unspecific and excluded from the candidates for further analyses. Among the 133 remaining proteins, we found FLII protein with 15 unique peptides specifically counted, further confirming the effectiveness of the immunoprecipitation. The other 132 proteins were considered to be the putative FLII-interacting partners.

Validation of interactions between FLII and the representative interacting partner

In order to confirm the interactions of FLII with its putative partners identified by mass spectrometry, we carried out experiments to confirm the co-immunoprecipitation of FLII with its several representative partners using corresponding specific antibodies, as shown in Fig. 2 for Importin β (karyopherin β), Nup88, Syncrip and EEF2. Due to the variable quality or suitability of the antibodies for co-immunoprecipitation assays, the experiments were performed either with endogenous or overexpressed Flag-tagged FLII as indicated when appropriate. Fig. 2A shows the interaction of the overexpressed FLII with Importin β using the anti-Flag antibody in Flag-FLII transfected H1299 cells (upper panel) and the interaction between endogenous FLII and Importin β in H1299 cells (lower panel). The interaction of FLII with Syncrip and Nup88 has been confirmed, respectively, by using the anti-Flag antibody in Flag-FLII transfected H1299 cells (Fig. 2B, upper panel) and by using the Nup88 antibody in H1299 cells (Fig. 2B, lower panel). As shown in Fig. 2C, we confirmed the interaction between FLII and EEF2 in Flag-FLII transfected H1299 cells. Among these confirmed representative partners, Importin β and Nup88 are, respectively, important nuclear transport receptor and component of the nuclear pore complex (NPC) involved in the nucleocytoplasmic transport. Particularly, both of them have been reported to be involved in the nuclear export of mRNA, protein and 60S ribosomal complex (2123). Syncrip is an RNA-binding protein involved in RNA metabolism, such as RNA stability, splicing, and translational control (2427). EEF2 (eukaryotic elongation factor 2), as its name indicates, is an essential factor for protein synthesis, promoting the GTP-dependent translocation of the nascent peptide from the A-site to the P-site of ribosome during the translational process (28).

Construction and functional enrichment analysis of the protein interaction network involving FLII and its interactors

The biological functions of all putative FLII binders were then categorized by consulting the genecards databases, we found that more than a half (74 out of the 132 total) of the putative FLII interactors are associated with RNA post-transcriptional modifications, RNA and protein nucleocytoplasmic transport and protein biosynthesis (Table I). To further assess the relevance of this result and to identify the biological processes and signaling pathways involving FLII and its interacting proteins, we used STRING program to build a protein interaction network comprising these putative interactors (not shown due to the impossibility of exporting a figure which could meet the publication standard). Among the total 133 proteins analyzed (nodes), 115 are connected with at least one other partner by at least one kind of interactions (edges). An important number of nodes are connected with its partner(s) by multiple edges. For the 133 nodes analyzed, the number of edges was 305, significantly superior to the expected value that is 164, resulting in an average node degree of 4.59, a clustering coefficient of 0.539, and a PPI enrichment P-value of 0. These network statistics clearly demonstrate that this protein interaction network has significantly more interactions than expected, and is therefore statistically reliable and functionally relevant. The functional enrichments in this network were determined according to biological process, molecular function, cellular component, KEGG pathways and PFAM protein domains (Table II). As expected, the results clearly indicate that the interactome of FLII is associated with RNA post-transcriptional regulations and functions, with the top ranked biological process being the gene expression, and the top molecular functions being the RNA/nucleic acid binding, supported by the top ranked KEGG pathways as RNA transport, spliceosome, protein processing and mRNA surveillance (Table II). These results, which depict a comprehensive interactome of FLII in H1299 cells, suggest strongly that FLII might play a role in the post-transcriptional regulation and function of RNA through interactions with the RNA-binding proteins and nucleoporins.

Table I

Flightless I-interacting proteins associated with RNA post-transcriptional regulation.

Table I

Flightless I-interacting proteins associated with RNA post-transcriptional regulation.

Protein IDsProtein namesGene namesFunction
IPI00893918Valyl-tRNA synthetaseVARSProtein translation
IPI00001639Karyopherin subunit β-1KPNB1Protein import into nucleus
IPI00001738Nuclear pore complex protein Nup88NUP88RNA translocation
IPI00877174 2′-5′-oligoadenylate synthase 3OAS3RNA binding
IPI00003704RNA-binding protein 4RBM4RNA binding
IPI00644708Nucleolysin TIARTIAL1RNA binding
IPI00005675NF-κ-B-repressing factorNKRFRNA binding
IPI00218187 Serine/threonine-protein phosphatase PP1-γ catalytic subunitPPP1CCRNA binding
IPI00006025Squamous cell carcinoma antigen recognized by T-cells 3SART3mRNA splicing
IPI00337397Nuclear pore complex protein Nup98NUP98mRNA export from nucleus
IPI00007818Cleavage and polyadenylation specificity factor subunit 3CPSF3mRNA export from nucleus
IPI00179713Insulin-like growth factor 2 mRNA-binding protein 2IMP2mRNA transport
IPI00010200Probable ATP-dependent RNA helicase YTHDC2YTHDC2RNA processing
IPI00010700Large proline-rich protein BAT2BAT2RNA binding
IPI00873899ATP-binding cassette sub-family F member 1ABCF1Protein translation
IPI00013877Heterogeneous nuclear ribonucleoprotein H3HNRNPH3RNA splicing, RNA processing
IPI00014474A-kinase anchor protein 8AKAP8RNA binding
IPI00015952Eukaryotic translation initiation factor 4γEIF4G2Translational initiation
IPI00016910Eukaryotic translation initiation factor 3 subunit CEIF3CTranslational initiation
IPI00017669SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1BAF57RNA binding
IPI0001812028S ribosomal protein S29DAP3RNA binding
IPI00018140Heterogeneous nuclear ribonucleoprotein QHNRPQ (Syncrip)RNA splicing, RNA processing, RNA binding
IPI00018522Protein arginine N-methyltransferase 1PRMT1RNA binding
IPI00019380Nuclear cap-binding protein subunit 1NCBP1mRNA export from nucleus, RNA splicing, RNA binding
IPI0002143526S protease regulatory subunit 7PSMC2Regulation of mRNA stability
IPI0092697726S protease regulatory subunit S10BPSMC6Regulation of mRNA stability
IPI00025491Eukaryotic initiation factor 4A-IEIF4A1RNA binding, translational initiation
IPI00026665Glutaminyl-tRNA synthetase variantQARSRNA binding, protein translation
IPI00026969SEC23-interacting proteinSEC23IPRNA binding
IPI00027107Elongation factor TuTUFMTranslational elongation, RNA binding
IPI00027415Probable ATP-dependent RNA helicase DHX36DHX36RNA binding
IPI00847793Dermcidin isoform 2DCDRNA binding
IPI00043407Trinucleotide repeat-containing gene 6C proteinTNRC6CTranslation regulation, RNA binding
IPI00384265Constitutive coactivator of PPAR-γ-like protein 1FAM120ARNA binding
IPI0007377928S ribosomal protein S35MRPS35RNA binding
IPI001001515′-3′ exoribonuclease 2XRN2RNA processing, RNA binding
IPI00106567WD repeat-containing protein 33WDR33mRNA processing, RNA binding
IPI00141318 Cytoskeleton-associated protein 4CKAP4RNA binding
IPI00163084Pre-mRNA-splicing factor SYF1XAB2mRNA processing, mRNA splicing
IPI00165434YLP motif-containing protein 1YLPM1RNA binding
IPI00168885Putative ATP-dependent RNA helicase DHX57DHX57RNA binding, RNA processing
IPI00171127 Ubiquitin-associated protein 2UBAP2RNA binding
IPI00646361Nuclear pore complex protein Nup214NUP214mRNA export from nucleus, regulation of mRNA stability
IPI00220609Nucleoporin SEH1-likeSEH1LmRNA export from nucleus
IPI00186290Elongation factor 2EEF2RNA binding
IPI00217413ATP-dependent RNA helicase DHX29DHX29RNA binding, RNA processing
IPI00217466Histone H1cHIST1H1DRNA binding
IPI00375144Serrate RNA effector molecule homologSRRTmRNA splicing, RNA binding
IPI00396171 Microtubule-associated protein 4MAP4RNA binding
IPI00941899Pyruvate kinase isozymes M1/M2PKM2RNA binding
IPI00549248NucleophosminNPM1RNA binding, regulation of translation
IPI00220834ATP-dependent DNA helicase 2 subunit 2XRCC5RNA binding
IPI00221106Splicing factor 3B subunit 2SF3B2mRNA processing, mRNA splicing, RNA binding
IPI00291939Structural maintenance of chromosomes protein 1ASMC1ARNA binding
IPI00910194Nuclear pore complex protein Nup153NUP153mRNA export from nucleus
IPI0029424228S ribosomal protein S31MRPS31RNA binding
IPI00294536Serine-threonine kinase receptor-associated proteinSTRAPmRNA processing, mRNA splicing, RNA binding
IPI00296337DNA-dependent protein kinase catalytic subunitPRKDCRNA binding
IPI00646058Scaffold attachment factor BSAFBregulation of mRNA processing, RNA binding
IPI00304692Heterogeneous nuclear ribonucleoprotein GRBMXmRNA processing, mRNA splicing
IPI00304925Heat shock 70 kDa protein 1HSPA1Aregulation of mRNA stability
IPI00853598Protein SEC13 homologSEC13mRNA transport
IPI00382470Heat shock protein HSP 90-αHSP90AA1RNA binding
IPI00940033Trinucleotide repeat-containing gene 6B proteinTNRC6BTranslation regulation
IPI00396435Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15DHX15mRNA processing, mRNA splicing
IPI00396485Elongation factor 1-α1EEF1A1translational elongation
IPI00418497Mitochondrial import inner membrane translocase subunit TIM50TIMM50RNA binding
IPI00453473Histone H4HIST1H4ARNA binding
IPI00455134Heterogeneous nuclear ribonucleoprotein A3HNRNPA3mRNA processing, mRNA splicing, RNA binding, mRNA transport
IPI00644127Isoleucyl-tRNA synthetaseIARSprotein translation
IPI00647635PERQ amino acid-rich with GYF domain-containing protein 2GIGYF2RNA binding, regulation of translation
IPI00784224zinc finger RNA-binding proteinZFRRNA binding
IPI00783872Caprin-1CAPRIN1RNA binding
IPI00871240Protein SCAF8RBM16mRNA processing, mRNA splicing

Table II

Functional enrichments in Flightless I-interacting network.

Table II

Functional enrichments in Flightless I-interacting network.

Pathway IDPathway descriptionCount in gene setFalse discovery rate
Biological process (GO)
 GO:0010467Gene expression617.44e-10
 GO:0006807Nitrogen compound metabolic process681.16e-07
 GO:0034641Cellular nitrogen compound metabolic process651.57e-07
 GO:0010033Response to organic substance419.68e-07
 GO:0043170Macromolecule metabolic process779.68e-07
Molecular function (GO)
 GO:0044822poly(A) RNA binding549.17e-30
 GO:0003723RNA binding574.45e-27
 GO:0003676Nucleic acid binding561.66e-08
 GO:0000166Nucleotide binding426.87e-08
 GO:0036094Small molecule binding434.56e-07
Cellular component (GO)
 GO:0031981Nuclear lumen681.16e-18
 GO:0005654Nucleoplasm621.89e-18
 GO:0044428Nuclear part701.89e-18
 GO:0070013Intracellular organelle lumen729.78e-12
 GO:0005634Nucleus842.02e-12
KEGG pathways
 03013RNA transport128.61e-08
 03040Spliceosome80.000217
 04141Protein processing in endoplasmic reticulum70.00885
 03015mRNA surveillance pathway50.0177
PFAM protein domains
 PF00076RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain)70.0306
 PF03144Elongation factor Tu domain 230.0466
Assessment of FLII functions in RNA trafficking and translation by high throughput sequencing

The nature of the FLII binders identified by us strongly pointed to an involvement of FLII in RNA post-transcriptional modification and trafficking in lung adenocarcinoma H1299 cells, we strived to verify this hypothesis. We have stably knocked down FLII in HBE cells by using a lentiviral shRNA vector, and in parallel, overexpressed Flag-FLII in H1299 cells by transient transfection. The effectiveness of the knockdown and overexpression of FLII respectively in HBE and H1299 cells was confirmed by both western blotting using anti-FLII and anti-Flag antibodies (Fig. 4A), and by qPCR (Fig. 3B). Furthermore, Boyden chamber assays were performed to confirm the functional impact of the alteration of FLII expression in these cells. As shown in Fig. 3C and D, FLII knockdown in HBE cells considerably stimulated their migration and invasion, whereas FLII overexpression showed inhibition ability.

High throughput sequencing was then used to assess the role of FLII in the overall biosynthesis and metabolism of the mRNA. We checked the effect of FLII knockdown and overexpression on the overall transcription of the genes. Total mRNA for both FLII knocked down HBE cells and H1299 cells overexpressing FLII, together with their corresponding control cells (respectively the empty vector-transfected and control virus-infected cells), were extracted and reverse transcribed into cDNA. High throughput sequencing was then performed to determine the mRNA species and levels in the various cells. Approximately 14,000 genes were quantified. As shown in Fig. 4, no significant effect of FLII knockdown or overexpression could be observed on the level of total mRNA levels in HBE or H1299 cells, suggesting that FLII does not affect the overall transcription of genes.

We then evaluated the impact of FLII knockdown or overexpression on the nucleocytoplasmic distribution of mRNA to investigate the possible role of FLII in the nucleocytoplasmic trafficking of mRNA. Cell fractionation experiments were performed with FLII knocked down HBE cells, FLII overexpressing H1299 cells, and the corresponding control cells described above, and the cytoplasmic and nuclear mRNA of each type of cells were separately extracted and subjected to high-throughput sequencing. The variations of the cytoplasmic and nuclear mRNA levels in FLII knockdown or over expression cells versus their control cells for each gene were analyzed as described in Materials and methods. Our results showed that both FLII overexpression in H1299 cells and its knockdown in HBE cells significantly affected the general nuclear-cytoplasmic distribution of mRNA (Fig. 6A–D). These two sets of data were then challenged against each other to compare the quantitative variation of mRNA for each single gene in the two experimental conditions (Fig. 5E). Among all genes whose nucleocytoplasmic distribution was affected by FLII overexpression or FLII knockdown, we considered as relevant those on which FLII knockdown and overexpression significantly exerted opposite effects, that is, either their level was significantly increased by FLII knockdown and decreased by FLII overexpression, or vice versa (Fig. 5E, black spots in the second and fourth quadrants). By this method, we eliminated the bias introduced by the transfection and infection systems we used in this study.

In order to examine the role of FLII on the translational process of mRNA, ribosome-nascent chain complex-associated mRNAs (RNC-mRNAs) from the same cells used for the studies described above were extracted and similarly quantified. Translatome sequencing has quantified over 12,000 genes in translating mRNA. Contrary to the results of the total mRNA levels, we found that FLII knockdown and overexpression significantly affected the level of RNC-mRNAs. Similarly as described above, we have compared the effect of FLII knockdown and overexpression on each gene analyzed and selected as potential FLII target genes those on which FLII knockdown and overexpression significantly exerted opposite effects (Fig. 6E, black spots in the second and fourth quadrants). As indicated, for 4% of the genes analyzed, the transcription was upregulated by FLII overexpression and downregulated by its knockdown, whereas for another 9.5%, the transcription was inversely regulated by FLII. These genes were then submitted to IPA program to assess their possible involvement in various biological processes and signaling pathways. Table III shows the top ranked diseases and biological functions, top networks and canonical pathways, upstream regulators, and the top regulator effect networks. As shown, the top ranked molecular and cellular functions are cellular movement and cellular growth and proliferation, and the top diseases and disorders associated with FLII regulated genes are dermatological diseases and conditions, organismal injury and abnormalities, and cancer. The top canonical pathways revealed to be the IL-12 signaling and production in macrophages and the VDR/RXR activation, whereas the top upstream regulator is estrogen receptor 1, and the top regulator effector network is NF-κB. Taken together, these results suggest that FLII might be involved in the determination of the cell fate, the regulation of development, the injury repair, the immune response and cancer by controlling the mRNA metabolism and trafficking of several groups of genes.

Table III

Functional categorization of Flightless I-regulated genes.

Table III

Functional categorization of Flightless I-regulated genes.

Top diseases and bio-functions
NameP-value# Molecules
Diseases and disorders
 Dermatological diseases and conditions 2.28E-02-7.41E-0855
 Organismal injury and abnormalities 2.68E-02-7.41E-08337
 Cancer 2.68E-02-3.48E-06329
 Reproductive system disease 2.28E-02-3.48E-06142
 Endocrine system disorders 2.28E-02-5.20E-06112
Molecular and cellular functions
 Cellular movement 2.45E-02-1.42E-0880
 Cellular growth and proliferation 2.73E-02-3.30E-07112
 Cell-to-cell signaling and interaction 2.85E-02-8.30E-0666
 Cell morphology 1.94E-02-2.46E-0539
 Cellular development 2.73E-02-2.46E-0599
Physiological system development and function
 Cardiovascular system development and function 2.71E-02-2.06E-0633
 Organismal development 2.71E-02-2.06E-0649
 Tissue development 2.73E-02-2.46E-0556
 Connective tissue development and function 2.71E-02-1.15E-0422
 Nervous system development and function 2.73E-02-1.09E-0312
Top networks
IDAssociated network functionsScore
1Cellular movement, cellular growth and proliferation, cell morphology40
2Cellular development, cellular growth and proliferation, cellular function and maintenance38
3Cellular movement, hematological system development and function, immune cell trafficking34
4Cellular movement, organismal injury and abnormalities, reproductive system disease24
5Cancer, organismal injury and abnormalities, reproductive system disease19
Top canonical pathways
NameP-valueOverlap
IL-12 signaling and production in macrophages1.08E-047.6%, 11/144
VDR/RXR activation1.14E-0410.4%, 8/77
Axonal guidance signaling3.68E-044.5%, 20/441
Production of nitric oxide and reactive oxygen species in macrophages1.26E-035.7%, 11/192
LPS/IL-1 mediated inhibition of RXR function2.37E-035.3%, 11/208
Top upstream regulators
Upstream regulatorP-value of overlapFalse discovery rate
ESR14.94E-06
Estrogen receptor6.22E-05
PDGF BB7.15E-05Activated
TNF9.44E-05
SMARCA41.49E-04Activated
Top regulator effect networks
IDRegulatorsDiseases and functionsConsistency score
1NF-κB (complex)Differentiation of cells (+1 more)3.13
2TGFB1Cell movement−6.935
3NF-κB (complex)Proliferation of cells−7.906
4ERBB2Invasion of tumor cell lines−12.522
5PDGF BBCell proliferation of tumor cell lines−15.497

Discussion

Growing evidence indicates that FLII plays important roles in multiple cellular processes (8,9,29). The characteristic feature of FLII being composed by two protein-protein interaction domains suggests that protein interactions might be key elements of the mechanisms governing its versatile functions. However, little has been done with success in the past to specifically and systematically identify the interacting partners of this protein. Two yeast two-hybrid screenings, respectively reported by Liu and yin in 1998 (15), and by Fong and de Couet in 1999 (16), have led to the identification of three homologous proteins as the interactors of FLII LRR domain, referred to as LRRFIP1 and LRRFIP2 (LRR FLII interacting protein 1 and 2) and FLAP (FLII associating protein). Unfortunately, neither study was able to depict, at the systemic level, the whole interactome of FLII, because all [4 clones, (15)] or most [12 from the 18, (16)] of the positive clones from these two screenings were derived from the genes encoding these three proteins. Besides, a few other proteins have also been independently reported to bind FLII in various contexts and with diverse functions, such as actin, BAF53, CARM1, ChREBP and Rac1 (10,15,3034). In this study, by combining immunoprecipitation and mass spectrometry analysis, we successfully identified 132 FLII interacting proteins and constructed the first putative interaction network of FLII in the cell. Interestingly, our data displayed striking directivity, in that more than a half of these candidate interactors are associated with RNA post-transcriptional regulation such as splicing, maturation, trafficking and protein synthesis (Table II). These results strongly suggested an undiscovered function of FLII in the regulation of RNA metabolism.

The target genes of FLII that we identified by using the RNC-bound RNA sequencing and IPA analysis encode mainly the proteins involved in the cellular processes such as cellular movement, cellular growth and proliferation, and the development at cellular, tissue, and organismal levels. They are also associated with the diseases and disorders such as dermatological diseases and conditions, organismal injury and abnormalities, and cancer. Moreover, the top ranked canonical pathways identified are IL-12 signaling and production in macrophages and VDR/RXR activation. These physiological and pathological processes and functions are very reminiscent of the reported functions of FLII, that are embryonic development, the regulation of wound repair, skin barrier development, the recovery after blistering and regulation of immune response, and more recently cancer (213). This confirmed, on one hand, the reliability of our approaches and the authenticity of our results, and on the other hand, suggested a novel mechanism of action of FLII, that is the overall post-transcriptional regulation of mRNA.

We find that FLII knockdown or overexpression affects at the same time the nucleus/cytoplasm ratio of mRNA and the RNC-bound mRNA amounts and species. The result of FLII interactome suggest that FLII might bind to both the proteins involved in the mRNA post-transcriptional modifications and translation, and those involved in mRNA trafficking, such as the nucleoporins. However, it remains to be determined whether FLII is involved in both the trafficking and the translational regulation of mRNA, or just play a role in mRNA trafficking which might impact on the subsequent translational process. No known RNA-binding domain has been found on FLII protein, which suggests that its function in the regulation of RNA trafficking or/and post-transcriptional regulation might be mediated by its interactions with numerous proteins involved in these biological processes. FLII might take part in the protein complexes associated with mRNA, and exerts its functions through direct and indirect interactions with various partners regulating one or several steps of mRNA post-transcriptional modification, trafficking and metabolism. For example, its interaction with Nup88 and Nup214 might be involved in the nuclear export of mRNP complexes since Nup214-Nup88 nucleoporin subcomplex has been shown to play an important role in the nuclear export of the mRNA and protein complexes (3538). Particularly, Nup214-Nup88 complex is required for the CRM1-mediated nuclear export of the 60S preribosomal subunit (36), hinting to the possible impact on the overall translation of mRNA. On the other hand, the interactions of FLII with the proteins playing roles in the translational regulation such as TIAL1 might also be functionally relevant. TIAL1, also named TIAR, was found to bind several mRNAs encoding translation factors such as eukaryotic initiation factor 4A (eIF4A) and eIF4E (translation initiation factors), eEF2 (a translation elongation factor), and c-Myc (which transcriptionally controls the expression of numerous translation regulatory proteins), suppressing their translation and participating to the global inhibition of translation machinery in response to low levels of short-wavelength uV irradiation (39). FLII possibly exerts its regulatory functions by interacting with these RNA-binding proteins through its N-terminal LRR domain, and with actin cytoskeleton via its C-terminal gelsolin related repeats, thereby mediating the traffic of the mRNP complexes along the actin filaments, and modulating the translational processes. In support of this hypothesis, we have been able to detect the interaction of the FLII LRR domain with Nup88 by in vitro GST pull-down assay (data not shown).

Guided by the results of FLII interactome obtained in this study, further studies allowing the confirmation and functional demonstration of these relevant interactions will provide new elements for the comprehensive understanding of the precise mechanisms of action of FLII.

Abbreviations:

FLII

flightless I

LRR

leucine-rich repeats

HBE

human bronchial epithelial

IPA

ingenuity pathway analysis

NSCLC

non-small cell lung cancer

RNC

ribosome-nascent chain complex

NPC

nuclear pore complex

rpkM

reads per kilobase per million reads

EEF2

eukaryotic elongation factor 2

Acknowledgments

This work was partially supported by National Program on Key Basic Research Project (973 Program) (grant no. 2011CB910700); Natural Science Foundation of China (grant no. 81322028); Natural Science Foundation of Guangdong Province (grant no. 2016A030313083; 2016A030313420); Guangzhou Science and Technology Project (20160701175); Fundamental Research Funds for the Central Universities (grant no. 21615407; 21609317); Science and Technology Program of Huadu District of Guangzhou, Guangdong Province (15-HDWS-016).

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July 2017
Volume 51 Issue 1

Print ISSN: 1019-6439
Online ISSN:1791-2423

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APA
Wang, C., Chen, K., Liao, S., Gu, W., Lian, X., Zhang, J. ... Liu, L. (2017). The flightless I protein interacts with RNA-binding proteins and is involved in the genome-wide mRNA post-transcriptional regulation in lung carcinoma cells. International Journal of Oncology, 51, 347-361. https://doi.org/10.3892/ijo.2017.3995
MLA
Wang, C., Chen, K., Liao, S., Gu, W., Lian, X., Zhang, J., Gao, X., Liu, X., Wang, T., He, Q., Zhang, G., Liu, L."The flightless I protein interacts with RNA-binding proteins and is involved in the genome-wide mRNA post-transcriptional regulation in lung carcinoma cells". International Journal of Oncology 51.1 (2017): 347-361.
Chicago
Wang, C., Chen, K., Liao, S., Gu, W., Lian, X., Zhang, J., Gao, X., Liu, X., Wang, T., He, Q., Zhang, G., Liu, L."The flightless I protein interacts with RNA-binding proteins and is involved in the genome-wide mRNA post-transcriptional regulation in lung carcinoma cells". International Journal of Oncology 51, no. 1 (2017): 347-361. https://doi.org/10.3892/ijo.2017.3995