Wei Han, Qing Xia, Bin Yin, and Xiao-zhong Peng*

1State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China

2State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing 100850, China

RIBOTRAP is an immnoaffinity tool to identify RNA-binding proteins (RBPs) that specifically bind to mRNAs of interest.1Multiple RBPs may associate with a given RNA and vice versa, each RBP may associate with a number of RNAs, and the RNA-RBP interactions are responsive to regulatory signals.2Although the characterization of mRNAs targeted by a specific RBP has been accelerated by the application of RBP immunoprecipitation-Chip (RIP-Chip),3reliable methods to describe the endogenous assembly of ribonucleoproteins (RNPs) are needed.

We previously reported that poly(C)-binding protein 2 (PCBP2), an important member in heterogeneous nuclear ribonucleoprotein (hnRNP) family, plays an oncoprotein-like role in human glioma tissues and cell lines. Using RIP-Chip analysis, we identified 35 mRNAs that preferentially bind PCBP2. Four-and-a-half LIM domain 3 (FHL3) has been confirmed as a novel binding target of PCBP2 and can inhibit glioma cells proliferation and anti-apoptosis ability. We further found that PCBP2 binds to the FHL3 mRNA 3’ untranslated region (UTR), and negatively regulates FHL3 expression through affecting its mRNA stability.4

In addition, FHL3 is a negative regulator of proliferation in breast cancer and hepatocellular carcinoma (HCC).5,6FHL3 protein was also reported to inhibit hypoxia-inducible factor 1 transcriptional activity in HCC cells and identified as a novel Angiogenin binding partner,7,8which suggested that FHL3 will be a key gene involved in cancer progression. Therefore it is meaningful to explore the regulatory mechanism of FHL3 itself, such as in the posttranscriptional level.

In this study, we applied a Ribotrap methodology in FHL3 mRNA 3’UTR specific purification of a target RNP by biotin pull-down assay and identified the proteins associated with FHL3 3’UTR in glioma cells, including PCBP2, by mass spectrometry, so as to lay the foundation for exploring the upstream molecular mechanism of FHL3 in glioma.

MATERIALS AND METHODS

Glioma cell culture and plasmids transfection

Human glioma cell line T98G was purchased from American Type Culture Collection and cultured according to the guidelines recommended by the center. Biotin acceptor peptide (BAP)-PCBP2 was constructed as previously described.4The plasmid was purified using the EndoFree Plasmid Maxi Kit (QIAGEN, Germantown, MD, USA) and transfected into T98G cells using VigoFect (Vigorous Biotechnology, Beijing).

Western blot

Whole-cell extracts were obtained by lysing cells in TNTE buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 10 mmol/L sodium pyrophosphate, 0.5% Triton X-100, 1 mmol/L sodium vanadate, and 25 mmol/L sodium fluoride) containing protease inhibitors (5 μg/ml phenylmethanesulfonyl fluoride, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, and 0.5 μg/ml aprotinin). The protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane (Amersham, USA) and detected using polyclonal rabbit anti-PCBP2 (1:2000, Beijing Aviva Systems Biology or MBL International Corporation), rabbit anti-FHL3 (1:500, Proteintech Group, Chicago, IL, USA), anti-PTBP1 (1:3000, generated by our laboratory) or mouse anti-β-actin (1:5000, Sigma, St. Louis, MO, USA) antibodies.

Biotin pull-down

Fragments located in the 3’UTR-A or -B of FHL3 mRNA were constructed into the pGEM-3zf vector containing a T7 promoter. The primers sequences were shown as follows: 3’UTR-A, (forward) 5’-CGGGATCCCAGGACTGTGGCTCCTTTTC-3’, (reverse) 5’-CCCAAGCTTGCACCCATAGGAGACCTGAA-3’; 3’UTR-B, (forward) 5’-CGGGATCCGGACTGTTCTCAGGCTTGAC-3’, (reverse) 5’-CCCAAGCTTCACACCGCTTTATTGCAGAATC-3’. The biotin-labeled sense RNA probes were synthesized in vitro using T7 RNA polymerase (Takara, Dalian, China). Cytoplasmic cell extracts were isolated from T98G cells using nuclear protein extraction reagents (NE-PER) (Thermo Scientific, Waltham, MA, USA). RNA affinity capture was subsequently conducted with streptavidinsepharose beads as previously described.9

Immunoprecipitation

For the immunoprecipitation assay, equal amounts (1200- 1500 μg) of the cell lysates were incubated with 1/100 (wt/wt) of rabbit IgG or the anti-PTBP1 antibody overnight at 4°C. Subsequently, the lysates were incubated with Protein A sepharose (Roche Co., Mannheim, Germany) while rotating for 3 hours at 4°C. Immunoprecipitates were washed four times with lysis buffer without the protease inhibitor, eluted in 20 μl of 6×loading buffer at 98°C and analyzed by immunoblot analysis.

Silver staining

To identifiy all the FHL3 mRNA 3’UTR specific binding proteins, the transcribed in vitro biotin-labeled RNA probes, FHL3 3’UTR-A and 3’UTR-B, were used as the templates for assembling bound proteins from T98G cells in biotin pull-down assay. Sliver staining presents the proteins associated with FHL3 mRNA 3’UTR-A or 3’UTR-B (the experimental group) compared with negative control. The protein samples from biotin pull-down were separated using 10% SDS-PAGE, and the gel was then incubated with the stationary liquid (50% methanol, 5% acetic acid) for 20 minutes. After washed with washing buffer for 10 minutes, the gel was further incubated with sensitization buffer (0.05 g Na2S2O3, 250 ml H2O) for 1 minute. It was later incubated with silver staining buffer for 20 minutes at 4°C and then with color reagent (4 g Na2CO3, 80 μl formaldehyde, 200 ml H2O) for 6 minutes.

Liquid chromatography-tandem mass spectrometry

The stained protein bands after silver staining on the gel were carved, digested, and analyzed by liquid chromatography- tandem mass spectrometry (LC-MS/MS) in the National Center of Biomedical Analysis. Sequence analysis was performed with MASCOT (version 2.2, Matrix Sciences, Boston, MA, USA) using the non-redundant protein database from Mascot’s web site (http://www.matrixscience. com/help/data_file_help.html). The bound proteins were screened according to ions scores, in which a number greater than 38 indicates identity or extensive homology (P<0.05).

Bioinformatical analysis

We utilized gene ontology (GO) to investigate the nature of the FHL3 3’UTR associated proteins identified by LC-MS/MS. The GO terms were analyzed using the web-based tool CapitalBio molecule annotation system (MAS V3.0, CapitalBio Corporation, Beijing, China). Molecular function, biological process and cellular component of the candidate proteins were analyzed.

RESULTS

Effect of PCBP2 on target FHL3 expression

The expression of FHL3 was decreased after overexpressing BAP-PCBP2 fusion construct in T98G glioma cell line (Fig. 1A). The fragments A and B located in the 3’UTR of FHL3 mRNA were predicted to contain potential binding sites by bioinformatical analysis (Fig. 1B). PCBP2 was not detected in the FHL3 3’UTR-B pull-down complex, while FHL3 3’UTR-A showed a strong binding of PCBP2 (Fig. 1C).

Candidate binding proteins to FHL3 3’UTR identified by LC-MS/MS

18 bands were selected in the experimental group (Fig. 2). Based on the ions scores and MASCOT database, 22 candidate proteins were identified after eliminating the same proteins (Table 1). Among the molecular functions, approximately 86.4% (19/22) and 50.0% (11/22) of the candidate proteins were related to the functions of protein binding and RNA binding, respectively. More than 50% of the proteins were localized in the nucleus (16/22, 72.7%) or cytoplasm (12/22, 54.5%) according to the result of cellular component analysis. The biological processes showed enrichment in RNA processing, export and stability (40.9% gene frequency), regulation of transcription (27.3% gene frequency), and cell grow and apoptosis (27.3% gene frequency) (Fig 3).

Possible interaction between PTBP1 and PCBP2

Among the 22 proteins identified by LC-MS/MS, we discovered another member of hnRNP family, PTBP1. It was also reported up-regulated in gliomas. With biotin pull-down assay, we confirmed that PTBP1 could combine with FHL3 3’UTR (Fig 4A). PTBP1 also interacted with P CBP2 as shown by immunoprecipitation (Fig 4B).

Figure 1. PCBP2 inhibits expression of FHL3 and binds to FHL3 mRNA 3’ untranslated region (UTR). A. Representative Western blot shows the decreased FHL3 protein level in biotin acceptor peptide (BAP)-PCBP2 overexpressing T98G cells. β-actin was used as a loading control. B. Schematic of FHL3 mRNA 3’UTR demonstrates the specific fragments A and B used as templates for the synthesis of biotin-labeled RNAs and the nucleotide positions amplified by PCR. C. Biotin pull-down analysis of complexes formed in vitro using biotin-labeled FHL3 3’UTR-A or 3’UTR-B and T98G whole-cell lysates and detected using anti-PCBP2 antibody. A nonsense sequence from pGEM-3zf vector was included as negative control.

Table 1. The result of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for FHL3 3’UTR associated proteins

Figure 2. Sliver staining of proteins which combined with FHL3 3’UTR-A, FHL3 3’UTR-B, or negative control by biotin pull-down. Input from T98G glioma cell lysate was used as a positive control. The arrows show the carved bands to be identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The numbers of carved bands (1-18) correspond with the results of LC-MS/MS in Table 1.

DISCUSSION

The 3’UTR of eukaryotic transcripts plays an important role in regulating gene expression in posttranscriptional level. In this region exist the 3’ end processing signals which instruct the 3’ end processing. 3’UTR not only controls mRNA stability and the rate of mRNA degradation, but also helps to determine mRNA localization, regulate translation initiation and translation efficiency as well as translation time. The interactions between 3’UTR and trans-acting factors can influence the expression of one or more genes, thus possibly leading to the occurrence of diseases. Regulatory sequence of mRNA 3’UTR and the specific bound proteins will become new molecular targets in drug design.10

In our previous study, we have confirmed that knockdown of PCBP2 can increase FHL3 protein level through stabilizing FHL3 mRNA.4In this study, 22 proteins associated with FHL3 3’UTR were identified by LC-MS/MS. Among them, the seventh band represented the PCBP2 protein, which was consistent with the results of our biotin pull-down assay. Eleven RBPs that we are most interested in in Table 1 are SFPQ, IGF2BP1, KHDRBS1, IGF2BP3, SYNCRIP, IGF2BP2, PTBP1, SERBP1, HNRNPH1, PCBP2, and HNRNPA2/B1. These proteins regulate RNA splicing, transport, stability and translation shuttling between the nucleus and cytoplasm. Other proteins also have important functions in cellular biological process. According to GO analysis, ILF3, TRIM28, RUVBL1, and PHB are involved in the regulation of transcription; CSE1L, NDUFS3 and EIF4G2 will modulate cell proliferation or apoptosis; while EEF1A1 and GNB2L1 are related to GTPase activity.

It is notable that 4 hnRNP proteins, PTBP1, hnRNP H1, hnRNP A2/B1, and PCBP2 were detected by LS-MS/MS in this study. Similar to PCBP2, the other 3 proteins are also overexpressed in glioblastomas and are correlated with poor prognosis.11-13

Figure 3. The candidate proteins are analyzed in terms of their gene ontology (GO) using molecule annotation system. FHL3 3’UTR-associated 22 proteins were annotated to the GO term “molecular function” (A), “cellular component” (B) and “biological process” (C) using the web-based tool MAS V3.0. The first three terms related to the largest gene number in each ontology were presented in the histogram.

Figure 4. PTBP1 can bind to FHL3 mRNA 3’UTR and interact with PCBP2. A. Biotin pull-down analysis of complexes formed in vitro using biotin-labeled FHL3 3’UTR-A or FHL3 3’UTR-B and T98G whole-cell lysates and detected using anti-PTBP1 antibody. B. T98G cell lysates were immunoprecipitated with IgG (control) and anti-PTBP1 antibody (experimental), then the precipitates were analyzed by Western blot with anti-PTBP1 and anti-PCBP2 antibodies. Immunoglobulin heavy and light chains are also shown.

We verified by using biotin pull-down that PTBP1 associated with FHL3 mRNA 3’UTR. Not only 3’UTR-A, but also 3’UTR-B could bind to PTBP1. A specific differential band at the FHL3 3’UTR-B paralleling to the fourth band compared to negative control was found in the silver staining PAGE gel. Immunoprecipitation experiments also revealed that PTBP1 can interact with PCBP2. We thus hypothesized that PTBP1 and PCBP2 may work together to involve in posttranscriptional regulation of FHL3 by forming a protein complex in glioma cells. In fact, PTBP1 has been reported to be associated with many hnRNP members. The complexes with PTBP1 and PCBP2 are required for the hepatitis C virus internal ribosome entry site-dependent initiation in mammalian cells.14,15The specific cooperative interactions among PTBP1/2, hnRNP H/F and KH-type splicing-regulatory protein regulate the splicing of c-src N1 exon in neuronal cells by binding to an intronic cluster of RNA regulatory elements.16In glioma cells, PTBP1 and the alternative splicing repressors hnRNP A1/A2 influence pyruvate kinase isoform expression and the switch to aerobic glycolysis.17

In conclusion, PCBP2, PTBP1 and other RBPs can bind to FHL3 3’UTR, forming a protein complex, and co-regulate FHL3 expression, thus affecting the proliferation and apoptosis of gliomas.

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