Identification of novel splicing variants from RON proto-oncogene pre-mRNA

  • Authors:
    • Heegyum Moon
    • Sunghee Cho
    • Xiaoming Yang
    • Jianhua Zhou
    • Tiing Jen Loh
    • Xuexiu Zheng
    • Haihong Shen
  • View Affiliations

  • Published online on: September 18, 2012
  • Pages: 2217-2220
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


RON is a proto-oncogene that induces cell dissociation, migration and matrix invasion. RON∆160, a splicing variant of RON, is a natural splicing product in colon cancers that is produced through skipping of exons 5 and 6 in alternative splicing process. RON∆160 promotes cellular transformation in vitro and tumor formation in vivo. We present, here, two novel splicing variants of RON in the partial splicing events that involve exons 5 and 6. The common facts of these two novel splicing variants are that exons 4-7 are included. In addition, intron 4 is spliced whereas intron 5 is included in both variants. The difference of these two isoforms is the inclusion or skipping of intron 6. In one variant intron 6 is included, but intron 6 is skipped in another variant. These two variants should be truncated but these proteins have not yet been detected.


RON receptor tyrosine kinase is a member of the MET proto-oncogene that induces cell dissociation, migration, and matrix invasion (13). Receptor tyrosine kinases consist of a large group of cell surface proteins with unique structure and biological activities (4,5). Abnormal accumulation and activation of receptor tyrosine kinases causes the initiation and progression of a variety of malignancies including tumors derived from breast and kidneys (6,7). The invasive growth features are controlled by a genetic program which is conserved in MET family members (8,9). Mature RON is a 180-kDa heterodimeric protein that is composed of a 40-kDa extracellular α chain and 150 kDa transmembrane β chain with intrinsic protein tyrosine kinase activity (10,11). α and β chains are proteolytic products of a 180-kDa RON precursor (12). RON is activatedby macrophage-stimulating protein (MSP), a serum protein that is constitutively expressed by liver cells as an inactive form and requires proteolytis conversion for receptor binding (1315). The binding of MSP to RON results in receptor autophosphorylation and upregulation of RON kinase activity, which in turn stimulates a number of intracellular pathways mediating MSP effects (16). In normal cells MSP-induced activation is a transient event, whereas in tumor cells RON activity is often constitutively upregulated (17). RON activation causes invasive growth and motility of certain epithelial tumor cells (18,19). A number off acts suggest that RON might be involved in the progression of certain epithelial malignancies, particularly at the stage of tumor metastasis (20).

It has been shown that receptor tyrosine kinases can be activated by a variety of mechanisms, including mutation, deletion, gene rearrangement and alternative splicing of pre-mRNA (21). Various RON protein isoforms are produced through alternative splicing of pre-mRNA (12). One of the protein variants is RonΔ160, a naturally occurring oncogenic form of RON identified in human colon cancers, which is produced from a splicing mRNA transcript by exons 5 and 6. RonΔ160 causes structural changes that lead to cellular transformation in vitro and tumor growth in vivo (22,23). Another variant RonΔ170 is a 170-kDa variant is generated by skipping of exon 19 in alternative splicing (24). RonΔ170 is kinase defective and acts as dominant negative agent that inhibits tumorigenic activities mediated by oncogenic variant RONΔ160 in colon cancer cells (24). RonΔ165 is produced by an in-frame deletion of exon 11 that affects the proteolytic process which results in the accumulation of single-chain pro-RonΔ165 in the cytoplasm (25). RonΔ155 is a derived from a combined deletion of exons 5, 6 and 11, the function of RonΔ155 is similar to RonΔ160 (25,26).

Here we show two novel splicing variants of RON proto-oncogene. From the sequence analysis it is proved they are partially spliced during the splicing events of exons 5 and 6. These two variants contain either intron 5, or introns 5 and 6, in addition to exons 4–7. The proteins encoded by these variants are not identified yet.

Materials and methods

Cell culture

HeLa and C33A cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and HCT116 and HT-29 cells were maintained in RPMI-1640 medium supplemented with 10% of Fetal Bovine Serum (FBS) at 37°C in a humidified 5% CO2 condition.


Total RNA was extracted using RiboEx (GeneAll) following the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed with a RON exon 8 specific primer (5′-TGGCACATAAAAGCTG-3′) using ImProm-II™ reverse transcriptase (Promega) following the manufacturer’s instructions. RON cDNA was amplified by PCR using RON exon 4-specific primer (5′-GTTTTCCAGGTACCTATCCAAG-3′) and RON exon 7 specific primer 5′-CGTGCTAGCAGACACTCAGTC-3′). The PCR products were loaded onto 2% agarose gel and visualized by staining with ethidium bromide solution. RT-PCR bands were purified and sequenced.


Novel RT-PCR products are produced from RNA fragment covering exons 4–7

In the pre-mRNA splicing of RON gene, RonΔ160 is produced due to skipping of exons 5 and 6. In order to compare the skipping/inclusion of exons 5 and 6 in different cell lines, we performed RT-PCR reaction using primers from exons 4 and 7. As expected we observed several splicing variants that had been previously described to include or exclude exons 5 and 6 in human colon cancer HT-29 and HCT116 cells (23). In addition to these two isoforms, a variant which includes exon 6, but not exon 5, is also produced in these two cell lines, consistent with previous results (27). To our surprise, we also found that two other PCR products which we name as F1 and F2, are also produced, as shown in Fig. 1. As compared with size markers on the agarose gel, the length of F1 is ~1,100 bp; the length of F2 is ~1,000 bp. On the other hand, in cervix cancer cell C33A, none of any RT-PCR product RON RNA is produced; consistent with the fact that RON is not expressed in cervix cells. In HeLa cells, the only RT-PCR products we have observed are variants that have exon 5 and 6 inclusion. F1 and F2 products are not found in HeLa and C33A cells.

F1 and F2 fragments are partially spliced product of RON pre-mRNA

In order to characterize two unknown pre-mRNA splicing products from exons 4 and 7 of RON, we performed sequence analysis. As shown in Fig. 2, we found that the RNA fragment, which corresponds to F1 RT-PCR product, contains exons 4 and 5, intron 5, exon 6, intron 6 and exon 7 (1103Base). Another RNA fragment, which corresponds to F2 RT-PCR product, contains exons 4 and 5, intron 5, exons 6 and 7 (1000Base). Therefore F1 is a partial splicing product in which only intron 4 is spliced while F2 is a product in which both introns 4 and 6 are spliced as shown in Fig. 3. F1 and F2 intron 5 are not spliced in either F1 or F2 products. We conclude that F1 and F2 are partially spliced product of RON pre-mRNA.

Intron 5 includes a stop codon

Both novel RNA variants which correspond to F1 and F2 contain intron 5. We analyzed intron 5 sequence. We found that intron 5 contains a stop codon at the position 63-nt from 5′ splice site of exon 5 in frame with exons 1–4. Therefore it is likely that F1 and F2 will encode truncated RON isoforms from exons 1–4, but not beyond exon 4.


The full length RON precursor is 180 kDa. But it has been well-documented that RON has several isoforms that are produced from alternative splicing or protein truncation. RonΔ160 without exons 5 and 6 is oncogenic while RonΔ170 is a dominant negative isoform of RON protein. The different effects of these two isoforms suggest that it is important to investigate how many isoforms and what isoforms are present in particular cells. In this study, we used RT-PCR and identified two additional RON RNA variants both of which include intron 5. The intron 5 sequence contains stop codons that prevent these two RON RNA variants to translate proteins beyond exon 4, leading to potential truncated isoforms of RON protein (from exons 1–4). The existence and possible functions of these isoforms should be investigated in the future.


This study was supported by Mid-career Researcher Program through National Research Foundation (NRF) grant (20110000188 and 2011-0016757) funded by the Ministry of Education, Science, and Technology (MEST), Korea; a Systems Biology Infrastructure Establishment Grant provided by GIST in 2011.



Ronsin C, Muscatelli F, Mattei MG and Breathnach R: A novel putative receptor protein tyrosine kinase of the met family. Oncogene. 8:1195–1202. 1993.PubMed/NCBI


Trusolino L and Comoglio PM: Scatter-factor and semaphorin receptors: cell signalling for invasive growth. Nature reviews Cancer. 2:289–300. 2002. View Article : Google Scholar : PubMed/NCBI


Wang MH, Wang D and Chen YQ: Oncogenic and invasive potentials of human macrophage-stimulating protein receptor, the RON receptor tyrosine kinase. Carcinogenesis. 24:1291–1300. 2003. View Article : Google Scholar : PubMed/NCBI


van der Geer P, Hunter T and Lindberg RA: Receptor protein-tyrosine kinases and their signal transduction pathways. AnnuRev Cell Biol. 10:251–337. 1994.PubMed/NCBI


Robertson SC, Tynan J and Donoghue DJ: RTK mutations and human syndromes: when good receptors turn bad. Trends Genet. 16:265–271. 2000. View Article : Google Scholar


Birchmeier W, Brinkmann V, Niemann C, et al: Role of HGF/SF and c-Met in morphogenesis and metastasis of epithelial cells. Ciba Found Symp. 212:230–240. 1997.PubMed/NCBI


Niranjan B, Buluwela L, Yant J, et al: HGF/SF: a potent cytokine for mammary growth, morphogenesis and development. Development. 121:2897–2908. 1995.PubMed/NCBI


Vande Woude GF, Jeffers M, Cortner J, Alvord G, Tsarfaty I and Resau J: Met-HGF/SF: tumorigenesis, invasion and metastasis. Ciba Found Symp. 212:119–154. 1997.PubMed/NCBI


Comoglio PM, Tamagnone L and Boccaccio C: Plasminogen-related growth factor and semaphorin receptors: a gene superfamily controlling invasive growth. Exp Cell Res. 253:88–99. 1999. View Article : Google Scholar : PubMed/NCBI


Xu XM, Zhou YQ and Wang MH: Mechanisms of cytoplasmic{beta}-catenin accumulation and its involvement in tumorigenic activities mediated by oncogenic splicing variant of the receptor originated from Nantes tyrosine kinase. J Biol Chem. 280:25087–25094. 2005.


Chen YQ, Zhou YQ, Angeloni D, Kurtz AL, Qiang XZ and Wang MH: Overexpression and activation of the RON receptor tyrosine kinase in a panel of human colorectal carcinoma cell lines. Exp Cell Res. 261:229–238. 2000. View Article : Google Scholar : PubMed/NCBI


Lu Y, Yao HP and Wang MH: Multiple variants of the RON receptor tyrosine kinase: biochemical properties, tumorigenic activities, and potential drug targets. Cancer Lett. 257:157–164. 2007. View Article : Google Scholar : PubMed/NCBI


Gaudino G, Follenzi A, Naldini L, et al: RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP. EMBO J. 13:3524–3532. 1994.PubMed/NCBI


Skeel A, Yoshimura T, Showalter SD, Tanaka S, Appella E and Leonard EJ: Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity. J Exp Med. 173:1227–1234. 1991. View Article : Google Scholar : PubMed/NCBI


Bezerra JA, Witte DP, Aronow BJ and Degen SJ: Hepatocyte-specific expression of the mouse hepatocyte growth factor-like protein. Hepatology. 18:394–399. 1993.PubMed/NCBI


Danilkovitch A and Leonard EJ: Kinases involved in MSP/RON signaling. J Leukoc Biol. 65:345–348. 1999.PubMed/NCBI


Danilkovitch-Miagkova A and Zbar B: Dysregulation of Met receptor tyrosine kinase activity in invasive tumors. J Clin Invest. 109:863–867. 2002. View Article : Google Scholar : PubMed/NCBI


Wang MH, Montero-Julian FA, Dauny I and Leonard EJ: Requirement of phosphatidylinositol-3 kinase for epithelial cell migration activated by human macrophage stimulating protein. Oncogene. 13:2167–2175. 1996.PubMed/NCBI


Santoro MM, Collesi C, Grisendi S, Gaudino G and Comoglio PM: Constitutive activation of the RON gene promotes invasive growth but not transformation. Mol Cell Biol. 16:7072–7083. 1996.PubMed/NCBI


Camp ER, Liu W, Fan F, Yang A, Somcio R and Ellis LM: RON, a tyrosine kinase receptor involved in tumor progression and metastasis. Ann Surg Oncol. 12:273–281. 2005. View Article : Google Scholar : PubMed/NCBI


Rodrigues GA and Park M: Oncogenic activation of tyrosine kinases. Curr Opin Genet Dev. 4:15–24. 1994. View Article : Google Scholar : PubMed/NCBI


Wang MH, Lee W, Luo YL, Weis MT and Yao HP: Altered expression of the RON receptor tyrosine kinase in various epithelial cancers and its contribution to tumourigenic phenotypes in thyroid cancer cells. J Pathol. 213:402–411. 2007. View Article : Google Scholar


Wang MH, Kurtz AL and Chen Y: Identification of a novel splicing product of the RON receptor tyrosine kinase in human colorectal carcinoma cells. Carcinogenesis. 21:1507–1512. 2000. View Article : Google Scholar : PubMed/NCBI


Wang MH, Lao WF, Wang D, Luo YL and Yao HP: Blocking tumorigenic activities of colorectal cancer cells by a splicing RON receptor variant defective in the tyrosine kinase domain. Cancer Biol Ther. 6:1121–1129. 2007. View Article : Google Scholar : PubMed/NCBI


Collesi C, Santoro MM, Gaudino G and Comoglio PM: A splicing variant of the RON transcript induces constitutive tyrosine kinase activity and an invasive phenotype. Mol Cell Biol. 16:5518–5526. 1996.


Zhou YQ, He C, Chen YQ, Wang D and Wang MH: Altered expression of the RON receptor tyrosine kinase in primary human colorectal adenocarcinomas: generation of different splicing RON variants and their oncogenic potential. Oncogene. 22:186–197. 2003. View Article : Google Scholar


Eckerich C, Schulte A, Martens T, Zapf S, Westphal M and Lamszus K: RON receptor tyrosine kinase in human gliomas: expression, function, and identification of a novel soluble splice variant. J Neurochem. 109:969–980. 2009. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

December 2012
Volume 28 Issue 6

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
Spandidos Publications style
Moon H, Cho S, Yang X, Zhou J, Loh TJ, Zheng X and Shen H: Identification of novel splicing variants from RON proto-oncogene pre-mRNA. Oncol Rep 28: 2217-2220, 2012
Moon, H., Cho, S., Yang, X., Zhou, J., Loh, T.J., Zheng, X., & Shen, H. (2012). Identification of novel splicing variants from RON proto-oncogene pre-mRNA. Oncology Reports, 28, 2217-2220.
Moon, H., Cho, S., Yang, X., Zhou, J., Loh, T. J., Zheng, X., Shen, H."Identification of novel splicing variants from RON proto-oncogene pre-mRNA". Oncology Reports 28.6 (2012): 2217-2220.
Moon, H., Cho, S., Yang, X., Zhou, J., Loh, T. J., Zheng, X., Shen, H."Identification of novel splicing variants from RON proto-oncogene pre-mRNA". Oncology Reports 28, no. 6 (2012): 2217-2220.