The CTBP2-PCIF1 complex regulates m6Am modification of mRNA in head and neck squamous cell carcinoma
Kang Li, Jie Chen, Caihua Zhang, Maosheng Cheng, Shuang Chen, Wei Song, Chunlong Yang, Rongsong Ling, Zhi Chen, Xiaochen Wang, Gan Xiong, Jieyi Ma, Yan Zhu, Quan Yuan, Qi Liu, Liang Peng, Qianming Chen, Demeng Chen
Kang Li, Jie Chen, Caihua Zhang, Maosheng Cheng, Shuang Chen, Wei Song, Chunlong Yang, Rongsong Ling, Zhi Chen, Xiaochen Wang, Gan Xiong, Jieyi Ma, Yan Zhu, Quan Yuan, Qi Liu, Liang Peng, Qianming Chen, Demeng Chen
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Research Article Cell biology Oncology

The CTBP2-PCIF1 complex regulates m6Am modification of mRNA in head and neck squamous cell carcinoma

Abstract

PCIF1 can mediate the methylation of N6,2′-O-dimethyladenosine (m6Am) in mRNA. Yet, the detailed interplay between PCIF1 and the potential cofactors and its pathological significance remain elusive. Here, we demonstrated that PCIF1-mediated cap mRNA m6Am modification promoted head and neck squamous cell carcinoma progression both in vitro and in vivo. CTBP2 was identified as a cofactor of PCIF1 to catalyze m6Am deposition on mRNA. CLIP-Seq data demonstrated that CTBP2 bound to similar mRNAs as compared with PCIF1. We then used the m6Am-Seq method to profile the mRNA m6Am site at single-base resolution and found that mRNA of TET2, a well-known tumor suppressor, was a major target substrate of the PCIF1-CTBP2 complex. Mechanistically, knockout of CTBP2 reduced PCIF1 occupancy on TET2 mRNA, and the PCIF1-CTBP2 complex negatively regulated the translation of TET2 mRNA. Collectively, our study demonstrates the oncogenic function of the epitranscriptome regulator PCIF1-CTBP2 complex, highlighting the importance of the m6Am modification in tumor progression.

Authors

Kang Li, Jie Chen, Caihua Zhang, Maosheng Cheng, Shuang Chen, Wei Song, Chunlong Yang, Rongsong Ling, Zhi Chen, Xiaochen Wang, Gan Xiong, Jieyi Ma, Yan Zhu, Quan Yuan, Qi Liu, Liang Peng, Qianming Chen, Demeng Chen

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Figure 3

CTBP2 and PCIF1 interact and co-catalyze m6Am modification on mRNA.

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CTBP2 and PCIF1 interact and co-catalyze m6Am modification on mRNA. (A) ...
(A) The CTBP2 peptide identified in MS analysis after coimmunoprecipitation assay. (B) Immunoprecipitation and immunoblotting analysis of the interaction between CTBP2 and PCIF1. (C) GST pull-down assay of recombinant GST-CTBP2 and FLAG-PCIF1 proteins. The coprecipitated CTBP2 and PCIF1 proteins were detected by Western blot with anti-GST and anti-FLAG antibodies. (D) Strategy of CTBP2 variant proteins for mapping interaction domains with PCIF1. (E and F) Mapping of the binding domain of CTBP2 shows the potential binding site. The lysates were immunoprecipitated with anti-FLAG antibodies, followed by immunoblotting with anti-HA and anti-FLAG antibodies. Anti-IgG antibody was used as a negative control. (G) Analysis of colocalization of CTBP2 (green) with PCIF1 (red) by double immunofluorescence staining in control cells, PCIF1-KO cells, and CTBP2-KO cells. Nuclei are stained with DAPI (blue). Scale bar: 2 μm. (H–J) Pearson’s correlation analysis showed a positive correlation between CTBP2 and PCIF1 expression according to TCGA data (H, n = 520), FAH-SYSU-Cohort1 (I, n = 57), and HS-SYSU-Cohort2 (J, n = 40). P value was calculated by Pearson’s correlation coefficient test. (K) Western blotting analyses detecting the CTBP2 expression in SCC25 control cells and CTBP2-KO cells. (L and M) LC-MS/MS quantification of the m6A/A ratio (L) and m6Am/A ratio (M) in mRNA obtained from HOK cells, control cells, PCIF1-KO cells, and CTBP2-KO cells (n = 3). P > 0.05, ***P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.