CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2 and promotes growth and metastasis of pancreatic cancer

Yi Huang,Long Huang,Cheng-Yu Liao,Jian-Fei Hu,Shi Chen,Can Yang,Yi-Feng Tian,Jing-Jing Pan,Zu-Wei Wang,Jiang-Zhi Chen,Jia-Qiang Zhang,Yao-Dong Wang,Qian Zhan,Bai-Yong Shen

doi:10.1186/s13045-021-01072-8

Abstract

BackgroundBoth aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear.MethodsRNA sequencing (RNA-seq) was performed using 15 pairs of pancreatic ductal adenocarcinoma (PDAC) tissues and corresponding normal tissues, and Cdc2-like kinases 1 (CLK1) was identified as a significantly upregulated alternative splicing related gene. Real-time quantitative PCR (qPCR) and western blotting were applied to determine the CLK1 levels. The prognostic value of CLK1 was elucidated by Immunohistochemistry (IHC) analyses in two independent PDAC cohorts. The functional characterizations and mechanistic insights of CLK1 in PDAC growth and metastasis were evaluated with PDAC cell lines and nude mice. SR-like splicing factors5250-Ser (SRSF5250-Ser) was identified as an important target phosphorylation site by phosphorylation mass spectrometry. Through transcriptome sequencing, Methyltransferase-like 14exon10 (METTL14exon10) and Cyclin L2exon6.3 skipping were identified as key alternative splicing events regulated by the CLK1-SRSF5 axis. RIP assays, RNA-pulldown and CLIP-qPCR were performed to confirm molecular interactions and the precise binding sites. The roles of the shift of METTL14exon 10 and Cyclin L2exon6.3 skipping were surveyed.ResultsCLK1 expression was significantly increased in PDAC tissues at both the mRNA and protein levels. High CLK1 expression was associated with poor prognosis. Elevated CLK1 expression promoted growth and metastasis of PC cells in vitro and in vivo. Mechanistically, CLK1 enhanced phosphorylation on SRSF5250-Ser, which inhibited METTL14exon10 skipping while promoted Cyclin L2exon6.3 skipping. In addition, aberrant METTL14exon 10 skipping enhanced the N6-methyladenosine modification level and metastasis, while aberrant Cyclin L2exon6.3 promoted proliferation of PDAC cells.ConclusionsThe CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2, which promotes growth and metastasis and regulates m6A methylation of PDAC cells. This study suggests the potential prognostic value and therapeutic targeting of this pathway in PDAC patients.

Highlights

Both aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear
Elevated expression of Cdc2-like kinases 1 (CLK1) in pancreatic cancer tissues correlated with worse prognosis of pancreatic ductal adenocarcinoma (PDAC) patients High-throughput sequencing was performed in specimens consisting of 15 pairs of paired PDAC and pancreatic normal tissues
While PANC-1 cells exhibited the highest level of CLK1, BxCP-3 cells showed the lowest level of CLK1 among these cancer cell lines (Fig. 1g)

Summary

Introduction

Both aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear. AS has been shown to play a crucial role in the complicated regulation of protein functions and the dysregulation of splicing factors is closely associated with initiation and progression of human cancers [7]. While the average human gene just produces three or more alternatively spliced mRNA isoforms, the tumor cells produce a significant redundant of splice variants [13, 14] These atypical splice variants reside at the core of human cancer development and progression. Since m6A modifications functions critically in homeostasis maintenance of cells, aberrant m6A modifications were widely reported to be associated with tumorgenesis and tumor development, in which process, the protein effects of “writer” METTL14 are significant. Exploration of METTL14 splicing mediated m6A RNA methylation in tumorigenesis and cancer development may be potentially interesting

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