Abstract
Background: Cyclin D1 regulates cyclin-dependent protein kinase activity of the cell cycle, and cyclin D1 alternative splicing generates a cyclin D1b isoform, acting as a mediator of aberrant cellular proliferation. As alternative splicing processes are sensitive to mechanical stimuli, whether the alternative splicing of cyclin D1 is regulated by mechanical stress and what kinds of factors may act as the regulator of mechano-induced alternative splicing remain unknown. Methods: The alternative splicing of Cyclin D1 was examined using reverse transcription polymerase chain reaction (RT-PCR) in osteoblast cell lines and keratinocyte cells loaded by a cyclic stretch. The expression of splicing factors and switching defective/sucrose non-fermenting (SWI/SNF) complex subunits were detected in stretched cells using real-time quantitative PCR (RT-qPCR). The protein interaction was tested by co-immunoprecipitation assay (Co-IP). Results: Cyclin D1 expression decreased with its splice variant upregulated in stretched cells. Serine/arginine-rich splicing factor 1 (SRSF1) and SWI/SNF complex subunit Brahma-related gene-1-associated factor 57 (BAF57), also named SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1 (SMARCE1), could respond to mechanical stimuli. Overexpression and knockdown experiments indicated the BAF57/SMARCE1 is probably a critical factor regulating the alternative splicing of cyclin D1. Co-IP showed an interaction between BAF57/SMARCE1 and SRSF1, implying a possible underlying mechanism of the regulator role of BAF57/SMARCE1 in the splicing process of cyclin D1. Conclusions: The splicing factor SRSF1 and BAF57/SMARCE1 are possibly responsible for the mechanical stress-induced alternative splicing of cyclin D1.
Highlights
Gene alternative splicing is a physiologic process that enormously enriches the proteome, which plays an important role in organ development and function disorder
Our previous study provided that insulin-like growth factor-1 (IGF-1) gene alternative splicing can be regulated by mechanical stress in osteoblast cells [9]
The interaction between splicing factors and splicing regulatory elements is crucial for the alternative splicing process, the research on splicing factors is insufficient for elucidating the molecular mechanisms whereby the gene alternative splicing is regulated by mechanical stress
Summary
Gene alternative splicing is a physiologic process that enormously enriches the proteome, which plays an important role in organ development and function disorder. Except for intrinsic factors, extracellular stimulus regulates gene alternative splicing, such as mechanical stimuli. Our previous study provided that insulin-like growth factor-1 (IGF-1) gene alternative splicing can be regulated by mechanical stress in osteoblast cells [9]. Tyagi [16] further found that Brm is associated with nascent pre-mRNPs, and influences the levels of alternatively processed mRNAs through the microarrays experiments This accumulating evidence indicates that SWI/SNF acts at the transcriptional level to regulate the amount of mRNAs synthesized from a given promoter, and exerts itself at the post-transcriptional level to modulate the type of alternative transcript produced. In this study, we screen the subunits of the SWI/SNF complex’s response to mechanical stress, and investigate the functions of the subunits on regulating mechanotransduction-mediated alternative splicing
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