Investigation for the Molecular Mechanism Involved in the Expression and Impact of Alternatively-Spliced Srsf3 and Map4k4 Variants on Brown Adipogenesis

Adipocytes compose prominent tissues that maintain the energy homeostasis of mammals. White adipose tissue (WAT) efficiently reserves the triglyceride as energy source, whereas brown adipose tissue (BAT) expenditures the energy through non-shivering thermogenesis in small rodents and infants. However, the development and function of BAT in adult human is largely unknown. Apart from the function, the knowledge of regulatory mechanism and factor involved in the development of BAT is also sparse. Alternative splicing (AS) functions as a prevalent mechanism in expanding the genetic diversity of eukaryotic cells. Approximately 95% of human genes generate more than one transcript through this meticulously controlled process. The interplay between splicing factors and cis-elements constitutes the molecular mechanism in programming the splicing profile in spatiotemporal manners. Deep RNA-sequencing (RNA-seq) is a well-established approach in identifying the spatialtemporally-regulated splicing network. In the previous works, we focused on the influence of RNA binding motif protein 4, a well-studied splicing factor, on the brown adipocyte-related splicing network. By using RNA-seq approach, the differential splicing profiles was identified in the RBM4-/- brown adipose tissues ac compared to the wild-type littermates. The impact of RBM4 and its specific candidates on brown adipogenesis was further characterized and the results were published in the BBA-Molecular Cell Research、RNA Biology、Scientific Reports 與BBA-Gene Regulatory Mechanism journal. By using RNA-seq approach, our recent results indicated the differential splicing profiles of Serine/arginine-rich splicing factor 3 (SRSF3) and MAPK kinase kinase kinase isoform 4 (MAP4K4) in the mature BAT as compared to that of embryonic BATs. Moreover, the differential influence of SRSF3 and MAP4K4 on UCP-1 is observed in brown adipocytes. Five major issues will be addressed in this project. First, the regulatory mechanism involved in the expression of alternatively-spliced SRSF3 and MAP4K4 transcripts during brown adipogenesis will be characterized. Second, the potential candidate of SRSF3 involved in brown adipogenesis will be further identified by using CLIP-seq approach. Third, the effect of MAP4K4 variants on the downstream signaling involved in the differentiation of brown adipocytes will be evaluated. Fourth, the impact of SRSF3 and MAP4K4 on the development or function of BATs will be characterized. Last, the SRSF3 or MAP4K4 knockout mice or C3H10T1/2 cells will be established. I anticipate the result of this project could bring a novel scope on the regulatory mechanism involved in the alternative splicing of SRSF3 and MAP4K4 gene during brown adipogenesis. The influence of SRSF3 or MAP4K4 variants on the development or function of BATs will be subsequently demonstrated. Moreover, these results could be applied to study the potential impact of SRSF3 or MAP4K4 variants on other cell type, tissue or related disease.