Cleavage and polyadenylation factors are potential regulators of adipogenesis

Abstract

Objective: Alternative polyadenylation (APA) is a co-transcriptional process that leads to isoform diversity in the 3' ends of mRNAs. APA is known to occur during differentiation, and its dysregulation is observed in diseases like cancer and autoimmune disorders. It has been previously reported that differentiation of 3T3-L1 cells to adipocytes leads to an overall lengthening of mRNAs, but the proteins involved in this regulation have not been identified. The expression levels of subunits of the cleavage and polyadenylation (C/P) complex can regulate the choice of poly(A) site, which in turn can affect different cellular activities. In this paper, we studied the change in levels of C/P proteins during 3T3-L1 differentiation.

Results: We observed that while the RNA expression of these proteins is unchanged during differentiation, the protein levels of some subunits do change, including a decrease in levels of CPSF73, the nuclease that cuts at the poly(A) site. However, overexpression of CPSF73 alone does not affect the efficiency and rate of differentiation.

Keywords: 3T3-L1; Adipogenesis; Cleavage and polyadenylation.

Fig. 1

Protein levels of cleavage and polyadenylation factors change during 3T3-L1 differentiation. (A) Representative cropped blots of protein levels of C/P factors from each complex are shown, along with quantifications (n = 3, mean ± SD) of protein levels at Day 7 relative to Day 0, where values above 1 indicate increased levels and values below 1 indicate decreased levels during differentiation. The protein levels were normalized to total protein levels. Significance testing was done using student’s unpaired two-tailed t-test with Welch’s correction, with p-values less than 0.05 considered significant and denoted with an asterisk. Full-length total-protein stained gels and uncropped blots are presented in Supplementary Figure S1. (B) Determination of change in mRNA expression of C/P factors during differentiation with threshold of log2-fold change of 1 and p-adjusted value < 0.05. P-adjusted values less than 0.05 are labelled with an asterisk.

Fig. 2

Overexpression of CPSF73 alone is not sufficient to affect differentiation efficiency. (A) CPSF73 is overexpressed in both undifferentiated and differentiated adipocytes compared to its control. Three replicates from Day 0 and from Day 8 of differentiation are shown. The FABP4 marker indicates successful differentiation, and the total protein stain (bottom panel) serves as loading control. Full-length total-protein stained gels and uncropped blots are presented in Supplementary Figure S2. (B) Oil Red O staining of Dox-induced control and CPSF73-ovexpressing 3T3-L1 cells at Day 0 and Day 8 of differentiation does not show a change in differentiation efficiency due to overexpression of CPSF73. (C) Overexpression of CPSF73 does not change the rate of 3T3-L1 differentiation. Expression of mRNAs of adipogenesis markers normalized to Rpl13A are shown relative to Day 0 of differentiation.