UBE3D Regulates mRNA 3′-End Processing and Maintains Adipogenic Potential in 3T3-L1 Cells

ABSTRACT

We have previously described the role of an essential Saccharomyces cerevisiae gene, important for cleavage and polyadenylation 1 (IPA1), in the regulation of gene expression through its interaction with Ysh1, the endonuclease subunit of the mRNA 3′-end processing complex. Through a similar mechanism, the mammalian homolog ubiquitin protein ligase E3D (UBE3D) promotes the migratory and invasive potential of breast cancer cells, but its role in the regulation of gene expression during normal cellular differentiation has not previously been described. In this study, we show that CRISPR/Cas9-mediated knockout of Ube3d in 3T3-L1 cells blocks their ability to differentiate into mature adipocytes. Consistent with previous studies in other cell types, Ube3d knockout leads to decreased levels of CPSF73 and global changes in cellular mRNAs indicative of a loss of 3′-end processing capacity. Ube3d knockout cells also display decreased expression of known preadipogenic markers. Overexpression of either UBE3D or CPSF73 rescues the differentiation defect and partially restores protein levels of these markers. These results support a model in which UBE3D is necessary for the maintenance of the adipocyte-committed state via its regulation of the mRNA 3′-end processing machinery.

KEYWORDS:

• RNA processing
• adipogenesis
• cell differentiation

Declaration of Interests

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

We acknowledge the Tufts University High Performance Compute Cluster (https://it.tufts.edu/high-performance-computing), which was utilized for the research reported in this paper. We thank Rebecca E. Batorsky at Tufts Technology Services for the help of RNA-seq data processing and Joel Graber for thoughtful discussion regarding analysis of RNA-seq data, as well as implementation of the APAlyzer and PAQR platforms. Matthew Zunitch provided invaluable expertise and technical assistance with immunofluorescent staining and subsequent quantification, for which we are deeply grateful. We also thank all members of the Moore laboratory for discussions and comments on the manuscript.

This work was supported by grants from the National Institutes of Health, USA (5R01AI152337 to C.L.M.), and the National Science Foundation (MCB-1244043 to C.L.M.).

Contributions of the authors to this article using the Contributor Roles Taxonomy (CRediT) are as follows: Daniel Heller-Trulli, Conceptualization (supporting), Formal Analysis (lead), Investigation (equal), Software (lead), Visualization (lead), Writing – Original Draft Preparation (lead), and Writing – Review & Editing (supporting). Huiyun Liu, Conceptualization (lead), Investigation (equal), Visualization (supporting), and Writing – Review & Editing (supporting). Srimoyee Mukherjee, Investigation (supporting) and Writing – Review & Editing (supporting). Claire L. Moore, Conceptualization (supporting), Funding Acquisition (lead), Supervision (lead), and Writing – Review & Editing (lead).