RESUMO
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.
Assuntos
Adipócitos , Adipogenia , Ubiquitina-Proteína Ligases , Animais , Camundongos , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Mamíferos/genética , Mamíferos/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cleavage by the endonuclease CPSF73 and polyadenylation of nascent RNA is an essential step in co-transcriptional mRNA maturation. Recent work has surprisingly identified CPSF73 as a promising drug target for inhibiting the growth of specific cancers, triggering further studies on understanding CPSF73 regulation and functions in cells. Here, we report that a HECT-like E3 ligase, UBE3D, participates in stabilizing CPFS73 protein by preventing its ubiquitin-mediated degradation by the proteasome. Depletion of UBE3D leads to CPSF73 downregulation, a pre-mRNA cleavage defect, and dysregulated gene expression in cells. UBE3D dysfunction or chemical inactivation of CPSF73 inhibited migration and invasion as well as stem cell renewal phenotypes in vitro in triple-negative breast cancer cells. In addition, genetic overexpression of CPSF73 promoted breast cancer stemness and knocking down CPSF73 inhibited stem cell renewal properties. Together, our findings indicate that targeting the pre-mRNA processing nuclease CPSF73 has potential for breast cancer therapy.
RESUMO
Mutation of the essential yeast protein Ipa1 has previously been demonstrated to cause defects in pre-mRNA 3' end processing and growth, but the mechanism underlying these defects was not clear. In this study, we show that the ipa1-1 mutation causes a striking depletion of Ysh1, the evolutionarily conserved endonuclease subunit of the 19-subunit mRNA Cleavage/Polyadenylation (C/P) complex, but does not decrease other C/P subunits. YSH1 overexpression rescues both the growth and 3' end processing defects of the ipa1-1 mutant. YSH1 mRNA level is unchanged in ipa1-1 cells, and proteasome inactivation prevents Ysh1 loss and causes accumulation of ubiquitinated Ysh1. Ysh1 ubiquitination is mediated by the Ubc4 ubiquitin-conjugating enzyme and Mpe1, which in addition to its function in C/P, is also a RING ubiquitin ligase. In summary, Ipa1 affects mRNA processing by controlling the availability of the C/P endonuclease and may represent a regulatory mechanism that could be rapidly deployed to facilitate reprogramming of cellular responses.
