Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation

Abstract

Ret finger protein-like 1 (RFPL1) is a primate-specific target gene of Pax6, a key transcription factor for pancreas, eye and neocortex development. However, its cellular activity remains elusive. In this article, we report that Pax6-elicited expression of the human (h)RFPL1 gene in HeLa cells can be enhanced by in vivo p53 binding to its promoter and therefore investigated the hypothesis that hRFPL1 regulates cell-cycle progression. Upon expression in these cells, hRFPL1 decreased cell number through a kinase-dependent mechanism as PKC activates and Cdc2 inhibits hRFPL1 activity. hRFPL1 antiproliferative activity led to an increased cell population in G 2 /M phase and specific cyclin B1 and Cdc2 downregulations, which were precluded by a proteasome inhibitor. Specifically, cytoplasm-localized hRFPL1 prevented cyclin B1 and Cdc2 accumulation during interphase. Consequently, cells showed a delayed entry into mitosis and cell-cycle lengthening resulting from a threefold increase in G 2 phase duration. Given previous reports that RFPL1 is expressed during cell differentiation, its impact on cell-cycle lengthening therefore provides novel insights into primate-specific development. The human Ret finger protein-like (hRFPL)1,2,3 genes (OMIM 605968, 605969, 605970) are recently identified targets of Pax6, which is notably a key transcription factor for pancreas, eye and neocortex development. [1][2][3] In this line, high hRFPL1,2,3 expression is found at the onset of neurogenesis in differentiating embryonic stem cells and in the developing neocortex. 4 In addition, the study of RFPL1,2,3 evolutionary history revealed that they are only found in Old World monkeys and great apes and show features believed to be important for human brain evolution. 4 Yet, the cellular activity of RFPL1,2,3 is still unknown. A murine RFPL (mRFPL) protein, encoded by an ancestral gene not belonging to the RFPL1,2,3 gene subfamily, 4 has also been cloned. 5 Previously reported as being expressed only in testis, ovaries and oocytes, 5,6 mRFPL has been shown to interact with the destruction box motif of cyclin B1 -the Cdc2 activating partner for driving germ cells through metaphases I and II 7 -and with proteins of the proteasome, speculating on mRFPL ability to elicit cyclin B1 degradation to control meiosis progression. 6 However, the fact that cyclin B1 and Cdc2 also form a key complex for controlling cell entry into mitosis 8 and our recent observations that the RFPL genes are also expressed in tissues in which cells divide mitotically 4 suggest that the RFPL proteins could regulate other aspects of cell division.We therefore examined RFPL-mediated control of mitotic cell-cycle progression by focusing on hRFPL1. Because no endogenously hRFPL1-expressing cell type suitable for this kind of study has been reported to date, we examined the influence of hRFPL1 gain of function on HeLa cells, a reference cell system for examining cell-cycle regulation. We report that hRFPL1 is an antiproliferative gene that controls G 2 -M phase transition, ...

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