Academic journal article Genetics

FBF and Its Dual Control of Gld-1 Expression in the Caenorhabditis Elegans Germline

Academic journal article Genetics

FBF and Its Dual Control of Gld-1 Expression in the Caenorhabditis Elegans Germline

Article excerpt

ABSTRACT

FBF, a PUF RNA-binding protein, is a key regulator of the mitosis/meiosis decision in the Caenorhabditis elegans germline. Genetically, FBF has a dual role in this decision: it maintains germ cells in mitosis, but it also facilitates entry into meiosis. In this article, we explore the molecular basis of that dual role. Previous work showed that FBF downregulates gld-1 expression to promote mitosis and that the GLD-2 poly(A) polymerase upregulates gld-1 expression to reinforce the decision to enter meiosis. Here we ask whether FBF can act as both a negative regulator and a positive regulator of gld-1 expression and also investigate its molecular mechanisms of control. We first show that FBF co-immunoprecipitates with gld-1 mRNA, a result that complements previous evidence that FBF directly controls gld-1 mRNA. Then we show that FBF represses gld-1 expression, that FBF physically interacts with the CCF-1/Pop2p deadenylase and can stimulate deadenylation in vitro, and that CCF-1 is partially responsible for maintaining low GLD-1 in the mitotic region. Finally, we show that FBF can elevate gld-1 expression, that FBF physically interacts with the GLD-2 poly(A) polymerase, and that FBF can enhance GLD-2 poly(A) polymerase activity in vitro. We propose that FBF can affect polyadenylation either negatively by its CCF-1 interaction or positively by its GLD-2 interaction.

PUF (Pumilio and FBF) RNA-binding proteins influence many aspects of development and physiology. In Drosophila and Caenorhabditis elegans, both Pumilio and FBF are required for maintenance of germline stem cells (Lin and Spradling 1997; Forbes and Lehmann 1998; Crittenden et al. 2002), and PUF proteins have been implicated in stem cell controls in other organisms, including humans (Wickens et al. 2002; Salvetti et al. 2005; Xu et al. 2007). In addition, PUF proteins influence embryonic patterning (Barker et al. 1992), germline sex determination (Zhang et al. 1997), and memory formation (Dubnau et al. 2003). A molecular understanding of PUF regulation will therefore affect a broad spectrum of critical biological processes.

This work focuses on C. elegans FBF ( fem-3 binding factor), a collective term for the nearly identical and largely redundant FBF-1 and FBF-2 proteins (Zhang et al. 1997). Biochemically, FBF-1 and FBF-2 bind the same RNA sequence, the FBF binding element (FBE) (Zhang et al. 1997; Bernstein et al. 2005), and also bind the same proteins, including GLD-3 (Eckmann et al. 2002). Genetically, fbf-1 and fbf-2 single mutants are virtually wild-type and fertile, but fbf-1 fbf-2 double mutants fail to maintain germline stem cells, fail to embark on oogenesis, and are sterile (Zhang et al. 1997; Crittenden et al. 2002; Lamont et al. 2004). Thus, FBF-1 and FBF-2 have similar biochemical activities in vitro and similar effects on the mitosis/meiosis decision.Work on PUF proteins in other organisms demonstrated that they repress mRNA activity, at least in part, by recruiting the deadenylation machinery (Goldstrohm et al. 2006, 2007), but the mechanism of FBF action has not yet been examined.

FBF promotes germline self-renewal by repressing regulators of meiotic entry (Figure 1A). Indeed, two regulatory branches control meiotic entry (Kadyk and Kimble 1998) and FBF represses an mRNA in each branch (Crittenden et al. 2002; Eckmann et al. 2004). One branch includes GLD-1, a translational repressor ( Jan et al. 1999; Lee and Schedl 2001; Marin and Evans 2003), and the other branch consists of GLD-2/ GLD-3, a translational activator and poly(A) polymerase (Wang et al. 2002; Suh et al. 2006). Meiotic entry is dramatically curtailed in double mutants that delete key components of both branches, but not in the single mutants (Kadyk and Kimble 1998; Eckmann et al. 2004; Hansen et al. 2004b). Of most relevance to this article, FBF directly represses gld-1 mRNA (Crittenden et al. 2002; Merritt et al. 2008), and GLD-2 directly activates gld-1 mRNA, a positive regulatory step that reinforces the decision to enter meiosis (Figure 1B) (Suh et al. …

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