Regulation of Germ Cell mRNPs by eIF4E:4EIP Complexes: Multiple Mechanisms, One Goal

Abstract

Translational regulation of mRNAs is critically important for proper gene expression in germ cells, gametes, and embryos. The ability of the nucleus to control gene expression in these systems may be limited due to spatial or temporal constraints, as well as the breadth of gene products they express to prepare for the rapid animal development that follows. During development germ granules are hubs of post-transcriptional regulation of mRNAs. They assemble and remodel messenger ribonucleoprotein (mRNP) complexes for translational repression or activation. Recently, mRNPs have been appreciated as discrete regulatory units, whose function is dictated by the many positive and negative acting factors within the complex. Repressed mRNPs must be activated for translation on ribosomes to introduce novel proteins into germ cells. The binding of eIF4E to interacting proteins (4EIPs) that sequester it represents a node that controls many aspects of mRNP fate including localization, stability, poly(A) elongation, deadenylation, and translational activation/repression. Furthermore, plants and animals have evolved to express multiple functionally distinct eIF4E and 4EIP variants within germ cells, giving rise to different modes of translational regulation.

Keywords: 4EIPs; RBPs; deadenylation; eIF4E; germ granules; mRNA decay; polyadenylation; translational control.

FIGURE 1

Germ granules as assembly sites for eIF4E:4EIP mRNP complexes to direct repression and activation of translation. In germ cells, mRNAs are exported out of the nucleus through the nuclear pore complex (NPC). As they exit they come under control by the germ granule environment. These conserved perinuclear structures act as hubs of post-transcriptional gene regulation, where they act as sites for the assembly and remodeling of diverse messenger ribonucleoprotein complexes (mRNPs). Germ granules are primarily sites of translational repression, as they do not contain ribosomes. However, recent evidence suggests that both repression and activation events may be set up here as a manner of mRNA sorting or licensing. Translation factors like eIF4E reside in complexes both within (repressed) and outside (activated) of the granule, potentially giving it a dual role. When eIF4E is in germ granules it binds to its cognate eIF4E-interacting protein (4EIP) in a complex with sequence specific 3′-UTR binding proteins. This complex prevents cap-dependent translation initiation by preventing eIF4G binding to eIF4E. Little is known about how the “decision” to activate translation is made, but our models suggests that a handoff from the granule to the cytoplasm occurs by mRNP remodeling. 4EIP is displaced by eIF4G and leads to initiation and recruitment to polyribosomes. Other factors participate in the repression to activation switch, like helicases (eIF4A, VASA, etc.) that unwind mRNA secondary structure, poly(A) polymerases (PAP) that extend 3′-poly(A) tails, and poly(A)-binding protein (PABP), which both protects transcripts and enhances translation via its interaction with eIF4G. Activating a repressed mRNP is the first step in producing novel gene products that can dictate the fate and function of germ cells.

FIGURE 2

Germ cell mRNPs use the same eIF4E:4EIP core but different RBPs for altered modes of local translation in the Drosophila oocyte. During the latter stages of Drosophila oogenesis many germline mRNPs like osk and nos are localized to and specifically translated in the germ plasm (posterior pole). These regulated mRNPs use the eIF4E:4EIP core, but each employs a unique mechanism to control mRNA stability, localization, and eventual translation. In the anterior region of the oocyte osk mRNPs are circularized by a core complex containing the oocyte eIF4E, Cup (4EIP), and Bruno (3′-UTR-binding protein). Cup prevents eIF4E:eIF4G binding while simultaneously recruiting the Ccr4-Not1 deadenylase complex. Because eIF4E:Cup also prevents de-capping and 5′-3′ decay by the Dcp2/Pacman complex, osk mRNA is protected and translationally silent while being shuttled to the posterior pole via microtubules. Similarly, nos mRNPs in the anterior region of the oocyte are circularized by a eIF4E:Cup:Smaug complex, which again prevents translation initiation and recruits Ccr4-Not1. Unlike osk, however, nos mRNA localizes by cytoplasmic streaming that is inefficient. Therefore, Smaug recruits Aubergine and associated piRNA machinery to degrade nos mRNA outside the germ plasm. Little is known about the events that promote translational activation of these two distinct mRNPs. However, it is likely that a remodeling occurs in which Cup is displaced by eIF4G leading to polyribosome recruitment. Additionally, Oskar protein translationally activates the nos mRNP, indicating a complex interplay of translational regulation regimens that ensure that germ cell determinants are synthesized in correct time and place.

FIGURE 3

Dynamic mRNP remodeling promotes alternate mRNA repression, poly(A) elongation, and ribosome recruitment during Xenopus oocyte maturation. In immature Xenopus oocytes, class II CPE mRNAs are translationally repressed by eIF4E:Maskin cap-binding complexes directed by the CPEB, CPSF, Symplekin 3′-UTR-binding module. These complexes also contain PABP, PARN, and GLD2 indicating the potential for both poly(A) elongation and deadenylation. At the 5′-end, Maskin (4EIP) inhibits cap-dependent initiation by competing with eIF4G for eIF4E binding. At the 3′-end PARN shortens the poly(A) tail. Although GLD2 is resident within the complex, PARN activity appears to be dominant. It also may be that CPE-containing mRNAs cycle between deadenylation and polyadenylation. Upon progesterone stimulation, CPEB is phosphorylated by Aurora A kinase or CaMKII which leads to remodeling of the complex and ejection of PARN. GLD2 then catalyzes extension of the mRNA poly(A) tail. Concurrently, class I CPE containing mRNAs are translated and lead to activation of CDK1. CPEB and Maskin are phosphorylated by CDK1, and PABP is recruited to the poly(A) tail for further stabilization. Maskin is displaced and PABP associates with eIF4G, allowing more opportunity for a productive eIF4E:eIF4G interaction. This stepwise remodeling leads to translational activation of mRNAs critical for germinal vesicle breakdown and maturation. In early Xenopus oocytes CPE containing mRNAs are repressed by an alternative eIF4EB1:4E-T cap-binding complex along with P body components (DDX6 and Rap55), CPEB, CPSF, and scaffold protein Symplekin. PARN is not found in this complex, suggesting that translational repression is caused by inhibition of cap-dependent initiation. P body components resident may also act to degrade CPE these early class II transcripts.