Marvin Wickens

A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line

The nematode Caenorhabditis elegans has two sexes, males and hermaphrodites. Hermaphrodites initially produce sperm but switch to producing oocytes. This switch appears to be controlled by the 3′ untranslated region of fem-3 messenger RNA. We have now identified a binding factor (FBF) which is a cytoplasmic protein that binds specifically to the regulatory region of fem-3 3′UTR and mediates the sperm/oocyte switch. The RNA-binding domain of FBF consists of a stretch of eight tandem repeats and two short flanking regions. This structural element is conserved in several proteins including Drosophila Pumilio, a regulatory protein that controls pattern formation in the fly by binding to a 3′UTR. We propose that FBF and Pumilio are members of a widespread family of sequence-specific RNA-binding proteins.

A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF (‘fem-3 mRNA binding factor’), were originally discovered as regulators of germline sex determination. Here we report that FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. We suggest that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family (‘Pumilio and FBF’) of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. We suggest that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells.

How the messenger got its tail: addition of poly(A) in the nucleus

Most mRNAs end in a poly(A) tail, the addition of which is catalysed by a poly(A) polymerase in conjunction with a distinct factor that provides specificity for mRNAs. The reaction is dynamic, involving separable initiation, elongation and termination phases. A companion article in next months TIBS will review the regulation of poly(A) addition and removal during early animal development.

A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans.

Messenger RNA regulation is a critical mode of controlling gene expression. Regulation of mRNA stability and translation is linked to controls of poly(A) tail length. Poly(A) lengthening can stabilize and translationally activate mRNAs, whereas poly(A) removal can trigger degradation and translational repression. Germline granules (for example, polar granules in flies, P granules in worms) are ribonucleoprotein particles implicated in translational control. Here we report that the Caenorhabditis elegans gene gld-2, a regulator of mitosis/meiosis decision and other germline events, encodes the catalytic moiety of a cytoplasmic poly(A) polymerase (PAP) that is associated with P granules in early embryos. Importantly, the GLD-2 protein sequence has diverged substantially from that of conventional eukaryotic PAPs, and lacks a recognizable RRM (RNA recognition motif)-like domain. GLD-2 has little PAP activity on its own, but is stimulated in vitro by GLD-3. GLD-3 is also a developmental regulator, and belongs to the Bicaudal-C family of RNA binding proteins. We suggest that GLD-2 is the prototype for a class of regulatory cytoplasmic PAPs that are recruited to specific mRNAs by a binding partner, thereby targeting those mRNAs for polyadenylation and increased expression.

Multifunctional deadenylase complexes diversify mRNA control

Dynamic changes of the lengths of mRNA poly(A) tails are catalysed by diverse deadenylase enzymes. Modulating the length of the poly(A) tail of an mRNA is a widespread means of controlling protein production and mRNA stability. Recent insights illuminate the specialized activities, biological functions and regulation of deadenylases. We propose that the recruitment of multifunctional deadenylase complexes provides unique opportunities to control mRNAs and that the heterogeneity of the deadenylase complexes is exploited to control translation and mRNA stability.

PUF proteins bind Pop2p to regulate messenger RNAs

PUF proteins, a family of RNA-binding proteins, interact with the 3′ untranslated regions (UTRs) of specific mRNAs to control their translation and stability. PUF protein action is commonly correlated with removal of the poly(A) tail of target mRNAs. Here, we focus on how PUF proteins enhance deadenylation and mRNA decay. We show that a yeast PUF protein physically binds Pop2p, which is a component of the Ccr4p–Pop2p–Not deadenylase complex, and that Pop2p is required for PUF repression activity. By binding Pop2p, the PUF protein simultaneously recruits the Ccr4p deadenylase and two other enzymes involved in mRNA regulation, Dcp1p and Dhh1p. We reconstitute regulated deadenylation in vitro and demonstrate that the PUF-Pop2p interaction is conserved in yeast, worms and humans. We suggest that the PUF-Pop2p interaction underlies regulated deadenylation, mRNA decay and repression by PUF proteins.

Multiple portions of poly(A)-binding protein stimulate translation in vivo

Translational stimulation of mRNAs during early development is often accompanied by increases in poly(A) tail length. Poly(A)‐binding protein (PAB) is an evolutionarily conserved protein that binds to the poly(A) tails of eukaryotic mRNAs. We examined PAB‘s role in living cells, using both Xenopus laevis oocytes and Saccharomyces cerevisiae, by tethering it to the 3′‐untranslated region of reporter mRNAs. Tethered PAB stimulates translation in vivo. Neither a poly(A) tail nor PABs poly(A)‐binding activity is required. Multiple domains of PAB act redundantly in oocytes to stimulate translation: the interaction of RNA recognition motifs (RRMs) 1 and 2 with eukaryotic initiation factor‐4G correlates with translational stimulation. Interaction with Paip‐1 is insufficient for stimulation. RRMs 3 and 4 also stimulate, but bind neither factor. The regions of tethered PAB required in yeast to stimulate translation and stabilize mRNAs differ, implying that the two functions are distinct. Our results establish that oocytes contain the machinery necessary to support PAB‐mediated translation and suggest that PAB may be an important participant in translational regulation during early development.

The Use of Xenopus Oocytes for the Expression of Cloned Genes

Publisher Summary This chapter discusses the use of Xenopus oocytes for the expression of cloned genes. The injection of amphibian oocytes was one of the first systems in which purified DNA was correctly transcribed and expressed as protein. An amphibian oocyte is a single large cell, surrounded by several thousand small follicle cells. It is in meiotic prophase, and active in RNA and protein synthesis, but totally inactive in DNA synthesis. The transcription of injected DNA takes place only in the nucleus or germinal vesicle of an oocyte, which is not normally visible. Gene isolation usually requires at least partially pure mRNA for the preparation of cDNA or for screening a genomic DNA library directly. As oocytes were first used for translating mRNA, cell-free systems have been greatly improved and generally have a lower background than oocytes.

PUF Protein-mediated Deadenylation Is Catalyzed by Ccr4p

PUF proteins control gene expression by binding to the 3′-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.

Analyzing mRNA–protein complexes using a yeast three-hybrid system

RNA-protein interactions are essential for the proper execution and regulation of every step in the life of a eukaryotic mRNA. Here we describe a three-hybrid system in which RNA-protein interactions can be analyzed using simple phenotypic or enzymatic assays in Saccharomyces cerevisiae. The system can be used to detect or confirm an RNA-protein interaction, to analyze RNA-protein interactions genetically, and to discover new protein or RNA partners when only one is known. Multicomponent complexes containing more than one protein can be detected, identified, and analyzed. We describe the method and how to use it, and discuss applications that bear particularly on eukaryotic mRNAs.