Poly(A)-binding proteins: Structure, domain organization, and activity regulation

RNA-binding proteins are of vital importance for mRNA functioning. Among these, poly(A)-binding proteins (PABPs) are of special interest due to their participation in virtually all mRNA-dependent events that is caused by their high affinity for A-rich mRNA sequences. Apart from mRNAs, PABPs interact with many proteins, thus promoting their involvement in cellular events. In the nucleus, PABPs play a role in polyadenylation, determine the length of the poly(A) tail, and may be involved in mRNA export. In the cytoplasm, they participate in regulation of translation initiation and either protect mRNAs from decay through binding to their poly(A) tails or stimulate this decay by promoting mRNA inter-actions with deadenylase complex proteins. This review presents modern notions of the role of PABPs in mRNA-dependent events; peculiarities of regulation of PABP amount in the cell and activities are also discussed.     

DAZ family proteins exist throughout male germ cell development and transit from nucleus to cytoplasm at meiosis in humans and mice

The human DAZ gene family is expressed in germ cells and consists of a cluster of nearly identical DAZ (deleted in azoospermia) genes on the Y chromosome and an autosomal homolog, DAZL (DAZ-like). Only the autosomal gene is found in mice. Y-chromosome deletions that encompass the DAZ genes are a common cause of spermatogenic failure in men, and autosomal homologs of DAZ are essential for testicular germ cell development in mice and DROSOPHILA: Previous studies have reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, we report here that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established.     

The roles of cytoplasmic poly(A)-binding proteins in regulating gene expression: a developmental perspective.

Poly(A)-binding proteins (PABPs) are central to the regulation of messenger RNA (mRNA) translation and stability; however, the roles and contributions of different PABP family members in controlling gene expression are not yet fully understood. In this paper, the current state of knowledge of the different cytoplasmic PABP proteins and their function in animal cells will be summarised, with particular reference to their roles in development. Possible regulatory mechanisms and potential new roles for these proteins in the control of specific mRNAs are also highlighted.     

Association of the mouse infertility factor DAZL1 with actively translating polyribosomes

The DAZ (Deleted in AZoospermia) gene family was isolated from a region of the human Y chromosome long arm that is deleted in about 10% of infertile men with idiopathic azoospermia. DAZ and an autosomal DAZ-like gene, DAZL1, are expressed in germ cells only. They encode proteins with an RNA recognition motif and with either a single copy (in DAZL1) or multiple copies (in DAZ) of a DAZ repeat. A role for DAZL1 and DAZ in spermatogenesis is supported by their homology to a Drosophila male infertility protein Boule and by sterility of Dazl1 knock-out mice. The biological function of these proteins remains unknown. We found that DAZL1 and DAZ bound similarly to various RNA homopolymers in vitro. We also used an antibody against the human DAZL1 to determine the subcellular localization of DAZL1 in mouse testis. The sedimentation profiles of DAZL1 in sucrose gradients indicate that DAZL1 is associated with polyribosomes, and further capture of DAZL1 on oligo(dT) beads demonstrates that the association is mediated through the binding of DAZL1 to poly(A) RNA. Our results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation.     

Identification of two novel proteins that interact with germ-cell-specific RNA-binding proteins DAZ and DAZL1

The human DAZ (deleted in azoospermia) gene family on the Y chromosome and an autosomal DAZ-like gene, DAZL1, encode RNA-binding proteins that are expressed exclusively in germ cells. Their role in spermatogenesis is supported by their homology with a Drosophila male infertility gene boule and sterility of Daz11 knock-out mice. While all mammals contain a DAZL1 homologue on their autosomes, DAZ homologues are present only on the Y chromosomes of great apes and Old World monkeys. The DAZ and DAZL1 proteins differ in the copy numbers of a DAZ repeat and the C-terminal sequences. We studied the interaction of DAZ and DAZL1 with other proteins as an approach to investigate functional similarity between these two proteins. Using DAZ as bait in a yeast two-hybrid system, we isolated two DAZAP (DAZ-associated protein) genes. DAZAP1 encodes a novel RNA-binding protein that is expressed most abundantly in the testis, and DAZAP2 encodes a ubiquitously expressed protein with no recognizable functional motif. DAZAP1 and DAZAP2 bind similarly to both DAZ and DAZL1 through the DAZ repeats. The DAZAP genes were mapped to chromosomal regions 19p13.3 and 2q33-q34, respectively, where no genetic diseases affecting spermatogenesis are known to map.     

Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis.

Poly(A)-binding proteins (PABPs) are the best characterized messenger RNA-binding proteins of eucaryotic cells and have been identified in diverse organisms such as mammals and yeasts. The in vitro poly(A)-binding properties of these proteins have been studied intensively; however, little is known about their function in cells. In this report, we show that sea urchin eggs have two molecular weight forms of PABP (molecular weights of 66,000 and 80,000). Each of these has at least five posttranslationally modified forms. Both sea urchin PABPs are found in approximately 1:1 ratios in both cytoplasmic and nuclear fractions of embryonic cells. Quantification in eggs and embryos revealed that sea urchin PABPs are surprisingly abundant, composing about 0.6% of total cellular protein. This is 50 times more than required to bind all the poly(A) in the egg based on the binding stoichiometry of 1 PABP per 27 adenosine residues. We found that density gradient centrifugation strips PABP from poly(A) and therefore underestimates the amount of PABP complexed to poly(A)+ RNA in cell homogenates. However, large-pore gel filtration chromatography could be used to separate intact poly(A)-PABP complexes from free PABP. Using the gel filtration method, we found that the threefold increase in poly(A) content of the egg after fertilization is paralleled by an approximate fivefold increase in the amount of bound PABP. Furthermore, both translated and nontranslated poly(A)+ RNAs appear to be complexed to PABP. As expected from the observation that PABPs are so abundant, greater than 95% of the PABP of the cell is uncomplexed protein.     

A network-based analysis of polyanion-binding proteins utilizing yeast protein arrays

The high affinity of certain cellular polyanions for many proteins (polyanion-binding proteins (PABPs)) has been demonstrated previously. It has been hypothesized that such polyanions may be involved in protein structure stabilization, stimulation of folding through chaperone-like activity, and intra- and extracellular protein transport as well as intracellular organization. The purpose of the proteomics studies reported here was to seek evidence for the idea that the nonspecific but high affinity interactions of PABPs with polyanions have a functional role in intracellular processes. Utilizing yeast protein arrays and five biotinylated cellular polyanion probes (actin, tubulin, heparin, heparan sulfate, and DNA), we identified proteins that interact with these probes and analyzed their structural and amino acid sequence requirements as well as their predicted functions in the yeast proteome. We also provide evidence for the existence of a network-like system for PABPs and their potential roles as critical hubs in intracellular behavior. This investigation takes a first step toward achieving a better understanding of the nature of polyanion-protein interactions within cells and introduces an alternative way of thinking about intracellular organization.     

Nanopore detachment kinetics of poly(A) binding proteins from RNA molecules reveals the critical role of C-terminus interactions

The ubiquitous and abundant cytoplasmic poly(A) binding protein (PABP) is a highly conserved multifunctional protein, many copies of which bind to the poly(A) tail of eukaryotic mRNAs to promote translation initiation. The N-terminus of PABP is responsible for the high binding specificity and affinity to poly(A), whereas the C-terminus is known to stimulate PABP multimerization on poly(A). Here, we use single-molecule nanopore force spectroscopy to directly measure interactions between poly(A) and PABPs. Both electrical and biochemical results show that the C-C domain interaction between two consecutive PABPs promotes cooperative binding. Up to now, investigators have not been able to probe the detailed polarity configuration (i.e., the internal arrangement of two PABPs on a poly(A) streak in which the C-termini face toward or away from each other). Our nanopore force spectroscopy system is able to distinguish the cooperative binding conformation from the noncooperative one. The ～50% cooperative binding conformation of wild-type PABPs indicates that the C-C domain interaction doubles the cooperative binding probability. Moreover, the longer dissociation time of a cooperatively bound poly(A)/PABP complex as compared with a noncooperatively bound one indicates that the cooperative mode is the most stable conformation for PABPs binding onto the poly(A). However, ～50% of the poly(A)/PABP complexes exhibit a noncooperative binding conformation, which is in line with previous studies showing that the PABP C-terminal domain also interacts with additional protein cofactors.     

Solution structure of the C-terminal domain from poly(A)-binding protein in Trypanosoma cruzi: a vegetal PABC domain

PABC is a phylogenetically conserved peptide-binding domain primarily found within the C terminus of poly(A)-binding proteins (PABPs). This domain recruits a series of translation factors including poly(A)-interacting proteins (Paip1 and Paip2) and release factor 3 (RF3/GSPT) to the initiation complex on mRNA. Here, we determine the solution structure of the Trypanosoma cruzi PABC domain (TcPABC), a representative of the vegetal class of PABP proteins. TcPABC is similar to human PABC (hPABC) and consists of five alpha-helices, in contrast to the four helices observed in PABC domains from yeast (yPABC) and hyper plastic disk proteins (hHYD). A mobile N-terminal helix is observed in TcPABC that does not pack against the core of the protein, as found in hPABC. Characteristic to all PABC domains, the last four helices of TcPABC fold into a right-handed super coil. TcPABC demonstrates high-affinity binding to PABP interacting motif-2 (PAM-2) and reveals a peptide-binding surface homologous to that of hPABC. Our results demonstrate the last four helices in TcPABC are sufficient for peptide recognition and we predict a similar binding mode in PABC domains. Furthermore, these results point to the presence of putative PAM-2 site-containing proteins in trypanosomes.