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.     

DAZAP1 interacts via its RNA-recognition motifs with the C-termini of other RNA-binding proteins

The turnover and translation of many human mRNAs is regulated by AU-rich elements present in their 3′untranslated region, which bind various trans acting factors. We previously identified a trans acting factor that interacts with these cis elements as DAZAP1 (deleted in Azoospermia (DAZ)-Associated Protein 1), whose interaction with the germ cell-specific protein DAZ was disrupted by the phosphorylation of DAZAP1. Here we have identified several other RNA-binding proteins as binding partners for DAZAP1 in non-germinal cells. Unlike DAZ, these interactions occur between the RNA recognition motifs of DAZAP1 and the C-termini of the binding partners and in a phosphorylation-independent manner. The results suggest that DAZAP1 is a component of complexes that are crucial for the degradation and silencing of mRNA.     

Phosphorylation of the ARE-binding protein DAZAP1 by ERK2 induces its dissociation from DAZ

A protein in RAW 264.7 macrophages, which became phosphorylated in response to LPS (lipopolysaccharide), was identified as the RNA-binding protein called DAZAP1 [DAZ (deleted in azoospermia)-associated protein 1]. The phosphorylation of this protein was prevented by specific inhibition of MKK1 [MAPK (mitogen-activated protein kinase) kinase 1], indicating that it was phosphorylated via the classical MAPK cascade. Further experiments showed that DAZAP1 was phosphorylated stoichiometrically in vitro by ERK2 (extracellular-signal-regulated protein kinase 2) at two Thr-Pro sequences (Thr269 and Thr315), and that both sites became phosphorylated in HEK-293 (human embryonic kidney 293) cells in response to PMA or EGF (epidermal growth factor), or RAW 264.7 macrophages in response to LPS. Phosphorylation induced by each stimulus was prevented by two structurally distinct inhibitors of MKK1 (PD184352 and U0126), demonstrating that DAZAP1 is a physiological substrate for ERK1/ERK2. The mutation of Thr269 and Thr315 to aspartate or the phosphorylation of these residues caused DAZAP1 to dissociate from its binding partner DAZ. DAZ interacts with PABP [poly(A)-binding protein] and thereby stimulates the translation of mRNAs containing short poly(A) tails [Collier, Gorgoni, Loveridge, Cooke and Gray (2005) EMBO J. 24, 2656-2666]. In the present study we have shown that DAZ cannot bind simultaneously to DAZAP1 and PABP, and suggest that the phosphorylation-induced dissociation of DAZ and DAZAP1 may allow the former to stimulate translation by interacting with PABP.     

Structure and RNA binding of the mouse Pumilio-2 Puf domain

Puf proteins control translation through the interaction of a C-terminal Puf domain with specific sequences present in the 3′ untranslated region of messenger RNAs. In Drosophila, binding of the protein Pumilio to mRNA leads to translational repression which is required for anterior/posterior patterning during embryogenesis. The vertebrate Pumilio homologue 2 (Pum2) has been implicated in controlling germ cell development through interactions with the RNA binding proteins deleted in azoospermia (DAZ), DAZ-like (DAZL) and BOULE. We present the 1.6 Å resolution X-ray crystal structure of the Puf domain from murine Pum2 and demonstrate that this domain is capable of binding with nanomolar affinity to RNA sequences from the hunchback Nanos response element (NRE) and a previously identified Pum2 binding element (PBE).     

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.     

Novel missense mutations of theDeleted-in-AZoospermia-Like (DAZL) gene in infertile women and men

Background  

TheDeleted-in-AZoospermia-Like (DAZL) gene has homologs required for germ cell development in many organisms. Recently, we showed that there are several common polymorphisms within theDAZL gene that are associated with age at ovarian failure/menopause and sperm count.     

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.     

Identification of a novel gene, DZIP (DAZ-interacting protein), that encodes a protein that interacts with DAZ (deleted in azoospermia) and is expressed in embryonic stem cells and germ cells

Evidence from diverse organisms, including humans, suggests that the DAZ (Deleted in Azoospermia) gene and a closely related homolog, DAZL (DAZ-like), are required early in germ cell development to maintain initial germ cell populations. Here we report the identification and characterization of the DZIP (DAZ-Interacting Protein) gene, which encodes at least three different protein isoforms that contain a C2H2 zinc-finger domain. The DZIP gene is expressed predominantly in human embryonic stem cells and fetal and adult germ cells; moreover, two DZIP protein isoforms colocalize with DAZ and/or DAZL proteins in these tissues. Finally, we provide evidence indicating that DZIP may associate with DAZ and its other cofactors in an RNA-binding protein complex that functions in both ES cells and germ cells.     

Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia

The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large‐scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2‐D gel electrophoresis approach. Protein spots were quantified and protein‐level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin‐1 and deleted in azoospermia‐associated protein (DAZAP‐1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP‐1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK‐1 and actin‐depolymerizing factor (ADF) in pleural ganglia and DAZAP‐1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel‐based mass spectrometrical method (FT‐MS). Taken together, protein‐level changes induced by the learning‐related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.     

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background

During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression.

Methodology/Principal Findings

Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430.

Conclusions/Significance

Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.     

Evolution of the <Emphasis Type="Italic">DAZ</Emphasis> gene and the <Emphasis Type="Italic">AZFc</Emphasis> region on primate Y chromosomes

Background  

The Azoospermia Factor c (AZFc) region of the human Y chromosome is a unique product of segmental duplication. It consists almost entirely of very long amplicons, represented by different colors, and is frequently deleted in subfertile men. Most of the AZFc amplicons have high sequence similarity with autosomal segments, indicating recent duplication and transposition to the Y chromosome. The Deleted in Azoospermia (DAZ) gene within the red-amplicon arose from an ancestral autosomal DAZ-like (DAZL) gene. It varies significantly between different men regarding to its copy number and the numbers of RNA recognition motif and DAZ repeat it encodes. We used Southern analyses to study the evolution of DAZ and AZFc amplicons on the Y chromosomes of primates.     

Mining protein networks for synthetic genetic interactions

Background  

The local connectivity and global position of a protein in a protein interaction network are known to correlate with some of its functional properties, including its essentiality or dispensability. It is therefore of interest to extend this observation and examine whether network properties of two proteins considered simultaneously can determine their joint dispensability, i.e., their propensity for synthetic sick/lethal interaction. Accordingly, we examine the predictive power of protein interaction networks for synthetic genetic interaction in Saccharomyces cerevisiae, an organism in which high confidence protein interaction networks are available and synthetic sick/lethal gene pairs have been extensively identified.     

Characterizing the neurite outgrowth inhibitory effect of Mani

Mani (myelin-associated neurite-outgrowth inhibitor) protein is implicated in both axonal guidance and axonal regeneration after central nervous system (CNS) injury. Here, we applied a neurite outgrowth assay, coupled with a siRNA-driven investigation and immunocytochemistry, to unveil Mani’s axonal outgrowth inhibitory effect in embryonic rat cortical primary neurons in vitro. We further demonstrate Mani’s neuronal localization in comparison with a principal subunit, Cdc27, of the anaphase promoting complex (APC). Considering the protein structure of Mani obtained via a series of bio-computational studies, we propose a Cdc27-Mani-APC-related signalling pathway may be involved in CNS axon regeneration.

Structured summary of protein interactions

MANIphysically interacts with DAZAP2 by two hybrid (View interaction)MANIphysically interacts with FAM168A by two hybrid (View interaction)MANIphysically interacts with YPEL2 by two hybrid (View interaction)MANIphysically interacts with TMTC1 by two hybrid (View interaction)     

Protein complex forming ability is favored over the features of interacting partners in determining the evolutionary rates of proteins in the yeast protein-protein interaction networks

Background  

Evolutionary rates of proteins in a protein-protein interaction network are primarily governed by the protein connectivity and/or expression level. A recent study revealed the importance of the features of the interacting protein partners, viz., the coefficient of functionality and clustering coefficient in controlling the protein evolutionary rates in a protein-protein interaction (PPI) network.     

DAZ家族新成员BOULE蛋白的结构与功能

BOULE蛋白是2001年发现的DAZ家族的新成员,是人类精子发生过程中减数分裂的关键调控因子. BOULE基因表达的改变或BOULE蛋白的缺乏可引起减数分裂阻滞和精子生成障碍,从而导致无精子症并产生不育. BOULE蛋白的一级结构中含有DAZ家族的特征结构域,包括DAZ重复和RNA结合域（RBM）,因此,将其列为继DAZ、DAZL之后DAZ家族的第3个成员.本文对BOULE的发现过程、结构和定位进行了总结回顾,并重点介绍了其在精子发生减数分裂中的作用及其作用机制.     

Interaction of the conserved meiotic regulators, BOULE (BOL) and PUMILIO-2 (PUM2)

Germ cell development is complex; it encompasses specification of germ cell fate, mitotic replication of early germ cell populations, and meiotic and postmeiotic development. Meiosis alone may require several hundred genes, including homologs of the BOULE (BOL) and PUMILIO (PUM) gene families. Both BOL and PUM homologs encode germ cell specific RNA binding proteins in diverse organisms where they are required for germ cell development. Here, we demonstrate that human BOL forms homodimers and is able to interact with a PUMILIO homolog, PUM2. We mapped the domain of BOL that is required for dimerization and for interaction with PUM2. We also show that BOL and PUM2 can form a complex on a subset of PUM2 RNA targets that is distinct from targets bound by PUM2 and another deleted in azoospermia (DAZ) family member, DAZ-like (DAZL). This suggests that RNA sequences bound by PUM2 may be determined by protein interactions. This data also suggests that although the BOL, DAZ, and DAZL proteins are all members of the same gene family, they may function in distinct molecular complexes during human germ cell development.     

Inactivation of NMD increases viability of <Emphasis Type="Italic">sup45</Emphasis> nonsense mutants in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.