Characterization of the mouse Dazap1 gene encoding an RNA-binding protein that interacts with infertility factors DAZ and DAZL

Background  

DAZAP1 (DAZ Associated Protein 1) was originally identified by a yeast two-hybrid system through its interaction with a putative male infertility factor, DAZ (Deleted in Azoospermia). In vitro, DAZAP1 interacts with both the Y chromosome-encoded DAZ and an autosome-encoded DAZ-like protein, DAZL. DAZAP1 contains two RNA-binding domains (RBDs) and a proline-rich C-terminal portion, and is expressed most abundantly in the testis. To understand the biological function of DAZAP1 and the significance of its interaction with DAZ and DAZL, we isolated and characterized the mouse Dazap1 gene, and studied its expression and the subcellular localization of its protein product.     

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.     

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.     

FATP1 Inhibits 11-cis Retinol Formation via Interaction with the Visual Cycle Retinoid Isomerase RPE65 and Lecithin:Retinol Acyltransferase

The isomerization of all-trans retinol (vitamin A) to 11-cis retinol in the retinal pigment epithelium (RPE) is a key step in the visual process for the regeneration of the visual pigment chromophore, 11-cis retinal. LRAT and RPE65 are recognized as the minimal isomerase catalytic components. However, regulators of this rate-limiting step are not fully identified and could account for the phenotypic variability associated with inherited retinal degeneration (RD) caused by mutations in the RPE65 gene. To identify new RPE65 partners, we screened a porcine RPE mRNA library using a yeast two-hybrid assay with full-length human RPE65. One identified clone (here named FATP1c), containing the cytosolic C-terminal sequence from the fatty acid transport protein 1 (FATP1 or SLC27A1, solute carrier family 27 member 1), was demonstrated to interact dose-dependently with the native RPE65 and with LRAT. Furthermore, these interacting proteins colocalize in the RPE. Cellular reconstitution of human interacting proteins shows that FATP1 markedly inhibits 11-cis retinol production by acting on the production of all-trans retinyl esters and the isomerase activity of RPE65. The identification of this new visual cycle inhibitory component in RPE may contribute to further understanding of retinal pathogenesis.     

DAZAP2 acts as specifier of the p53 response to DNA damage

The DNA damage-responsive tumor suppressors p53 and HIPK2 are well established regulators of cell fate decision-making and regulate the cellular sensitivity to DNA-damaging drugs. Here, we identify Deleted in Azoospermia-associated protein 2 (DAZAP2), a small adaptor protein, as a novel regulator of HIPK2 and specifier of the DNA damage-induced p53 response. Knock-down or genetic deletion of DAZAP2 strongly potentiates cancer cell chemosensitivity both in cells and in vivo using a mouse tumour xenograft model. In unstressed cells, DAZAP2 stimulates HIPK2 polyubiquitination and degradation through interplay with the ubiquitin ligase SIAH1. Upon DNA damage, HIPK2 site-specifically phosphorylates DAZAP2, which terminates its HIPK2-degrading function and triggers its re-localization to the cell nucleus. Interestingly, nuclear DAZAP2 interacts with p53 and specifies target gene expression through modulating a defined subset of p53 target genes. Furthermore, our results suggest that DAZAP2 co-occupies p53 response elements to specify target gene expression. Collectively, our findings propose DAZAP2 as novel regulator of the DNA damage-induced p53 response that controls cancer cell chemosensitivity.     

DAZ Family Proteins,Key Players for Germ Cell Development

DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution.     

Combined deletion of DAZ2 and DAZ4 copies of Y chromosome DAZ gene is associated with male infertility in Tunisian men

The relationship between male infertility and AZFc micro-deletions that remove multiple genes of the Y chromosome varies among countries and populations. The purpose of this study was to analyze the prevalence and the characteristics of different Deleted in azoospermia (DAZ) gene copy deletions and their association with spermatogenic failure and male infertility in Tunisian men. 241 infertile men (30.7% azoospermic (n = 74), 31.5% oligozoospermic (n = 76) and 37.7% normozoospermic (n = 91)) and 115 fertile healthy males who fathered at least one child were included in the study. Three DAZ-specific single nucleotide variant loci and six bi-allelic DAZ-SNVs (I–VI) were analyzed using polymerase chain reaction (PCR)–restriction fragment length polymorphism and PCR. Our findings showed high frequencies of infertile men (73.85%) and controls (78.26%) having only three DAZ gene copies (DAZ1/DAZ2/DAZ3 or DAZ1/DAZ3/DAZ4 variants); so deletion of DAZ2 or DAZ4 were frequent both in infertile (36.5% and 37.3%, respectively) and fertile groups (33.9% and 44.3%, respectively) and removing DAZ4 copy was significantly more frequent in oligospermic than in normospermic men (p = 0.04) in infertile group. We also report for the first time that simultaneous deletion of both DAZ2 and DAZ4 copies was significantly more common in infertile men (12.4%) than in fertile men (4.3%) (p = 0.01). However, deletions of DAZ1/DAZ2 and DAZ3/DAZ4 clusters were very rare. Analysis of DAZ gene copies in Tunisian population, suggested that the simultaneous deletion of DAZ2 and DAZ4 gene copies is associated with male infertility, and that oligospermia seems to be promoted by removing DAZ4 copy.     

Male germ cell-specific protein Trs4 binds to multiple proteins

Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners.     

Coevolution within and between Regulatory Loci Can Preserve Promoter Function Despite Evolutionary Rate Acceleration

Phenotypes that appear to be conserved could be maintained not only by strong purifying selection on the underlying genetic systems, but also by stabilizing selection acting via compensatory mutations with balanced effects. Such coevolution has been invoked to explain experimental results, but has rarely been the focus of study. Conserved expression driven by the unc-47 promoters of Caenorhabditis elegans and C. briggsae persists despite divergence within a cis-regulatory element and between this element and the trans-regulatory environment. Compensatory changes in cis and trans are revealed when these promoters are used to drive expression in the other species. Functional changes in the C. briggsae promoter, which has experienced accelerated sequence evolution, did not lead to alteration of gene expression in its endogenous environment. Coevolution among promoter elements suggests that complex epistatic interactions within cis-regulatory elements may facilitate their divergence. Our results offer a detailed picture of regulatory evolution in which subtle, lineage-specific, and compensatory modifications of interacting cis and trans regulators together maintain conserved gene expression patterns.     

HRP-2, the Caenorhabditis elegans Homolog of Mammalian Heterogeneous Nuclear Ribonucleoproteins Q and R, Is an Alternative Splicing Factor That Binds to UCUAUC Splicing Regulatory Elements

Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.Alternative pre-mRNA splicing is a mechanism for generating multiple mRNA isoforms from a single gene. This process can allow a gene to encode for more than one protein isoform. For some genes, it is a mechanism for regulating message stability through production of alternative mRNA isoforms that are substrates for the nonsense-mediated mRNA decay pathway (1). The majority of human genes undergo alternative splicing (2), and the process can be regulated in tissue-specific and developmental stage-specific manners. Current models propose that cis elements on the pre-mRNA, in exons and introns, serve as recognition sites for trans-acting protein factors that bind to the pre-mRNA and regulate assembly of the splicing machinery, thus regulating splice site choice (3).In recent years, a number of groups have employed bioinformatics techniques to identify cis splicing regulatory elements (4). These techniques include using multiple interspecies sequence alignments to identify conserved intronic regions, identification of short sequences in exons that are bounded by weak consensus splice sites, and identification of common intronic sequences flanking similarly regulated alternative exons (5–9). These efforts have added many new sequences to the list of known and potential splicing regulators. The identification of the protein factor partners for these sequences will be important for understanding their function in alternative splicing regulation.Experimental approaches have identified alternative splicing factors that interact with specific cis elements (10), but the number of trans factors discovered still lags behind the number of newly identified cis element partners. Some examples of well-characterized cis element/trans-acting factor interactions include the NOVA K homology domain splicing factor binding to the sequence UCAY (11), the FOX splicing factors binding to the sequence UGCAUG (12–14), and hnRNP³ F/H proteins binding to the sequence GGGG (15, 16). By using cross-linking immunoprecipitation followed by large scale sequencing, entire catalogs of RNAs that the splicing factors NOVA, SF2/ASF, and FOX2 bind to in vivo have been determined (17–19). These approaches have led to models for how the proteins binding to their cis regulatory elements may alter splicing. These models include a role for the relative position of a cis element to an alternative cassette exon in determining alternative exon inclusion or skipping (18, 19).In a previous bioinformatics analysis of evolutionarily conserved intronic sequences flanking alternatively spliced exons, we identified the hexamer sequence UCUAUC as a novel splicing regulatory element (8). UCUAUC is found flanking both sides of alternative exon 16 of the unc-52 gene of Caenorhabditis elegans. Genetic analysis of a class of viable unc-52 mutants led to the discovery that exons 16–18 are alternative cassette exons and that every combination of skipping and inclusion of these three exons occurs (20). This splicing is regulated by the alternative splicing factor MEC-8 (21). Fig. 1A shows a schematic diagram of the alternatively spliced region of unc-52, with the MEC-8-enhanced alternative splicing events indicated. Using an unc-52 splicing reporter trans gene containing alternative exons 15–19, we previously reported that alternative splicing is regulated by the intronic motif UCUAUC in the intron downstream of exon 16 (8). In addition we showed that this element works cooperatively with a UGCAUG hexamer (the consensus FOX-1-binding site) in the upstream intron to regulate alternative splicing (8).Open in a separate windowFIGURE 1.RNA affinity chromatography identifies HRP-2 as binding to UCUAUC elements. A, schematic representation of the alternatively spliced region of unc-52 (adapted from Ref. 21). The alternative splicing events promoted by MEC-8 are indicated by bold lines. The lines next to introns 15 and 16 are the sites of the UCUAUC elements in those introns whose sequences were used in the RNA affinity chromatography. B, table showing sequences of RNAs immobilized to beads in the RNA affinity chromatography experiment. C, Coomassie-stained SDS-PAGE analysis of RNA affinity chromatography. C. elegans embryo extract was incubated with the different immobilized RNA substrates listed on top of the gel. Proteins identified by mass spectrometry are listed to the right of the gel, with arrows pointing to coincident protein bands. D, the left panel shows the silver stain result for the RNA affinity chromatography experiment. Each lane represents a different immobilized substrate, as indicated above. The band corresponding to HRP-2 is indicated by an arrow. The right panel is an immunoblot of the same gel using anti-HRP-2 polyclonal antibody. E, anti-HRP-2 immunoblot of an RNA affinity chromatography experiment for the indicated substrates.In this study, we report the results of a biochemical identification of a protein factor from C. elegans that binds to the UCUAUC intronic splicing regulatory element. We transcribed different short RNA sequences containing the UCUAUC element in its native intronic context, or as part of a repeating unit, and immobilized these onto agarose beads. After passing embryo extracts across these beads, we found that the protein HRP-2, the C. elegans homolog of the mammalian hnRNP Q/R proteins, binds to this sequence with high affinity. By using RNAi to reduce the level of HRP-2 in worms, we observed changes in alternative splicing of unc-52 and lin-10, two genes that contain UCUAUC elements in introns flanking alternative exons. We propose that HRP-2 is an alternative splicing factor that works through the UCUAUC intronic elements to regulate alternative splicing.