Pumilio Homolog Research Articles

WTAP-induced N6-methyladenosine of PD-L1 blocked T-cell-mediated antitumor activity under hypoxia in colorectal cancer.

N6-Methyladenosine (m6A) is a important process regulating gene expression post-transcriptionally. Programmed death ligand 1 (PD-L1) is a major immune inhibitive checkpoint that facilitates immune evasion and is expressed in tumor cells. In this research we discovered that Wilms' tumor 1-associated protein (WTAP) degradation caused by ubiquitin-mediated cleavage in cancer cells (colorectal cancer, CRC) under hypoxia was inhibited by Pumilio homolog 1 (PUM1) directly bound to WTAP. WTAP enhanced PD-L1 expression in a way that was m6A-dependent. m6A "reader," Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) identified methylated PD-L1 transcripts and subsequently fixed its mRNA. Additionally, we found that T-cell proliferation and its cancer cell-killing effects were prevented by overexpression of WTAP invitro and invivo. Overexpression prevented T cells from proliferating and killing CRC by maintaining the expression of PD-L1. Further evidence supporting the WTAP-PD-L1 regulatory axis was found in human CRC and organoid tissues. Tumors with high WTAP levels appeared more responsive to anti-PD1 immunotherapy, when analyzing samples from patients undergoing treatment. Overall, our findings demonstrated a novel PD-L1 regulatory mechanism by WTAP-induced mRNA epigenetic regulation and the possible application of targeting WTAP as immunotherapy for tumor hypoxia.

High-throughput quantitation of protein-RNA UV-crosslinking efficiencies as a predictive tool for high-confidence identification of RNA-binding proteins.

UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions, however, makes attribution of RNA-binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA-binding protein (RBP) confidence scoring metric (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high- and low-confidence candidate RBPs. Although RCS has utility, we sought a direct metric for quantification and comparative evaluation of protein-RNA interactions. Here we propose the use of protein-specific UV-crosslinking efficiency (%CL), representing the molar fraction of a protein that is crosslinked to RNA, for functional evaluation of candidate RBPs. Application to the HeLa RNA interactome yielded %CL values for 1097 proteins. Remarkably, %CL values span over five orders of magnitude. For the HeLa RNA interactome, %CL values comprise a range from high efficiency, high specificity interactions, e.g., the Elav protein HuR and the Pumilio homolog Pum2, with %CL values of 45.9 and 24.2, respectively, to very low efficiency and specificity interactions, for example, the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and alpha-enolase, with %CL values of 0.0016, 0.006, and 0.008, respectively. We further extend the utility of %CL through prediction of protein domains and classes with known RNA-binding functions, thus establishing it as a useful metric for RNA interactome analysis. We anticipate that this approach will benefit efforts to establish functional RNA interactomes and support the development of more predictive computational approaches for RBP identification.

RNA binding protein PUM2 promotes hepatocellular carcinoma proliferation and apoptosis via binding to the 3'UTR of BTG3.

Pumilio homolog 2 (PUM2) is an RNA-binding protein that functions as an oncogene in various types of cancer. However, its role in hepatocellular carcinoma (HCC) has remained to be fully elucidated. In the present study, the role of PUM2 was investigated in HCC and its regulation was assessed by examining its binding to the 3′-untranslated region (UTR) of B-cell translocation gene 3 (BTG3). The expression levels of PUM2 were determined in datasets from the UALCAN and Cancer Cell Line Encyclopedia databases. Furthermore, Gene Expression Profiling Interactive Analysis was used to analyze overall survival in patients with HCC. Reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses were then performed to detect the expression levels of PUM2 and BTG3 in HCC cells. Cell proliferation was assessed using Cell Counting Kit-8 and colony-formation assays. The induction of cell apoptosis was evaluated using TUNEL and western blotting assays. StarBase and RNA-Protein Interaction Prediction were used to determine the possible direct interaction between PUM2 and BTG3. The interaction between PUM2 and BTG3 was then verified by luciferase reporter and RNA-binding protein immunoprecipitation assays. The results indicated that PUM2 expression was upregulated in HCC tissues and cells and that it was associated with the prognosis of patients with HCC. PUM2 silencing inhibited the proliferation and promoted the apoptosis of Huh-7 cells. In addition, PUM2 was confirmed to directly bind to the 3′UTR of BTG3. Downregulation of BTG3 reversed the effects of PUM2 silencing on cell proliferation and apoptosis in Huh-7 cells. Collectively, the results suggested that PUM2 regulated HCC cell proliferation and apoptosis via interacting with BTG3, which may provide a novel therapeutic strategy for the treatment of human HCC.

CAFs-Derived Exosomal miRNA-130a Confers Cisplatin Resistance of NSCLC Cells Through PUM2-Dependent Packaging.

PurposeChemoresistance is a significant barrier to the treatment and management of non-small cell lung cancer (NSCLC). Exosomes play an essential role in intercellular communication. Understanding the mechanism underlying the role of tumor stroma, especially cancer-associated fibroblasts (CAFs), during chemoresistance would significantly contribute to the clinical application of chemotherapy agents.ResultsIn this study, we demonstrated that NSCLC-derived CAFs were innately resistant to cisplatin treatment and CAFs-conditioned medium significantly promoted the survival rate of NSCLC cells after cisplatin treatment. Additionally, CAFs-derived exosomes were taken up by NSCLC cells. Moreover, exosomal miRNA-130a was transferred from CAFs to recipient NSCLC cells and knockdown of miRNA-130a reversed the effect of CAFs-derived exosomes during chemoresistance of NSCLC cells. Furthermore, pumilio homolog 2 (PUM2), a RNA-binding protein, mediated the packaging of miRNA-130a into exosomes. The overexpression and knockdown of PUM2 promoted and inhibited tumor growth of xenograft mice, respectively.ConclusionTaken together, these results suggest that CAFs-derived exosomes confer cisplatin resistance of NSCLC cells through transferring miRNA-130a and that PUM2 is a critical factor for packaging miRNA-130a into exosomes. This study indicates that CAFs-derived exosomal miRNA-130a may be a potential therapeutic target for cisplatin resistance in NSCLC.

LncRNA TTN‑AS1 promotes endometrial cancer by sponging miR‑376a‑3p.

Increasing research has demonstrated that lncRNAs participate in the development of multiple cancer types. However, the role of TTN-AS1 in endometrial cancer (EC) remains unknown. The present study aimed to explore the function of titin-antisense RNA1 (TTN-AS1) in EC progression and the underlying mechanisms. qRT-PCR was performed to assess the TTN-AS1 expression patterns in EC tissues and cell lines. Loss of function experiments were carried out to estimate the effects of TTN-AS1 on EC cell proliferation, migration and invasion. To reveal the underlying mechanisms, informatics tools were used to predict the targets. Rescue experiments were performed to investigate the TTN-AS1-regulated miR-376a-3p/pumilio homolog 2 (PUM2) axis involved. The results of the present study revealed that TTN-AS1 was highly expressed in both EC tissues and cell lines, and TTN-AS1 knockdown inhibited EC cell proliferation, migration and invasion. With respect to the mechanisms, miR-376a-3p was revealed to be targeted by TTN-AS1, and reversed the effects on EC development induced by TTN-AS1. In addition, PUM2 was positively regulated by TTN-AS1, and miR-376a-3p mediated the regulation between them. Furtherly, in vivo experiments confirmed the results. Collectively, TTN-AS1 enhanced EC cell proliferation and metastasis by targeting the miR-376a-3p/PUM2 axis, which may shed light on EC diagnosis and treatment.

MiR-411-5p acts as a tumor suppressor in non-small cell lung cancer through targeting PUM1.

The aim of this study was to investigate the expression of micro ribonucleic acid-411-5P (miR-411-5p) in non-small cell lung cancer (NSCLC), and to explore the effect of miR-411-5p on the biological behavior of NSCLC cells as well as the underlying molecular mechanism. Quantitative Real Time- Polymerase Chain Reaction (qRT-PCR) was used to detect the expression level of miR-411-5p in NSCLC tissues and cells. MiR-411-5p mimics and relevant controls were transfected into NSCLC cells according to the instructions of Lipidosome 2000. Transfected cells were divided into the experimental group and the control group. The transfection efficiency of each group was detected by qRT-PCR. After miR-411-5p overexpression, methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, and transwell assay were used to detect the biological changes of cells in each group. Bioinformatics predicted that pumilio homolog 1 (PUM1) was the target gene of miR-411-5p. Subsequently, the mRNA and protein expression level of PUM1 in each group was detected by qRT-PCR and Western blotting, respectively. Dual-luciferase reporter assay was used to validate the target regulatory relationship between miR-411-5p and PUM1. The results of qRT-PCR showed that miR-411-5p was relatively lowly expressed in NSCLC tissues and cells. After miR-411-5p overexpression, MTT results revealed that the proliferation of NSCLC cells was decreased. Flow cytometry results indicated that the apoptosis rate of NSCLC cells was increased, and cell cycle was arrested in the G0-G1 phase. Meanwhile, the transwell assay demonstrated that the migration and invasion abilities of NSCLC cells were decreased. Bioinformatics predicted that PUM1 was the target gene of miR-411-5p. After miR-411-4p was overexpressed in NSCLC cells, qRT-PCR and Western blotting showed that both the mRNA and protein expression levels of PUM1 were up-regulated. Moreover, dual-luciferase reporter assay demonstrated that miR-411-5p could significantly inhibit the luciferase activity of wild-type PUM1-3'-untranslated region (3'-UTR). However, it exhibited no effect on the luciferase activity of cells transfected with mutant plasmids. MiR-411-5p may be involved in regulating the biological function of NSCLC cells via targeting PUM1. In addition, miR-411-5p may serve as a potential target for the molecular therapy of NSCLC.

Pumilio2regulates synaptic plasticity via translational repression of synaptic receptors in mice

PUMILIO 2 (PUM2) is a member of Pumilio and FBF (PUF) family, an RNA binding protein family with phylogenetically conserved roles in germ cell development. The Drosophila Pumilio homolog is also required for dendrite morphogenesis and synaptic function via translational control of synaptic proteins, such as glutamate receptors, and recent mammalian studies demonstrated a similar role in neuronal culture with associated motor and memory abnormalities in vivo. Importantly, transgenic mice with PUM2 knockout show prominent epileptiform activity, and patients with intractable temporal lobe epilepsy and mice with pilocarpine-induced seizures have decreased neuronal PUM2, possibly leading to further seizure susceptibility. However, how PUM2 influences synaptic function in vivo and, subsequently, seizures is not known. We found that PUM2 is highly expressed in the brain, especially in the temporal lobe, and knockout of Pum2 (Pum2–/–) resulted in significantly increased pyramidal cell dendrite spine and synapse density. In addition, multiple proteins associated with excitatory synaptic function, including glutamate receptor 2 (GLUR2), are up-regulated in Pum2–/– mice. The expression of GLUR2 protein but not mRNA is increased in the Pum2–/– mutant hippocampus, Glur2 transcripts are increased in mutant polysome fractions, and overexpression of PUM2 led to repression of reporter expression containing the 3′Untranslated Region (3′UTR) of Glur2, suggesting translation of GLUR2 was increased in the absence of Pum2. Overall, these studies provide a molecular mechanism for the increased temporal lobe excitability observed with PUM2 loss and suggest PUM2 might contribute to intractable temporal lobe epilepsy.

Inferring Methionine Sulfoxidation and serine Phosphorylation crosstalk from Phylogenetic analyses

BackgroundThe sulfoxidation of methionine residues within the phosphorylation motif of protein kinase substrates, may provide a mechanism to couple oxidative signals to changes in protein phosphorylation. Herein, we hypothesize that if the residues within a pair of phosphorylatable-sulfoxidable sites are functionally linked, then they might have been coevolving. To test this hypothesis a number of site pairs previously detected on human stress-related proteins has been subjected to analysis using eukaryote ortholog sequences and a phylogenetic approach.ResultsOverall, the results support the conclusion that in the eIF2α protein, serine phosphorylation at position 218 and methionine oxidation at position 222, belong to the same functional network. First, the observed data were much better fitted by Markovian models that assumed coevolution of both sites, with respect to their counterparts assuming independent evolution (p-value = 0.003). Second, this conclusion was robust with respect to the methods used to reconstruct the phylogenetic relationship between the 233 eukaryotic species analyzed. Third, the co-distribution of phosphorylatable and sulfoxidable residues at these positions showed multiple origins throughout the evolution of eukaryotes, which further supports the view of an adaptive value for this co-occurrence. Fourth, the possibility that the coevolution of these two sites might be due to structure-driven compensatory mutations was evaluated. The results suggested that factors other than those merely structural were behind the observed coevolution. Finally, the relationship detected between other modifiable site pairs from ataxin-2 (S814-M815), ataxin-2-like (S211-M215) and Pumilio homolog 1 (S124-M125), reinforce the view of a role for phosphorylation-sulfoxidation crosstalk.ConclusionsFor the four stress-related proteins analyzed herein, their respective pairs of PTM sites (phosphorylatable serine and sulfoxidable methionine) were found to be evolving in a correlated fashion, which suggests a relevant role for methionine sulfoxidation and serine phosphorylation crosstalk in the control of protein translation under stress conditions.

Elucidation of novel budding yeast separase mutants.

The mitotic separase cleaves Scc1 in cohesin to allow sister chromatids to separate from each other upon anaphase onset. Separase is also required for DNA damage repair. Here, we isolated and characterized 10 temperature-sensitive (ts) mutants of separase ESP1 in the budding yeast Saccharomyces cerevisiae. All mutants were defective in sister chromatid separation at the restricted temperature. Some esp1-ts mutants were hypersensitive to the microtubule poison benomyl and/or the DNA-damaging agent bleomycin. Overexpression of securin alleviated the growth defect in some esp1-ts mutants, whereas it rather exacerbated it in others. The Drosophila Pumilio homolog MPT5 was isolated as a high-dosage suppressor of esp1-ts cells. We discuss various features of separase based on these findings.

Validation of endogenous control reference genes for normalizing gene expression studies in endometrial carcinoma.

Real-time quantitative RT-PCR (qRT-PCR) is a powerful technique used for the relative quantification of target genes, using reference (housekeeping) genes for normalization to ensure the generation of accurate and robust data. A systematic examination of the suitability of endogenous reference genes for gene expression studies in endometrial cancer tissues is absent. The aims of this study were therefore to identify and evaluate from the thirty-two possible reference genes from a TaqMan(®) array panel their suitability as an internal control gene. The mathematical software packages geNorm qBasePLUS identified Pumilio homolog 1 (Drosophila) (PUM1), ubiquitin C (UBC), phosphoglycerate kinase (PGK1), mitochondrial ribosomal protein L19 (MRPL19) and peptidylpropyl isomerase A (cyclophilin A) (PPIA) as the best reference gene combination, whilst NormFinder identified MRPL19 as the best single reference gene, with importin 8 (IPO8) and PPIA being the best combination of two reference genes. BestKeeper ranked MRPL19 as the most stably expressed gene. In addition, the study was validated by examining the relative expression of a test gene, which encodes the cannabinoid receptor 1 (CB1). A significant difference in CB1 mRNA expression between malignant and normal endometrium using MRPL19, PPIA, and IP08 in combination was observed. The use of MRPL19, IPO8 and PPIA was identified as the best reference gene combination for the normalization of gene expression levels in endometrial carcinoma. This study demonstrates that the arbitrary selection of endogenous control reference genes for normalization in qRT-PCR studies of endometrial carcinoma, without validation, risks the production of inaccurate data and should therefore be discouraged.

A pumilio homolog in Polycelis sp.

Pumilio proteins (PUMs), members of the pumilio/fem-3 mRNA-binding factor (PUF) family, are eukaryote-specific RNA-binding proteins. We isolated a 2,048-basepair cDNA fragment of a pumilio homolog from the planarian flatworm Polycelis sp. This pumilio protein (PyPUM) contains a conserved pumilio homology domain (PUM-HD) consisting of eight repeats and two flanking half repeats. PyPUM shows high similarity to Dugesia japonica pumilio (DjPUM) from another planarian D. japonica, and their PUM-HD also shows high similarity to each other. Furthermore, our data showed that there is a flatworm-specific spacer between repeats 7 and 8. Phylogenetic analysis showed that PyPUM has a closer relationship to other PUM homologs from flatworms. These results provide a foundation for future functional studies of pumilio gene in Polycelis sp.

Comparative analysis of reference gene stability in human mesenchymal stromal cells during osteogenic differentiation

Mesenchymal stromal cells (MSCs) are one of the most frequently used cell sources for tissue engineering strategies. Cultivation of osteogenic MSCs is a prerequisite for cell-based concepts that aim at bone regeneration. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis is a commonly used method for the examination of mRNA expression levels. However, data on suitable reference genes for osteogenically cultivated MSCs is scarce. Hence, the aim of the study was to compare the regulation of different potential reference genes in osteogenically stimulated MSCs. Human MSCs were isolated from bone marrow aspirates of N = 6 hematologically healthy individuals, expanded by polystyrene-adherence, and maintained with and without osteogenic supplements for 14 days. Cellular proliferation and osteogenic differentiation were assessed by total DNA quantification, cell-specific alkaline phosphatase (ALP) activity and by qualitative staining for ALP and alizarin red, respectively. mRNA expression levels of N = 32 potential reference genes were quantified using the human Endogenous Control TaqMan® assays. mRNA expression stability was calculated using geNorm. The combined use of the most stable reference genes and DNA-damage-inducible alpha, Pumilio homolog 1, and large ribosomal protein P0 significantly improved gene expression accuracy as compared to the use of the commonly used reference genes beta actin and glyceraldehyde-3-phosphate dehydrogenase during qRT-PCR-based target gene expression analysis of osteogenically stimulated MSCs.

PUF-8, a Pumilio Homolog, Inhibits the Proliferative Fate in theCaenorhabditis elegansGermline

Stem cell populations are maintained by keeping a balance between self-renewal (proliferation) and differentiation of dividing stem cells. Within the Caenorhabditis elegans germline, the key regulator maintaining this balance is the canonical Notch signaling pathway, with GLP-1/Notch activity promoting the proliferative fate. We identified the Pumilio homolog, PUF-8, as an inhibitor of the proliferative fate of stem cells in the C. elegans germline. puf-8(0) strongly enhances overproliferation of glp-1(gf) mutants and partially suppresses underproliferation of a weak glp-1(lf) mutant. The germline tumor that is formed in a puf-8(0); glp-1(gf) double mutant is due to a failure of germ cells to enter meiotic prophase. puf-8 likely inhibits the proliferative fate through negatively regulating GLP-1/Notch signaling or by functioning parallel to it.

Pumilio-2 Function in the Mouse Nervous System

Coordinated mRNA translation at the synapse is increasingly recognized as a critical mechanism for neuronal regulation. Pumilio, a translational regulator, is known to be involved in neuronal homeostasis and memory formation in Drosophila. Most recently, the mammalian Pumilio homolog Pumilio-2 (Pum2) has been found to play a role in the mammalian nervous system, in particular in regulating morphology, arborization and excitability of neuronal dendrites, in vitro. However, the role of Pum2 in vivo remains unclear. Here, we report our investigation of the functional and molecular consequences of Pum2 disruption in vivo using an array of neurophysiology, behavioral and gene expression profiling techniques. We used Pum2-deficient mice to monitor in vivo brain activity using EEG and to study behavior traits, including memory, locomotor activity and nesting capacities. Because of the suspected role of Pum2 in neuronal excitability, we also examined the susceptibility to seizure induction. Finally, we used a quantitative gene expression profiling assay to identify key molecular partners of Pum2. We found that Pum2-deficient mice have abnormal behavioral strategies in spatial and object memory test. Additionally, Pum2 deficiency is associated with increased locomotor activity and decreased body weight. We also observed environmentally-induced impairment in nesting behavior. Most importantly, Pum2-deficient mice showed spontaneous EEG abnormalities and had lower seizure thresholds using a convulsing dosage of pentylenetetrazole. Finally, some genes, including neuronal ion channels, were differentially expressed in the hippocampus of Pum2-deficient mice. These findings demonstrate that Pum2 serves key functions in the adult mammalian central nervous system encompassing neuronal excitability and behavioral response to environmental challenges.

Biochemical Characterization of Pumilio1 and Pumilio2 in Xenopus Oocytes

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.

A structural-based statistical approach suggests a cooperative activity of PUM1 and miR-410 in human 3'-untranslated regions

BackgroundMicro (mi)RNAs comprise a large family of small non-coding RNAs that are thought to regulate a large fraction of protein-coding genes. Generally, miRNAs downregulate messenger (m)RNA expression by binding to the 3' untranslated regions (UTRs) of the RNA molecules. An important factor for binding specificity is the matching in the seed region. In addition, target site accessibility is thought to be crucial for efficient repression of miRNA targets. Several recent studies indicated that miRNA repression can be facilitated by RNA-binding proteins. In this study, we examine the conjecture that RNA-binding proteins are involved in ushering miRNAs to bind targets that are initially less accessible.ResultsWe analyzed human 3'-UTR sequences containing potential binding sites of 153 conserved miRNA families, and ranked sequences around the sites according to their miRNA accessibility. By applying a rank-based motif search tool to these miRNA targets, we found motifs that are enriched among less accessible targets. As expected from our ranking method, most of the significant motifs were GC-rich. However, one AU-rich motif was found to be enriched among miR-410 less accessible targets. This motif resembles the Pumilio homolog 1 (PUM1) consensus binding site. We observed a stronger enrichment of the PUM1 motif in conserved targets than in non-conserved targets; moreover, the enrichment of this motif was found to be conserved in a subset of placental mammals. Further, we analyzed publicly available gene expression data, and found that the mutual expression of PUM1 and miR-410 has a greater negative influence on the expression of low accessibility targets than on other targets, an effect that was stronger than when considering both miR-410 and PUM1 separately.ConclusionsTaken together, our findings suggest a cooperative relationship between miR-410 and PUM1 in regulating human highly structured 3'-UTRs. This kind of cooperation can allow a second level of regulation of such targets. Considering cases in which miRNAs bind low accessibility targets may help to improve current miRNA prediction tools and to obtain a better understanding of the mechanisms underlying miRNA regulation activity.

The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3′UTR-mediated repression of the let-7 microRNA target gene, hbl-1

The Puf family of RNA-binding proteins directs cell fates by regulating gene expression at the level of translation and RNA stability. Here, we report that the Caenorhabditis elegans pumilio homolog, puf-9, controls the differentiation of epidermal stem cells at the larval-to-adult transition. Genetic analysis reveals that loss-of-function mutations in puf-9 enhance the lethality and heterochronic phenotypes caused by mutations in the let-7 microRNA (miRNA), while suppressing the heterochronic phenotypes of lin-41, a let-7 target and homolog of Drosophila Brat. puf-9 interacts with another known temporal regulator hbl-1, the Caenorhabditis elegans ortholog of hunchback. We present evidence demonstrating that puf-9 is required for the 3′UTR-mediated regulation of hbl-1, in both the hypodermis and the ventral nerve cord. Finally, we show that this regulation is dependent on a region of the hbl-1 3′UTR that contains putative Puf family binding sites as well as binding sites for the let-7 miRNA family, suggesting that puf-9 and let-7 may mediate hypodermal seam cell differentiation by regulating common targets.

P3-441: Expression of mRNAs regulating local translation in synaptic terminals of MCI and early ad brain

Recent evidence suggests Alzheimer's Disease (AD) begins with subtle alterations of synaptic function producing memory deficits prior to histopathological changes. Decreased expression of presynaptic proteins before synapse loss is part of a continuum of modifications during clinical progression of dementia. However, activity–dependant expression of proteins locally translated in both pre–and post–synaptic terminals, and mRNA binding proteins regulating their translation, have not been assessed in the AD brain. Perturbation of synaptic protein synthesis could result from modifications in structure and function of the pre–synaptic terminal and/or direct effects of Ab peptides at the post–synaptic terminal. To compare expression of mRNAs locally translated at the synapse in MCI/early AD versus controls. Patients were grouped primarily according to MMSE but also adjusted for amyloid burden. Frontal cortex (Area 9) was obtained post–mortem, from 8 age–matched controls, 6 MCI/early AD and 10 moderate–to–severe AD patients. Synaptoneurosomes were prepared and mRNA extracted for Affymetrix microarray profiling and quantitative PCR. Clustering of genes and fold change data was prepared with the Bioconductor package. Gene annotation and gene ontology groupings were identified using EASE (http://david.niaid.nih.gov/david/ease.htm). We screened mRNA sequences of changed genes for 3∼ UTR regulatory motifs, such as the cytoplasmic polyadenylation element (CPE), which regulate local translation at the synapse. In MCI there is up–regulation of genes involved in synaptic transmission, transport (including vesicle–mediated transport) and cell recognition, and down–regulation of cell–cycle genes, (p<0.01). Approximately 50% of up–regulated genes code for proteins localized to the membrane. We found 50 putative CPE–containing mRNAs out of 304 up–regulated (16%) and 22 of 157 down–regulated (14%). Genes encoding the mRNA–binding proteins pumilio homolog and HuD, and some corresponding mRNAs they regulate, were also up–regulated in MCI. Synaptoneurosome samples were evaluated for in vitro translation using biotin–labeled tRNA. Immunoblots revealed biotin labeled aCAMKII in all synaptosomal preparations tested. Thus, using human synaptoneurosomes, we have enriched a group of mRNAs with regulatory sequences involved in de novo protein synthesis at the synapse. Microarray profiling reveals that some of these genes are impacted in MCI brain and may be implicated in functional impairment during AD progression.

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.

Statistical modeling for selecting housekeeper genes

Statistical models are presented for selecting the best housekeepers to normalize quantitative data within a given tissue type and across different types of tissue samples.