PAZ Domain Research Articles

In vivo structure–function analysis of human Dicer reveals directional processing of precursor miRNAs

Dicer is an RNase III family endoribonuclease and haploinsufficient tumor suppressor that processes mature miRNAs from the 5' (5p) or 3' (3p) arm of hairpin precursors. In murine Dicer knockout fibroblasts, we expressed human Dicer with point mutations in the RNase III, helicase, and PAZ domains and characterized miRNA expression by Northern blot and massively parallel sequencing of small RNAs. We report that inactivation of the RNase IIIA domain results in complete loss of 3p-derived mature miRNAs, but only partial reduction in 5p-derived mature miRNAs. Conversely, inactivation of the RNase IIIB domain by mutation of D1709, a residue mutated in a subset of nonepithelial ovarian cancers, results in complete loss of 5p-derived mature miRNAs, including the tumor-suppressive let-7 family, but only partial reduction in 3p-derived mature miRNAs. Mutation of the PAZ domain results in global reduction of miRNA processing, while mutation of the Walker A motif in the helicase domain of Dicer does not alter miRNA processing. These results provide insight into the biochemical activity of human Dicer in vivo and, furthermore, suggest that mutation of the clinically relevant residue D1709 within the RNase IIIB results in a uniquely miRNA-haploinsufficient state in which the let-7 family of tumor suppressor miRNAs is lost while a complement of 3p-derived miRNAs remains expressed.

Argonaute identity defines the length of mature mammalian microRNAs

MicroRNAs (miRNAs) are 19- to 25-nt-long non-coding RNAs that regulate gene expression by base-pairing with target mRNAs and reducing their stability or translational efficiency. Mammalian miRNAs function in association with four closely related Argonaute proteins, AGO1–4. All four proteins contain the PAZ and the MID domains interacting with the miRNA 3′ and 5′ termini, respectively, as well as the PIWI domain comprising an mRNA ‘slicing’ activity in the case of AGO2 but not AGO1, AGO3 and AGO4. However, the slicing mode of the miRNA-programmed AGO2 is rarely realized in vivo and the four Argonautes are thought to play largely overlapping roles in the mammalian miRNA pathway. Here, we show that the average length of many miRNAs is diminished during nervous system development as a result of progressive shortening of the miRNA 3′ ends. We link this modification with an increase in the fractional abundance of Ago2 in the adult brain and identify a specific structural motif within the PAZ domain that enables efficient trimming of miRNAs associated with this but not the other three Argonautes. Taken together, our data suggest that mammalian Argonautes may define the length and possibly biological activity of mature mammalian miRNAs in a developmentally controlled manner.

Local and global effects of Mg2+ on Ago and miRNA-target interactions

Three magnesium ions (Mg(2+)), named Mg1 (in Mid domain), Mg2 and Mg3 (both in PIWI domain), located at the small RNA binding domain of Argonaute (Ago) protein, are important for sequence-specific miRNA-target interactions. Such conjunction between the Ago protein and miRNA raises the question: How do Mg(2+) ions participate in the recognition process of miRNA by Ago or its target. Furthermore, it is still unclear whether the Mg(2+) ions contribute to the local or global stability of the miRNA complex. In this work, we have performed a series of 16 independent molecular dynamic simulations (MD) to characterize the functions of Mg(2+), hydration patterns and the conformational events involved in the miRNA-target interactions. The cross correlation analysis shows that Mg1 and Mg2 significantly enhance a locally cooperated movement of the PAZ, PIWI and Mid domains with the average correlation coefficient of ∼0.65, producing an "open-closed" motion (rotation Angle, 46.5°) between the PAZ and PIWI domains. Binding of Mg3 can globally stabilize the whole Ago protein with the average RMSD of ∼0.34 Å, compared with the systems in absence of Mg3 (average RMSD = ∼0.43 Å). Three structural water molecules surrounding the Mg(2+)-binding regions also stabilize these ions, thus facilitating the recognition of miRNA to its target. In addition, the thermodynamic analysis also verifies the positive contribution of all three Mg(2+) to the binding of miRNA to Ago, as well as the importance Mg2 plays in the cleavage of the miRNA targets.

Human chorionic gonadotropin suppresses expression of Piwis in common carp (Cyprinus carpio) ovaries

Piwi proteins are required for germline maintenance and gonad development. In this study, the cDNAs encoding Piwil1 and Piwil2 were cloned and sequenced from the common carp. The full-length cDNA of Piwil1 and Piwil2 were 3114 and 3421bp, encoding 858 and 1034 amino acids including PAZ domain and PIWI domain, respectively. In addition, the Piwil1 and Piwil2 proteins shared high homology with other teleosts. Reverse transcriptase PCR revealed that the Piwi mRNAs were exclusively expressed in adult testes and ovaries. Using real-time PCR, expression study of different developmental profiles showed that Piwil1 and Piwil2 were down-regulated during pre-ovulation. Further, human chorionic gonadotropin treatment in ovaries (in vivo) and in cultured ovaries cells (in vitro) resulted in down-regulation of Piwi RNAs. These results suggest that the decreased expression which was regulated by hormone plays a crucial role during ovarian differentiation and development.

Piwil2 Suppresses P53 by Inducing Phosphorylation of Signal Transducer and Activator of Transcription 3 in Tumor Cells

Piwi proteins have been implicated in germ cell proliferation, differentiation, germline stem cell maintenance and transposon control in germline from Drosophila to mammals. The Piwi-like2 (piwil2) gene is mainly expressed in testis or embryonic cells among normal tissues but widely expressed in tumors. However, it remains to be fully determined through which mechanism piwil2 is involved in tumorigenesis. Here we report that Human piwil2, or Hili represses the tumor suppressor P53 in human cancer cells. Immunoprecipitation analysis shows that Piwil2 can directly associate with Signal Transducer and Activator of Transcription 3 (STAT3) protein via its PAZ domain and form a Piwil2/STAT3/c-Src triple protein-protein complex. Furthermore, STAT3 is phosphorylated by c-Src and translocated to nucleus, then binds to P53 promoter and represses its transcription. The present study demonstrated that Piwil2 plays a role in anti-apoptosis in tumor cells possessing P53 as a positive regulator of STAT3 signaling pathway, providing novel sights into roles of Piwil2 in tumorigenesis.

Cucumber mosaic virus suppressor 2b binds to AGO4‐related small RNAs and impairs AGO4 activities

Cucumber mosaic virus suppressor 2b (CMV2b) is a nuclear viral suppressor that interferes with local and systemic silencing and inhibits AGO1 slicer activity. CMV2b-mediated transgene hypomethylation and its localization in Cajal bodies suggests a role of CMV2b in RNA-directed DNA methylation (RdDM). However, its direct involvement in RdDM, or its binding with small RNAs (sRNAs) in vivo is not yet established. Here, we show that CMV2b binds both microRNAs (miRNAs) and small interfering RNAs (siRNAs) in vivo. sRNA sequencing data from the CMV2b immunocomplex revealed its preferential binding with 24-nt repeat-associated siRNAs. We provide evidence that CMV2b also has direct interaction with the AGO4 protein by recognizing its PAZ and PIWI domains. Subsequent analysis of AGO4 functions revealed that CMV2b reduced AGO4 slicer activity and the methylation of several loci, accompanied by the augmented accumulation of 24-nt siRNAs in Arabidopsis inflorescences. Intriguingly, CMV2b also regulated an AGO4-related epiallele independently of its catalytic potential, which further reinforces the repressive effects of CMV2b on AGO4 activity. Collectively, our results demonstrate that CMV2b can counteract AGO4-related functions. We propose that by adopting novel counter-host defense strategies against AGO1 and AGO4 proteins, CMV creates a favorable cellular niche for its proliferation.

TOPAZ1, a novel germ cell-specific expressed gene conserved during evolution across vertebrates.

BackgroundWe had previously reported that the Suppression Subtractive Hybridization (SSH) approach was relevant for the isolation of new mammalian genes involved in oogenesis and early follicle development. Some of these transcripts might be potential new oocyte and granulosa cell markers. We have now characterized one of them, named TOPAZ1 for the Testis and Ovary-specific PAZ domain gene.Principal FindingsSheep and mouse TOPAZ1 mRNA have 4,803 bp and 4,962 bp open reading frames (20 exons), respectively, and encode putative TOPAZ1 proteins containing 1,600 and 1653 amino acids. They possess PAZ and CCCH domains. In sheep, TOPAZ1 mRNA is preferentially expressed in females during fetal life with a peak during prophase I of meiosis, and in males during adulthood. In the mouse, Topaz1 is a germ cell-specific gene. TOPAZ1 protein is highly conserved in vertebrates and specifically expressed in mouse and sheep gonads. It is localized in the cytoplasm of germ cells from the sheep fetal ovary and mouse adult testis.ConclusionsWe have identified a novel PAZ-domain protein that is abundantly expressed in the gonads during germ cell meiosis. The expression pattern of TOPAZ1, and its high degree of conservation, suggests that it may play an important role in germ cell development. Further characterization of TOPAZ1 may elucidate the mechanisms involved in gametogenesis, and particularly in the RNA silencing process in the germ line.

Modeling and analysis of Schistosoma Argonaute protein molecular spatial conformation

Objective To analyze the amino acid sequence composition, secondary structure, the spatial conformation of its domain and other characteristics of Argonaute protein. Methods Bioinformatics tools and the internet server were used. Firstly, the amino acid sequence composition features of the Argonaute protein were analyzed, and the phylogenetic tree was constructed. Secondly, Argonaute protein's distribution of secondary structure and its physicochemical properties were predicted. Lastly, the protein functional expression form of the domain group was established through the Phyre-based analysis on the spatial conformation of Argonaute protein domains. Results 593 amino acids were encoded by Argonaute protein, the phylogenetic tree was constructed, and Argonaute protein's distribution of secondary structure and its physicochemical properties were obtained through analysis. In addition, the functional expression form which comprised the N-terminal PAZ domain and C-terminal Piwi domain for the Argonaute protein was obtained with Phyre. Conclusions The information relationship between the structure and function of the Argonaute protein can be initially established with bioinformatics tools and the internet server, and this provides the theoretical basis for further clarifying the function of Schistosoma Argonaute protein.

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Argonaute proteins (AGOs) are essential effectors in RNA-mediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein. Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5' end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing.

Structural insights into piRNA recognition by the human PIWI‐like 1 PAZ domain

Major advancements have been made in the past decade towards understanding the regulatory role of small non-coding RNAs (ncRNAs) in diverse development processes1. An integral part of this regulation is the conserved PIWI/Argonaute protein family. Phylogenetic analysis indicates that this protein family can be divided into two sub-families: the Piwi sub-family, named after Drosophila melanogaster protein PIWI (P-element induced wimpy testis), and the Ago sub-family, based on its founding member in Arabidopsis thaliana (Ago). Ago proteins are involved in RNA-mediated gene silencing complexes and utilize short guide RNAs to target specific nucleic acids1-4, whereas Piwi proteins bind to a novel class of ncRNAs called PIWI-interacting RNAs (piRNAs) that play diverse functions in germline development and gametogenesis5-11. Piwi/Ago proteins have a molecular weight of about 90 kDa and are defined by three protein motifs: the PAZ, MID and PIWI domains. The N-terminal PAZ domain is accountable for binding the 3′-end of the guide strand RNA; the middle MID domain is responsible for binding the 5′-phosphate RNA; and the following C-terminal PIWI domain has an RNase H endonuclease fold that is responsible for the RNA cleavage. The best-characterized small RNA partners of Ago proteins are microRNAs (miRNAs). Ago proteins utilize miRNAs to silence genes post-transcriptionally or use small-interfering RNAs (siRNAs) in both transcription and post-transcription silencing mechanisms12-17. However, Piwi proteins are restricted to germline cells and stem cells, and have been found to interact with a novel class of piRNAs (∼28-33 nucleotides) longer than miRNAs and siRNAs (∼20 nucleotides), suggesting novel functions distinct from those of Ago proteins18-22. piRNAs are 28 to 33 nucleotides in length and have been characterized by the cloning of small RNAs from anti-Piwi immunoprecipitates prepared from mammalian testes7-9. As the proportion of repeat elements able to generate piRNAs is actually smaller within the piRNA regions than the frequency of repeat sequences in the mouse genome, they are not believed to be derived from repeat sequences. It is believed that piRNAs are processed from single-stranded primary transcripts that are transcribed from defined genomic regions and have a preference for a uridine at their 5′ terminal6-10,23. Mammalian piRNAs are a highly complex mix of sequences, with tens of thousands of distinct piRNAs generated from the 50 to 100 defined primary transcripts. This may suggest that mammalian piRNAs, unlike miRNAs, are not processed in a precise manner. However, approximately 20% of all piRNA sequences were cloned three or more times, and many piRNA sequences from the same strand are partially overlapping, suggesting a quasi-random mechanism. The mechanism of biogenesis of D. melanogaster rasiRNAs is beginning to be elucidated, and may offer parallels for a specific mode of processing for piRNAs as well. piRNA biogenesis is thought to be Dicer-independent and they appear to be 2′-O'methylated at their 3′ terminal24-26. Between mammals, mature piRNAs are not conserved; however, the genomic regions, from which they derive, in particular the promoter sequences, are conserved. Mammalian piRNAs are strongly expressed in the male germline, their total number per cell obtained from testis tissue reaching up to two million – about 10-fold higher than the miRNA content of these cells. Despite their importance in developmental biology, a mechanistic understanding of the biogenesis of piRNAs is lacking, and the specific functional role PIWI proteins play in piRNA generation remains elusive. To understand the molecular basis of piRNA recognition by PIWI proteins, we have determined the three-dimensional solution structures of the human PIWI-like 1 (hPIWIL1) PAZ domain in its free form, as well as bound to a ssRNA (5′-pUGACA) oligonucleotide using nuclear magnetic resonance (NMR) spectroscopy methods.

Double Take on Piwi Protein/piRNA Complex Structure

Two recent studies by the Carlomagno/Pillai labs (Simon etal., 2011) and the Patel group (Tian etal., 2011) report the structures of complexes of Piwi PAZ domains with piRNA, utilizing two major methods in structural biology, NMR spectroscopy, and X-ray crystallography, and derive very similar structures with similar resolution.

Recognition of 2′- O-Methylated 3′-End of piRNA by the PAZ Domain of a Piwi Protein

Piwi proteins are germline-specific Argonautes that associate with small RNAs called Piwi-interacting RNAs (piRNAs), and together with these RNAs are implicated in transposon silencing. The PAZ domain of Argonaute proteins recognizes the 3'-end of the RNA, which in the case of piRNAs is invariably modified with a 2'-O-methyl group. Here, we present the solution structure of the PAZ domain from the mouse Piwi protein, MIWI, in complex with an 8-mer piRNA mimic. The methyl group is positioned in a hydrophobic cavity made of conserved amino acids from strand β7 and helix α3, where it is contacted by the side chain of methionine-382. Our structure is similar to that of Ago-PAZ, but subtle differences illustrate how the PAZ domain has evolved to accommodate distinct 3' ends from a variety of RNA substrates.

Structural and Binding Study of Modified siRNAs with the Argonaute 2 PAZ Domain by NMR Spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.

Piwi‐expressing hemoblasts serve as germline stem cells during postembryonic germ cell specification in colonial ascidian, Botryllus primigenus

Animals that propagate asexually are exciting models to investigate the cellular system, which produces germline cells constitutively throughout life. The present research investigated whether piwi was a germline-specific marker in the colonial ascidian Botryllus primigenus. An approximately 2.8 kb long cDNA fragment was cloned and termed BpPiwi, since the obtained amino acid sequence (874 aa) contained PAZ and PIWI domains. BpPiwi was expressed specifically by germline cells such as the loose cell mass (germline precursor cells), oocytes, spermatogonia, and spermatocytes. In addition, BpPiwi transcripts were also detected in some coelomic cells in the hemocoel and tunic vessels. BpPiwi(+) coelomic cells possessed similar morphological features to hemoblasts (stem cells). The concentration of BpPiwi(+) cells was found to be significantly lower than that obtained for hemoblasts suggesting that BpPiwi(+) cells comprise a fraction of hemoblasts. Further, the ability of BpPiwi(+) cells to serve as somatic stem cells was examined. No BpPiwi signals were detected from somatic hemoblasts forming vascular buds. The genetic knockdown of BpPiwi induced by siRNA injection resulted in the formation of a defective germline precursor. These results suggest that BpPiwi(+) hemoblasts reside in the hemocoel and tunic vessels and function as germline stem cells in the postembryonic colony. Based on the findings of the characterization of three effective germline genes piwi, vasa, and nanos, we propose that germline stem cells reside as BpPiwi(+)/BpVas(-)/BpNos(+) hemoblasts in B.primigenus.

Cloning, characterization and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids

Over the last years an expanding family of small non-coding RNAs (sRNA) has been identified in eukaryotic genomes which behave as sequence-specific triggers for mRNA degradation, translation repression, heterochromatin formation and genome stability. To achieve their effectors functions, sRNAs associate with members of the Argonaute protein family. Argonaute proteins are segregated into three paralogous groups: the AGO-like subfamily, the PIWI-like subfamily, and the WAGO subfamily (for Worm specific AGO). Detailed phylogenetic analysis of the small RNA-related machinery components revealed that they can be traced back to the common ancestor of eukaryotes. However, this machinery seems to be lost or excessively simplified in some unicellular organisms such as Saccharomyces cerevisiae, Trypanosoma cruzi, Leishmania major and Plasmodium falciparum which are unable to utilize dsRNA to trigger degradation of target RNAs. We reported here a unique ORF encoding for an AGO/PIWI protein in T. cruzi which was expressed in all stages of its life cycle at the transcript as well as the protein level. Database search for remote homologues, revealed the presence of a divergent PAZ domain adjacent to the well supported PIWI domain. Our results strongly suggested that this unique AGO/PIWI protein from T. cruzi is a canonical Argonaute in terms of its domain architecture. We propose to reclassify all Argonaute members from trypanosomatids as a distinctive phylogenetic group representing a new subfamily of Argonaute proteins and propose the generic designation of AGO/PIWI-tryp to identify them. Inside the Trypanosomatid-specific node, AGO/PIWI-tryps were clearly segregated into two paralog groups designated as AGO-tryp and PIWI-tryp according to the presence or absence of a functional link with RNAi-related phenomena, respectively.

Systematic deletion and site-directed mutagenesis of FHVB2 establish the role of C-terminal amino acid residues in RNAi suppression

Viruses and siRNA/miRNA machinery of the host cell interact in diverse ways with the virus encoded RNAi suppressor proteins. These interactions have implications on the replication and pathogenicity of the virus and also on the immune response of the host. Suppressor protein B2 of insect Flock House Virus (FHVB2), has been shown to mediate RNAi suppression via N-terminal region by directly binding to dsRNA. We have previously shown that FHVB2 protein also interacts with host Dicer protein via its PAZ domain. In the present study, we performed systematic mutagenesis studies to map the FHVB2 regions involved in mediating suppression of RNAi. Progressive deletion of 17 amino acids from N- and C-terminii of FHVB2 resulted in cumulative decrease in RNAi suppression activity of FHVB2. The deletion of 17 amino acids from the C-terminus resulted in more reduction in RNAi suppression in comparison to the N-terminal deletions. Subsequently, we generated 17 successive point mutants of FHVB2 C-terminus and evaluated the RNAi suppression activity for each of the point mutants. Each point mutation resulted in a significant reduction in RNAi suppression activity of FHVB2. These results provide evidence for the role of C-terminal of FHVB2 in RNAi suppression.

Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein

Argonaute (AGO) proteins are core components of RNA-induced silencing complexes and have essential roles in RNA-mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino-terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE-2 from Neurospora crassa. This domain adopts a Rossmann-like fold and recognizes the 5'-terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5'-nucleotide-binding site shares common residues with a second, adjacent ligand-binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.

Identification of two piwi genes and their expression profile in honeybee, Apis mellifera

Piwi genes play an important role in regulating spermatogenesis and oogenesis because they participate in the biogenesis of piRNAs, a new class of noncoding RNAs. However, these genes are not well understood in most insects. To understand the function of piwi genes in honeybee reproduction, we amplified two full-length piwi-like genes, Am-aub and Am-ago3. Both the cloned Am-aub and Am-ago3 genes contained typical PAZ and PIWI domains and active catalytic motifs "Asp-Asp-Asp/His/Glu/Lys," suggesting that the two piwi-like genes possessed slicer activity. We examined the expression levels of Am-aub and Am-ago3 in workers, queens, drones, and female larvae by quantitative PCR. Am-aub was more abundant than Am-ago3 in all the tested samples. Both Am-aub and Am-ago3 were highly expressed in drones but not in workers and queens. The significant finding was that the larval food stream influenced the expression of Piwi genes in adult honeybees. This helps to understand the nutritional control of reproductive status in honeybees at the molecular level.

Abstract 3946: Stem cell protein piwil2 enhances nucleotide excision repair through regulating chromatin remodeling following DNA damage

Abstract The piwi family gene, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. Three piwi homologs have been identified in the mouse genome. Amongst these, mili (piwil2) protein expression appears to be associated with tumorigenesis as evidenced by the facts that piwil2 stably expresses in pre-cancerous stem cells and also appears at variable levels in different human and animal tumor cell lines. Based on our preliminary data showing that piwil2 is induced in normal cultured human fibroblasts in vitro by physical and chemical genotoxic agents, we hypothesized that piwil2 might function in DNA damage response. Here, we demonstrated that mammalian cells lacking piwil2 are hypersensitive to cisplatin and UV irradiation. In addition, nucleotide excision repair (NER) efficiency is significantly decreased in the absence of piwil2 as assessed by the removal of cisplatin-induced intra-strand cross-links (Pt-GG) and UV-induced cyclobutane pyrimidine dimers (CPDs) from genomic DNA. Although quantitative real-time PCR analysis indicated higher transcript levels of various NER proteins in piwil2 knockout mouse embryo fibroblasts (MEFs), the Western blotting did not show an obvious difference in the NER protein levels between mili+/+ and mili−/− MEFs. Since piwil2 is believed to orchestrate chromatin remodeling, we reasoned that low NER efficiency in mili−/− MEFs may result from disrupted chromatin remodeling in response to UV or cisplatin induced DNA damage. Thus, chromatin relaxation and histone acetylation was analyzed in these MEFs following treatment with UV or cisplatin. MEFs lacking piwil2 showed less sensitivity to micrococcal nuclease (MNase) digestion compared with piwil2-proficient MEFs, indicating a compact chromatin structure in these cells. Moreover, in contrast to mili+/+ MEFs, mili−/− MEFs exhibited decreased acetylated Histone H3 upon both UV and cisplatin treatment. This decrease could be blocked by histone deacetylase (HDAC) inhibition indicating that piwil2 may function in damage-induced chromatin remodeling via inhibiting the HDAC activity. In summary, our data suggests that piwli2 can participate in the NER through regulating chromatin relaxation which could be a pivotal factor in genotoxin-induced tumorigenesis as well as efficacy of cancer therapeutics. (Supported by NIH grants CA93413, ES2388 and ES12991 to AAW). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3946.

Systematic studies on the recognition of 3′‐end methylated small silencing RNAs by PAZ domains

The PAZ domain is a conserved RNA‐binding module that is only found in two core components of RNA silencing, Argonaute and Dicer. Previous studies showed that the PAZ domain specifically recognizes 3′‐end 2‐nucleotide overhang of small RNAs. Recently, it has been demonstrated that many small silencing RNAs, such as miRNAs and siRNAs in plants, piRNAs in animals and siRNAs in Drosophila, are methylated on the 2′‐OH of the 3′ terminal nucleotide. This kind of modification resulted in a reduction of binding affinity between the small RNAs and the PAZ domain from human AGO1. In order to further understand the function of the PAZ domain from different Argonaute proteins, systematic binding and structural studies of PAZ domains derived from two subfamilies of Argonaute proteins (AGO and PIWI) and four species (human, fly, fish and worm) are carried out. Results from different binding assays (fluorescence anisotropy and isothermal titration calorimetry) indicate that PAZ domains from human and Drosophila AGO1 proteins bind to 3′‐end methylated small RNAs with reduced binding affinities but PAZ domains from Drosophila AGO2 and other PIWI subfamily Argonaute proteins bind to 3′‐end methylated small RNAs with enhanced binding affinities, which consist with deeper and wider pockets found in PAZ domains from Drosophila AGO2 and PIWI subfamily proteins. Structural comparison implies that PAZ domains from divergent Argonaute proteins may have distinct functions.