P-element-induced Wimpy Testis Proteins Research Articles

PIWI-RNAs Small Noncoding RNAs with Smart Functions: Potential Theranostic Applications in Cancer.

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a new class of small noncoding RNAs (ncRNAs) that bind components of the PIWI protein family. piRNAs are specifically expressed in different human tissues and regulate important signaling pathways. Aberrant expressions of piRNAs and PIWI proteins have been associated with tumorigenesis and cancer progression. Recent studies reported that piRNAs are contained in extracellular vesicles (EVs), nanosized lipid particles, with key roles in cell-cell communication. EVs contain several bioactive molecules, such as proteins, lipids, and nucleic acids, including emerging ncRNAs. EVs are one of the components of liquid biopsy (LB) a non-invasive method for detecting specific molecular biomarkers in liquid samples. LB could become a crucial tool for cancer diagnosis with piRNAs as biomarkers in a precision oncology approach. This review summarizes the current findings on the roles of piRNAs in different cancer types, focusing on potential theranostic applications of piRNAs contained in EVs (EV-piRNAs). Their roles as non-invasive diagnostic and prognostic biomarkers and as new therapeutic options have been also discussed.

P-Element-Induced Wimpy Testis Proteins and P-Element-Induced Wimpy Testis-Interacting RNAs Expression in Ovarian Cancer Stem Cells.

Background: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a type of noncoding RNA and are predominantly expressed in germline cells. piRNAs function as gene regulators and potential biomarkers for the development of a number of malignancies. The biological importance of piRNAs in ovarian cancer is still unknown. In this study, we investigated the expression of piRNAs in ovarian cancer stem cells and compared it with that in adherent cells. Methods: To assess changes in the expression levels of PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2, we used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis. Changes in piRNA expression levels in ovarian cancer stem cells were analyzed using Arraystar piRNA microarray screening. Gene Ontology (GO) enrichment analysis was conducted to determine the potential functions of piRNAs. Results: Using microarray analysis, we identified a cohort of differentially expressed piRNAs. Fifteen piRNAs, including DQ570763 and DQ597396, were downregulated, and 58 piRNAs were upregulated when compared with those in adherent A2780 and SKOV3 cells (p > 0.05, >2.0, respectively). GO functions of the downregulated piRNAs (DQ570763 and DQ570797) suggest that their roles are commonly associated with the Golgi apparatus. In addition, A2780-SP and SKOV3-SP cells had higher PIWIL3 and PIWIL4 mRNA levels than adherent cells (A2780 and SKOV3). Moreover, we determined, using receiver operating characteristic plot, that the expression level of PIWIL4 was lower in responders than in nonresponders after treatment with platins in patients with ovarian cancer. Finally, in ovarian cancer, PIWIL4 expression was associated with somatic mutations of dynein axonemal heavy chain 2, signal induced proliferation associated 1 like 2, YTH N6-methyladenosine RNA-binding protein 1, TBC1 domain family member 8, and LPS responsive Beige-like anchor protein. Conclusion: Our study showed that PIWI proteins and piRNAs are potential diagnostic and prognostic biomarkers for ovarian cancer.

The Regulation and Role of piRNAs and PIWI Proteins in Cancer

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are regulatory small non-coding RNAs that participate in transposon inactivation, chromatin regulation, and endogenous gene regulation. Numerous genetic and epigenetic factors regulate cell proliferation and tumor metastasis. PIWI proteins and piRNAs have been revealed to function in regulating upstream or downstream of oncogenes or tumor-suppressor genes in cancer tissues. In the present review, we summarize major recent findings in uncovering the regulation and role of PIWI proteins and piRNAs in tumorigenesis and highlight some of the promising applications of specific piRNAs in cancer therapeutics and as cancer biomarkers.

Critical Roles of PIWIL1 in Human Tumors: Expression, Functions, Mechanisms, and Potential Clinical Implications.

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a class of small non-coding RNA molecules that are 24–31 nucleotides in length. PiRNAs are thought to bind to PIWI proteins (PIWL1-4, a subfamily of Argonaute proteins), forming piRNA/PIWI complexes that influence gene expression at the transcriptional or post-transcriptional levels. However, it has been recently reported that the interaction of PIWI proteins with piRNAs does not encompass the entire function of PIWI proteins in human tumor cells. PIWIL1 (also called HIWI) is specifically expressed in the testis but not in other normal tissues. In tumor tissues, PIWIL1 is frequently overexpressed in tumor tissues compared with normal tissues. Its high expression is closely correlated with adverse clinicopathological features and shorter patient survival. Upregulation of PIWIL1 drastically induces tumor cell proliferation, epithelial-mesenchymal transition (EMT), invasion, cancer stem-like properties, tumorigenesis, metastasis and chemoresistance, probably via piRNA-independent mechanisms. In this article, we summarize the current existing literature on PIWIL1 in human tumors, including its expression, biological functions and regulatory mechanisms, providing new insights into how the dysregulation of PIWIL1 contributes to tumor initiation, development and chemoresistance through diverse signaling pathways. We also discuss the most recent findings on the potential clinical applications of PIWIL1 in cancer diagnosis and treatment.

PIWIL1 interacting RNA piR-017061 inhibits pancreatic cancer growth via regulating EFNA5.

PIWI (P element induced wimpy testis) integrating RNAs (piRNAs) are small non-coding RNAs with the length of approximately 30 nucleotides that plays crucial roles in germ cells and adult stem cells. Recently, accumulating data have shown that piRNA and PIWI proteins are involved in tumorigenesis. However, the roles of PIWI proteins and piRNAs in pancreatic cancer are still elusive. Here, we showed that piR-017061 is significantly downregulated in pancreatic cancer patients' samples and pancreatic cancer cell lines. Furthermore, we studied the function of piR-017061 in pancreatic cancer and our data revealed that piR-017061 inhibits pancreatic cancer cell growth in vitro and in vivo. Moreover, we analyzed the genomic loci around piR-017061 and identified EFNA5 as a novel target of piR-017061. Importantly, our data further revealed a direct binding between piR-017061 and EFNA5 mRNA mediated by PIWIL1. Mechanically, piR-017061 cooperates with PIWIL1 to facilitate EFNA5 mRNA degradation and loss of piR-017061 results in accumulation of EFNA5 which facilitates pancreatic cancer development. Hence, our data provided novel insights into PIWI/piRNA-mediated gene regulation and their function in pancreatic cancer. Since PIWI proteins and piRNA predominately express in germline and cancer cells, our study provided novel therapeutic strategy for pancreatic cancer treatment.

PIWI-interacting RNAs in human cancer.

P-element-induced wimpy testis (PIWI) interacting RNAs (piRNAs) are a class of small regulatory RNAs mechanistically similar to but much less studied than microRNAs and small interfering RNAs. Today the best understood function of piRNAs is transposon control in animal germ cells, which has earned them the name 'guardians of the germline'. Several molecular/cellular characteristics of piRNAs, including high sequence diversity, lack of secondary structures, and target-oriented generation seem to serve this purpose. Recently, aberrant expressions of piRNAs and PIWI proteins have been implicated in a variety of malignant tumors and associated with cancer hallmarks such as cell proliferation, inhibited apoptosis, invasion, metastasis and increased stemness. Researchers have also demonstrated multiple mechanisms of piRNA-mediated target deregulation associated with cancer initiation, progression or dissemination. We review current research findings on the biogenesis, normal functions and cancer associations of piRNAs, highlighting their potentials as cancer diagnostic/prognostic biomarkers and therapeutic tools. Whenever applicable, we draw connections with other research fields to encourage intercommunity conversations. We also offer recommendations and cautions regarding the general process of cancer-related piRNA studies and the methods/tools used at each step. Finally, we call attention to some issues that, if left unsolved, might impede the future development of this field.

Lesson from a Fab-enabled co-crystallization study of TDRD2 and PIWIL1.

The interaction of Tudor domain-containing proteins (TDRDs) with P-element-induced wimpy testis (PIWI) proteins plays critical roles in transposon silencing and spermatogenesis. Most human TDRDs recognize PIWI proteins in a methylarginine-dependent manner via their extended Tudor (eTudor) domains, except TDRD2, which prefers an unmethylated PIWI protein. In order to illustrate the recognition of unmethylated PIWI proteins by TDRD2, we extensively tried co-crystallization of the TDRD2 eTudor with different PIWIL1 peptides, but to no avail. Recombinant antigen-binding fragments (Fabs) have been used to crystallize some difficult proteins in the past, so we generated Fab against the TDRD2 eTudor protein using a phage-display antibody library, and one of these Fab fragments indeed facilitated the co-crystallization of TDRD2 and PIWIL1. Structural analysis of Fab, the TDRD2 eTudor domain in complex with an unmethylated PIWIL1-derived peptide revealed that the PIWIL1 residues G3 through R8 bound between the Tudor core and SN domain of TDRD2. The C-terminal residues of the PIWIL1 peptide were not resolved, presumably due to steric competition with the heavy chain of the Fab. We propose Fab-assisted crystallization as a tool not only for structural studies of single proteins, but also for analysis of interactions between proteins and their ligands in cases where co-crystallization of native protein complexes fails.

Identification and characterization of dysregulated P-element induced wimpy testis-interacting RNAs in head and neck squamous cell carcinoma.

It is clear that alcohol consumption is a major risk factor in the pathogenesis of head and neck squamous cell carcinoma (HNSCC); however, the molecular mechanism underlying the pathogenesis of alcohol-associated HNSCC remains poorly understood. The aim of the present study was to identify and characterize P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) and PIWI proteins dysregulated in alcohol-associated HNSCC to elucidate their function in the development of this cancer. Using next generation RNA-sequencing (RNA-seq) data obtained from 40 HNSCC patients, the piRNA and PIWI protein expression of HNSCC samples was compared between alcohol drinkers and non-drinkers. A separate piRNA expression RNA-seq analysis of 18 non-smoker HNSCC patients was also conducted. To verify piRNA expression, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed on the most differentially expressed alcohol-associated piRNAs in ethanol and acetaldehyde-treated normal oral keratinocytes. The correlation between piRNA expression and patient survival was analyzed using Kaplan-Meier estimators and multivariate Cox proportional hazard models. A comparison between alcohol drinking and non-drinking HNSCC patients demonstrated that a panel of 3,223 piRNA transcripts were consistently detected and differentially expressed. RNA-seq analysis and in vitro RT-qPCR verification revealed that 4 of these piRNAs, piR-35373, piR-266308, piR-58510 and piR-38034, were significantly dysregulated between drinking and non-drinking cohorts. Of these four piRNAs, low expression of piR-58510 and piR-35373 significantly correlated with improved patient survival. Furthermore, human PIWI-like protein 4 was consistently upregulated in ethanol and acetaldehyde-treated normal oral keratinocytes. These results demonstrate that alcohol consumption may cause dysregulation of piRNA expression in HNSCC and in vitro verifications identified 4 piRNAs that may be involved in the pathogenesis of alcohol-associated HNSCC.

Epigenetic roles of PIWI‑interacting RNAs (piRNAs) in cancer metastasis (Review).

P‑element‑induced wimpy testis (PIWI)‑interacting RNAs (piRNAs) are epigenetic‑related short ncRNAs that participate in chromatin regulation, transposon silencing, and modification of specific gene sites. These epigenetic factors or alterations are also involved in the growth of a variety of human cancers, including lung, breast, and colon cancer. Accumulating evidence has revealed that tumor metastasis and invasion involve genetic and epigenetic factors. Cancer metastasis is characterized by epigenetic alterations including DNA methylation and histone modification. Changes in DNA methylation, H3K9me3 heterochromatin and transposable elements have been detected in several cancers. piRNAs may function in gene silencing and gene modification upstream or downstream of oncogenes in cancer cell lines or cancer tissues. In addition to piRNAs, PIWI proteins can be used as biomarkers for prognosis, diagnosis and clinical evaluation and may be factors in cancer metastasis. Here, we elucidated the possible mechanisms by which piRNAs regulate cancer metastasis, including but not restricted to influencing DNA and histone methylation and transposable elements.

Aberrant Expression of PIWIL1 and PIWIL2 and Their Clinical Significance in Ductal Breast Carcinoma.

P-Element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) are involved in epigenetic regulation of gene expression in germline cells. Aberrant expression of piRNA and PIWI proteins have been identified in various types of tumour cells. The aim of this study was to evaluate the expression profiles of PIWI-like protein-1, -2 (PIWIL1 and PIWIL2), their immunohistochemical (IHC) characteristics in ductal breast cancer, and determine their correlation with clinicopathological parameters of this type of cancer. Material for IHC studies comprised of 101 invasive ductal carcinoma (IDC) cases and 31 mastopathy tissues. Frozen fragments of paired tissue specimens (tumour and adjacent non-malignant breast tissue) taken from 55 patients with IDC and 18 samples of mastopathy were used for molecular studies using real-time polymerase chain reaction (RT-PCR). A statistically significantly higher level of PIWIL1 and PIWIL2 was found in IDC compared to mastopathy samples (p≤0.0001). Increased expression of PIWIL1 was correlated with increased PIWIL2 expression in breast cancer tissue. Surprisingly, PIWIL1 mRNA was detected only in cancer and mastopathy, but was not found in most normal breast tissues, although it is noteworthy that the PIWIL2 mRNA level was statistically significantly lower in mastopathy and IDC samples compared to normal breast tissues. Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development.

Structural basis for arginine methylation-independent recognition of PIWIL1 by TDRD2

The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain-PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain-PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2-PIWIL1 interaction compromise piRNA maturation via 3'-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.

PIWI-interacting RNAs as novel regulators of pancreatic beta cell function.

P-element induced Wimpy testis (PIWI)-interacting RNAs (piRNAs) are small non-coding RNAs that interact with PIWI proteins and guide them to silence transposable elements. They are abundantly expressed in germline cells and play key roles in spermatogenesis. There is mounting evidence that piRNAs are also present in somatic cells, where they may accomplish additional regulatory tasks. The aim of this study was to identify the piRNAs expressed in pancreatic islets and to determine whether they are involved in the control of beta cell activities. piRNA profiling of rat pancreatic islets was performed by microarray analysis. The functions of piRNAs were investigated by silencing the two main Piwi genes or by modulating the level of selected piRNAs in islet cells. We detected about 18,000 piRNAs in rat pancreatic islets, many of which were differentially expressed throughout islet postnatal development. Moreover, we identified changes in the level of several piRNAs in the islets of Goto-Kakizaki rats, a well-established animal model of type 2 diabetes. Silencing of Piwil2 or Piwil4 genes in adult rat islets caused a reduction in the level of several piRNAs and resulted in defective insulin secretion and increased resistance of the cells to cytokine-induced cell death. Furthermore, overexpression in the islets of control animals of two piRNAs that are upregulated in diabetic rats led to a selective defect in glucose-induced insulin release. Our results provide evidence for a role of PIWI proteins and their associated piRNAs in the control of beta cell functions, and suggest a possible involvement in the development of type 2 diabetes. Data have been deposited in Gene Expression Omnibus repository under the accession number GSE93792. Data can be accessed via the following link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ojklueugdzehpkv&acc=GSE93792.

The meaning of PIWI proteins in cancer development.

Cancer is a histologically and genetically heterogeneous population of tumor cells that exhibits distinct molecular profiles determined by epigenetic alterations. P-element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) have been previously demonstrated to be involved in epigenetic regulation in germline cells. Recently, reactivation of PIWI expression, primarily PIWI-like protein 1 and 2, through aberrant DNA methylation resulting in genomic silencing has been identified in various types of tumors. It has been suggested that the PIWI-piRNA complex contributes to cancer development and progression by promoting a stem-like state of cancer cells, or cancer stem cells (CSCs). It has been identified that CSCs represent the cells that have undergone epithelial-mesenchymal transition (EMT) and acquired metastatic capacities. However, the molecular association between the EMT process and the stem-cell state remains unclear. Further extensive characterization of CSCs in individual types of tumors is required to identify specific markers for the heterogeneous population of CSCs and therefore selectively target CSCs. Previous studies indicate a reciprocal regulation between PIWI proteins and a complex signaling network linking markers characterized for CSCs and transcription factors involved in EMT. In the present review, studies of PIWI function are summarized, and the potential involvement of PIWI proteins in cancer development and progression is discussed.

The Role of PIWIL4, an Argonaute Family Protein, in Breast Cancer

P-element-induced wimpy testis (PIWI) proteins bind to PIWI-interacting RNAs and play key roles in the biogenesis and functions of PIWI-interacting RNAs. It has been reported that PIWI proteins are essential for stem cell self-renewal and germline development in diverse organisms and that they are ectopically expressed in multiple forms of cancer. However, the role of PIWI in cancer remains elusive. Here we report that one of the four PIWI proteins in humans, PIWIL4, is highly expressed in both breast cancer tissues and the cytoplasm of MDA-MB-231 cells derived from breast cancer. Reducing PIWIL4 expression drastically impairs the migration ability of MDA-MB-231 cells, significantly increases their apoptosis, and mildly affects their proliferation. Our transcriptome and proteome analysis reveal that these functions are at least partially achieved via the PIWIL4 regulation of TGF-β and FGF signaling pathways and MHC class II proteins. These findings suggest that PIWIL4 may serve as a potential therapeutic target for breast cancer.

Abstract A29: PIWI-like proteins and piRNAs in breast cancer cell proliferation and migration

Abstract Breast cancer is the major cause of cancer-related death in women globally. With the revelation of key molecular pathways modulating all aspects of the breast cancer, the pharmaceutical development and clinical treatment have achieved significant progress. However, patients are still threatened by serious issues, including drug resistance, relapse and metastasis. In addition, a reliable and comprehensive system of early detection of the tumor is still lacking. As such, we set out to identify potential signaling pathway that could serve as novel drug target and diagnostic indicator. Recently, the PIWI (P-element-induced wimpy testis)-like proteins are gaining more interest as prospective biomarkers and targets for therapeutic development because of their re-expression in cancer cells. Initially, the PIWI-like proteins were regarded as germline-specific regulators. They have been revealed to exert important epigenetic functions in germ cells, including the suppression of retrotransposons, the formation of heterochromatin, and the maintenance of DNA methylation status. Surprisingly, in various human tumors, the abnormal expression of PIWI-like proteins was readily observed. The in vitro data showed that the repression of these proteins significantly slowed down the tumor growth, raising an intriguing question whether the PIWI-like proteins and the associated small RNAs, piRNAs, contribute to the breast cancer initiation and progression. In this study, we investigated the expression PIWI-like proteins and piRNAs in three different breast cell lines. It was found that in the normal mammalian epithelial cell line, MCF12A, there was a general lack of PIWI-like expression, which was unaffected by the addition of estrogen. In addition, there was no indication of piRNA generation and accumulation in MCF12A. However, in two breast cancer cell lines, MCF7 and MDA-MB-231, we detected robust expression of PIWI proteins and piRNAs. The knockdown of individual PIWI-like homologs significantly impacted two important indicators of the cancer, rapid proliferation and migration capacity. Furthermore, we found that the depletion of PIWI-like homologs repressed the expression of key regulators of pro-growth genes, such as CCND1, while enhancing those anti-proliferation factors and metastasis inhibitors, such as Bcl-2, p27, p53 and Oct-4. The data indicate that PIWI-like proteins could contribute to cancer formation and progression. We will further explore whether they exert these regulatory functions at the epigenetic level, just like in germ cells. Also, a comprehensive profiling of piRNAs in breast cancer will provide further clue about the clinical potential of using the PIWI/piRNA pathway as a screening indicator. Note: This abstract was not presented at the conference. Citation Format: Jing Yi Lee, Wai Cheong Yip, Qidong Hu. PIWI-like proteins and piRNAs in breast cancer cell proliferation and migration. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A29.

Induction of DNA Methylation by Artificial piRNA Production in Male Germ Cells

Global DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1-3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important forde novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4-6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7-10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11-16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNMT3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells.

PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) are found in animal germlines and are essential for fertility, but their functions outside of the gonad are not well understood. The cnidarian Hydra is a simple metazoan with well-characterized stem/progenitor cells that provides a unique model for analysis of PIWI function. Here we report that Hydra has two PIWI proteins, Hydra PIWI (Hywi) and Hydra PIWI-like (Hyli), both of which are expressed in all Hydra stem/progenitor cells, but not in terminally differentiated cells. We identified ∼15 million piRNAs associated with Hywi and/or Hyli and found that they exhibit the ping-pong signature of piRNA biogenesis. Hydra PIWI proteins are strictly cytoplasmic and thus likely act as posttranscriptional regulators. To explore this function, we generated a Hydra transcriptome for piRNA mapping. piRNAs map to transposons with a 25- to 35-fold enrichment compared with the abundance of transposon transcripts. By sequencing the small RNAs specific to the interstitial, ectodermal, and endodermal lineages, we found that the targeting of transposons appears to be largely restricted to the interstitial lineage. We also identified putative nontransposon targets of the pathway unique to each lineage. Finally we demonstrate that hywi function is essential in the somatic epithelial lineages. This comprehensive analysis of the PIWI-piRNA pathway in the somatic stem/progenitor cells of a nonbilaterian animal suggests that this pathway originated with broader stem cell functionality.

Conserved functional characteristics of the PIWI family members in chicken germ cell lineage

The P-element-induced wimpy testis (PIWI) protein family, one of two subfamily members of Argonautes that regulate gene expression by associating with small noncoding RNAs, plays a role in maintaining the integrity of germ cells and stem cells. PIWI proteins have been identified and characterized in many species, including humans, mice, and zebrafish, but not as yet in avian species. In this study, chicken PIWI-like protein 1 (CIWI) and 2 (CILI) were identified from a 25-wk-old adult male testis cDNA library. Similar to mammals, CIWI and CILI have two conserved functional domains (PIWI/Argonaute/Zwille [PAZ] and PIWI), which bind the 3′ end of single-stranded RNAs and the RNase H domain. The similarities between the PAZ and PIWI domain in CIWI were 65% and 80% to those of humans, and 67% and 80% to those of mice, respectively. In addition, the similarities between PAZ and PIWI in CILI were 60% and 61% to humans, and 60% and 63% to mice, respectively. Notably, the PIWI domain in the PIWIL1 protein showed high similarity between mammals and birds (80% between chickens and humans vs. 80% between chickens and mice), but the PIWI domain in PIWIL2 was less similar compared with PIWIL1 (61% between chickens and humans vs. 63% between chickens and mice). Reverse transcription polymerase chain reaction, quantitative real-time polymerase chain reaction, and in situ hybridization were conducted to analyze the expression patterns of PIWI family genes during gametogenesis in adult chickens during gonad development in the embryonic stage. CIWI and CILI showed germ cell-specific expression in both males and females, and CIWI and CILI mRNAs were distinctively expressed in different cell stages of spermatogenesis in testis. Using RNA interference, knockdowns of CIWI and CILI resulted in the increased expression of Chicken Repetitive 1 (CR1) Elements in primordial germ cells. Thus, the CIWI and CILI proteins of the PIWI family are conserved in avian species and play a critical role in germ line development and gametogenesis.

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.

How does the Royal Family of Tudor rule the PIWI-interacting RNA pathway?

PIWI (P-element-induced wimpy testis) proteins are a subset of the Argonaute proteins and are expressed predominantly in the germlines of a variety of organisms, including Drosophila and mammals. PIWI proteins associate specifically with PIWI-interacting RNAs (piRNAs), small RNAs that are also expressed predominantly in germlines, and silence transposable DNA elements and other genes showing complementarities to the sequences of associated piRNAs. This mechanism helps to maintain the integrity of the genome and the development of gametes. PIWI proteins have been shown recently to contain symmetrical dimethyl arginines (sDMAs), and this modification is mediated by the methyltransferase PRMT5 (also known as Dart5 or Capsuleen). It was then demonstrated that multiple members of the Tudor (Tud) family of proteins, which are necessary for gametogenesis in both flies and mice, associate with PIWI proteins specifically through sDMAs in various but particular combinations. Although Tud domains in Tud family members are known to be sDMA-binding modules, involvement of the Tudor family at the molecular level in the piRNA pathway has only recently come into focus.