PIWI-like Proteins Research Articles

Molecular cloning, gene expression and methylation status analysis of PIWIL1 in cattle-yaks and the parental generation

The PIWI-like protein 1 (PIWIL1) plays an important role in spermatogenesis and transposon control. In mammals, the absence of the PIWIL1 gene can lead to over-expression of retrotransposons, spermatogenetic arrest, and male infertility. To assess whether the PIWIL1 gene is associated with the male sterility of cattle-yaks, we examined PIWIL1 and LINE-1 (long interspersed element-1) retrotransposon mRNA expression and DNA methylation patterns in the testes of cattle, yaks, and cattle-yaks. Our results indicate that PIWIL1 mRNA expression in the testes of cattle-yaks is significantly lower than that of cattle or yaks. Correlating with decreased gene expression, we also found that the methylation levels of the PIWIL1 gene in cattle-yaks were significantly higher than in cattle or yaks. However, even though LINE-1 element expressed higher mRNA levels in the testes of cattle-yaks than in their parents, DNA methylation patterns were un-altered. Our results suggest that the higher methylation levels of the PIWIL1 gene in cattle-yaks coincides with decreased PIWIL1 mRNA expression, thereby affecting the transposon silencing mechanism and possibly contributing to cattle-yak male sterility.

Novel Features of a PIWI-Like Protein Homolog in the Parasitic Protozoan Leishmania

In contrast to nearly all eukaryotes, the Old World Leishmania species L. infantum and L. major lack the bona fide RNAi machinery genes. Interestingly, both Leishmania genomes code for an atypical Argonaute-like protein that possesses a PIWI domain but lacks the PAZ domain found in Argonautes from RNAi proficient organisms. Using sub-cellular fractionation and confocal fluorescence microscopy, we show that unlike other eukaryotes, the PIWI-like protein is mainly localized in the single mitochondrion in Leishmania. To predict PIWI function, we generated a knockout mutant for the PIWI gene in both L. infantum (Lin) and L. major species by double-targeted gene replacement. Depletion of PIWI has no effect on the viability of insect promastigote forms but leads to an important growth defect of the mammalian amastigote lifestage in vitro and significantly delays disease pathology in mice, consistent with a higher expression of the PIWI transcript in amastigotes. Moreover, amastigotes lacking PIWI display a higher sensitivity to apoptosis inducing agents than wild type parasites, suggesting that PIWI may be a sensor for apoptotic stimuli. Furthermore, a whole-genome DNA microarray analysis revealed that loss of LinPIWI in Leishmania amastigotes affects mostly the expression of specific subsets of developmentally regulated genes. Several transcripts encoding surface and membrane-bound proteins were found downregulated in the LinPIWI(−/−) mutant whereas all histone transcripts were upregulated in the null mutant, supporting the possibility that PIWI plays a direct or indirect role in the stability of these transcripts. Although our data suggest that PIWI is not involved in the biogenesis or the stability of small noncoding RNAs, additional studies are required to gain further insights into the role of this protein on RNA regulation and amastigote development in Leishmania.

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.

Piwil2 modulates the proliferation and metastasis of colon cancer via regulation of matrix metallopeptidase 9 transcriptional activity

Piwi-like protein 2 (Piwil2) has recently emerged as a putative oncogene which is amplified in several human malignancies. However, the role of Piwil2 in colon cancer remains poorly understood. The aim of this study was to investigate the clinical and pathological significance of Piwil2, and the possible role in the proliferation and metastasis of colon cancer. Primary colon cancer paired with adjacent normal colon tissue and lymph node metastasis (LNM) lesions in 66 patients' tissue microarrays (TMA) were used to determine the expression of Piwil2. Knocked down Piwil2 expression in SW620 and SW480 colon cancer cell lines was performed to evaluate the role of Piwil2 in cell proliferation, invasion, metastasis in vitro and tumorigenicity in vivo. The possible roles of Piwil2 in the regulation of a 2 kb matrix metallopeptidase 9 (MMP9) promoter fragment and on the regulation of apoptotic pathways were evaluated by using a luciferase reporter construct and Western blots, respectively. Significantly higher expression levels of Piwil2 were observed in primary colon cancer tissue and in LNM in comparison with normal colon mucosa. Piwil2 expression significantly correlated with more aggressive clinical and pathological parameters with poorer five-year metastasis-free survival and overall survival. Piwil2 silencing significantly reduced cancer cell proliferation, colony formation ability and increased apoptosis in vitro and inhibited tumor growth in vivo. Piwil2 knockdown also attenuated migration and invasion of colon cancer cells via modulation of MMP9 transcriptional activities. Our results indicate that Piwil2 moderates the proliferation and metastasis potential of colon cancer.

Structural studies on piRNA recognition by mammalian PIWI-like Argonaute proteins

Structural studies on piRNA recognition by mammalian PIWI-like Argonaute proteins

Small RNA-mediated control of retrotrotransposons in Drosophila

In the Drosophila ovary, the I-element (a NLR retrotransposon) and gypsy are both controlled by piRNAs (small RNAs associated with Piwi-like proteins); but there are important variations on this common theme, according to the cell lineage where it operates: the germline (for the I-element) versus the ovarian somatic cells (for gypsy). We are taking advantage of these differences to study the piRNA biogenesis, the epigenetic effects of the maternal transmission of piRNAs and the mechanism of piRNA-mediated regulation.

A C. elegans Piwi, PRG-1, Regulates 21U-RNAs during Spermatogenesis

Epigenetic regulation by diverse classes of small RNAs is mediated by the highly conserved Argonaute/Piwi family of proteins. Although Argonautes are broadly expressed, the Piwi subfamily primarily functions in the germ line. Piwi proteins are associated with germline-specific ribonucleoprotein (RNP) granules in Drosophila, zebrafish, and mouse. Depending on the species and on the specific family member, Piwis play important roles in spermatogenesis and/or in maintaining germ cell and stem cell totipotency. Piwis bind to a newly discovered class of small RNAs, called piRNAs. C. elegans contains a large set of Argonaute/Piwi-related proteins, including two closely related to piwi called prg-1 and prg-2. The function of prg-1 and prg-2 and whether piRNAs exist in C. elegans is unknown. Here, we demonstrate that the Piwi-like protein PRG-1 is localized to P granules in germ cells entering spermatogenesis and is required for successful spermatogenesis. Loss of prg-1 causes a marked reduction in expression of a subset of mRNAs expressed during spermatogenesis, and prg-1 mutant sperm exhibit extensive defects in activation and fertilization. Moreover, prg-1 activity is required for the presence of the small RNAs called 21U-RNAs. Our data suggest that PRG-1 promotes expression, processing, or stability of 21U-RNAs, which, in turn or in concert with PRG-1, promote proper expression of spermatogenesis transcripts.

Life beyond cleavage: the case of Ago2 and hematopoiesis

A recent article in The Economist (June 16, 2007) describes research on RNA as the 21st century’s Big Bang in biology. The “stars” of the RNA universe are small regulatory RNAs known as microRNAs (miRNAs). These ∼22-nt-long RNAs were discovered in 1993 (Lee et al. 1993; Wightman et al. 1993), but their role in biology remained obscure until 2001, when the Tuschl, Ambros, and Bartel laboratories identified hundreds of miRNAs in different organisms (Lagos-Quintana et al. 2001; Lau et al. 2001; Lee and Ambros 2001). Functional analyses of these miRNAs and their associated complexes subsequently led to the discovery of RNA-guided genesilencing pathways as important novel principles in controlling gene expression. miRNAs indeed regulate the expression of a large proportion of eukaryotic protein-coding genes at the post-transcriptional level by sequence-specific binding to the 3 untranslated region (3 UTR) of mRNAs (Lim et al. 2005). To date, RNA silencing is known to play an important role in diverse biological phenomena such as development, stem cell maintenance, cell proliferation, survival, differentiation, metabolism, and cancer (Kloosterman and Plasterk 2006). The primary transcripts of miRNA genes are mainly processed by the nuclear RNase III enzyme Drosha into stem–loop-structured miRNA precursors (pre-miRNAs) (Lee et al. 2003; Ruby et al. 2007), which are transported via the Exportin-5 receptor to the cytoplasm (Yi et al. 2003). The cytoplasmic RNase III enzyme Dicer cleaves the pre-miRNAs into double-stranded (ds) RNAs of ∼22 nt (Bernstein et al. 2001; Hutvagner et al. 2001). One strand of these dsRNA intermediates associates as a mature miRNA with a member of the Argonaute protein family to generate an active ribonucleoprotein complex known as RNA-Induced Silencing Complex (RISC). Within this effector complex, both the miRNA and the Argonaute protein fulfill specific roles. The singlestranded miRNA recognizes the target mRNA with full or partial base-pair complementarity. The Argonaute protein helps the miRNA in searching for its target mRNA and, upon recognition, can either cleave it or remain tethered to the mRNA to repress its translation and/or regulate its stability. While the biochemistry of the miRNA-guided cleavage reactions is well understood (Martinez and Tuschl 2004; Schwarz et al. 2004), it is still controversial to what extent RISC interferes with the initiation or elongation of translation or controls mRNA stability by sequestration into cytoplasmic processing bodies (P-bodies) (Kiriakidou et al. 2007; Peters and Meister 2007; Pillai et al. 2007). The Argonaute (Ago) proteins can be subdivided into Ago-like and Piwi-like subfamilies. The mammalian Piwi-like proteins are specifically expressed during spermatogenesis, where they bind a novel set of small RNAs termed Piwi-interacting RNAs (piRNAs) (Aravin et al. 2006; Seto et al. 2007). In contrast, the Ago proteins are broadly expressed in somatic cells, associate with miRNAs and are key actors in different RNA silencing pathways (Peters and Meister 2007; Tolia and Joshua-Tor 2007). The Ago gene family consists of four (AGO1– AGO4) and five (Ago1–Ago5) members in human and mouse, respectively (Peters and Meister 2007). However, only the Ago2 protein displays endonucleolytic or “Slicer” activity and can therefore execute miRNA-directed cleavage of target mRNA, provided that the basepairing between the Ago2-associated miRNA and the mRNA sequence is perfect (Liu et al. 2004; Meister et al. 2004). In case of partial complementarity, the Ago2 protein fails to cleave, but instead interferes with translation of the target mRNA via its translational repression activity. In addition to Ago2, other mammalian Ago proteins are also part of miRNA effector complexes that mediate translational inhibition of target mRNAs (Liu et al. 2004; Meister et al. 2004). Gene disruption in the mouse demonstrated that the Ago2 protein is essential for embryonic development (Liu et al. 2004). To study the function of Ago2 in adult hematopoiesis, O’Carroll et al. (2007) have used conditional gene inactivation to bypass the embryonic lethality and report in this issue of Genes & Development that Ago2 is a key regulator of B-lymphoid and erythroid development. Retroviral rescue experiments surprisingly revealed that the Slicer endonuclease activity, which is a unique and defining feature of Ago2, is dispensable for hematopoietic developCorresponding author. E-MAIL busslinger@imp.ac.at; FAX (43/1) 79730-223150. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1591407.

Identification and characterization of Piwi subfamily in insects

As a subfamily of Argonaute proteins, Piwi is poorly understood compared with Ago subfamily until recent discovery of Piwi protein interacting with piRNA. We did a large scale screening of insect genomes to identify piwi-like genes. Full or partial cDNA sequences were obtained by EST elongation and GENSCAN. We found that the exon numbers were totally different between vertebrates and invertebrates, approximately 20 exons in mammals but only 6–9 exons in insects. This infers either intron insertion or loss occurred during evolution. Characterized PAZ, c-terminal PIWI domains exist in almost all predicted Piwi-like proteins. We found six conserved motifs, which contain active catalytic triad “Asp-Asp-His/Lys” required for slicer activity. The expression of siwi1 and siwi2 in Bombyx mori were verified with RT-PCR. Phylogenetic tree inferred by Bayesian algorithm indicates invertebrate Piwi-like proteins are classified into three clades, of which Ago3 clade is closer to mammalian Piwi proteins.

The induction of H3K9 methylation by PIWIL4 at the p16Ink4a locus

The field of epigenetics has made progress by the identification of the small RNA-mediated epigenetic modification. However, little is known about the key proteins. Here, we report that the human PIWI-like family is a candidate protein that is involved in the pathway responsible for chromatin remodeling. The PIWI-like family proteins, expressed as the Flag-fusion proteins, formed a bulky body and localized to the nuclear periphery. Transient transfection of PIWI-like 4 (PIWIL4), only member of the PIWI-like family that was ubiquitously expressed in human tissues, induced histone H3 lysine 9 methylation at the p16Ink4a (CDKN2A) locus. The elevated level of histone methylation resulted in the downregulation of the p16Ink4a gene. These results suggest PIWIL4 plays important roles in the chromatin-modifying pathway in human somatic cells.

On the origin and functions of RNA-mediated silencing: from protists to man

Double-stranded RNA has been shown to induce gene silencing in diverse eukaryotes and by a variety of pathways. We have examined the taxonomic distribution and the phylogenetic relationship of key components of the RNA interference (RNAi) machinery in members of five eukaryotic supergroups. On the basis of the parsimony principle, our analyses suggest that a relatively complex RNAi machinery was already present in the last common ancestor of eukaryotes and consisted, at a minimum, of one Argonaute-like polypeptide, one Piwi-like protein, one Dicer, and one RNA-dependent RNA polymerase. As proposed before, the ancestral (but non-essential) role of these components may have been in defense responses against genomic parasites such as transposable elements and viruses. From a mechanistic perspective, the RNAi machinery in the eukaryotic ancestor may have been capable of both small-RNA-guided transcript degradation as well as transcriptional repression, most likely through histone modifications. Both roles appear to be widespread among living eukaryotes and this diversification of function could account for the evolutionary conservation of duplicated Argonaute-Piwi proteins. In contrast, additional RNAi-mediated pathways such as RNA-directed DNA methylation, programmed genome rearrangements, meiotic silencing by unpaired DNA, and miRNA-mediated gene regulation may have evolved independently in specific lineages.