Haifan Lin

miwi, a Murine Homolog of piwi, Encodes a Cytoplasmic Protein Essential for Spermatogenesis

The piwi family genes are crucial for stem cell self-renewal, RNA silencing, and translational regulation in diverse organisms. However, their function in mammals remains unexplored. Here we report the cloning of a murine piwi gene (miwi) essential for spermatogenesis. miwi encodes a cytoplasmic protein specifically expressed in spermatocytes and spermatids. miwi(null) mice display spermatogenic arrest at the beginning of the round spermatid stage, resembling the phenotype of CREM, a master regulator of spermiogenesis. Furthermore, mRNAs of ACT (activator of CREM in testis) and CREM target genes are downregulated in miwi(null) testes. Whereas MIWI and CREM do not regulate each others expression, MIWI complexes with mRNAs of ACT and CREM target genes. Hence, MIWI may control spermiogenesis by regulating the stability of these mRNAs.

Mili, a mammalian member of piwi family gene, is essential for spermatogenesis

The piwi family genes, 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. To reveal the function of the mammalian homolog of piwi, we produced and analyzed mice with targeted mutations in the Mili gene, which is one of three mouse homologs of piwi. Spermatogenesis in the MILI-null mice was blocked completely at the early prophase of the first meiosis, from the zygotene to early pachytene, and the mice were sterile. However, primordial germ cell development and female germ cell production were not disturbed. Furthermore, MILI bound to MVH, which is an essential factor during the early spermatocyte stage. The similarities in the phenotypes of the MILI- and MVH-deficient mice and in the physical binding properties of MILI and MVH indicate a functional association of these proteins in post-transcriptional regulation. These data indicate that MILI is essential for the differentiation of spermatocytes.

MicroRNAs: key regulators of stem cells.

The hallmark of a stem cell is its ability to self-renew and to produce numerous differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new dimension of gene regulation in controlling stem cell fate and behaviour.

The Biogenesis and Function of PIWI Proteins and piRNAs: Progress and Prospect

The evolutionarily conserved Argonaute/PIWI (AGO/PIWI, also known as PAZ-PIWI domain or PPD) family of proteins is crucial for the biogenesis and function of small noncoding RNAs (ncRNAs). This family can be divided into AGO and PIWI subfamilies. The AGO proteins are ubiquitously present in diverse tissues. They bind to small interfering RNAs (siRNAs) and microRNAs (miRNAs). In contrast, the PIWI proteins are predominantly present in the germline and associate with a novel class of small RNAs known as PIWI-interacting RNAs (piRNAs). Tens of thousands of piRNA species, typically 24-32 nucleotide (nt) long, have been found in mammals, zebrafish, and Drosophila. Most piRNAs appear to be generated from a small number of long single-stranded RNA precursors that are often encoded by repetitive intergenic sequences in the genome. PIWI proteins play crucial roles during germline development and gametogenesis of many metazoan species, from germline determination and germline stem cell (GSC) maintenance to meiosis, spermiogenesis, and transposon silencing. These diverse functions may involve piRNAs and may be achieved via novel mechanisms of epigenetic and posttranscriptional regulation.

An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster.

Heterochromatin, representing the silenced state of transcription, consists largely of transposon-enriched and highly repetitive sequences. Implicated in heterochromatin formation and transcriptional silencing in Drosophila are Piwi (P-element induced wimpy testis) and repeat-associated small interfering RNAs (rasiRNAs). Despite this, the role of Piwi in rasiRNA expression and heterochromatic silencing remains unknown. Here we report the identification and characterization of 12,903 Piwi-interacting RNAs (piRNAs) in Drosophila, showing that rasiRNAs represent a subset of piRNAs. We also show that Piwi promotes euchromatic histone modifications and piRNA transcription in subtelomeric heterochromatin (also known as telomere-associated sequence, or TAS), on the right arm of chromosome 3 (3R-TAS). Piwi binds to 3R-TAS and a piRNA uniquely mapped to 3R-TAS (3R-TAS1 piRNA). In piwi mutants, 3R-TAS loses euchromatic histone modifications yet accumulates heterochromatic histone modifications and Heterochromatin Protein 1a (HP1a). Furthermore, the expression of both the 3R-TAS1 piRNA and a white reporter gene in 3R-TAS becomes suppressed. A P element inserted 128 base pairs downstream of the 3R-TAS1 piRNA coding sequence restores the euchromatic histone modifications of 3R-TAS and the expression of 3R-TAS1 piRNA in piwi mutants, as well as partly rescuing their defects in germline stem-cell maintenance. These observations suggest that Piwi promotes the euchromatic character of 3R-TAS heterochromatin and its transcriptional activity, opposite to the known roles of Piwi and the RNA-mediated interference pathway in epigenetic silencing. This activating function is probably achieved through interaction with at least 3R-TAS1 piRNA and is essential for germline stem-cell maintenance.

MIWI associates with translational machinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis

Noncoding small RNAs have emerged as important regulators of gene expression at both transcriptional and posttranscriptional levels. Particularly, microRNA (miRNA)-mediated translational repression involving PIWI/Argonaute family proteins has been widely recognized as a novel mechanism of gene regulation. We previously reported that MIWI, a murine PIWI family member, is required for initiating spermiogenesis, a process that transforms round spermatids into mature sperm. MIWI is a cytoplasmic protein present in spermatocytes and round spermatids, and it is required for the expression of its target mRNAs involved in spermiogenesis. Most recently, we discovered a class of noncoding small RNAs called PIWI-interacting RNAs (piRNAs) that are abundantly expressed during spermiogenesis in a MIWI-dependent fashion. Here, we show that MIWI associates with both piRNAs and mRNAs in cytosolic ribonucleoprotein and polysomal fractions. As polysomes increase in early spermiogenesis, MIWI increases in polysome fractions. Moreover, MIWI associates with the mRNA cap-binding complex. Interestingly, MIWI is required for the expression of not only piRNAs but also a subset of miRNAs, despite the presence of Dicer. These results suggest that MIWI has a complicated role in the biogenesis and/or maintenance of two distinct types of small RNAs. Together, our results indicate that MIWI, a PIWI subfamily protein, uses piRNA as the major, but not exclusive, binding partner, and it is associated with translational machinery.

Role for piRNAs and Noncoding RNA in de Novo DNA Methylation of the Imprinted Mouse Rasgrf1 Locus

Small, noncoding PIWI-interacting RNAs regulate the imprinting of a mouse gene. Genomic imprinting causes parental origin–specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.

Molecular characterization of hiwi, a human member of the piwi gene family whose overexpression is correlated to seminomas

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Drosophila melanogaster and C. elegans to Arabidopsis. Here we report the molecular characterization of hiwi, a human member of the piwi gene family. hiwi maps to the long arm of chromosome 12, band 12q24.33, a genomic region that displays genetic linkage to the development of testicular germ cell tumors of adolescents and adults (TGCTs), i.e., seminomas and nonseminomas. In addition, gain of this chromosomal region has been found in some TGCTs. hiwi encodes a 3.6 kb mRNA that is expressed abundantly in the adult testis. It encodes a highly basic 861-amino-acid protein that shares significant homology throughout its entire length with other members of the PIWI family proteins in Drosophila, C. elegans and mammals. In normal human testes, hiwi is specifically expressed in germline cells, with its expression detectable in spermatocytes and round spermatids during spermatogenesis. No expresssion was observed in testicular tumors of somatic origin, such as Sertoli cell and Leydig cell tumors. Enhanced expression was found in 12 out of 19 sampled testicular seminomas–tumors originating from embryonic germ cells with retention of germ cell phenotype. In contrast, no enhanced expression was detected in 10 nonseminomas–testicular tumors that originate from the same precursor cells as seminomas yet have lost their germ cell characteristics. Finally, no enhanced expression was detected in four spermatocytic seminomas–testicular tumors that most likely originate from germ cells capable of partial meiosis. Thus, hiwi is specifically expressed in both normal and malignant spermatogenic cells in a maturation stage-dependent pattern, in which it might function in germ cell proliferation.

The Role of PIWI and the miRNA Machinery in Drosophila Germline Determination

BACKGROUND The germ plasm has long been demonstrated to be necessary and sufficient for germline determination, with translational regulation playing a key role in the process. Beyond this, little is known about molecular activities underlying germline determination. RESULTS We report the function of Drosophila PIWI, DICER-1, and dFMRP (Fragile X Mental Retardation Protein) in germline determination. PIWI is a maternal component of the polar granule, a germ-plasm-specific organelle essential for germline specification. Depleting maternal PIWI does not affect OSK or VASA expression or abdominal patterning but leads to failure in pole-plasm maintenance and primordial-germ-cell (PGC) formation, whereas doubling and tripling the maternal piwi dose increases OSK and VASA levels correspondingly and doubles and triples the number of PGCs, respectively. Moreover, PIWI forms a complex with dFMRP and DICER-1, but not with DICER-2, in polar-granule-enriched fractions. Depleting DICER-1, but not DICER-2, also leads to a severe pole-plasm defect and a reduced PGC number. These effects are also seen, albeit to a lesser extent, for dFMRP, another component of the miRISC complex. CONCLUSIONS Because DICER-1 is required for the miRNA pathway and DICER-2 is required for the siRNA pathway yet neither is required for the rasiRNA pathway, our data implicate a crucial role of the PIWI-mediated miRNA pathway in regulating the levels of OSK, VASA, and possibly other genes involved in germline determination in Drosophila.

Uniting Germline and Stem Cells: The Function of Piwi Proteins and the piRNA Pathway in Diverse Organisms

The topipotency of the germline is the full manifestation of the pluri- and multipotency of embryonic and adult stem cells, thus the germline and stem cells must share common mechanisms that guarantee their multipotentials in development. One of the few such known shared mechanisms is represented by Piwi proteins, which constitute one of the two subfamilies of the Argonaute protein family. Piwi proteins bind to Piwi-interacting RNAs (piRNAs) that are generally 26 to 31 nucleotides in length. Both Piwi proteins and piRNAs are most abundantly expressed in the germline. Moreover, Piwi proteins are expressed broadly in certain types of somatic stem/progenitor cells and other somatic cells across animal phylogeny. Recent studies indicate that the Piwi-piRNA pathway mediates epigenetic programming and posttranscriptional regulation, which may be responsible for its function in germline specification, gametogenesis, stem cell maintenance, transposon silencing, and genome integrity in diverse organisms.