Toshiaki Watanabe

Bio: Toshiaki Watanabe is an academic researcher from Yale University. The author has contributed to research in topics: Piwi-interacting RNA & Argonaute. The author has an hindex of 25, co-authored 35 publications receiving 5732 citations. Previous affiliations of Toshiaki Watanabe include Central Institute for Experimental Animals & Graduate University for Advanced Studies.

Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes

Abstract: RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both approximately 25-27-nucleotide Piwi-interacting RNAs (piRNAs) and approximately 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes

Abstract: Silencing of transposable elements occurs during fetal gametogenesis in males via de novo DNA methylation of their regulatory regions. The loss of MILI (miwi-like) and MIWI2 (mouse piwi 2), two mouse homologs of Drosophila Piwi, activates retrotransposon gene expression by impairing DNA methylation in the regulatory regions of the retrotransposons. However, as it is unclear whether the defective DNA methylation in the mutants is due to the impairment of de novo DNA methylation, we analyze DNA methylation and Piwi-interacting small RNA (piRNA) expression in wild-type, MILI-null, and MIWI2-null male fetal germ cells. We reveal that defective DNA methylation of the regulatory regions of the Line-1 (long interspersed nuclear elements) and IAP (intracisternal A particle) retrotransposons in the MILI-null and MIWI2-null male germ cells takes place at the level of de novo methylation. Comprehensive analysis shows that the piRNAs of fetal germ cells are distinct from those previously identified in neonatal and adult germ cells. The expression of piRNAs is reduced under MILI- and MIWI2-null conditions in fetal germ cells, although the extent of the reduction differs significantly between the two mutants. Our data strongly suggest that MILI and MIWI2 play essential roles in establishing de novo DNA methylation of retrotransposons in fetal male germ cells.

Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes

Abstract: RNA interference (RNAi) is a sequence-specific gene regulatory mechanism conserved among diverse eukaryotes. The sequence specificity in RNAi is determined by a family of 18- to 30-nucleotide (nt) regulatory small RNAs (for review, see Aravin and Tuschl 2005). Two major classes of endogenous small RNAs have been characterized: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs—the best-characterized endogenous small RNAs in eukaryotes—have been identified in diverse plants and animals, and are mainly involved in development and differentiation. miRNAs are processed from miRNA precursors (pre-miRNAs) with a stem-loop structure and regulate gene expression through translational repression or mRNA cleavage (for reviews, see Ambros 2004; Bartel 2004; He and Hannon 2004; Du and Zamore 2005). siRNAs are generated from long double-stranded RNA (dsRNA) and are mainly involved in defense against molecular parasites including viruses, transposons, and transgenes through RNAi (Sijen and Plasterk 2003; Shi et al. 2004). Endogenous siRNAs have been classified into at least three subclasses: repeat-associated siRNAs (rasiRNAs), trans-acting siRNAs (ta-siRNAs), and siRNAs derived from natural antisense transcripts (nat-siRNAs) (Lippman and Martienssen 2004; Peragine et al. 2004; Borsani et al. 2005). rasiRNAs corresponding to repetitive elements repress the repeat sequences at the transcriptional or post-transcriptional level and maintain a centromeric heterochromatic structure (Lippman and Martienssen 2004). rasiRNAs have been cloned and sequenced in Schizosaccharomyces pombe, Trypanosoma brucei, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, and Arabidopsis thaliana, but not in mammals (for review, see Aravin and Tuschl 2005). In D. melanogaster and D. rerio, however, the lengths of rasiRNAs are longer than those of miRNAs (Ambros et al. 2003; Chen et al. 2005). ta-siRNAs are processed from dsRNAs synthesized by an endogenous RNA-dependent RNA polymerase (RdRp) utilizing endogenous mRNAs as template (Peragine et al. 2004). nat-siRNAs are processed from dsRNAs formed between endogenous sense and antisense transcripts (Borsani et al. 2005). In addition to miRNA and siRNA, several classes of small RNAs have been reported. scanRNAs (scnRNAs; 27–30 nt) in Tetrahymena thermophila are thought to be derived from dsRNA precursors and guide DNA elimination (Mochizuki et al. 2002; Lee and Collins 2006). small RNAs (23–24 nt) in T. thermophila are mapped to the genome in clusters and are oriented in the same direction (Lee and Collins 2006). X-cluster small RNAs in C. elegans are derived from a locus on Chromosome X extending ∼2 kb and are oriented in the same direction (Ambros et al. 2003). Several factors including DCR-1, Dicer-related helicase DRH-1, the RdRp RRF-1, and the exonuclease ERI-1 are reported to be required for the accumulation of X-cluster small RNAs (Duchaine et al. 2006). The roles of 23- to 24-nt small RNAs in T. thermophila and X-cluster small RNAs in C. elegans remain unknown. Key factors required for the biogenesis and function of these small RNAs are Dicer and Argonaute proteins (for review, see Tomari and Zamore 2005). Dicer is an RNaseIII-like enzyme that recognizes dsRNAs, including pre-miRNAs, and processes them into double-stranded small RNAs (Hutvagner et al. 2001). The Dicer-generated double-stranded small RNAs are recruited by Argonaute, and then a strand (called the passenger strand) is released from Argonaute and the other strand (called the guide strand) remains associated with Argonaute as a guide to regulate gene expression (Matranga et al. 2005). Based on the amino acid sequence alignments, the Argonaute protein family has been subdivided into two subfamilies, referred to as the Argonaute and Piwi families (Carmell et al. 2002). Mice have four Argonaute family genes (AGO1–4) and three Piwi family genes (Miwi, Mili, and Piwil4). AGO1–4, which are ubiquitously expressed in many tissues (Lu et al. 2005), recruit miRNAs (Liu et al. 2004). Piwi family genes are expressed predominantly in male germline cells (Kuramochi-Miyagawa et al. 2001) and have crucial roles in spermatogenesis. Disruption of Miwi causes spermatogenic arrest at the beginning of the round spermatid stage (Deng and Lin 2002). Spermatogenesis in Mili-null mice is blocked completely at the early prophase of the first meiosis (Kuramochi-Miyagawa et al. 2004). The molecular functions and associated small RNAs of Piwi family proteins remain unknown. Whether endogenous siRNAs are present in mouse is unclear for three reasons: (1) There has been no report of siRNA cloning in mammals; (2) there is no evidence for the presence of RdRp activity in mammals, which generates dsRNAs, namely the precursors of siRNAs; and (3) induction of the interferon pathway by dsRNAs usually results in cell death, suggesting that mammalian cells may not tolerate dsRNAs (Elbashir et al. 2001a). However, in mouse oocytes and preimplantation embryos the interferon response is suppressed, and injection of long dsRNAs results in specific reduction in the amount of target mRNAs (Svoboda et al. 2000; Yan et al. 2005). Furthermore, retrotransposons and their antisense transcripts are expressed in mouse preimplantation embryos (Peaston et al. 2004; Svoboda et al. 2004a), and knockdown of Dicer in mouse preimplantation embryos results in a 50% increase in IAP and MuERV-L retrotransposons (Svoboda et al. 2004a). These studies suggest that endogenous dsRNA-induced RNAi can occur in mouse oocytes and early embryos. In mammals, hundreds of miRNAs have been identified by extensive small RNA cloning and bioinformatic analyses (Lagos-Quintana et al. 2001; Houbaviy et al. 2003; Lim et al. 2003). However, other classes of small RNAs have not been studied. In this study, as a step to obtain a whole picture of the small RNA population in mammals, we have cloned and sequenced small RNAs from oocytes and testes. We identified two classes of small RNAs other than miRNAs. One class comprised ∼22-nt retrotransposon-derived siRNAs in oocytes, which showed characteristics of small RNAs associated with RNAi. The other class comprised novel 26- to 30-nt germline small RNAs (gsRNAs) that were present in male germ cells and had some interesting features that were distinct from those of siRNAs and miRNAs. The features of the two classes of small RNAs suggest their distinct origins and functions and the existence of two separate small RNA pathways in the mouse germline.

The Mechanism Selecting the Guide Strand from Small RNA Duplexes is Different Among Argonaute Proteins

Abstract: Double-stranded RNA induces RNA silencing and is cleaved into 21-24 nt small RNA duplexes by Dicer enzyme. A strand of Dicer-generated small RNA duplex (called the guide strand) is then selected by a thermodynamic mechanism to associate with Argonaute (AGO) protein. This AGO-small RNA complex functions to cleave mRNA, repress translation or modify chromatin structure in a sequence-specific manner. Although a model plant, Arabidopsis thaliana, contains 10 AGO genes, their roles and molecular mechanisms remain obscure. In this study, we analyzed the roles of Arabidopsis AGO2 and AGO5. Interestingly, the 5' nucleotide of small RNAs that associated with AGO2 was mainly adenine (85.7%) and that with AGO5 was mainly cytosine (83.5%). Small RNAs that were abundantly cloned from the AGO2 immunoprecipitation fraction (miR163-LL, which is derived from the Lower Left of mature miR163 in pre-miR163, and miR390) and from the AGO5 immunoprecipitation fraction (miR163-UL, which is derived from the Upper Left of mature miR163 in pre-miR163, and miR390(*)) are derived from the single small RNA duplexes, miR163-LL/miR163-UL and miR390/miR390(*). Each strand of the miR163-LL/miR163-UL duplex is selectively sorted to associate with AGO2 or AGO5 in a 5' nucleotide-dependent manner rather than in a thermodynamic stability-dependent manner. Furthermore, we showed that both AGO2 and AGO5 have the ability to bind cucumber mosaic virus-derived small RNAs. These results clearly indicate that the mechanism selecting the guide strand is different among AGO proteins and that multiple AGO genes are involved in anti-virus defense in plants.

Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus.

Abstract: 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.

MicroRNA signatures in human cancers

Abstract: MicroRNA (miRNA ) alterations are involved in the initiation and progression of human cancer. The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target protein- coding genes involved in cancer.

MicroRNA signatures in human cancers

Abstract: MicroRNA (miRNA) alterations are involved in the initiation and progression of human cancer. The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target protein- coding genes involved in cancer.

Non-coding RNAs in human disease

Abstract: The role of non-coding RNAs (ncRNAs) in disease is best understood for microRNAs in cancer. However, there is increasing interest in the disease-related roles of other ncRNAs — including piRNAs, snoRNAs, T-UCRs and lncRNAs — and in using this knowledge for therapy.

Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals

Abstract: There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified ~1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFκB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.

Palladium(II)-catalyzed C-H activation/C-C cross-coupling reactions: versatility and practicality.

Abstract: Pick your Pd partners: A number of catalytic systems have been developed for palladium-catalyzed CH activation/CC bond formation. Recent studies concerning the palladium(II)-catalyzed coupling of CH bonds with organometallic reagents through a PdII/Pd0 catalytic cycle are discussed (see scheme), and the versatility and practicality of this new mode of catalysis are presented. Unaddressed questions and the potential for development in the field are also addressed. In the past decade, palladium-catalyzed CH activation/CC bond-forming reactions have emerged as promising new catalytic transformations; however, development in this field is still at an early stage compared to the state of the art in cross-coupling reactions using aryl and alkyl halides. This Review begins with a brief introduction of four extensively investigated modes of catalysis for forming CC bonds from CH bonds: PdII/Pd0, PdII/PdIV, Pd0/PdII/PdIV, and Pd0/PdII catalysis. A more detailed discussion is then directed towards the recent development of palladium(II)-catalyzed coupling of CH bonds with organometallic reagents through a PdII/Pd0 catalytic cycle. Despite the progress made to date, improving the versatility and practicality of this new reaction remains a tremendous challenge.