Details of a Researcher - Iwasaki, Yuka

Papers - Iwasaki, Yuka

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Clinical Utility of SARS-CoV-2 Whole Genome Sequencing in Deciphering Source of Infection

Toshiki Takenouchi, Yuka W Iwasaki, Sei Harada, Hirotsugu Ishizu, Yoshifumi Uwamino, Shunsuke Uno, Asami Osada, Kodai Abe, Naoki Hasegawa, Mitsuru Murata, Toru Takebayashi, Koichi Fukunaga, Hideyuki Saya, Yuko Kitagawa, Masayuki Amagai, Haruhiko Siomi, Kenjiro Kosaki, Keio Donner Project

Journal of Hospital Infection In press 2020.10

ISSN 0195-6701

COVID-19 caused by SARS-CoV-2 is a worldwide problem. From the standpoint of hospital infection control, determining the source of infection is critical. We conducted the present study to evaluate the efficacy of using whole genome sequencing to determine the source of infection in hospitalized patients who do not have a clear infectious contact history. Recently, we encountered two seemingly separate COVID-19 clusters in a tertiary hospital. Whole viral genome sequencing distinguished the two clusters according to the viral haplotype. However, the source of infection was unclear in 14 patients with COVID-19 who were clinically unlinked to clusters #1 or #2. These patients, who had no clear history of infectious contact within the hospital (“undetermined source of infection”), had haplotypes similar to those in cluster #2 but did not have two of the mutations used to characterize cluster #2, suggesting that these 14 cases of “undetermined source of infection” were not derived from cluster #2. Whole viral genome sequencing can be useful for confirming that sporadic COVID-19 cases with an undetermined source of infection are indeed not part of clusters at the institutional level.

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Nuclear RNA export factor variant initiates piRNA‐guided co‐transcriptional silencing

Murano K, Iwasaki YW, Ishizu H, Mashiko A, Shibuya A, Kondo S, Adachi S, Suzuki S, Saito K, Natsume T, Siomi MC, Siomi H

the EMBO journal e102870 e102870 2019.08

ISSN 1460-2075

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing.

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Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification

Sadahiro, T., Isomi, M., Muraoka, N., Kojima, H., Haginiwa, S., Kurotsu, S., Tamura, F., Tani, H., Tohyama, S., Fujita, J., Miyoshi, H., Kawamura, Y., Goshima, N., Iwasaki, Y. W., Murano, K., Saito, K., Oda, M., Andersen, P., Kwon, C., Uosaki, H., Nishizono, H., Fukuda, K. and Ieda, M.

Cell Stem Cell 23 ( 3 ) 382 - 395 e5 2018.09

ISSN 1875-9777

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.

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Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis

Nishida, K. M., Sakakibara, K., Iwasaki, Y. W., Yamada, H., Murakami, R., Murota, Y., Kawamura, T., Kodama, T., Siomi, H. and Siomi, M. C.

Nature 555 ( 7695 ) 260 - 264 2018.03

ISSN 1476-4687

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines. piRNA 3' end formation in the silkworm Bombyx mori has been shown to be mediated by the 3'-to-5' exonuclease Trimmer (Trim; known as PNLDC1 in mammals), and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi. However, it remains unclear whether the Zucchini (Zuc) endonuclease and Nibbler (Nbr) 3'-to-5' exonuclease, both of which have pivotal roles in piRNA biogenesis in Drosophila, are required for piRNA processing in other species. Here we show that the loss of Zuc in Bombyx had no effect on the levels of Trim and Nbr, but resulted in the aberrant accumulation of piRNA intermediates within the Papi complex, and that these were processed to form mature piRNAs by recombinant Zuc. Papi exerted its RNA-binding activity only when bound with PIWI and phosphorylated, suggesting that complex assembly involves a hierarchical process. Both the 5' and 3' ends of piRNA intermediates within the Papi complex showed hallmarks of PIWI 'slicer' activity, yet no phasing pattern was observed in mature piRNAs. The loss of Zuc did not affect the 5'- and 3'-end formation of the intermediates, strongly supporting the idea that the 5' end of Bombyx piRNA is formed by PIWI slicer activity, but independently of Zuc, whereas the 3' end is formed by the Zuc endonuclease. The Bombyx piRNA biogenesis machinery is simpler than that of Drosophila, because Bombyx has no transcriptional silencing machinery that relies on phased piRNAs.

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Profiling Open Chromatin Structure in the Ovarian Somatic Cells Using ATAC-seq

Murano, K., Iwasaki, Y. W. and Siomi, H.

Methods Mol Biol 1680 165 - 177 2018

ISSN 1940-6029

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) was recently established as a method to profile open chromatin, which overcomes the sample size limitations of the alternative methods DNase/MNase-seq. To investigate the role of Piwi in heterochromatin formation around transposable element loci, we have used ATAC-seq to examine chromatin accessibility at target transposable elements in a Drosophila cultured cell line, ovarian somatic cells (OSCs). In this chapter, we describe our method to profile open chromatin structure in OSCs using ATAC-seq.

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Deep sequencing and high-throughput analysis of PIWI-associated small RNAs

Iwasaki, Y. W., Ishino, K. and Siomi, H.

Methods 126 66 - 75 2017.08

ISSN 1095-9130

Small RNAs are now known to be major regulatory factors of gene expression. Emerging methods based on deep-sequencing have enabled the analysis of small RNA expression in a high-throughput manner, leading to the identification of large numbers of small RNAs in various species. Moreover, profiling small RNA data together with transcriptome data enables transcriptional and post-transcriptional regulation mediated by small RNAs to be hypothesized. Here, we isolated PIWIL1 (MIWI)-associated small RNAs from mouse testes, and performed small RNA-seq analysis. In addition, directional RNA-seq was performed using Piwil1 mutant mouse testes. Using these data, we describe protocols for analyzing small RNA-seq reads to obtain profiles of small RNAs associated with PIWI proteins. We also present bioinformatic protocols for analyzing RNA-seq reads that aim to annotate expression of piRNA clusters and identify genes regulated by piRNAs.

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Piwi Modulates Chromatin Accessibility by Regulating Multiple Factors Including Histone H1 to Repress Transposons

Iwasaki, Y. W., Murano, K., Ishizu, H., Shibuya, A., Iyoda, Y., Siomi, M. C., Siomi, H. and Saito, K.

Mol Cell 63 ( 3 ) 408 - 19 2016.08

ISSN 1097-4164

PIWI-interacting RNAs (piRNAs) mediate transcriptional and post-transcriptional silencing of transposable element (TE) in animal gonads. In Drosophila ovaries, Piwi-piRNA complexes (Piwi-piRISCs) repress TE transcription by modifying the chromatin state, such as by H3K9 trimethylation. Here, we demonstrate that Piwi physically interacts with linker histone H1. Depletion of Piwi decreases H1 density at a subset of TEs, leading to their derepression. Silencing at these loci separately requires H1 and H3K9me3 and heterochromatin protein 1a (HP1a). Loss of H1 increases target loci chromatin accessibility without affecting H3K9me3 density at these loci, while loss of HP1a does not impact H1 density. Thus, Piwi-piRISCs require both H1 and HP1a to repress TEs, and the silencing is correlated with the chromatin state rather than H3K9me3 marks. These findings suggest that Piwi-piRISCs regulate the interaction of chromatin components with target loci to maintain silencing of TEs through the modulation of chromatin accessibility.

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Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells

Sumiyoshi, T., Sato, K., Yamamoto, H., Iwasaki, Y. W., Siomi, H. and Siomi, M. C.

Genes Dev 30 ( 14 ) 1617 - 22 2016.07

ISSN 1549-5477

In Drosophila germ cells, PIWI-interacting RNAs (piRNAs) are amplified through a PIWI slicer-dependent feed-forward loop termed the ping-pong cycle, yielding secondary piRNAs. However, the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been available. Here, we show that CRISPR-mediated loss of function of lethal (3) malignant brain tumor [l(3)mbt] leads to ectopic activation of the germ-specific ping-pong cycle in ovarian somatic cells. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation. This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis in Drosophila.

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piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals

Parrish, N. F., Fujino, K., Shiromoto, Y., Iwasaki, Y. W., Ha, H., Xing, J., Makino, A., Kuramochi-Miyagawa, S., Nakano, T., Siomi, H., Honda, T. and Tomonaga, K.

RNA 21 ( 10 ) 1691 - 703 2015.10

ISSN 1469-9001

Endogenous bornavirus-like nucleoprotein elements (EBLNs) are sequences within vertebrate genomes derived from reverse transcription and integration of ancient bornaviral nucleoprotein mRNA via the host retrotransposon machinery. While species with EBLNs appear relatively resistant to bornaviral disease, the nature of this association is unclear. We hypothesized that EBLNs could give rise to antiviral interfering RNA in the form of PIWI-interacting RNAs (piRNAs), a class of small RNA known to silence transposons but not exogenous viruses. We found that in both rodents and primates, which acquired their EBLNs independently some 25-40 million years ago, EBLNs are present within piRNA-generating regions of the genome far more often than expected by chance alone (P = 8 x 10(-3)-6 x 10(-8)). Three of the seven human EBLNs fall within annotated piRNA clusters and two marmoset EBLNs give rise to bona fide piRNAs. In both rats and mice, at least two of the five EBLNs give rise to abundant piRNAs in the male gonad. While no EBLNs are syntenic between rodent and primate, some of the piRNA clusters containing EBLNs are; thus we deduce that EBLNs were integrated into existing piRNA clusters. All true piRNAs derived from EBLNs are antisense relative to the proposed ancient bornaviral nucleoprotein mRNA. These observations are consistent with a role for EBLN-derived piRNA-like RNAs in interfering with ancient bornaviral infection. They raise the hypothesis that retrotransposon-dependent virus-to-host gene flow could engender RNA-mediated, sequence-specific antiviral immune memory in metazoans analogous to the CRISPR/Cas system in prokaryotes.

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Krimper Enforces an Antisense Bias on piRNA Pools by Binding AGO3 in the Drosophila Germline

Sato, K., Iwasaki, Y. W., Shibuya, A., Carninci, P., Tsuchizawa, Y., Ishizu, H., Siomi, M. C. and Siomi, H.

Mol Cell 59 ( 4 ) 553 - 63 2015.08

ISSN 1097-4164

Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. Here we show that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3's compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. We propose that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs.

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Somatic Primary piRNA Biogenesis Driven by cis-Acting RNA Elements and trans-Acting Yb

Ishizu, H., Iwasaki, Y. W., Hirakata, S., Ozaki, H., Iwasaki, W., Siomi, H. and Siomi, M. C.

Cell Rep 12 ( 3 ) 429 - 40 2015.07

ISSN 2211-1247

Primary piRNAs in Drosophila ovarian somatic cells arise from piRNA cluster transcripts and the 3' UTRs of a subset of mRNAs, including Traffic jam (Tj) mRNA. However, it is unclear how these RNAs are determined as primary piRNA sources. Here, we identify a cis-acting 100-nt fragment in the Tj 3' UTR that is sufficient for producing artificial piRNAs from unintegrated DNA. These artificial piRNAs were effective in endogenous gene transcriptional silencing. Yb, a core component of primary piRNA biogenesis center Yb bodies, directly bound the Tj-cis element. Disruption of this interaction markedly reduced piRNA production. Thus, Yb is the trans-acting partner of the Tj-cis element. Yb-CLIP revealed that Yb binding correlated with somatic piRNA production but Tj-cis element downstream sequences produced few artificial piRNAs. We thus propose that Yb determines primary piRNA sources through two modes of action: primary binding to cis elements to specify substrates and secondary binding to downstream regions to increase diversity in piRNA populations.

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Respective functions of two distinct Siwi complexes assembled during PIWI-interacting RNA biogenesis in Bombyx germ cells

Nishida, K. M., Iwasaki, Y. W., Murota, Y., Nagao, A., Mannen, T., Kato, Y., Siomi, H. and Siomi, M. C.

Cell Rep 10 ( 2 ) 193 - 203 2015.01

ISSN 2211-1247

PIWI-interacting RNA (piRNA) biogenesis consists of two sequential steps: primary piRNA processing and the ping-pong cycle that depends on reciprocal Slicer-mediated RNA cleavage by PIWI proteins. However, the molecular functions of the factors involved remain elusive. Here, we show that RNAs cleaved by a Bombyx mori PIWI, Siwi, remain bound to the protein upon cleavage but are released by a DEAD box protein BmVasa. BmVasa copurifies with Siwi but not another PIWI BmAgo3. A lack of BmVasa does not affect primary piRNA processing but abolishes the ping-pong cycle. Siwi also forms a complex with BmSpn-E and BmQin. This complex is physically separable from the Siwi/BmVasa complex. BmSpn-E, unlike BmVasa, is necessary for primary piRNA production. We propose a model for piRNA biogenesis, where the BmSpn-E/BmQin dimer binds Siwi to function in primary piRNA processing, whereas BmVasa, by associating with Siwi, ensures target RNA release upon cleavage to facilitate the ping-pong cycle.

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Tudor-domain containing proteins act to make the piRNA pathways more robust in Drosophila

Sato, K., Iwasaki, Y. W., Siomi, H. and Siomi, M. C.

Fly (Austin) 9 ( 2 ) 86 - 90 2015

ISSN 1933-6942

PIWI-interacting RNAs (piRNAs), a subset of small non-coding RNAs enriched in animal gonads, repress transposons by assembling with PIWI proteins to form potent gene-silencing RNP complexes, piRISCs. Accumulating evidence suggests that piRNAs are produced through three interdependent pathways; the de novo primary pathway, the ping-pong pathway, and the phased primary pathway. The de novo primary pathway in Drosophila ovaries produces primary piRNAs for two PIWI members, Piwi and Aub. Aub then initiates the ping-pong pathway to produce secondary piRNAs for AGO3. AGO3-slicer dependent cleavage subsequently produces secondary piRNAs for Aub. Trailer products of AGO3-slicer activity are consumed by the phased primary pathway to increase the Piwi-bound piRNA population. All these pathways are regulated by a number of piRNA factors in a highly coordinated fashion. Recent studies show that two Tudor-domain containing piRNA factors, Krimper (Krimp) and Qin/Kumo, play crucial roles in making Aub-AGO3 heterotypic ping-pong robust. This maintains the levels of piRNAs loaded onto Piwi and Aub to efficiently repress transposons at transcriptional and post-transcriptional levels, respectively.

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PIWI-Interacting RNA: Its Biogenesis and Functions

Iwasaki, Y. W., Siomi, M. C. and Siomi, H.

Annu Rev Biochem 84 405 - 33 2015

ISSN 1545-4509

PIWI-interacting RNAs (piRNAs) are a class of small RNAs that are 24-31 nucleotides in length. They associate with PIWI proteins, which constitute a germline-specific subclade of the Argonaute family, to form effector complexes known as piRNA-induced silencing complexes, which repress transposons via transcriptional or posttranscriptional mechanisms and maintain germline genome integrity. In addition to having a role in transposon silencing, piRNAs in diverse organisms function in the regulation of cellular genes. In some cases, piRNAs have shown transgenerational inheritance to pass on the memory of "self" and "nonself," suggesting a contribution to various cellular processes over generations. Many piRNA factors have been identified; however, both the molecular mechanisms leading to the production of mature piRNAs and the effector phases of gene silencing are still enigmatic. Here, we summarize the current state of our knowledge on the biogenesis of piRNA, its biological functions, and the underlying mechanisms.

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miRNA regulatory ecosystem in early development

Iwasaki, Y. W. and Siomi, H.

Mol Cell 56 ( 5 ) 615 - 6 2014.12

ISSN 1097-4164

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Epigenetic silencing of miR-210 increases the proliferation of gastric epithelium during chronic Helicobacter pylori infection

Kiga, K., Mimuro, H., Suzuki, M., Shinozaki-Ushiku, A., Kobayashi, T., Sanada, T., Kim, M., Ogawa, M., Iwasaki, Y. W., Kayo, H., Fukuda-Yuzawa, Y., Yashiro, M., Fukayama, M., Fukao, T. and Sasakawa, C.

Nat Commun 5 4497 2014.09

ISSN 2041-1723

Persistent colonization of the gastric mucosa by Helicobacter pylori (Hp) elicits chronic inflammation and aberrant epithelial cell proliferation, which increases the risk of gastric cancer. Here we examine the ability of microRNAs to modulate gastric cell proliferation in response to persistent Hp infection and find that epigenetic silencing of miR-210 plays a key role in gastric disease progression. Importantly, DNA methylation of the miR-210 gene is increased in Hp-positive human gastric biopsies as compared with Hp-negative controls. Moreover, silencing of miR-210 in gastric epithelial cells promotes proliferation. We identify STMN1 and DIMT1 as miR-210 target genes and demonstrate that inhibition of miR-210 expression augments cell proliferation by activating STMN1 and DIMT1. Together, our results highlight inflammation-induced epigenetic silencing of miR-210 as a mechanism of induction of chronic gastric diseases, including cancer, during Hp infection.

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Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset, a model primate

Hirano, T., Iwasaki, Y. W., Lin, Z. Y., Imamura, M., Seki, N. M., Sasaki, E., Saito, K., Okano, H., Siomi, M. C. and Siomi, H.

RNA 20 ( 8 ) 1223 - 37 2014.08

ISSN 1469-9001

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.

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Yb integrates piRNA intermediates and processing factors into perinuclear bodies to enhance piRISC assembly

Murota, Y., Ishizu, H., Nakagawa, S., Iwasaki, Y. W., Shibata, S., Kamatani, M. K., Saito, K., Okano, H., Siomi, H. and Siomi, M. C.

Cell Rep 8 ( 1 ) 103 - 13 2014.07

ISSN 2211-1247

PIWI-interacting RNAs (piRNAs) direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. We suggest that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells.

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DmGTSF1 is necessary for Piwi-piRISC-mediated transcriptional transposon silencing in the Drosophila ovary

Ohtani, H., Iwasaki, Y. W., Shibuya, A., Siomi, H., Siomi, M. C. and Saito, K.

Genes Dev 27 ( 15 ) 1656 - 61 2013.08

ISSN 1549-5477

The Piwi-piRNA (PIWI-interacting RNA) complex (Piwi-piRISC) in Drosophila ovarian somatic cells represses transposons transcriptionally to maintain genome integrity; however, the underlying mechanisms remain obscure. Here, we reveal that DmGTSF1, a Drosophila homolog of gametocyte-specific factor 1 (GTSF1) (which is required for transposon silencing in mouse testes), is necessary for Piwi-piRISC to repress target transposons and neighboring genes. DmGTSF1 depletion affected neither piRNA biogenesis nor nuclear import of Piwi-piRISC. DmGTSF1 mutations caused derepression of transposons and loss of ovary follicle layers, resulting in female infertility. We suggest that DmGTSF1, a nuclear Piwi interactor, is an integral factor in Piwi-piRISC-mediated transcriptional silencing.

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Global microRNA elevation by inducible Exportin 5 regulates cell cycle entry

Iwasaki, Y. W., Kiga, K., Kayo, H., Fukuda-Yuzawa, Y., Weise, J., Inada, T., Tomita, M., Ishihama, Y. and Fukao, T.

RNA 19 ( 4 ) 490 - 7 2013.04

ISSN 1469-9001

Proper regulation of gene expression during cell cycle entry ensures the successful completion of proliferation, avoiding risks such as carcinogenesis. The microRNA (miRNA) network is an emerging molecular system regulating multiple genetic pathways. We demonstrate here that the global elevation of miRNAs is critical for proper control of gene expression program during cell cycle entry. Strikingly, Exportin 5 (XPO5) is promptly induced during cell cycle entry by a PI3K-dependent post-transcriptional mechanism. Inhibition of XPO5 induction interfered with global miRNA elevation and resulted in a proliferation defect associated with delayed G1/S transition. During cell cycle entry, XPO5 therefore plays a paramount role as a critical molecular hub controlling the gene expression program through global regulation of miRNAs. Our data suggest that XPO5-mediated global miRNA elevation might be involved in a broad range of cellular events associated with cell cycle control.

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Systems Biology Reveals MicroRNA-Mediated Gene Regulation

Watanabe, Y. and Kanai, A.

Front Genet 2 29 2011

ISSN 1664-8021

MicroRNAs (miRNAs) are members of the small non-coding RNAs, which are principally known for their functions as post-transcriptional regulators of target genes. Regulation by miRNAs is triggered by the translational repression or degradation of their complementary target messenger RNAs (mRNAs). The growing number of reported miRNAs and the estimate that hundreds or thousands of genes are regulated by them suggest a magnificent gene regulatory network in which these molecules are embedded. Indeed, recent reports have suggested critical roles for miRNAs in various biological functions, such as cell differentiation, development, oncogenesis, and the immune responses, which are mediated by systems-wide changes in gene expression profiles. Therefore, it is essential to analyze this complex regulatory network at the transcriptome and proteome levels, which should be possible with approaches that include both high-throughput experiments and computational methodologies. Here, we introduce several systems-level approaches that have been applied to miRNA research, and discuss their potential to reveal miRNA-guided gene regulatory systems and their impacts on biological functions.

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Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals

Takane, K., Fujishima, K., Watanabe, Y., Sato, A., Saito, N., Tomita, M. and Kanai, A.

BMC Genomics 11 101 2010.02

ISSN 1471-2164

BACKGROUND: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution. RESULTS: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells. CONCLUSIONS: The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.

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Perspective in the evolution of human microRNAs: copy number expansion and acquisition of target gene specialization

Watanabe, Y., Tomita, M. and Kanai, A.

Prog Theor Phys Suppl 173 219 - 228 2008

ISSN 0375-9687

A novel class of small, noncoding RNAs called microRNAs (miRNAs) was recently identified, and up to 20%–30% of human genes are said to be regulated by the members of this class of RNA. miRNAs regulate their target genes by imperfect binding to the 3′ untranslated region (UTR) of the gene transcript, and computational predictions of the binding between miRNAs and target gene transcripts have been performed in order to determine which genes are regulated by these RNAs. Phylogenetic analysis has also been used as a powerful tool to predict miRNA target genes. Much emphasis has therefore been placed on studying the phylogenetic conservation and evolution of this novel type of gene regulator, although there still is much that is not known. Here, we propose a hypothesis of how human miRNAs optimized their gene regulation by adjusting their transcript levels, and how they evolved through specific selection of their target genes. We analyzed the correlation between the conservation of miRNAs among species and three features: the number of transcripts, the formation of duplicates, and the number of target genes. The number of miRNA transcripts and the formation of duplicates increased as the conservation rate increased. In contrast, the number of target genes decreased as the conservation rate increased. Therefore, we propose that miRNAs gradually gain an ability to regulate specific target genes when such regulation has a positive effect on the organism. As its pool of target genes is refined, the ability of an miRNA to regulate the genes may be stabilized by an increase in the miRNA transcript number and the formation of duplicates.

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Computational analysis of microRNA-mediated antiviral defense in humans

Watanabe, Y., Kishi, A., Yachie, N., Kanai, A. and Tomita, M.

FEBS Lett 581 ( 24 ) 4603 - 10 2007.10

ISSN 0014-5793

Recent studies have proposed the interesting perspective that viral gene expression is downregulated by host microRNAs (miRNAs), small non-coding RNAs well known as post-transcriptional gene regulators. We computationally predicted human miRNA target sites within 228 human-infecting and 348 invertebrate-infecting virus genomes, and we observed that human-infecting viruses were more likely than invertebrate-infecting ones to be targeted by human miRNAs. We listed 62 possible human miRNA-targeted viruses from 6 families, most of which consisted of single-stranded RNA viruses. These results suggest that miRNAs extensively mediate antiviral defenses in humans.

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Computational methods for microRNA target prediction

Watanabe, Y., Tomita, M. and Kanai, A.

Methods Enzymol 427 65 - 86 2007

ISSN 0076-6879

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Large numbers of miRNAs have been identified in a wide range of species, and most of them are known to downregulate translation of messenger RNAs (mRNAs) via imperfect binding of the miRNA to a specific site or sites in the 3' untranslated region (UTR) of the mRNA. Identification of genes targeted by miRNAs is widely believed to be an important step toward understanding the role of miRNAs in gene regulatory networks. As part of the effort to understand interactions between miRNAs and their targets, computational algorithms have been developed based on observed rules for features such as the degree of hybridization between the two RNA molecules. These in silico approaches provide important tools for miRNA target detection, and together with experimental validation, help to reveal regulated targets of miRNAs. Here, we summarize the knowledge that has been accumulated about the principles of target recognition by miRNAs and the currently available computational methodologies for prediction of miRNA target genes.

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Computational analysis of microRNA targets in Caenorhabditis elegans

Watanabe, Y., Yachie, N., Numata, K., Saito, R., Kanai, A. and Tomita, M.

Gene 365 2 - 10 2006.01

ISSN 0378-1119

MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide (nt) non-coding RNAs that post-transcriptionally regulate the expression of target genes via hybridization to target mRNA. Using known pairs of miRNA and target mRNA in Caenorhabditis elegans, we first performed computational analysis for specific hybridization patterns between these two RNAs. We counted the numbers of perfectly complementary dinucleotide sequences and calculated the free energy within complementary base pairs of each dinucleotide, observed by sliding a 2-nt window along all nucleotides of the miRNA-mRNA duplex. We confirmed not only strong base pairing within the 5' region of miRNAs (nts 1-8) in C. elegans, but also the required mismatch within the central region (nt 9 or nt 10), and we found weak binding within the 3' region (nts 13-14). We also predicted 687 possible miRNA target transcripts, many of which are thought to be involved in C. elegans development, by combining the above mentioned hybridization tendency with the following analyses: (1) prediction of the miRNA-mRNA duplex with free-energy minimization; (2) identification of the complementary pattern within the miRNA-mRNA duplex; (3) conservation of target sites between C. elegans and C. briggsae, a related soil nematode; and (4) extraction of mRNA candidates with multiple target sites. Rigorous tests using shuffled miRNA controls supported these predictions. Our results suggest that miRNAs recognize their target mRNAs by their hybridization pattern and that many target mRNAs may be regulated through a combination of several specific miRNA target sites in C. elegans.

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