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Article ; Online: The dual role of Spn-E in supporting heterotypic ping-pong piRNA amplification in silkworms.

Izumi, Natsuko / Shoji, Keisuke / Negishi, Lumi / Tomari, Yukihide

2024

Abstract: The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping- ... ...

Abstract	The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping-pong cycle, is mediated by the two PIWI proteins, Siwi and BmAgo3, in silkworms. However, the detailed molecular mechanism of the ping-pong cycle remains largely unclear. Here, we show that Spindle-E (Spn-E), a putative ATP-dependent RNA helicase, is essential for BmAgo3-dependent production of Siwi-bound piRNAs in the ping-pong cycle and that this function of Spn-E requires its ATPase activity. Moreover, Spn-E acts to suppress homotypic Siwi-Siwi ping-pong, but this function of Spn-E is independent of its ATPase activity. These results highlight the dual role of Spn-E in facilitating proper heterotypic ping-pong in silkworms.
Language	English
Publishing date	2024-04-17
Publishing country	England
Document type	Journal Article
ZDB-ID	2020896-0
ISSN	1469-3178 ; 1469-221X
ISSN (online)	1469-3178
ISSN	1469-221X
DOI	10.1038/s44319-024-00137-2
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Zs.MG 41: Show issues

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Article ; Online: The piRNA cluster torimochi is an expanding transposon in cultured silkworm cells.

Shoji, Keisuke / Umemura, Yusuke / Katsuma, Susumu / Tomari, Yukihide

PLoS genetics

2023 Volume 19, Issue 2, Page(s) e1010632

Abstract: PIWI proteins and PIWI-interacting RNAs (piRNAs) play a central role in repressing transposable elements in animal germ cells. It is thought that piRNAs are mainly produced from discrete genomic loci named piRNA clusters, which often contain many "dead" ... ...

Abstract	PIWI proteins and PIWI-interacting RNAs (piRNAs) play a central role in repressing transposable elements in animal germ cells. It is thought that piRNAs are mainly produced from discrete genomic loci named piRNA clusters, which often contain many "dead" transposon remnants from past invasions and have heterochromatic features. In the genome of silkworm ovary-derived cultured cells called BmN4, a well-established model for piRNA research, torimochi was previously annotated as a unique and specialized genomic region that can capture transgenes and produce new piRNAs bearing a trans-silencing activity. However, the sequence identity of torimochi has remained elusive. Here, we carefully characterized torimochi by utilizing the updated silkworm genome sequence and the long-read sequencer MinION. We found that torimochi is in fact a full-length gypsy-like LTR retrotransposon, which is exceptionally active and has massively expanded its copy number in BmN4 cells. Many copies of torimochi in BmN4 cells have features of open chromatin and the ability to produce piRNAs. Therefore, torimochi may represent a young, growing piRNA cluster, which is still "alive" and active in transposition yet capable of trapping other transposable elements to produce de novo piRNAs.
MeSH term(s)	Animals ; Female ; Piwi-Interacting RNA ; RNA, Small Interfering/genetics ; Bombyx/genetics ; DNA Transposable Elements ; Cells, Cultured ; Argonaute Proteins/genetics ; Germ Cells/metabolism
Chemical Substances	Piwi-Interacting RNA ; RNA, Small Interfering ; DNA Transposable Elements ; Argonaute Proteins
Language	English
Publishing date	2023-02-09
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2186725-2
ISSN	1553-7404 ; 1553-7390
ISSN (online)	1553-7404
ISSN	1553-7390
DOI	10.1371/journal.pgen.1010632
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Life of RISC: Formation, action, and degradation of RNA-induced silencing complex.

Iwakawa, Hiro-Oki / Tomari, Yukihide

Molecular cell

2021 Volume 82, Issue 1, Page(s) 30–43

Abstract: Small RNAs regulate a wide variety of biological processes by repressing the expression of target genes at the transcriptional and post-transcriptional levels. To achieve these functions, small RNAs form RNA-induced silencing complex (RISC) together with ...

Abstract	Small RNAs regulate a wide variety of biological processes by repressing the expression of target genes at the transcriptional and post-transcriptional levels. To achieve these functions, small RNAs form RNA-induced silencing complex (RISC) together with a member of the Argonaute (AGO) protein family. RISC is directed by its bound small RNA to target complementary RNAs and represses their expression through mRNA cleavage, degradation, and/or translational repression. Many different factors fine-tune RISC activity and stability-from guide-target RNA complementarity to the recruitment of other protein partners to post-translational modifications of RISC itself. Here, we review recent progress in understanding RISC formation, action, and degradation, and discuss new, intriguing questions in the field.
MeSH term(s)	Animals ; Argonaute Proteins/genetics ; Argonaute Proteins/metabolism ; Humans ; MicroRNAs/genetics ; MicroRNAs/metabolism ; Protein Binding ; Protein Stability ; Proteolysis ; RNA/genetics ; RNA/metabolism ; RNA Stability ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Small Interfering/genetics ; RNA, Small Interfering/metabolism ; RNA-Induced Silencing Complex/genetics ; RNA-Induced Silencing Complex/metabolism
Chemical Substances	Argonaute Proteins ; MicroRNAs ; RNA, Messenger ; RNA, Small Interfering ; RNA-Induced Silencing Complex ; RNA (63231-63-0)
Language	English
Publishing date	2021-12-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	1415236-8
ISSN	1097-4164 ; 1097-2765
ISSN (online)	1097-4164
ISSN	1097-2765
DOI	10.1016/j.molcel.2021.11.026
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Article ; Online: My encounter with RNA.

Tomari, Yukihide

RNA (New York, N.Y.)

2015 Volume 21, Issue 4, Page(s) 747–748

MeSH term(s)	Biocatalysis ; Nucleic Acid Conformation ; RNA/chemistry ; RNA/genetics ; RNA/metabolism
Chemical Substances	RNA (63231-63-0)
Language	English
Publishing date	2015-04
Publishing country	United States
Document type	Journal Article
ZDB-ID	1241540-6
ISSN	1469-9001 ; 1355-8382
ISSN (online)	1469-9001
ISSN	1355-8382
DOI	10.1261/rna.050278.115
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Article ; Online: Non-gonadal somatic piRNA pathways ensure sexual differentiation, larval growth, and wing development in silkworms.

Kiuchi, Takashi / Shoji, Keisuke / Izumi, Natsuko / Tomari, Yukihide / Katsuma, Susumu

PLoS genetics

2023 Volume 19, Issue 9, Page(s) e1010912

Abstract: PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in ... ...

Abstract	PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. Here, we generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. Our results highlight the roles of non-gonadal somatic piRNAs in B. mori development.
MeSH term(s)	Animals ; Bombyx/genetics ; Larva/genetics ; Sex Differentiation ; Piwi-Interacting RNA ; Drosophila
Chemical Substances	Piwi-Interacting RNA
Language	English
Publishing date	2023-09-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2186725-2
ISSN	1553-7404 ; 1553-7390
ISSN (online)	1553-7404
ISSN	1553-7390
DOI	10.1371/journal.pgen.1010912
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Fusion with heat-resistant obscure (Hero) proteins have the potential to improve the molecular property of recombinant proteins.

Morimoto, Eri / Tsuboyama, Kotaro / Tomari, Yukihide

PloS one

2022 Volume 17, Issue 6, Page(s) e0270097

Abstract: Although recombinant proteins are widely used in biotechnology and pharmaceutical industries, improving their solubility and stability is often a challenging issue. We recently discovered a class of highly unstructured heat-resistant obscure (Hero) ... ...

Abstract	Although recombinant proteins are widely used in biotechnology and pharmaceutical industries, improving their solubility and stability is often a challenging issue. We recently discovered a class of highly unstructured heat-resistant obscure (Hero) proteins, which function to protect other "client" proteins in trans from various stresses in vitro and in vivo. Here, we show that fusion of Hero proteins in cis can enhance the molecular property of recombinant proteins. Fusion with Hero11 improved the otherwise challenging production of TAR DNA-binding protein of 43 kDa (TDP-43) in Escherichia coli. Moreover, fusing with Hero9 strongly protected the activity of firefly luciferase bearing destabilizing mutations against heat and other stress conditions. These data suggest that Hero proteins have the potential to be used as versatile stabilization tags for recombinant protein production.
MeSH term(s)	Biotechnology ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Hot Temperature ; Recombinant Fusion Proteins/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Solubility
Chemical Substances	Recombinant Fusion Proteins ; Recombinant Proteins
Language	English
Publishing date	2022-06-17
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0270097
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Revisiting the Glass Treatment for Single-Molecule Analysis of ncRNA Function.

Shen, Shuting / Naganuma, Masahiro / Tomari, Yukihide / Tadakuma, Hisashi

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2509, Page(s) 209–231

Abstract: Single-molecule imaging is a powerful method for unveiling precise molecular mechanisms. Particularly, single-molecule analysis with total internal reflection fluorescence (TIRF ) microscopy has been successfully applied to the characterization of ... ...

Abstract	Single-molecule imaging is a powerful method for unveiling precise molecular mechanisms. Particularly, single-molecule analysis with total internal reflection fluorescence (TIRF ) microscopy has been successfully applied to the characterization of molecular mechanisms in ncRNA studies. Tracing interactions at the single-molecule level have elucidated the intermediate states of the reaction, which are hidden by ensemble averaging in combinational biochemical approaches, and clarified the key steps of the interaction. However, applying a single-molecule technique to ncRNA analysis still remains a challenge, requiring laborious trial and error to identify a suitable glass surface passivation method. In this chapter, we revisit the major glass surface passivation methods using polyethylene glycol (PEG) treatment and summarize a detailed protocol for single-molecule analysis of the dicing process of Dcr-2, which may apply piRNA studies in the future.
MeSH term(s)	Glass/chemistry ; Microscopy, Fluorescence/methods ; Nanotechnology ; Polyethylene Glycols/chemistry ; Single Molecule Imaging/methods
Chemical Substances	Polyethylene Glycols (3WJQ0SDW1A)
Language	English
Publishing date	2022-07-07
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2380-0_13
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Life of RISC: Formation, action, and degradation of RNA-induced silencing complex

Iwakawa, Hiro-oki / Tomari, Yukihide

Molecular cell. 2022 Jan. 06, v. 82, no. 1

2022

Abstract	Small RNAs regulate a wide variety of biological processes by repressing the expression of target genes at the transcriptional and post-transcriptional levels. To achieve these functions, small RNAs form RNA-induced silencing complex (RISC) together with a member of the Argonaute (AGO) protein family. RISC is directed by its bound small RNA to target complementary RNAs and represses their expression through mRNA cleavage, degradation, and/or translational repression. Many different factors fine-tune RISC activity and stability—from guide-target RNA complementarity to the recruitment of other protein partners to post-translational modifications of RISC itself. Here, we review recent progress in understanding RISC formation, action, and degradation, and discuss new, intriguing questions in the field.
Keywords	cells ; complementary RNA ; fields ; gene expression ; gene targeting ; post-translational modification ; proteins ; transcription (genetics)
Language	English
Dates of publication	2022-0106
Size	p. 30-43.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1415236-8
ISSN	1097-4164 ; 1097-2765
ISSN (online)	1097-4164
ISSN	1097-2765
DOI	10.1016/j.molcel.2021.11.026
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Fusion with heat-resistant obscure (Hero) proteins have the potential to improve the molecular property of recombinant proteins

Eri Morimoto / Kotaro Tsuboyama / Yukihide Tomari

PLoS ONE, Vol 17, Iss

2022 Volume 6

Abstract	Although recombinant proteins are widely used in biotechnology and pharmaceutical industries, improving their solubility and stability is often a challenging issue. We recently discovered a class of highly unstructured heat-resistant obscure (Hero) proteins, which function to protect other “client” proteins in trans from various stresses in vitro and in vivo. Here, we show that fusion of Hero proteins in cis can enhance the molecular property of recombinant proteins. Fusion with Hero11 improved the otherwise challenging production of TAR DNA-binding protein of 43 kDa (TDP-43) in Escherichia coli. Moreover, fusing with Hero9 strongly protected the activity of firefly luciferase bearing destabilizing mutations against heat and other stress conditions. These data suggest that Hero proteins have the potential to be used as versatile stabilization tags for recombinant protein production.
Keywords	Medicine ; R ; Science ; Q
Subject code	580
Language	English
Publishing date	2022-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The two Gtsf paralogs in silkworms orthogonally activate their partner PIWI proteins for target cleavage.

Izumi, Natsuko / Shoji, Keisuke / Kiuchi, Takashi / Katsuma, Susumu / Tomari, Yukihide

RNA (New York, N.Y.)

2022

Abstract: The PIWI-interacting RNA (piRNA) pathway is a protection mechanism against transposons in animal germ cells. Most PIWI proteins possess piRNA-guided endonuclease activity, which is critical for silencing transposons and producing new piRNAs. Gametocyte- ... ...

Abstract	The PIWI-interacting RNA (piRNA) pathway is a protection mechanism against transposons in animal germ cells. Most PIWI proteins possess piRNA-guided endonuclease activity, which is critical for silencing transposons and producing new piRNAs. Gametocyte-specific factor 1 (Gtsf1), an evolutionarily conserved zinc finger protein, promotes catalysis by PIWI proteins. Many animals have multiple Gtsf1 paralogs; however, their respective roles in the piRNA pathway are not fully understood. Here, we dissected the roles of Gtsf1 and its paralog Gtsf1-like (Gtsf1L) in the silkworm piRNA pathway. We found that Gtsf1 and Gtsf1L preferentially bind the two silkworm PIWI paralogs, Siwi and BmAgo3, respectively, and facilitate the endonuclease activity of each PIWI protein. This orthogonal activation effect was further supported by specific reduction of BmAgo3-bound Masculinizer piRNA and Siwi-bound Feminizer piRNA, the unique piRNA pair required for silkworm feminization, upon depletion of Gtsf1 and Gtsf1L, respectively. Our results indicate that the two Gtsf paralogs in silkworms activate their respective PIWI partners, thereby facilitating the amplification of piRNAs.
Language	English
Publishing date	2022-11-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	1241540-6
ISSN	1469-9001 ; 1355-8382
ISSN (online)	1469-9001
ISSN	1355-8382
DOI	10.1261/rna.079380.122
Database	MEDical Literature Analysis and Retrieval System OnLINE

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ab Jg. 2022: Lesesaal (EG)

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