Biology subjects

Shoji, K.

Publications and source records attributed to Shoji, K..

10 recordsLinked to original sources

bioRxiv2026

Theoretical framework and experimental demonstration of sustainable in vitro regeneration of major translation factors EF-Tu and IF3

The development of molecular systems capable of self-regeneration, much like living organisms, is a major goal in synthetic biology. Previous efforts to expand the repertoire of regenerating proteins have relied on empirical optimization without a theoretical foundation, fundamentally limiting the scalability of this approach. Here, we developed the first theoretical framework, based on measurable parameters of translational proteins, to rationally predict the dilution rate that enables sustainable regeneration and the steady-state translation level. Using this framework, we successfully demonstrated sustainable regeneration of EF-Tu, the most abundant translation factor, for up to 15 rounds of serial dilution. We further extended this framework to the co-regeneration of multiple translation factors and demonstrated sustainable co-regeneration of EF-Tu and IF3, another major essential translation protein, for up to 20 rounds. These results establish a rational methodology for systematically expanding the number of regenerating proteins, providing a clear path toward the realization of a fully self-regenerating system.

Synthetic Biology↗

bioRxiv2026

A two-step selection method for in vitro evolution of translational proteins

Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.

synthetic biology↗

bioRxiv2026

An anti-aggregation region of the SGS3 N-terminal IDR is essential for secondary siRNA biogenesis

Secondary siRNA biogenesis amplifies small RNA signals from target transcripts and plays a pivotal role in plant development and defense responses. The RNA-binding protein SGS3 is essential for this pathway, recruiting RNA-dependent RNA polymerase 6 (RDR6) to Argonaute-small RNA-bound targets. The N-terminal intrinsically disordered region (IDR) of SGS3, which contains a prion-like domain (PrLD), has been reported to drive liquid-liquid phase separation, forming siRNA bodies, and to be required for secondary siRNA production. However, the molecular mechanism by which the N-terminal IDR contributes to secondary siRNA production remains unclear. Here, using in vitro reconstitution and in planta assays, we show that the N-terminal IDR comprises two functional modules: the PrLD and a negatively charged region (NCR). The PrLD is required for phase separation and siRNA body formation but is dispensable for secondary siRNA production. In contrast, mutations in the NCR caused SGS3 to form abnormally large cytoplasmic assemblies and markedly impaired secondary siRNA production. These results suggest that the N-terminal IDR helps maintain SGS3 in a functional, soluble state that supports efficient secondary siRNA biogenesis.

molecular biology↗

bioRxiv2025

The Masc-PSI complex directly induces male-type doublesex splicing in silkworms

The WZ sex determination system is found in a diverse range of animals, including lepidopteran insects. In the silkworm Bombyx mori, a lepidopteran model insect, females carry the W chromosome with the feminizing gene Feminizer (Fem), which is the source of a female-specific PIWI-interacting RNA (piRNA). The Fem piRNA-PIWI complex cleaves an mRNA encoding the masculinizing protein Masculinizer (BmMasc), resulting in the production of the female-type splice variant of Bombyx mori doublesex (Bmdsx), which is the master genetic switch of B. mori sex differentiation. In contrast, in males, BmMasc induces the production of the male-type Bmdsx splice variant (BmdsxM). However, the molecular mechanism through which BmMasc transduces the masculinizing signal to the male-specific Bmdsx splicing event remains unknown. In this study, we showed that BmMasc physically interacts with Bombyx mori P-element somatic inhibitor (BmPSI), that is a RNA binding protein required for BmdsxM expression. BmMasc overexpression resulted in the production of BmdsxM in B. mori ovary-derived BmN-4 cells, but this induction was severely inhibited in BmPSI-knocked down cells, indicating that BmPSI is essential for the masculinizing activity of BmMasc. We also identified that BmMasc-containing protein complex was associated with a region of Bmdsx pre-mRNA spanning introns 2 to 4, particularly around the junctions of intron 2 with exon 3 and intron 3 with exon 4. Exons 3 and 4 of Bmdsx are female-specific units and are skipped in BmdsxM. Together with a previous report of the binding of BmPSI to the CE1 sequence located in exon 4 of Bmdsx, the current results strongly suggest that the BmMasc-BmPSI complex is required for male-specific exon skipping in Bmdsx pre-mRNA through its binding to the female-specific Bmdsx introns and exons.

molecular biology↗

bioRxiv2024

Autonomous Shaping of the piRNA Sequence Repertoire by Competition between Adjacent Ping-Pong Sites

PIWI-interacting RNAs (piRNAs) are crucial for silencing transposable elements (TEs). In many species, piRNAs are generated via a complex process known as the ping-pong pathway, which couples TE cleavage with new piRNA amplification. However, the biological significance of this complexity and its impact on the piRNA sequence repertoire remain unclear. Here, we systematically compared piRNA production patterns in two closely related silkworm cell lines and found significant changes in their piRNA sequence repertoire. Importantly, the changeability of this repertoire showed a strong negative correlation with the efficiency of piRNA biogenesis. This can be explained by competition between adjacent ping-pong sites, as supported by our mathematical modeling. Moreover, this competition can rationalize how piRNAs autonomously avoid deleterious mismatches to target TEs. These findings unveil the intrinsic plasticity and adaptability of the piRNA system to combat diverse TE sequences and highlight the universal power of competition and self-amplification to drive autonomous optimization.

molecular biology↗

bioRxiv2024

A potential role of inefficient and non-specific piRNA production from the whole transcriptome

PIWI-interacting RNAs (piRNAs) safeguard germline genomes from invasive genetic elements such as transposons. While long-term silencing relies on sequence-specific mechanisms such as ping-pong amplification and the incorporation of invader fragments into genomic piRNA clusters, how newly invading elements are initially recognized as "non-self" remains unresolved. Here, we show that low levels of sense-stranded piRNAs are broadly generated from the transcriptome in silkworms, flies, and mice, in direct proportion to RNA abundance and largely independent of canonical piRNA biogenesis pathways. This process can directly sample invaders: in silkworm cells, extremely abundant Bombyx mori latent virus RNAs enter this pathway. Reanalysis of recently endogenized retroviruses reveals distinct stages of adaptation in vivo--mouse AKV remains abundance-coupled, whereas koala KoRV-A has progressed into ping-pong amplification. We propose that this inefficient, non-selective, abundance-coupled pathway constitutes a naive germline surveillance system that seeds an initial piRNA pool for later recruitment into more efficient, specificity-conferring silencing pathways.

genetics↗

bioRxiv2023

The dual role of Spn-E in supporting heterotypic ping-pong piRNA amplification in silkworms

The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers 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.

molecular biology↗

bioRxiv2022

The piRNA cluster torimochi is an expanding transposon in cultured silkworm cells

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. (185 words)

genomics↗

bioRxiv2022

The two Gtsf paralogs in silkworms orthogonally activate their partner PIWI proteins for target cleavage

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.

biochemistry↗

bioRxiv2022

The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1-10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5' nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5' adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5' A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.

molecular biology↗