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Haruhiko Siomi

Publications and source records attributed to Haruhiko Siomi.

17 recordsLinked to original sources
PubMed2026

A single-cell meta-analysis evidences transposable element dysregulation in sex-based differences in Parkinson's disease.

Transposable elements (TEs) (mobile genetic elements comprising ∼45% of the human genome) have recently emerged as potential contributors to Parkinson's disease (PD); however their role and sex-specific impact remain poorly understood. Here, we present the first integrative meta-analysis of TE expression across 4 substantia nigra single-nucleus RNA-seq datasets, comprising a total of 66 donors, generating a cell-type-resolved atlas of TE dysregulation in PD. We identified widespread TE activation across major brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglia), with marked upregulation of L1s in neurons and HERVs in oligodendrocytes. Sex-stratified analyses revealed distinct male- and female-biased TE signatures, indicating regulatory programs uniquely affected in each sex, including MIR elements in microglia and Alu subfamilies in neurons. Correlation and genomic proximity analyses also uncovered TE-gene associations linked to important PD pathways such as neuroinflammation or myelination. Collectively, our study positions TEs as potential sex-modulated contributors to PD pathology and also provides a public web resource (PATOSS) to explore PD-associated TE transcriptional deregulation.

Parkinson's disease↗
PubMed2006

Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome.

In Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, is essential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved in gene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is known about the molecular mechanisms of Piwi-dependent gene silencing. Here we show that endogenous Piwi immunopurified from ovary specifically associates with small RNAs of 25-29 nucleotides in length. Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs (miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive targets.

Animals↗
PubMed2006

In vitro precursor microRNA processing assays using Drosophila Schneider-2 cell lysates.

Recent studies have shown that the microRNA (miRNA) pathway is an evolutionarily conserved endogenous pathway that is important for normal development. Mature miRNAs are excised from precursors in a stepwise process and subsequently incorporated into an RNA-induced silencing complex (RISC), which mediates either cleavage of the target messenger RNA (mRNA) or translational repression, depending on the complementarity between the miRNA and its target mRNA. In this chapter, we describe in vitro precursor (pre)-miRNA processing assays using Drosophila Schneider-2 (S2) cell lysates and immunopurified materials.

Animals↗
PubMed2005

Slicer function of Drosophila Argonautes and its involvement in RISC formation.

Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage ("Slicer") activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here we show that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. We show that upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as "Slicer" of the passenger strand of the siRNA duplex.

Adenosine Triphosphate↗
PubMed2005

Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells.

microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Here we show that Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs.

Amino Acid Sequence↗
PubMed2005

A potential link between transgene silencing and poly(A) tails.

Argonaute proteins function in gene silencing induced by double-stranded RNA (dsRNA) in various organisms. In Drosophila, the Argonaute proteins AGO1 and AGO2 have been implicated in post-transcriptional gene-silencing (PTGS)/RNA interference (RNAi). In this study, we found that AGO1 and AGO2 depletion caused the accumulation of multicopied enhanced green fluorescence protein (EGFP) transgene transcripts in Drosophila S2 cells. Depletion of AGO1, the essential factor for miRNA biogenesis, led to an increased transcriptional rate of the transgenes. In contrast, depletion of AGO2, the essential factor for siRNA-directed RNAi, resulted in EGFP mRNA stabilization with concomitant shortening of the EGFP mRNA poly(A) tail. Our findings suggest that AGO1 and AGO2 mediate multicopied transgene silencing by different mechanisms. Intriguingly, Dicer2 depletion phenocopies AGO2 depletion, with an increase in EGFP protein levels and shortening of the EGFP mRNA poly(A) tail. The possibility that AGO2 and Dicer2 involve, at least in part, poly(A) length maintenance of transgene mRNA suggests a potentially important link between transgene silencing and poly(A) tails.

Animals↗
PubMed2004

Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways.

In mammalian cells, both microRNAs (miRNAs) and small interfering RNAs (siRNAs) are thought to be loaded into the same RNA-induced silencing complex (RISC), where they guide mRNA degradation or translation silencing depending on the complementarity of the target. In Drosophila, Argonaute2 (AGO2) was identified as part of the RISC complex. Here we show that AGO2 is an essential component for siRNA-directed RNA interference (RNAi) response and is required for the unwinding of siRNA duplex and in consequence assembly of siRNA into RISC in Drosophila embryos. However, Drosophila embryos lacking AGO2, which are siRNA-directed RNAi-defective, are still capable of miRNA-directed target RNA cleavage. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, which is dispensable for siRNA-directed target RNA cleavage, is required for mature miRNA production that impacts on miRNA-directed RNA cleavage. The association of AGO1 with Dicer-1 and pre-miRNA also suggests that AGO1 is involved in miRNA biogenesis. Our findings show that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism and suggest that there are inherent differences between siRNA-initiated RISCs and miRNA-initiated RISCs in Drosophila.

Animals↗
PubMed2004

RNA interference: a new mechanism by which FMRP acts in the normal brain? What can Drosophila teach us?

Fragile X syndrome is the most common heritable form of mental retardation caused by loss-of-function mutations in the FMR1 gene. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. Recent work in Drosophila melanogaster has shown that the fly homolog of FMR1 (dFMR1) plays an important role in regulating neuronal morphology, which may underlie the observed deficits in behaviors of dFMR1 mutant flies. Biochemical analysis has revealed that dFMR1 forms a complex that includes ribosomal proteins and, surprisingly, Argonaute2 (AGO2), an essential component of the RNA-induced silencing complex (RISC) that mediates RNA interference (RNAi) in Drosophila. dFMR1 also associates with Dicer, another essential processing enzyme of the RNAi pathway. Moreover, both a micro-RNA (miRNA) and short interfering RNAs (siRNAs) can coimmunoprecipitate with dFMR1. Together these findings suggest that dFMR1 functions in an RNAi-related apparatus to regulate the expression of its target genes at the level of translation. These findings raise the possibility that Fragile X syndrome may be the result of a protein synthesis abnormality caused by a defect in an RNAi-related apparatus. Because the core mechanisms of complex behaviors such as learning and memory and circadian rhythms appear to be conserved, studies of Fragile X syndrome using Drosophila as a model provide an economy-of-scale for identifying biological processes that likely underlie the abnormal morphology of dendritic spines and behavioral disturbances observed in Fragile X patients.

Animals↗
PubMed2002

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins.

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.

Amino Acid Sequence↗
PubMed2002

A role for the Drosophila fragile X-related gene in circadian output.

Mutations that abolish expression of an X-linked gene, FMR1, result in the pathogenesis of fragile X syndrome, the most common form of inherited mental retardation. To understand the normal function of the FMR1 protein, we have produced fly strains bearing deletions in a Drosophila homolog of FMR1 (dfmr1). Since fragile X patients show a number of abnormal behaviors including sleep problems, we investigated whether a loss-of-function mutation of dfmr1 affect circadian behavior. Here we show that under constant darkness (DD), a lack of dfmr1 expression causes arrhythmic locomotor activity, but in light:dark cycles, their behavioral rhythms appear normal. In addition, the clock-controlled eclosion rhythm is normal in DFMR1-deficient flies. These results suggest that DFMR1 plays a critical role in the circadian output pathway regulating locomotor activity in Drosophila.

Animals↗
PubMed2002

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

Amino Acid Motifs↗
PubMed2002

Casein kinase II phosphorylates the fragile X mental retardation protein and modulates its biological properties.

Fragile X syndrome is caused by loss of FMR1 protein expression. FMR1 binds RNA and associates with polysomes in the cytoplasm; thus, it has been proposed to function as a regulator of gene expression at the posttranscriptional level. Posttranslational modification of FMR1 had previously been suggested to regulate its activity, but no experimental support for this model has been reported to date. Here we report that FMR1 in Drosophila melanogaster (dFMR1) is phosphorylated in vivo and that the homomer formation and the RNA-binding activities of dFMR1 are modulated by phosphorylation in vitro. Identification of a protein phosphorylating dFMR1 showed it to be Drosophila casein kinase II (dCKII). dCKII directly interacts with and phosphorylates dFMR1 in vitro. The phosphorylation site in dFMR1 was identified as Ser406, which is highly conserved among FMR1 family members from several species. Using mass spectrometry, we established that Ser406 of dFMR1 is indeed phosphorylated in vivo. Furthermore, human FMR1 (hFMR1) is also phosphorylated in vivo, and alteration of the conserved Ser500 in hFMR1 abolishes phosphorylation by CKII in vitro. These studies support the model that the biological functions of FMR1, such as regulation of gene expression, are likely regulated by its phosphorylation.

Amino Acid Sequence↗