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Results for “RNA-Induced Silencing Complex”

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At least 19 recordsLinked to original sources
PubMed2001

Argonaute2, a link between genetic and biochemical analyses of RNAi.

Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs ( approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. Here, we report the biochemical purification of the RNAi effector nuclease from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems.

Amino Acid Sequence↗
PubMed2001

Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila.

Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)"[1-4]; this process is called "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC)[16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.

5' Untranslated Regions↗
PubMed2026

LncRNA DNAJC3-AS1 promotes gastric cancer malignancy through miR-576-5p-mediated upregulation of LYPLA1.

Long non-coding RNAs (lncRNAs) are involved in tumor progression, but the role of lnc-DNAJC3-AS1 in gastric cancer (GC) remains unclear. This study aimed to investigate the biological function and regulatory mechanism of lnc-DNAJC3-AS1 in GC. Reverse transcription quantitative PCR (RT-qPCR) was used to detect the expression levels of lnc-DNAJC3-AS1, miR-576-5p, and LYPLA1, and Western blot was used to analyze protein expression. The Cancer Genome Atlas (TCGA) database and clinical samples were used to evaluate their clinical relevance. Cell viability, cell cycle distribution, apoptosis, migration, and invasion were assessed using Cell Counting Kit-8 (CCK-8), flow cytometry, wound-healing, Transwell, and immunofluorescence assays. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP), fluorescence in situ hybridization (FISH), and rescue assays were performed to explore the potential regulatory relationship among lnc-DNAJC3-AS1, miR-576-5p, and LYPLA1. A xenograft tumor model was also established to evaluate the role of lnc-DNAJC3-AS1 in vivo. The results showed that lnc-DNAJC3-AS1 and LYPLA1 were upregulated, whereas miR-576-5p was downregulated in GC tissues and cells. Knockdown of lnc-DNAJC3-AS1 inhibited GC cell viability, migration, and invasion, induced G0/G1 phase arrest and apoptosis, and suppressed tumor growth in vivo. Mechanistically, lnc-DNAJC3-AS1 was mainly localized in the cytoplasm and was associated with miR-576-5p-related RNA-induced silencing complex (RISC) complexes. MiR-576-5p targeted LYPLA1, and restoration of miR-576-5p or knockdown of LYPLA1 partially attenuated the effects of lnc-DNAJC3-AS1 overexpression on GC cell phenotypes and LYPLA1 enzymatic activity. These findings suggest that lnc-DNAJC3-AS1 promotes GC progression, at least in part, through the miR-576-5p/LYPLA1 pathway, providing a potential target for GC treatment.

Gastric cancer↗
PubMed2001

Two mouse piwi-related genes: miwi and mili.

Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. We cloned mouse homologues of piwi by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologues MIWI and MILI showed that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili were found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. Our data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production.

Amino Acid Sequence↗
PubMed2001

Stem cells: so what's in a niche?

Much effort is being invested in defining the intrinsic and extrinsic factors that control stem cell maintenance and proliferation. Recent studies have identified a signaling hierarchy involved in coordinating the proliferation of germ line and somatic stem cells in the Drosophila ovary.

Animals↗
PubMed2001

RNA interference: it's a small RNA world.

Short RNAs regulate gene expression in many species. Some are generated from any double-stranded RNA and degrade complementary RNAs; others are encoded by genes and repress specific mRNAs. Both, it turns out, are processed and handled by similar proteins. These pathways offer a glimpse into a world of small RNAs.

Animals↗
PubMed1999

Stem cells: A tale of two kingdoms.

Homologous genes have recently been shown to regulate stem cell maintenance in animals and plants. This discovery should facilitate elucidation of the poorly understood factors that control stem cell maintenance and differentiation.

Animals↗
PubMed2000

A global profile of germline gene expression in C. elegans.

We used DNA microarrays to profile gene expression patterns in the C. elegans germline and identified 1416 germline-enriched transcripts that define three groups. The sperm-enriched group contains an unusually large number of protein kinases and phosphatases. The oocyte-enriched group includes potentially new components of embryonic signaling pathways. The germline-intrinsic group, defined as genes expressed similarly in germlines making only sperm or only oocytes, contains a family of piwi-related genes that may be important for stem cell proliferation. Finally, examination of the chromosomal location of germline transcripts revealed that sperm-enriched and germline-intrinsic genes are nearly absent from the X chromosome, but oocyte-enriched genes are not.

Animals↗
PubMed2001

Yb modulates the divisions of both germline and somatic stem cells through piwi- and hh-mediated mechanisms in the Drosophila ovary.

The coordinated division of distinctive types of stem cells within an organ is crucial for organogenesis and homeostasis. Here we show genetic interactions among fs(1)Yb (Yb), piwi, and hedgehog (hh) that regulate the division of both germline stem cells (GSCs) and somatic stem cells (SSCs), the two constituent stem cell populations of the Drosophila ovary. Yb is required for both GSC and SSC divisions; loss of Yb function eliminates GSCs and reduces SSC division, while Yb overexpression increases GSC number and causes SSC overproliferation. We also show that Yb acts via the piwi- and hh-mediated signaling pathways that emanate from the same signaling cells to control GSC and SSC division, respectively. hh signaling also has a minor effect in GSC division.

Animals↗
PubMed2002

RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila.

Two types of transgene silencing were found for the Alcohol dehydrogenase (Adh) transcription unit. Transcriptional gene silencing (TGS) is Polycomb dependent and occurs when Adh is driven by the white eye color gene promoter. Full-length Adh transgenes are silenced posttranscriptionally at high copy number or by a pulsed increase over a threshold. The posttranscriptional gene silencing (PTGS) exhibits molecular hallmarks typical of RNA interference (RNAi), including the production of 21--25 bp length sense and antisense RNAs homologous to the silenced RNA. Mutations in piwi, which belongs to a gene family with members required for RNAi, block PTGS and one aspect of TGS, indicating a connection between the two types of silencing.

ATP-Binding Cassette Transporters↗
PubMed2002

Molecular characterization of hiwi, a human member of the piwi gene family whose overexpression is correlated to seminomas.

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Drosophila melanogaster and C. elegans to Arabidopsis. Here we report the molecular characterization of hiwi, a human member of the piwi gene family. hiwi maps to the long arm of chromosome 12, band 12q24.33, a genomic region that displays genetic linkage to the development of testicular germ cell tumors of adolescents and adults (TGCTs), i.e., seminomas and nonseminomas. In addition, gain of this chromosomal region has been found in some TGCTs. hiwi encodes a 3.6 kb mRNA that is expressed abundantly in the adult testis. It encodes a highly basic 861-amino-acid protein that shares significant homology throughout its entire length with other members of the PIWI family proteins in Drosophila, C. elegans and mammals. In normal human testes, hiwi is specifically expressed in germline cells, with its expression detectable in spermatocytes and round spermatids during spermatogenesis. No expresssion was observed in testicular tumors of somatic origin, such as Sertoli cell and Leydig cell tumors. Enhanced expression was found in 12 out of 19 sampled testicular seminomas-tumors originating from embryonic germ cells with retention of germ cell phenotype. In contrast, no enhanced expression was detected in 10 nonseminomas-testicular tumors that originate from the same precursor cells as seminomas yet have lost their germ cell characteristics. Finally, no enhanced expression was detected in four spermatocytic seminomas-testicular tumors that most likely originate from germ cells capable of partial meiosis. Thus, hiwi is specifically expressed in both normal and malignant spermatogenic cells in a maturation stage-dependent pattern, in which it might function in germ cell proliferation.

Amino Acid Sequence↗
PubMed2002

ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos.

Double-stranded RNA (dsRNA) triggers homology-dependent posttranscriptional gene interference (RNAi) in a diverse range of eukaryotic organisms, in a process mechanistically related to viral and transgene-mediated cosuppression. RNAi is characterized by the conversion of long dsRNA into approximately 21-25-nt small interfering RNAs (siRNA) that guide the degradation of homologous mRNA. Many of the genes required for siRNA production and target mRNA degradation are widely conserved. Notably, members of the Argonaute-like gene family from Arabidopsis, Caenorhabditis elegans, Drosophila, and Neurospora have been genetically and/or biochemically identified as components of the RNAi/cosuppression pathway. We show here that mutations in the Drosophila Argonaute1 (AGO1) gene suppress RNAi in embryos. This defect corresponds to a reduced ability to degrade mRNA in response to dsRNA in vitro. Furthermore, AGO1 is not required for siRNA production in vitro nor can the introduction of siRNA bypass AGO1 mutants in vivo. These data suggest that AGO1 functions downstream of siRNA production.

ATP-Binding Cassette Transporters↗
PubMed1998

A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal.

Germ-line stem cells (GSCs) serve as the source for gametogenesis in diverse organisms. We cloned and characterized the Drosophila piwi gene and showed that it is required for the asymmetric division of GSCs to produce and maintain a daughter GSC but is not essential for the further differentiation of the committed daughter cell. Genetic mosaic and RNA in situ analyses suggest that piwi expression in adjacent somatic cells regulates GSC division. piwi encodes a highly basic novel protein, well conserved during evolution. We isolated piwi homologs in Caenorhabditis elegans and humans and also identified Arabidopsis piwi-like genes known to be required for meristem cell maintenance. Decreasing C. elegans piwi expression reduces the proliferation of GSC-equivalent cells. Thus, piwi represents a novel class of genes required for GSC division in diverse organisms.

Amino Acid Sequence↗
PubMed2002

hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing.

The endogenous Delta 12-desaturase gene (FAD2) in Arabidopsis was targeted for silencing using seed-specific cosuppression (CS), hairpin (HP) RNA (hpRNA), and intron-spliced HP (iHP) constructs. The iHP construct, incorporating the 120-bp 3'-untranslated region of the FAD2 gene, gave the highest degree of silencing. In some iHP lines Delta 12-desaturase activity was reduced to levels as low as those in the null fad2-1 mutant, and every primary transformant showed a pronounced reduction in FAD2 activity. One highly silenced iHP line was propagated for five generations and showed no reversion or diminution in its degree of silencing. About 75% of plants transformed with the HP construct, targeting the FAD2 coding region, gave dramatically reduced Delta 12-desaturase activity, whereas approximately 50% of plants transformed with the CS construct, containing the same coding region sequence, showed silencing at a much less profound level. In all three types of constructs, the degree of silencing was increased when the transgenes were homozygous, but this was much more pronounced for the CS constructs. All three types of construct could give a single locus that was capable of effective silencing, but in the one such CS line where this was the case, the locus had a complex insertion pattern. This is consistent with the concept that posttranscriptional gene silencing is induced by double-stranded, or self-complementary, RNA that is formed in cases of CS by complex insertion patterns at a single locus and that the most effective way of generating profoundly silenced plants is by the use of constructs that encode hpRNAs. Furthermore, these results demonstrate for the first time, to our knowledge, that iHP constructs targeted against an endogenous seed-expressed gene are clearly able to generate phenotypic changes that are inherited stably over several generations, making this approach a reliable technique for genetic modification of seed quality and possibly other traits in agricultural plants.

Arabidopsis↗
PubMed2002

Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase.

RNA silencing is a sequence-specific RNA degradation process that follows the recognition of double-stranded RNA. Here, we show that virus vectors carrying parts of a green fluorescent protein (GFP) transgene targeted RNA silencing in Nicotiana benthamiana and Arabidopsis against the entire GFP RNA. These results indicate that there was spreading of RNA targeting from the initiator region into the adjacent 5' and 3' regions of the target gene. Spreading was accompanied by methylation of the corresponding GFP DNA. It also was dependent on transcription of the transgene and on the putative RNA-dependent RNA polymerase, SDE1/SGS2. These findings indicate that SDE1/SGS2 produces double-stranded RNA using the target RNA as a template. RNA silencing of ribulose-1,5-bisphosphate carboxylase/oxygenase and phytoene desaturase was not associated with the spreading of RNA targeting or DNA methylation, indicating that these endogenous RNAs are not templates for SDE1/SGS2.

Arabidopsis↗