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Cell Apr 2021
Topics: Argonaute Proteins; Humans; Infertility, Male; Male
PubMed: 33861958
DOI: 10.1016/j.cell.2021.03.003 -
Molecular Cell Aug 2017In miRNA-mediated gene silencing, the physical interaction between human Argonaute (hAgo) and GW182 (hGW182) is essential for facilitating the downstream silencing of...
In miRNA-mediated gene silencing, the physical interaction between human Argonaute (hAgo) and GW182 (hGW182) is essential for facilitating the downstream silencing of the targeted mRNA. GW182 can interact with hAgo via three of the GW/WG repeats in its Argonaute-binding domain: motif-1, motif-2, and the hook motif. The structure of hAgo1 in complex with the hook motif of hGW182 reveals a "gate"-like interaction that is critical for GW182 docking into one of hAgo1's tryptophan-binding pockets. We show that hAgo1 and hAgo2 have a single GW182-binding site and that miRNA binding increases hAgo's affinity to GW182. With target binding occurring rapidly, this ensures that only mature RISC would be recruited for silencing. Finally, we show that hGW182 can recruit up to three copies of hAgo via its three GW motifs. This may explain the observed cooperativity in miRNA-mediated gene silencing.
Topics: Animals; Argonaute Proteins; Autoantigens; Binding Sites; Binding, Competitive; Crystallography, X-Ray; Eukaryotic Initiation Factors; Gene Silencing; Humans; MicroRNAs; Molecular Docking Simulation; Mutation; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; RNA-Binding Proteins; Sf9 Cells; Structure-Activity Relationship; Transfection; RNA, Guide, CRISPR-Cas Systems
PubMed: 28781232
DOI: 10.1016/j.molcel.2017.07.007 -
Current Biology : CB Sep 2017To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary...
To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells [1]. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors [2-12] but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. Our data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. Our study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing [13, 14] and Drosophila PAF1 opposing PIWI/piRNA-directed silencing.
Topics: Animals; Argonaute Proteins; Drosophila Proteins; Drosophila melanogaster; Female; Gene Silencing; Ovary; RNA, Small Interfering
PubMed: 28844648
DOI: 10.1016/j.cub.2017.07.052 -
Cell Reports Mar 2023The clearance of untranslated mRNAs by Argonaute proteins is essential for embryonic development in metazoans. However, it is currently unknown whether similar processes...
The clearance of untranslated mRNAs by Argonaute proteins is essential for embryonic development in metazoans. However, it is currently unknown whether similar processes exist in unicellular eukaryotes. The ciliate Paramecium tetraurelia harbors a vast array of PIWI-clade Argonautes involved in various small RNA (sRNA) pathways, many of which have not yet been investigated. Here, we investigate the function of a PIWI protein, Ptiwi08, whose expression is limited to a narrow time window during development, concomitant with the start of zygotic transcription. We show that Ptiwi08 acts in an endogenous small interfering RNA (endo-siRNA) pathway involved in the clearance of untranslated mRNAs. These endo-siRNAs are found in clusters that are strictly antisense to their target mRNAs and are a subset of siRNA-producing clusters (SRCs). Furthermore, the endo-siRNAs are 2'-O-methylated by Hen1 and require Dcr1 for their biogenesis. Our findings suggest that sRNA-mediated developmental mRNA clearance extends beyond metazoans and may be a more widespread mechanism than previously anticipated.
Topics: RNA, Small Interfering; RNA Interference; Paramecium; RNA, Messenger; RNA, Double-Stranded; Argonaute Proteins
PubMed: 36870062
DOI: 10.1016/j.celrep.2023.112213 -
Nature Communications Mar 2024Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite...
Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the 'upstream sequence transcription complex' (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.
Topics: Animals; Piwi-Interacting RNA; RNA, Small Interfering; Casein Kinase II; Phosphorylation; Soy Foods; Argonaute Proteins; Drosophila Proteins; Drosophila melanogaster
PubMed: 38548791
DOI: 10.1038/s41467-024-46882-9 -
ELife Oct 2021microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although...
microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. We previously showed that repressing the production of one Argonaute protein, Ago2, by Trim71 is important for mouse embryonic stem cells (mESCs) self-renewal (Liu et al., 2021). Here, we show that among the four Argonaute proteins in mammals, Ago2 is the major developmentally regulated Argonaute protein in mESCs. Moreover, in pluripotency, besides the Trim71-mediated regulation of (Liu et al., 2021), also repress . Specific inhibition of this microRNA-mediated repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.
Topics: Animals; Argonaute Proteins; Cell Differentiation; Cell Line; Cell Lineage; Cell Proliferation; Cell Self Renewal; Gene Expression Regulation, Developmental; Mice; MicroRNAs; Mouse Embryonic Stem Cells; Transcription Factors
PubMed: 34596044
DOI: 10.7554/eLife.72289 -
PLoS Genetics Nov 2023PIWI proteins and their associated piRNAs act to silence transposons and promote gametogenesis. Murine PIWI proteins MIWI, MILI, and MIWI2 have multiple arginine and...
PIWI proteins and their associated piRNAs act to silence transposons and promote gametogenesis. Murine PIWI proteins MIWI, MILI, and MIWI2 have multiple arginine and glycine (RG)-rich motifs at their N-terminal domains. Despite being known as docking sites for the TDRD family proteins, the in vivo regulatory roles for these RG motifs in directing PIWI in piRNA biogenesis and spermatogenesis remain elusive. To investigate the functional significance of RG motifs in mammalian PIWI proteins in vivo, we genetically engineered an arginine to lysine (RK) point mutation of a conserved N-terminal RG motif in MIWI in mice. We show that this tiny MIWI RG motif is indispensable for piRNA biogenesis and male fertility. The RK mutation in the RG motif disrupts MIWI-TDRKH interaction and impairs enrichment of MIWI to the intermitochondrial cement (IMC) for efficient piRNA production. Despite significant overall piRNA level reduction, piRNA trimming and maturation are not affected by the RK mutation. Consequently, MiwiRK mutant mice show chromatoid body malformation, spermatogenic arrest, and male sterility. Surprisingly, LINE1 transposons are effectively silenced in MiwiRK mutant mice, indicating a LINE1-independent cause of germ cell arrest distinctive from Miwi knockout mice. These findings reveal a crucial function of the RG motif in directing PIWI proteins to engage in efficient piRNA production critical for germ cell progression and highlight the functional importance of the PIWI N-terminal motifs in regulating male fertility.
Topics: Male; Mice; Animals; Piwi-Interacting RNA; Testis; RNA, Small Interfering; Spermatogenesis; Proteins; Mice, Knockout; Arginine; Argonaute Proteins; Mammals
PubMed: 37956204
DOI: 10.1371/journal.pgen.1011031 -
Molecules and Cells Aug 2016Post-translational modifications (PTMs) of proteins are essential to increase the functional diversity of the proteome. By adding chemical groups to proteins, or... (Review)
Review
Post-translational modifications (PTMs) of proteins are essential to increase the functional diversity of the proteome. By adding chemical groups to proteins, or degrading entire proteins by phosphorylation, glycosylation, ubiquitination, neddylation, acetylation, lipidation, and proteolysis, the complexity of the proteome increases, and this then influences most biological processes. Although small RNAs are crucial regulatory elements for gene expression in most eukaryotes, PTMs of small RNA microprocessor and RNA silencing components have not been extensively investigated in plants. To date, several studies have shown that the proteolytic regulation of AGOs is important for host-pathogen interactions. DRB4 is regulated by the ubiquitin-proteasome system, and the degradation of HYL1 is modulated by a de-etiolation repressor, COP1, and an unknown cytoplasmic protease. Here, we discuss current findings on the PTMs of microprocessor and RNA silencing components in plants.
Topics: Arabidopsis Proteins; Argonaute Proteins; Gene Expression Regulation, Plant; Host-Pathogen Interactions; MicroRNAs; Plants; Protein Processing, Post-Translational; Proteolysis; RNA Interference; RNA-Binding Proteins; Ubiquitin
PubMed: 27440184
DOI: 10.14348/molcells.2016.0085 -
Nature Feb 2017MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary...
MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
Topics: Amino Acid Sequence; Argonaute Proteins; CRISPR-Cas Systems; Casein Kinase II; Gene Silencing; HCT116 Cells; Humans; MicroRNAs; Phosphoprotein Phosphatases; Phosphorylation; RNA, Messenger; Substrate Specificity
PubMed: 28114302
DOI: 10.1038/nature21025 -
PloS One 2016Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins,...
Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins, among them human Argonaute2 (hAgo2) and Ago from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an Ago-associated complementary guide strand. Here we present an in-depth kinetic side-by-side analysis of hAgo2 and MjAgo guide and target substrate binding as well as target strand cleavage, which enabled us to disclose similarities and differences in the mechanistic pathways as a function of the chemical nature of the substrate. Testing all possible guide-target combinations (i.e. RNA/RNA, RNA/DNA, DNA/RNA and DNA/DNA) with both Ago variants we demonstrate that the molecular mechanism of substrate association is highly conserved among archaeal-eukaryotic Argonautes. Furthermore, we show that hAgo2 binds RNA and DNA guide strands in the same fashion. On the other hand, despite striking homology between the two Ago variants, MjAgo cannot orientate guide RNA substrates in a way that allows interaction with the target DNA in a cleavage-compatible orientation.
Topics: Argonaute Proteins; DNA; DNA Cleavage; Humans; Kinetics; Methanocaldococcus; Oligonucleotides; Protein Binding; Substrate Specificity; RNA, Guide, CRISPR-Cas Systems
PubMed: 27741323
DOI: 10.1371/journal.pone.0164695