-
Nucleic Acids Research Sep 2023Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have...
Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have revolutionized our ability to engineer genomes across diverse eukaryotic species. However, key challenges, including delivery, specificity and targeting organellar genomes, pose barriers to translational applications. Here, we use peptide nucleic acids (PNAs) to facilitate precise DNA strand invasion and unwinding, enabling prokaryotic Argonaute (pAgo) proteins to specifically bind displaced single-stranded DNA and introduce site-specific double-strand breaks (DSBs) independent of the target sequence. We named this technology PNA-assisted pAgo editing (PNP editing) and determined key parameters for designing PNP editors to efficiently generate programable site-specific DSBs. Our design allows the simultaneous use of multiple PNP editors to generate multiple site-specific DSBs, thereby informing design considerations for potential in vitro and in vivo applications, including genome editing.
Topics: CRISPR-Cas Systems; DNA; DNA Breaks, Double-Stranded; Gene Editing; Genome; Peptide Nucleic Acids; Argonaute Proteins
PubMed: 37560931
DOI: 10.1093/nar/gkad655 -
Cold Spring Harbor Perspectives in... Mar 2019MicroRNAs (miRNAs) posttranscriptionally regulate gene expression by repressing protein synthesis and exert a broad influence over development, physiology, adaptation,... (Review)
Review
MicroRNAs (miRNAs) posttranscriptionally regulate gene expression by repressing protein synthesis and exert a broad influence over development, physiology, adaptation, and disease. Over the past two decades, great strides have been made toward elucidating how miRNAs go about shutting down messenger RNA (mRNA) translation and promoting mRNA decay.
Topics: 3' Untranslated Regions; Animals; Argonaute Proteins; Gene Regulatory Networks; Gene Silencing; Humans; MicroRNAs; Protein Domains
PubMed: 29959194
DOI: 10.1101/cshperspect.a032771 -
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 siteĀ 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 -
Plant Reproduction Jun 2022Meiosis is a specialized cell division that is key for reproduction and genetic diversity in sexually reproducing plants. Recently, different RNA silencing pathways have...
Meiosis is a specialized cell division that is key for reproduction and genetic diversity in sexually reproducing plants. Recently, different RNA silencing pathways have been proposed to carry a specific activity during meiosis, but the pathways involved during this process remain unclear. Here, we explored the subcellular localization of different ARGONAUTE (AGO) proteins, the main effectors of RNA silencing, during male meiosis in Arabidopsis thaliana using immunolocalizations with commercially available antibodies. We detected the presence of AGO proteins associated with posttranscriptional gene silencing (AGO1, 2, and 5) in the cytoplasm and the nucleus, while AGOs associated with transcriptional gene silencing (AGO4 and 9) localized exclusively in the nucleus. These results indicate that the localization of different AGOs correlates with their predicted roles at the transcriptional and posttranscriptional levels and provide an overview of their timing and potential role during meiosis.
Topics: Arabidopsis; Arabidopsis Proteins; Argonaute Proteins; Meiosis
PubMed: 34812935
DOI: 10.1007/s00497-021-00434-z -
Nature Communications Jun 2023Argonaute protein (AGO) in association with small RNAs is the core machinery of RNA silencing, an essential mechanism for precise development and defense against...
Argonaute protein (AGO) in association with small RNAs is the core machinery of RNA silencing, an essential mechanism for precise development and defense against pathogens in many organisms. Here, we identified two AGOs in rice anthers, AGO1b and AGO1d, that interact with phased small interfering RNAs (phasiRNAs) derived from numerous long non-coding RNAs. Moreover, 3D-immunoimaging and mutant analysis indicated that rice AGO1b and AGO1d cell type-specifically regulate anther development by acting as mobile carriers of these phasiRNAs from the somatic cell layers to the germ cells in anthers. Our study also highlights a new mode of reproductive RNA silencing via the specific nuclear and cytoplasmic localization of three AGOs, AGO1b, AGO1d, and MEL1, in rice pollen mother cells.
Topics: Argonaute Proteins; RNA, Plant; Gene Expression Regulation, Plant; RNA, Small Interfering; Oryza
PubMed: 37286636
DOI: 10.1038/s41467-023-38881-z -
Methods in Molecular Biology (Clifton,... 2021RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing...
RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.
Topics: Arabidopsis; Arabidopsis Proteins; Argonaute Proteins; Gene Expression Regulation, Plant; High-Throughput Nucleotide Sequencing; RNA, Plant; RNA, Small Untranslated
PubMed: 33175381
DOI: 10.1007/978-1-0716-0880-7_11 -
Acta Biochimica Et Biophysica Sinica May 2015It is generally accepted that PIWI proteins are predominately expressed in the germline but absent in somatic tissues. Their best-characterized role is to suppress... (Review)
Review
It is generally accepted that PIWI proteins are predominately expressed in the germline but absent in somatic tissues. Their best-characterized role is to suppress transposon expression, which ensures genomic stability in the germline. However, increasing evidence has suggested that PIWI proteins are linked to the hallmarks of cancer defined by Weinberg and Hanahan, such as cell proliferation, anti-apoptosis, genomic instability, invasion and metastasis. This provides new possibilities for anticancer therapies through the targeting of PIWI proteins, which may have fewer side effects due to their potential classification as a CTA (cancer/testis antigen). Furthermore, PIWI has been proposed to act as a diagnostic and prognostic marker for many types of cancer, and even to differentiate early- and late-stage cancers. We herein summarize the latest progress in this exciting field, hoping to encourage new investigations of PIWIs in cancer biology that will help to develop new therapeutics for clinical application.
Topics: Argonaute Proteins; Carcinogenesis; Disease Progression; Humans; Neoplasms
PubMed: 25854579
DOI: 10.1093/abbs/gmv018 -
Aging Nov 2019PiRNAs are a small class of non-coding small RNAs newly discovered in recent years. Millions of piRNAs have been discovered to date, and more than 20,000 piRNA genes... (Review)
Review
PiRNAs are a small class of non-coding small RNAs newly discovered in recent years. Millions of piRNAs have been discovered to date, and more than 20,000 piRNA genes have been found in the human genome. Due to the relatively small number of studies related to piRNA, our understanding of piRNAs is very limited. Currently, the clear biological function of piRNAs is transposon mobilization inhibition by promoting transcript degradation and regulating chromatin formation. In addition, piRNAs can form piRNA-PIWI protein complexes with some members of the PIWI branch of the Argonaute protein. Based on these biological functions, piRNAs and PIWI proteins are important in maintaining the genomic integrity of germline cells. Because of this, the popularity of piRNAs research has been focused on its role in germline cells for a long time after the discovery of piRNAs. As the field of research expands, there is growing evidence that piRNAs and PIWI proteins are abnormally expressed in various types of cancers, which may be potential cancer biomarkers and cancer therapeutic targets. In this review, we will focus on the relationship between piRNAs and PIWI proteins and cancers based on previous research, as well as their significance in cancer detection, grading and treatment.
Topics: Animals; Argonaute Proteins; DNA Transposable Elements; Gene Amplification; Humans; Neoplasms; RNA Interference; RNA, Small Interfering
PubMed: 31727866
DOI: 10.18632/aging.102417 -
Nucleic Acid Therapeutics Feb 2012Small RNAs are a commonly used tool for gene silencing and a promising platform for nucleic acid drug development. They are almost exclusively used to silence gene... (Review)
Review
Small RNAs are a commonly used tool for gene silencing and a promising platform for nucleic acid drug development. They are almost exclusively used to silence gene expression post-transcriptionally through degradation of mRNA. Small RNAs, however, can have a broader range of function by binding to Argonaute proteins and associating with complementary RNA targets in the nucleus, including long noncoding RNAs (lncRNAs) and pre-mRNA. Argonaute-RNA complexes can regulate nuclear events like transcription, genome maintenance, and splicing. Thousands of lncRNAs and alternatively spliced pre-mRNA isoforms exist in humans, and these RNAs may serve as natural targets for regulation and therapeutic intervention. This review describes nuclear mechanisms for Argonaute proteins and small RNAs, new pathways for sequence-specific targeting, and the potential for therapeutic development of small RNAs with nuclear targets.
Topics: Animals; Argonaute Proteins; Gene Expression Regulation; Humans; RNA Interference; RNA, Nuclear
PubMed: 22283730
DOI: 10.1089/nat.2011.0330 -
Frontiers in Endocrinology 2022The piRNA (PIWI-interacting RNA) is P-Element induced wimpy testis (PIWI)-interacting RNA which is a small molecule, non-coding RNA with a length of 24-32nt. It was... (Review)
Review
The piRNA (PIWI-interacting RNA) is P-Element induced wimpy testis (PIWI)-interacting RNA which is a small molecule, non-coding RNA with a length of 24-32nt. It was originally found in germ cells and is considered a regulator of germ cell function. It can interact with PIWI protein, a member of the Argonaute family, and play a role in the regulation of gene transcription and epigenetic silencing of transposable factors in the nucleus. More and more studies have shown that piRNAs are abnormally expressed in a variety of cancer tissues and patient fluids, and may become diagnostic tools, therapeutic targets, staging markers, and prognostic evaluation tools for cancer. This article reviews the recent research on piRNA and summarizes the structural characteristics, production mechanism, applications, and its role in urological tumors, to provide a reference value for piRNA to regulate urological tumors.
Topics: Humans; Argonaute Proteins; Epigenesis, Genetic; Piwi-Interacting RNA; RNA, Small Interfering; Urologic Neoplasms
PubMed: 36733811
DOI: 10.3389/fendo.2022.1054216