Argonaute

The Argonaute protein family, first discovered for its evolutionarily conserved stem cell function, ^[1] plays a central role in RNA silencing processes as essential components of the RNA-induced silencing complex (RISC). RISC is responsible for the gene silencing phenomenon known as RNA interference (RNAi). ^[2] Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). Small RNAs guide Argonaute proteins to their specific targets through sequence complementarity (base pairing), which then leads to mRNA cleavage, translation inhibition, and/or the initiation of mRNA decay. ^[3]

Contents

• RNA interference
• Functional domains and mechanism
• Family members
• Disease and therapeutic tools
• Biotechnological applications of prokaryotic Argonaute proteins
• References
• External links

The name of this protein family is derived from a mutant phenotype resulting from mutation of AGO1 in Arabidopsis thaliana , which was likened by Bohmert et al. to the appearance of the pelagic octopus Argonauta argo . ^[4]

Argonaute Piwi domain
An argonaute protein from Pyrococcus furiosus. PDB 1U04 . PIWI domain is on the right, PAZ domain to the left.
Identifiers
Symbol	Piwi
Pfam	PF02171
InterPro	IPR003165
PROSITE	PS50822
CDD	cd02826
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

RNA interference

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, via either destruction of specific mRNA molecules or suppressing translation. ^[5] RNAi has a significant role in defending cells against parasitic nucleotide sequences ^{[ citation needed ]}. In eukaryotes, including animals, RNAi is initiated by the enzyme Dicer. Dicer cleaves long double-stranded RNA (dsRNA, often found in viruses and small interfering RNA) molecules into short double stranded fragments of around 20 nucleotide siRNAs. The dsRNA is then separated into two single-stranded RNAs (ssRNA) – the passenger strand and the guide strand. Subsequently, the passenger strand is degraded, while the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome of the RNAi is post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute, that lies in the core of RNA-induced silencing complex.

Argonaute proteins are the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. ^[6] Theoretically the dicer produces short double-stranded fragments so there should be also two functional single-stranded siRNA produced. But only one of the two single-stranded RNA here will be utilized to base pair with target mRNA. It is known as the guide strand, which is incorporated into the Argonaute protein and leads gene silencing. The other single-stranded RNA, named the passenger strand, is degraded during the RNA-induced silencing complex process. ^[7]

Once the Argonaute is associated with the small RNA, the enzymatic activity conferred by the PIWI domain cleaves only the passenger strand of the small interfering RNA. RNA strand separation and incorporation into the Argonaute protein are guided by the strength of the hydrogen bond interaction at the 5′-ends of the RNA duplex, known as the asymmetry rule. Also the degree of complementarity between the two strands of the intermediate RNA duplex defines how the miRNA are sorted into different types of Argonaute proteins.

In animals, Argonaute associated with miRNA binds to the 3′-untranslated region of mRNA and prevents the production of proteins in various ways. The recruitment of Argonaute proteins to targeted mRNA can induce mRNA degradation. The Argonaute-miRNA complex can also affect the formation of functional ribosomes at the 5′-end of the mRNA. The complex here competes with the translation initiation factors and/or abrogate ribosome assembly. Also, the Argonaute-miRNA complex can adjust protein production by recruiting cellular factors such as peptides or post translational modifying enzymes, which degrade the growing of polypeptides. ^[8]

In plants, once de novo double-stranded (ds) RNA duplexes are generated with the target mRNA, an unknown RNase-III-like enzyme produces new siRNAs, which are then loaded onto the Argonaute proteins containing PIWI domains, lacking the catalytic amino acid residues, which might induce another level of specific gene silencing.

Functional domains and mechanism

The Argonaute (AGO) gene family encodes six characteristic domains: N- terminal (N), Linker-1 (L1), PAZ, Linker-2 (L2), Mid, and a C-terminal PIWI domain. ^[8]

The PAZ domain is named for Drosophila Piwi, Arabidopsis Argonaute-1, and Arabidopsis Zwille (also known as pinhead, and later renamed argonaute-10), where the domain was first recognized to be conserved. The PAZ domain is an RNA binding module that recognizes single-stranded 3′ ends of siRNA, miRNA and piRNA, in a sequence independent manner.

PIWI is named after the Drosophila Piwi protein. Structurally resembling RNaseH, the PIWI domain is essential for the target cleavage. The active site with aspartate–aspartate–glutamate triad harbors a divalent metal ion, necessary for the catalysis. Family members of AGO that lost this conserved feature during evolution lack the cleavage activity. In human AGO, the PIWI motif also mediates protein-protein interaction at the PIWI box, where it binds to Dicer at an RNase III domain. ^[9]

At the interface of PIWI and Mid domains sits the 5′ phosphate of a siRNA, miRNA or piRNA, which is found essential in the functionality. Within Mid lies a MC motif, a homologue structure proposed to mimic the cap-binding structure motif found in eIF4E. It was later found that the MC motif is not involved in mRNA cap binding ^[8]

Family members

In humans, there are eight AGO family members, some of which are investigated intensively. However, even though AGO1–4 are capable of loading miRNA, endonuclease activity and thus RNAi-dependent gene silencing exclusively belongs to AGO2. Considering the sequence conservation of PAZ and PIWI domains across the family, the uniqueness of AGO2 is presumed to arise from either the N-terminus or the spacing region linking PAZ and PIWI motifs. ^[9]

Several AGO family members in plants also attract study. AGO1 is involved in miRNA related RNA degradation, and plays a central role in morphogenesis. In some organisms, it is strictly required for epigenetic silencing. It is regulated by miRNA itself. AGO4 does not involve in RNAi directed RNA degradation, but in DNA methylation and other epigenetic regulation, through small RNA (smRNA) pathway. AGO10 is involved in plant development. AGO7 has a function distinct from AGO 1 and 10, and is not found in gene silencing induced by transgenes. Instead, it is related to developmental timing in plants. ^[10]

Disease and therapeutic tools

Argonaute proteins were reported to be associated with cancers. ^{[11] [12]} For the diseases that are involved with selective or elevated expression of particular identified genes, such as pancreatic cancer, the high sequence specificity of RNA interference might make it suitable to be a suitable treatment, particularly appropriate for combating cancers associated with mutated endogenous gene sequences. It has been reported several tiny non-coding RNAs(microRNAs) are related with human cancers, like miR-15a and miR-16a are frequently deleted and/or down-regulated in patients. Even though the biological functions of miRNAs are not fully understood, the roles for miRNAs in the coordination of cell proliferation and cell death during development and metabolism have been uncovered. It is trusted that the miRNAs can direct negative or positive regulation at different levels, which depends on the specific miRNAs and target base pair interaction and the cofactors that recognize them. ^[13]

Because it has been widely known that many viruses have RNA rather than DNA as their genetic material and go through at least one stage in their life cycle when they make double-stranded RNA, RNA interference has been considered to be a potentially evolutionarily ancient mechanism for protecting organisms from viruses. The small interfering RNAs produced by Dicer cause sequence specific, post-transcriptional gene silencing by guiding an endonuclease, the RNA-induced silencing complex (RISC), to mRNA. This process has been seen in a wide range of organisms, such as Neurospora fungus (in which it is known as quelling), plants (post-transcriptional gene silencing) and mammalian cells(RNAi). If there is a complete or near complete sequence complementarity between the small RNA and the target, the Argonaute protein component of RISC mediates cleavage of the target transcript, the mechanism involves repression of translation predominantly^{[ citation needed ]}.

Biotechnological applications of prokaryotic Argonaute proteins

In 2016, a group from Hebei University of Science and Technology reported genome editing using a prokaryotic Argonaute protein from Natronobacterium gregoryi. However, evidence for application of Argonaute proteins as DNA-guided nucleases for genome editing have been questioned, with the retraction of the claim from the leading journal. ^[14] In 2017, a group from University of Illinois reported using a prokaryotic Argonaute protein taken from Pyrococcus furiosus (PfAgo) along with guide DNA to edit DNA in vitro as artificial restriction enzymes. ^[15] PfAgo based artificial restriction enzymes were also used for storing data on native DNA sequences via enzymatic nicking. ^[16]

Related Research Articles

Small RNA (sRNA) are polymeric RNA molecules that are less than 200 nucleotides in length, and are usually non-coding. RNA silencing is often a function of these molecules, with the most common and well-studied example being RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA) and its subspecies repeat associated small interfering RNA (rasiRNA). Small RNA "is unable to induce RNAi alone, and to accomplish the task it must form the core of the RNA–protein complex termed the RNA-induced silencing complex (RISC), specifically with Argonaute protein".

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded non-coding RNA molecules, typically 20–24 base pairs in length, similar to microRNA (miRNA), and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading messenger RNA (mRNA) after transcription, preventing translation. It was discovered in 1998 by Andrew Fire at the Carnegie Institution for Science in Washington, D.C. and Craig Mello at the University of Massachusetts in Worcester.

Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene. Being part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively. These fragments are approximately 20–25 base pairs long with a two-base overhang on the 3′-end. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. RISC has a catalytic component Argonaute, which is an endonuclease capable of degrading messenger RNA (mRNA).

The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which functions in gene silencing via a variety of pathways at the transcriptional and translational levels. Using single-stranded RNA (ssRNA) fragments, such as microRNA (miRNA), or double-stranded small interfering RNA (siRNA), the complex functions as a key tool in gene regulation. The single strand of RNA acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in defense against viral infections, as it is triggered by the presence of double-stranded RNA (dsRNA).

Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules expressed in animal cells. piRNAs form RNA-protein complexes through interactions with piwi-subfamily Argonaute proteins. These piRNA complexes are mostly involved in the epigenetic and post-transcriptional silencing of transposable elements and other spurious or repeat-derived transcripts, but can also be involved in the regulation of other genetic elements in germ line cells.

Piwi genes were identified as regulatory proteins responsible for stem cell and germ cell differentiation. Piwi is an abbreviation of P-elementInduced WImpy testis in Drosophila. Piwi proteins are highly conserved RNA-binding proteins and are present in both plants and animals. Piwi proteins belong to the Argonaute/Piwi family and have been classified as nuclear proteins. Studies on Drosophila have also indicated that Piwi proteins have no slicer activity conferred by the presence of the Piwi domain. In addition, Piwi associates with heterochromatin protein 1, an epigenetic modifier, and piRNA-complementary sequences. These are indications of the role Piwi plays in epigenetic regulation. Piwi proteins are also thought to control the biogenesis of piRNA as many Piwi-like proteins contain slicer activity which would allow Piwi proteins to process precursor piRNA into mature piRNA.

RNA-induced transcriptional silencing (RITS) is a form of RNA interference by which short RNA molecules – such as small interfering RNA (siRNA) – trigger the downregulation of transcription of a particular gene or genomic region. This is usually accomplished by posttranslational modification of histone tails which target the genomic region for heterochromatin formation. The protein complex that binds to siRNAs and interacts with the methylated lysine 9 residue of histones H3 (H3K9me2) is the RITS complex.

RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA (dsRNA). RNA silencing mechanisms are conserved among most eukaryotes. The most common and well-studied example is RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA) and its subspecies repeat associated small interfering RNA (rasiRNA).

RNA activation (RNAa) is a small RNA-guided and Argonaute (Ago)-dependent gene regulation phenomenon in which promoter-targeted short double-stranded RNAs (dsRNAs) induce target gene expression at the transcriptional/epigenetic level. RNAa was first reported in a 2006 PNAS paper by Li et al. who also coined the term "RNAa" as a contrast to RNA interference (RNAi) to describe such gene activation phenomenon. dsRNAs that trigger RNAa have been termed small activating RNA (saRNA). Since the initial discovery of RNAa in human cells, many other groups have made similar observations in different mammalian species including human, non-human primates, rat and mice, plant and C. elegans, suggesting that RNAa is an evolutionarily conserved mechanism of gene regulation.

Protein argonaute-2 is a protein that in humans is encoded by the EIF2C2 gene.

Protein argonaute-1 is a protein that in humans is encoded by the EIF2C1 gene.

Trans-acting siRNA are a class of small interfering RNA (siRNA) that repress gene expression through post-transcriptional gene silencing in land plants. Precursor transcripts from TAS loci are polyadenylated and converted to double-stranded RNA, and are then processed into 21-nucleotide-long RNA duplexes with overhangs. These segments are incorporated into an RNA-induced silencing complex (RISC) and direct the sequence-specific cleavage of target mRNA. Ta-siRNAs are classified as siRNA because they arise from double-stranded RNA (dsRNA).

RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. The detailed study of each of these seemingly different processes elucidated that the identity of these phenomena were all actually RNAi. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNAi in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense therapy for gene suppression. Antisense RNA produced intracellularly by an expression vector may be developed and find utility as novel therapeutic agents.

Transposon silencing is a form of transcriptional gene silencing targeting transposons. Transcriptional gene silencing is a product of histone modifications that prevent the transcription of a particular area of DNA. Transcriptional silencing of transposons is crucial to the maintenance of a genome. The “jumping” of transposons generates genomic instability and can cause extremely deleterious mutations. Transposable element insertions have been linked to many diseases including hemophilia, severe combined immunodeficiency, and predisposition to cancer. The silencing of transposons is therefore extremely critical in the germline in order to stop transposon mutations from developing and being passed on to the next generation. Additionally, these epigenetic defenses against transposons can be heritable. Studies in Drosophila, Arabidopsis thaliana, and mice all indicate that small interfering RNAs are responsible for transposon silencing. In animals, these siRNAS and piRNAs are most active in the gonads.

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

RDE-1 (RNAi-DEfective 1) is a primary Argonaute protein required for RNA-mediated interference (RNAi) in Caenorhabditis elegans. The rde-1 gene locus was first characterized in C. elegans mutants resistant to RNAi, and is a member of a highly conserved Piwi gene family that includes plant, Drosophila, and vertebrate homologs.

RNA silencing suppressor p19 is a protein expressed from the ORF4 gene in the genome of tombusviruses. These viruses are positive-sense single-stranded RNA viruses that infect plant cells, in which RNA silencing forms a widespread and robust antiviral defense system. The p19 protein serves as a counter-defense strategy, specifically binding the 19- to 21-nucleotide double-stranded RNAs that function as small interfering RNA (siRNA) in the RNA silencing system. By sequestering siRNA, p19 suppresses RNA silencing and promotes viral proliferation. The p19 protein is considered a significant virulence factor and a component of an evolutionary arms race between plants and their pathogens.

DCL2 is a gene in plants that codes for the DCL2 protein, a ribonuclease III enzyme involved in processing exogenous double-stranded RNA (dsRNA) into 22 nucleotide small interference RNAs (siRNAs).

References

External links

• starBase database: a database for exploring microRNA–mRNA interaction maps from Argonaute CLIP-Seq(HITS-CLIP, PAR-CLIP) and Degradome-Seq data.