Argonaute protein family plays a central role in RNA silencing processes, as essential catalytic components of the RNA-induced silencing complex (RISC). RISC complex is responsible for the gene silencing phenomenon known as RNA interference (RNAi). 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 or translation inhibition.
|Argonaute Piwi domain|
|Argonaute Paz domain|
The RNA interference (RNAi) was first reported in 1995 by Guo and Kemphues, and similar pathways collectively referred to as RNA silencing were discovered in plants and fungi. The beginning of people’s understanding of the mechanism of RNA silencing began only in 1998 with the experiments of Fire and colleagues demonstrating that double-stranded RNA triggered RNAi. RNA silencing pathways process long RNAs into small RNAs that direct the repression of transcription or translation of nucleic acid targets with sequence corresponding to the small RNAs. These single-stranded RNAs, referred to as guide strands, are incorporated into RNA silencing effectors complexes such as the RNA-induced silencing complex (RISC). These RNA silencing effector complexes contain Argonaute family proteins.
Argonaute in RNA interference
RNA interference(RNAi) is a significant biological process, in which the RNA molecules inhibit gene expression. The typical way of this process is causing the destruction of specific mRNA molecules. The RNA interference has a significant role in defending cells against parasitic nucleotide sequences. In many eukaryotes, including animals, the RNA interference pathway is found, and it is initiated by enzyme Dicer. Dicer cleaves long double-stranded 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. Consequently, 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 RISC.
Argonaute proteins are the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. 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, incorporated into the Argonaute protein and leads gene silencing. The other single-stranded named passenger strand is degraded during the RNA-induced silencing complex process.
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 stand 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 effect the formation of functional ribosomes at the 5’-end of the mRNA. The complex here compete 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.
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 of Argonautes and Mechanism
The PAZ domain is named after proteins PIWI, AGO, and Zwille, whereby it is found to conserved. The PAZ domain is an RNA binding module that recognizes the 3' end of both siRNA and miRNA, in a sequence independent manner. Consequently, it targets the mRNA for cleavage or translation inhibition by base-pairing interaction.
The Drosophila PIWI protein gave its name to this characteristic motif. Structurally resembles 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 will lack the cleavage activity. In human AGO, the PIWI motif also mediate protein-protein interaction at the PIWI box, where it binds to Dicer at one of the RNase III domain.
At the interface of PIWI and Mid domains sits the 5' phosphate of a siRNA or miRNA, which is found essential in the functionality. Within Mid lies a MC motif, a homologue structure to the cap structure motif found in eIF4E. It is later proved that the MC motif is involved in binding cap structure and consequently, translation control.
In human, there are eight AGO family members, some of which are investigated intensively. However, even though AGO1-4 are capable of loading miRNA, endonuclease activity, thus RNAi dependent gene silencing is exclusively found with 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.
Several AGO family in plants also attracts tremendous effort of studying. AGO1 is clearly involved in miRNA related RNA degradation, and plays a central role in morphogenesis. In some organisms, it is strictly required for epigenetic silencing. Interestingly, 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.
Disease and Therapeutic Tools
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.
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-transcriptionl 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.
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