RDE-1

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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. [1]

Contents

Role in RNAi pathway

Exogenous RNAi Pathway in C. elegans Exogenous RNAi Pathway in C. elegans, edited.svg
Exogenous RNAi Pathway in C. elegans

Primary siRNA-argonaute complex

Upon uptake into the cell, exogenous trigger dsRNA is bound by RDE-4 and cleaved into 21-25nt primary siRNA by a Dicer complex that includes RDE-1. [2] Primary siRNA binding to RDE-1 then promotes the formation of the RNA-induced silencing complex (RISC). Unlike in other Argonautes characterized in Drosophila [3] and humans, [4] the catalytic RNase H motif in RDE-1 has not been shown to exhibit Slicer activity of the target transcript. Instead, RNase H activity in RDE-1 is primarily facilitates siRNA maturation through cleavage of the passenger strand. [5]

Secondary siRNA-argonaute complex

The primary Argonaute complex recruits an RNA-dependent RNA polymerase (RdRP) to generate secondary siRNAs, triggering an amplification response. Secondary siRNAs are bound by degenerate secondary Argonautes, which are then directly involved in target transcript degradation. [6] However, RDE-1 shows no stable association with the more abundant secondary siRNAs. [7]

Whereas rde-4 deficiency can be rescued by high concentrations of trigger dsRNA, [8] and secondary Argonaute exhibit functional redundancy, [6] there has been no evidence that RNA-mediated silencing can be reinstated in rde-1 deficient mutants. To this extent, RDE-1 appears to have a qualitatively distinct activity in the exogenous RNAi pathway. [7]

Structure

Canonical Argonaute proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and PIWI domains. PAZ and MID orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing. The PIWI domain folds into an RNase H-like structure, and contains the conserved catalytic triad “DDH” (two aspartate residues, one histidine residue). [9] The crystal structure of RDE-1 has not been formally elucidated, but can be assumed to closely resemble its human homologs.

Passenger strand degradation

Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation. [10] However, RDE-1 has not been implicated in mRNA-cleavage activity.

Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA. [5] Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity. [5] Thus, RDE-1's RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.

See also

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.

<span class="mw-page-title-main">Small interfering RNA</span> Biomolecule

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.

<span class="mw-page-title-main">Dicer</span> Enzyme that cleaves double-stranded RNA (dsRNA) into short dsRNA fragments

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).

<span class="mw-page-title-main">Ribonuclease III</span> Class of enzymes

Ribonuclease III (RNase III or RNase C)(BRENDA 3.1.26.3) is a type of ribonuclease that recognizes dsRNA and cleaves it at specific targeted locations to transform them into mature RNAs. These enzymes are a group of endoribonucleases that are characterized by their ribonuclease domain, which is labelled the RNase III domain. They are ubiquitous compounds in the cell and play a major role in pathways such as RNA precursor synthesis, RNA Silencing, and the pnp autoregulatory mechanism.

<span class="mw-page-title-main">Argonaute</span> Protein that plays a role in RNA silencing process

The Argonaute protein family, first discovered for its evolutionarily conserved stem cell function, 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). 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, which then leads to mRNA cleavage, translation inhibition, and/or the initiation of mRNA decay.

<span class="mw-page-title-main">Drosha</span> Ribonuclease III enzyme

Drosha is a Class 2 ribonuclease III enzyme that in humans is encoded by the DROSHA gene. It is the primary nuclease that executes the initiation step of miRNA processing in the nucleus. It works closely with DGCR8 and in correlation with Dicer. It has been found significant in clinical knowledge for cancer prognosis and HIV-1 replication.

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.

<span class="mw-page-title-main">Piwi</span>

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).

Small temporal RNA regulates gene expression during roundworm development by preventing the mRNAs they bind from being translated. In contrast to siRNA, stRNAs downregulate expression of target RNAs after translation initiation without affecting mRNA stability. Nowadays, stRNAs are better known as miRNAs.

esiRNA or Endoribonuclease-prepared siRNAs are a mixture of siRNA oligos resulting from cleavage of long double-stranded RNA (dsRNA) with an endoribonuclease such as Escherichia coli RNase III or Dicer.

<span class="mw-page-title-main">EIF2C2</span> Protein-coding gene in the species Homo sapiens

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

<span class="mw-page-title-main">EIF2C1</span> Protein-coding gene in the species Homo sapiens

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).

<span class="mw-page-title-main">RNA interference</span> Biological process of gene regulation

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.

<span class="mw-page-title-main">DCL2</span> Dicer-like gene in plants

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

  1. ↑ Tabara, H.; Sarkissian, M.; Kelly, W. G.; Fleenor, J.; Grishok, A.; Timmons, L.; Fire, A.; & Mello, C. C. (Oct 1999). "The rde-1 gene, RNA interference and transposon silencing in C. elegans". Cell. 99 (2): 123–32. doi: 10.1016/S0092-8674(00)81644-X . PMID   10535731.
  2. ↑ Tabara, H.; Yigit, E.; Siomi, H.; Mello, C.; Mello, C. C. (Jun 2002). "The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1 and a DExH-box helicase to direct RNAi in C. elegans". Cell. 109 (7): 861–871. doi: 10.1016/S0092-8674(02)00793-6 . PMID   12110183.
  3. ↑ Hammond SM, Bernstein E, Beach D, Hannon GJ (Mar 2000). "An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells". Nature. 404 (6775): 293–296. doi:10.1038/35005107. PMID   10749213. S2CID   9091863.
  4. ↑ Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T (Jul 2004). "Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs". Mol Cell. 15 (2): 185–197. doi: 10.1016/j.molcel.2004.07.007 . PMID   15260970.
  5. 1 2 3 Steiner, F. A.; Okihara, K. L.; Hoogstrate, S. W.; Sijen, T.; Ketting, R. F. (Feb 2009). "RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans". Nat Struct Mol Biol. 16 (2): 207–11. doi:10.1038/nsmb.1541. PMID   19151723. S2CID   22909313.
  6. 1 2 Boisvert, M.E.; Simard, M.J (2008). "RNAi pathway in C. elegans: the argonautes and collaborators". Curr. Top. Microbiol. Immunol. Current Topics in Microbiology and Immunology. 320: 21–36. doi:10.1007/978-3-540-75157-1_2. ISBN   978-3-540-75156-4. PMID   18268838.
  7. 1 2 Yigit E, Batista PJ, Bei Y, Pang KM, Chen CC, Tolia NH, Joshua-Tor L, Mitani S, Simard MJ, Mello CC (2006). "Analysis of the C. elegans Argonaute Family Reveals that Distinct Argonautes Act Sequentially during RNAi". Cell. 127 (4): 747–57. doi: 10.1016/j.cell.2006.09.033 . PMID   17110334.
  8. ↑ Habig JW, Aruscavage PJ, Bass BL (2008). "In C. elegans, high levels of dsRNA allow RNAi in the absence of RDE-4". PLOS ONE. 3 (12): e4052. doi: 10.1371/journal.pone.0004052 . PMC   2603325 . PMID   19112503.
  9. ↑ Song, J.J.; Smith, S.K.; Hannon, G.J.; Joshua-Tor, L. (Sep 2004). "Crystal structure of Argonaute and its implications for RISC slicer activity". Science. 305 (5689): 1434–7. doi: 10.1126/science.1102514 . PMID   15284453. S2CID   38557910.
  10. ↑ Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ (Sep 2004). "Argonaute2 is the catalytic engine of mammalian RNAi". Science. 305 (5689): 1437–41. doi: 10.1126/science.1102513 . PMID   15284456. S2CID   2778088.
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