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.
Ribonuclease is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 and 3.1 classes of enzymes.
Ribonuclease H is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/DNA substrate via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from bacteria to archaea to eukaryotes.
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.
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).
Ribonuclease L or RNase L, known sometimes as ribonuclease 4 or 2'-5' oligoadenylate synthetase-dependent ribonuclease — is an interferon (IFN)-induced ribonuclease which, upon activation, destroys all RNA within the cell. RNase L is an enzyme that in humans is encoded by the RNASEL gene.
Ribonuclease III (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.
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.
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 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.
The degradosome is a multiprotein complex present in most bacteria that is involved in the processing of ribosomal RNA and the degradation of messenger RNA and is regulated by Non-coding RNA. It contains the proteins RNA helicase B, RNase E and Polynucleotide phosphorylase.
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 a 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 polymerase IV is an enzyme that synthesizes small interfering RNA (siRNA) in plants, which silence gene expression. RNAP IV belongs to a family of enzymes that catalyze the process of transcription known as RNA Polymerases, which synthesize RNA from DNA templates. Discovered via phylogenetic studies of land plants, genes of RNAP IV are thought to have resulted from multistep evolution processes that occurred in RNA Polymerase II phylogenies. Such an evolutionary pathway is supported by the fact that RNAP IV is composed of 12 protein subunits that are either similar or identical to RNA polymerase II, and is specific to plant genomes. Via its synthesis of siRNA, RNAP IV is involved in regulation of heterochromatin formation in a process known as RNA directed DNA Methylation (RdDM).
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.
RDE-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.
The microprocessor complex is a protein complex involved in the early stages of processing microRNA (miRNA) and RNA interference (RNAi) in animal cells. The complex is minimally composed of the ribonuclease enzyme Drosha and the dimeric RNA-binding protein DGCR8, and cleaves primary miRNA substrates to pre-miRNA in the cell nucleus. Microprocessor is also the smaller of the two multi-protein complexes that contain human Drosha.
DCL1 is a gene in plants that codes for the DCL1 protein, a ribonuclease III enzyme involved in processing double-stranded RNA (dsRNA) and microRNA (miRNA). Although DCL1, also called Endoribonuclease Dicer homolog 1, is named for its homology with the metazoan protein Dicer, its role in miRNA biogenesis is somewhat different, due to substantial differences in miRNA maturation processes between plants and animals, as well due to additional downstream plant-specific pathways, where DCL1 paralogs like DCL4 participate, such Trans-acting siRNA biogenesis.
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.
DCL3 is a gene in plants that codes for the DCL3 protein, a ribonuclease III enzyme involved in plants specific pathway RNA-directed DNA methylation. Where DCL3 cleaves endogenous double-stranded RNAs into 24 nucleotide small interfering RNAs. The main difference to other DCLs is the dsRNA source, which precursor for DCL3 is generally transcribed in heterochromatic regions by the RNA polymerase complex, RNA polymerase IV, producing single-stranded RNA roughly of 30 to 45 nucleotides in length, which are converted into dsRNA by RNA-dependent RNA polymerase 2. Once cleaved by DCL3, the 24-nt siRNA strand is loaded into AGO4, which interacts with Pol V–transcribed long noncoding RNAs and recruits domains-rearranged methylase 2, facilitating DNA methylation.
