RNA interference (also called "RNA-mediated interference", abbreviated RNAi) is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. Conserved in most eukaryotic organisms, the RNAi pathway is thought to have evolved as a form of innate immunity against viruses and also plays a major role in regulating development and genome maintenance.
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The RNAi pathway is initiated by the enzyme dicer, which cleaves double-stranded RNA (dsRNA) to short double-stranded fragments of 20–25 base pairs. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and base-pairs with complementary sequences. The most well-studied outcome of this recognition event is a form of post-transcriptional gene silencing. This occurs when the guide strand base pairs with a messenger RNA (mRNA) molecule and induces degradation of the mRNA by argonaute, the catalytic component of the RISC complex. The short RNA fragments are known as small interfering RNA (siRNA) when they derive from exogenous sources and microRNA (miRNA) when they are produced from RNA-coding genes in the cell's own genome. The RNAi pathway has been particularly well-studied in certain model organisms such as the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the flowering plant Arabidopsis thaliana.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms; synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.
Historically, RNA interference was known by other names, including post transcriptional gene silencing, transgene silencing, and quelling. Only after these apparently-unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. RNAi has also been confused with antisense suppression of gene expression, which does not act catalytically to degrade mRNA but instead involves single-stranded RNA fragments physically binding to mRNA and blocking translation. In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm C. elegans, which they published in 1998.
DNA stores the information for protein synthesis and RNA carries out the instructions encoded in DNA, most biological activities are carried out by proteins. The accurate synthesis of proteins thus is critical to the proper functioning of cells and organisms.
the linear order of amino acids in each protein determines its three-dimensional structure and activity. For this reason, assembly of amino acids in their correct order, as encoded in DNA, is the key to production of functional proteins.
Three kinds of RNA molecules perform different but cooperative functions in protein synthesis
1. Messenger RNA (mRNA) carries the genetic information copied from DNA in the form of a series of three-base code “words,” each of which specifies a particular amino acid.
2. Transfer RNA (tRNA) is the key to deciphering the code words in mRNA. Each type of amino acid has its own type of tRNA, which binds it and carries it to the growing end of a polypeptide chain if the next code word on mRNA calls for it. The correct tRNA with its attached amino acid is selected at each step because each specific tRNA molecule contains a three-base sequence that can base-pair with its complementary code word in the mRNA.
3. Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. These complex structures, which physically move along an mRNA molecule, catalyze the assembly of amino acids into protein chains. They also bind tRNAs and various accessory molecules necessary for protein synthesis. Ribosomes are composed of a large and small subunit, each of which contains its own rRNA molecule or molecules.
Translation is the whole process by which the base sequence of an mRNA is used to order and to join the amino acids in a protein. The three types of RNA participate in this essential protein-synthesizing pathway in all cells; in fact, the development of the three distinct functions of RNA was probably the molecular key to the origin of life.
RNA contains ribonucleotides of adenine, cytidine, guanine, and uracil; DNA contains deoxyribonucleotides of adenine, cytidine, guanine, and thymine. Because 4 nucleotides, taken individually, could represent only 4 of the 20 possible amino acids in coding the linear arrangement in proteins, a group of nucleotides is required to represent each amino acid. The code employed must be capable of specifying at least 20 words (i.e., amino acids).
If two nucleotides were used to code for one amino acid, then only 16 (or 42) different code words could be formed, which would be an insufficient number. However, if a group of three nucleotides is used for each code word, then 64 (or 43) code words can be formed. Any code using groups of three or more nucleotides will have more than enough units to encode 20 amino acids. Many such coding systems are mathematically possible. However, the actual genetic code used by cells is a triplet code, with every three nucleotides being “read” from a specified starting point in the mRNA. Each triplet is called a codon. Of the 64 possible codons in the genetic code, 61 specify individual amino acids and three are stop codons. most amino acids are encoded by more than one codon. Only two — methionine and tryptophan — have a single codon; at the other extreme, leucine, serine, and arginine are each specified by six different codons. The different codons for a given amino acid are said to be synonymous. The code itself is termed degenerate, which means that it contains redundancies.
Synthesis of all protein chains in prokaryotic and eukaryotic cells begins with the amino acid methionine. In most mRNAs, the start (initiator) codon specifying this aminoterminal methionine is AUG. In a few bacterial mRNAs, GUG is used as the initiator codon, and CUG occasionally is used as an initiator codon for methionine in eukaryotes. The three codons UAA, UGA, and UAG do not specify amino acids but constitute stop (terminator) signals that mark the carboxyl terminus of protein chains in almost all cells. The sequence of codons that runs from a specific start site to a terminating codon is called a reading frame. This precise linear array of ribonucleotides in groups of three in mRNA specifies the precise linear sequence of amino acids in a protein and also signals where synthesis of the protein chain starts and stops.
Because the genetic code is a commaless, overlapping triplet code, a particular mRNA theoretically could be translated in three different reading frames. Indeed some mRNAs have been shown to contain overlapping information that can be translated in different reading frames, yielding different polypeptides.The vast majority of mRNAs, however, can be read in only one frame because stop codons encountered in the other two possible reading frames terminate translation before a functional protein is produced. Another unusual coding arrangement occurs be- cause of frameshifting. In this case the protein-synthesizing machinery may read four nucleotides as one amino acid and then continue reading triplets, or it may back up one base and read all succeeding triplets in the new frame until termination of the chain occurs. These frameshifts are not common events, but a few dozen such instances are known
The vast majority of mRNAs, however, can be read in only one frame because stop codons encountered in the other two possible reading frames terminate translation before a functional protein is produced. Another unusual coding arrangement occurs be- cause of frameshifting. In this case the protein-synthesizing machinery may read four nucleotides as one amino acid and then continue reading triplets, or it may back up one base and read all succeeding triplets in the new frame until termination of the chain occurs. These frameshifts are not common events, but a few dozen such instances are known
The Folded Structure of tRNA Promotes Its Decoding Functions
understanding the flow of genetic information from DNA to protein was to determine how the nucleotide sequence of mRNA is converted into the amino acid sequence of protein. This decoding process requires two types of adapter molecules: tRNAs and enzymes called aminoacyl-tRNA synthetases.
All tRNAs have two functions: to be chemically linked to a particular amino acid and to base-pair with a codon in mRNA so that the amino acid can be added to a growing peptide chain. Each tRNA molecule is recognized by one and only one of the 20 aminoacyl-tRNA synthetases. Likewise, each of these enzymes links one and only one of the 20 amino acids to a particular tRNA, forming an aminoacyl-tRNA. Once its correct amino acid is attached, a tRNA then recognizes a codon in mRNA, thereby delivering its amino acid to the growing polypeptide.
Ribosomes Are Protein-Synthesizing Machines
If the many components that participate in translating mRNA had to interact in free solution, the likelihood of simultaneous collisions occurring would be so low that the rate of amino acid polymerization would be very slow. The efficiency of translation is greatly increased by the binding of the mRNA and the individual aminoacyl-tRNAs to the most abundant RNA-protein complex in the cell — the ribosome. This two-part machine directs the elongation of a polypeptide at a rate of three to five amino acids added per second. Small proteins of 100 – 200 amino acids are therefore made in a minute or less. On the other hand, it takes 2 to 3 hours to make the largest known protein, titin, which is found in muscle and contains 30,000 amino acid residues. The machine that accomplishes this task must be precise and persistent.
A ribosome is composed of several different ribosomal RNA (rRNA) molecules and more than 50 proteins, organized into a large subunit and a small subunit. The proteins in the two subunits differ, as do the molecules of rRNA. The small ribosomal subunit contains a single rRNA molecule, referred to as small rRNA; the large subunit contains a molecule of large rRNA and one molecule each of two much smaller rRNAs in eukaryotes.The lengths of the rRNA molecules, the quantity of proteins in each subunit, and consequently the sizes of the subunits differ in prokaryotic and eukaryotic cells. (The small and large rRNAs are about 1500 and 3000 nucleotides long in bacteria and about 1800 and 5000 nucleotides long in humans.) Perhaps of more interest than these differences are the great structural and functional similarities among ribosomes from all species. This consistency is another reflection of the common evolutionary origin of the most basic constituents of living cells.
Transcription is the process through which a DNA sequence is enzymatically copied by an RNA polymerase to produce a complementary RNA. In other words, it is the transfer of genetic information from DNA into RNA. In the case of protein-encoding DNA, transcription is the beginning of the process that ultimately leads to the translation of the genetic code (via the mRNA intermediate) into a functional peptide or protein. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying DNA; therefore, transcription has a lower copying fidelity than DNA replication.
As in DNA replication, transcription proceeds in the 5' → 3' direction (i.e. the old polymer is read in the 3' → 5' direction and the new, complementary fragments are generated in the 5' → 3' direction). Transcription is divided into 3 stages: initiation, elongation and termination.
Initiation in prokaryotes
Transcription of RNA differs from DNA synthesis in that only one strand of DNA, the template strand, is used to make mRNA. Because transcription only proceeds in the 5' → 3' direction, it follows that the DNA template strand that is used must be oriented in 3' → 5' (complementary) direction. The strand that is not used as a template strand is called the non-template strand. Thus, DNA exists as a double strand, whereas RNA only exists as a single strand. The difference is due to the fact that DNA replication is semi-conservative, while transcription results in de novo production of a single strand of RNA.
Transcription begins with the binding of RNA polymerase to the promoter in DNA. The RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β' subunit, and 1 ω subunit. At the start of initiation, the core enzyme is associated with a sigma factor (number 70) that aids in finding the appropriate -35 and -10 basepairs downstream of promoter sequences. Unlike DNA replication, transcription does not need a primer to start. The DNA unwinds and produces a small open complex and synthesis begins on only the template strand.
Elongation
Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases, so many mRNA molecules can be produced from a single copy of the gene. This step also involves a proofreading mechanism that can replace an incorrectly added RNA molecule.
Termination
Upon seeing a termination codon within the DNA template, RNA transcription can stop by forming a secondary hairpin loop that lets it come off the DNA template. Alternatively, another protein designated "Rho" can pull the mRNA away from polymerase
Prokaryotic vs. eukaryotic transcription
Prokaryotic transcription occurs in the cytoplasm alongside translation.
Eukaryotic transcription is primarily localized to the nucleus, where it is separated from the cytoplasm (where translation occurs) by the nuclear membrane.
Transcription factories
Active transcription units are clustered in the nucleus, in discrete sites called ‘transcription factories’. Such sites could be visualized after allowing engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U), and immuno-labeling the tagged nascent RNA. Transcription factories can also be localized using fluorescence in situ hybridization, or marked by antibodies directed against polymerases. There are ~10,000 factories in the nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000 polymerase III factories. Each polymerase II factory contains ~8 polymerases. As most active transcription units are associated with only one polymerase, each factory will be associated with ~8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a ‘cloud’ around the factory.
Transcription initiation complex
Transcription factors mediate the binding of RNA polymerase and the initiation of transcription. The RNA polymerase only binds to the promoter after certain transcription factors are assembled. The completed assembly of transcription factors and RNA polymerase bound to the promoter is called the transcription initiation complex.
Reverse transcription
Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA in order to see a cell's genome. The main enzyme responsible for this type of transcription is called reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesising a complementary DNA strand (cDNA) to the viral RNA genome. An associated enzyme, ribonuclease H, digests the RNA strand and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure. This cDNA is integrated into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral proteins which reassemble into new viral particles. Subsequently, the host cell undergoes programmed cell death (apoptosis).
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated.
siRNAs were first discovered by David Baulcombe's group in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants". Shortly thereafter, in 2001, synthetic siRNAs were shown to be able to induce RNAi in mammalian cells by Thomas Tuschl and colleagues in a paper published in Nature. This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development.
Structure
siRNAs have a well-defined structure: a short (usually 21-nt) double strand of RNA (dsRNA) with 2-nt 3' overhangs on either end:
Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs.SiRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. This has made siRNAs an important tool for gene function and drug target validation studies in the post-genomic era.
