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A hairpin loop from a pre-mRNA. Highlighted are the nucleobases (green) and the ribose-phosphate backbone (blue). Note that this is a single strand of RNA that folds back upon itself.
Ribonucleic acid (RNA) is a polymeric molecule. It is implicated in various biological roles in coding, decoding, regulation, and expression of genes. DNA and RNA are nucleic acids, and, along with proteins and carbohydrates, constitute the three major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more often found in nature as a single-strand folded unto itself, rather than a paired double-strand. Cellular organisms use messenger RNA (mRNA) to convey genetic information (using the letters G, A, U, and C to denote the nitrogenous bases guanine, adenine, uracil and cytosine) that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.
Some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these active processes is protein synthesis, a universal function whereby mRNA molecules direct the assembly of proteins on ribosomes. This process uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal RNA (rRNA) links amino acids together to form proteins.
Comparison with DNA
Three-dimensional representation of the 50S ribosomal subunit. RNA is in ochre, protein in blue. The active site is in the middle (red).
The chemical structure of RNA is very similar to that of DNA, but differs in three main ways:
- Unlike double-stranded DNA, RNA is a single-stranded molecule in many of its biological roles and has a much shorter chain of nucleotides. However, RNA can, by complementary base pairing, form intrastrand double helixes, as in tRNA.
- While DNA contains deoxyribose, RNA contains ribose (in deoxyribose there is no hydroxyl group attached to the pentose ring in the 2' position). These hydroxyl groups make RNA less stable than DNA because it is more prone to hydrolysis.
- The complementary base to adenine is not thymine, as it is in DNA, but rather uracil, which is an unmethylated form of thymine.[1]
Structure
Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'. A base is attached to the 1' position, in general, adenine (A), cytosine (C), guanine (G), or uracil (U). Adenine and guanine are purines, cytosine and uracil are pyrimidines. A phosphate group is attached to the 3' position of one ribose and the 5' position of the next. The phosphate groups have a negative charge each at physiological pH, making RNA a charged molecule (polyanion). The bases form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil.[5] However, other interactions are possible, such as a group of adenine bases binding to each other in a bulge,[6] or the GNRA tetraloop that has a guanine–adenine base-pair.[5]
An important structural feature of RNA that distinguishes it from DNA is the presence of a hydroxyl group at the 2' position of the ribose sugar. The presence of this functional group causes the helix to adopt the A-form geometry rather than the B-form most commonly observed in DNA.[7] This results in a very deep and narrow major groove and a shallow and wide minor groove.[8] A second consequence of the presence of the 2'-hydroxyl group is that in conformationally flexible regions of an RNA molecule (that is, not involved in formation of a double helix), it can chemically attack the adjacent phosphodiester bond to cleave the backbone.[9]
RNA is transcribed with only four bases (adenine, cytosine, guanine and uracil),[10] but these bases and attached sugars can be modified in numerous ways as the RNAs mature. Pseudouridine (Ψ), in which the linkage between uracil and ribose is changed from a C–N bond to a C–C bond, and ribothymidine (T) are found in various places (the most notable ones being in the TΨC loop of tRNA).[11] Another notable modified base is hypoxanthine, a deaminated adenine base whose nucleoside is called inosine (I). Inosine plays a key role in the wobble hypothesis of the genetic code.[12]
There are more than 100 other naturally occurring modified nucleosides,[13] The greatest structural diversity of modifications can be found in tRNA,[14] while pseudouridine and nucleosides with 2'-O-methylribose often present in rRNA are the most common.[15] The specific roles of many of these modifications in RNA are not fully understood. However, it is notable that, in ribosomal RNA, many of the post-transcriptional modifications occur in highly functional regions, such as the peptidyl transferase center and the subunit interface, implying that they are important for normal function.[16]
The functional form of single-stranded RNA molecules, just like proteins, frequently requires a specific tertiary structure. The scaffold for this structure is provided by secondary structural elements that are hydrogen bonds within the molecule. This leads to several recognizable "domains" of secondary structure like hairpin loops, bulges, and internal loops.[17] Since RNA is charged, metal ions such as Mg2+ are needed to stabilise many secondary and tertiary structures.[18]
The naturally occurring enantiomer of RNA is D-RNA composed of D-ribonucleotides. All chirality centers are located in the D-ribose. By the use of L-ribose or rather L-ribonucleotides, L-RNA can be synthesized. L-RNA is much more stable against degradation by RNase.[19]
Synthesis
Synthesis of RNA is usually catalyzed by an enzyme—RNA polymerase—using DNA as a template, a process known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA (usually found "upstream" of a gene). The DNA double helix is unwound by the helicase activity of the enzyme. The enzyme then progresses along the template strand in the 3’ to 5’ direction, synthesizing a complementary RNA molecule with elongation occurring in the 5’ to 3’ direction. The DNA sequence also dictates where termination of RNA synthesis will occur.[20]Primary transcript RNAs are often modified by enzymes after transcription. For example, a poly(A) tail and a 5' cap are added to eukaryotic pre-mRNA and introns are removed by the spliceosome.
There are also a number of RNA-dependent RNA polymerases that use RNA as their template for synthesis of a new strand of RNA. For instance, a number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their genetic material.[21] Also, RNA-dependent RNA polymerase is part of the RNA interference pathway in many organisms.[22]
Types of RNA
Overview
Messenger RNA (mRNA) is the RNA that carries information from DNA to the ribosome, the sites of protein synthesis (translation) in the cell. The coding sequence of the mRNA determines the amino acid sequence in the protein that is produced.[23] However, many RNAs do not code for protein (about 97% of the transcriptional output is non-protein-coding in eukaryotes[24][25][26][27]).
These so-called non-coding RNAs ("ncRNA") can be encoded by their own genes (RNA genes), but can also derive from mRNA introns.[28] The most prominent examples of non-coding RNAs are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation.[1] There are also non-coding RNAs involved in gene regulation, RNA processing and other roles. Certain RNAs are able to catalyse chemical reactions such as cutting and ligating other RNA molecules,[29] and the catalysis of peptide bond formation in the ribosome;[4] these are known as ribozymes.
In translation
Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes, the protein synthesis factories in the cell. It is coded so that every three nucleotides (a codon) correspond to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes its introns—non-coding sections of the pre-mRNA. The mRNA is then exported from the nucleus to the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA. In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is being transcribed from DNA. After a certain amount of time the message degrades into its component nucleotides with the assistance of ribonucleases.[23]Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain through hydrogen bonding.[28]
Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time.[23] Nearly all the RNA found in a typical eukaryotic cell is rRNA.
Transfer-messenger RNA (tmRNA) is found in many bacteria and plastids. It tags proteins encoded by mRNAs that lack stop codons for degradation and prevents the ribosome from stalling.[30]
Regulatory RNAs
Several types of RNA can downregulate gene expression by being complementary to a part of an mRNA or a gene's DNA. MicroRNAs (miRNA; 21-22 nt) are found in eukaryotes and act through RNA interference (RNAi), where an effector complex of miRNA and enzymes can cleave complementary mRNA, block the mRNA from being translated, or accelerate its degradation.[31][32]While small interfering RNAs (siRNA; 20-25 nt) are often produced by breakdown of viral RNA, there are also endogenous sources of siRNAs.[33][34] siRNAs act through RNA interference in a fashion similar to miRNAs. Some miRNAs and siRNAs can cause genes they target to be methylated, thereby decreasing or increasing transcription of those genes.[35][36][37] Animals have Piwi-interacting RNAs (piRNA; 29-30 nt) that are active in germline cells and are thought to be a defense against transposons and play a role in gametogenesis.[38][39]
Many prokaryotes have CRISPR RNAs, a regulatory system similar to RNA interference.[40] Antisense RNAs are widespread; most downregulate a gene, but a few are activators of transcription.[41] One way antisense RNA can act is by binding to an mRNA, forming double-stranded RNA that is enzymatically degraded.[42] There are many long noncoding RNAs that regulate genes in eukaryotes,[43] one such RNA is Xist, which coats one X chromosome in female mammals and inactivates it.[44]
An mRNA may contain regulatory elements itself, such as riboswitches, in the 5' untranslated region or 3' untranslated region; these cis-regulatory elements regulate the activity of that mRNA.[45] The untranslated regions can also contain elements that regulate other genes.[46]
In RNA processing
Many RNAs are involved in modifying other RNAs. Introns are spliced out of pre-mRNA by spliceosomes, which contain several small nuclear RNAs (snRNA),[1] or the introns can be ribozymes that are spliced by themselves.[47] RNA can also be altered by having its nucleotides modified to other nucleotides than A, C, G and U. In eukaryotes, modifications of RNA nucleotides are in general directed by small nucleolar RNAs (snoRNA; 60-300 nt),[28] found in the nucleolus and cajal bodies. snoRNAs associate with enzymes and guide them to a spot on an RNA by basepairing to that RNA. These enzymes then perform the nucleotide modification. rRNAs and tRNAs are extensively modified, but snRNAs and mRNAs can also be the target of base modification.[48][49] RNA can also be methylated.[50][51]
RNA genomes
Like DNA, RNA can carry genetic information. RNA viruses have genomes composed of RNA that encodes a number of proteins. The viral genome is replicated by some of those proteins, while other proteins protect the genome as the virus particle moves to a new host cell. Viroids are another group of pathogens, but they consist only of RNA, do not encode any protein and are replicated by a host plant cell's polymerase.[52]In reverse transcription
Reverse transcribing viruses replicate their genomes by reverse transcribing DNA copies from their RNA; these DNA copies are then transcribed to new RNA. Retrotransposons also spread by copying DNA and RNA from one another,[53] and telomerase contains an RNA that is used as template for building the ends of eukaryotic chromosomes.[54]Double-stranded RNA
Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all cells. dsRNA forms the genetic material of some viruses (double-stranded RNA viruses). Double-stranded RNA such as viral RNA or siRNA can trigger RNA interference in eukaryotes, as well as interferon response in vertebrates.[55][56][57][58]Key discoveries in RNA biology
Research on RNA has led to many important biological discoveries and numerous Nobel Prizes. Nucleic acids were discovered in 1868 by Friedrich Miescher, who called the material 'nuclein' since it was found in the nucleus.[59] It was later discovered that prokaryotic cells, which do not have a nucleus, also contain nucleic acids. The role of RNA in protein synthesis was suspected already in 1939.[60] Severo Ochoa won the 1959 Nobel Prize in Medicine (shared with Arthur Kornberg) after he discovered an enzyme that can synthesize RNA in the laboratory.[61] However, the enzyme discovered by Ochoa (polynucleotide phosphorylase) was later shown to be responsible for RNA degradation, not RNA synthesis. In 1956 Alex Rich and David Davies hybridized two separate strands of RNA to form the first crystal of RNA whose structure could be determined by X-ray crystallography.[62]
The sequence of the 77 nucleotides of a yeast tRNA was found by Robert W. Holley in 1965,[63] winning Holley the 1968 Nobel Prize in Medicine (shared with Har Gobind Khorana and Marshall Nirenberg). In 1967, Carl Woese hypothesized that RNA might be catalytic and suggested that the earliest forms of life (self-replicating molecules) could have relied on RNA both to carry genetic information and to catalyze biochemical reactions—an RNA world.[64][65]
During the early 1970s, retroviruses and reverse transcriptase were discovered, showing for the first time that enzymes could copy RNA into DNA (the opposite of the usual route for transmission of genetic information). For this work, David Baltimore, Renato Dulbecco and Howard Temin were awarded a Nobel Prize in 1975. In 1976, Walter Fiers and his team determined the first complete nucleotide sequence of an RNA virus genome, that of bacteriophage MS2.[66]
In 1977, introns and RNA splicing were discovered in both mammalian viruses and in cellular genes, resulting in a 1993 Nobel to Philip Sharp and Richard Roberts. Catalytic RNA molecules (ribozymes) were discovered in the early 1980s, leading to a 1989 Nobel award to Thomas Cech and Sidney Altman. In 1990, it was found in Petunia that introduced genes can silence similar genes of the plant's own, now known to be a result of RNA interference.[67][68]
At about the same time, 22 nt long RNAs, now called microRNAs, were found to have a role in the development of C. elegans.[69] Studies on RNA interference gleaned a Nobel Prize for Andrew Fire and Craig Mello in 2006, and another Nobel was awarded for studies on the transcription of RNA to Roger Kornberg in the same year. The discovery of gene regulatory RNAs has led to attempts to develop drugs made of RNA, such as siRNA, to silence genes.[70]
