Messenger RNA

The brief existence of an mRNA molecule begins with transcription, and ultimately ends in degradation. During its life, an mRNA molecule may also be processed, edited, and transported prior to translation.. Transcription Transcription is the process by which genetic information stored in DNA is copied into RNA by the enzyme RNA polymerase. During transcription, RNA polymerase binds to a promoter sequence on the DNA and synthesizes a complementary RNA strand (mRNA) from the DNA template. In eukaryotes, transcription occurs within the cell nucleus. The initial product of transcription is not functional mRNA but is termed precursor mRNA or pre-mRNA. This pre-mRNA must undergo extensive processing (including 5' capping, splicing to remove non-coding introns, and 3' polyadenylation) to become mature mRNA. Eukaryotic pre-mRNA processing Processing of mRNA differs greatly among eukaryotes, bacteria, and archaea. Eukaryotic pre-mRNA, however, requires several processing steps before its transport to the cytoplasm and its translation by the ribosome. Splicing The extensive processing of eukaryotic pre-mRNA that leads to the mature mRNA is the RNA splicing, a mechanism by which introns or outrons (non-coding regions) are removed and exons (coding regions) are joined. 5' cap addition A ''5' cap'' (also termed an RNA cap, an RNA 7-methylguanosine cap, or an RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of a eukaryotic messenger RNA shortly after the start of transcription. The 5' cap consists of a terminal 7-methylguanosine residue that is linked through a 5'-5'-triphosphate bond to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5' end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. Editing In some instances, an mRNA molecule is edited, which changes the nucleotide composition of the transcript. A prominent example in humans involves the apolipoprotein B mRNA. In certain tissues, RNA editing of this transcript creates a premature stop codon, which results in the production of a shorter protein variant. Another well studied mechanism is A-to-I (adenosine-to-inosine) editing. This reaction is catalyzed by ADAR enzymes (adenosine deaminase acting on RNA) and typically occurs within double-stranded RNA regions. A-to-I editing may occur in both coding sequences and untranslated regions. Through these modifications, the process can affect protein recoding, RNA structure, and gene regulation. Polyadenylation Polyadenylation is the covalent linkage of a polyadenylyl moiety to a messenger RNA molecule. In eukaryotic organisms most messenger RNA (mRNA) molecules are polyadenylated at the 3' end, but recent studies have shown that short stretches of uridine (oligouridylation) are also common. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs during and/or immediately after transcription of DNA into RNA. If this site is altered, cleavage and polyadenylation can shift to a downstream poly(A) site, producing an abnormally long and unstable mRNA. Transport Another difference between eukaryotes and prokaryotes is mRNA transport. Because eukaryotic transcription and translation is compartmentally separated, eukaryotic mRNAs must be exported from the nucleus to the cytoplasm—a process that may be regulated by different signaling pathways. Mature mRNAs are recognized by their processed modifications and then exported through the nuclear pore by binding to the cap-binding proteins CBP20 and CBP80, as well as the transcription/export complex (TREX). Multiple mRNA export pathways have been identified in eukaryotes. In spatially complex cells, some mRNAs are transported to particular subcellular destinations. In mature neurons, certain mRNA are transported from the soma to dendrites. One site of mRNA translation is at polyribosomes selectively localized beneath synapses. The mRNA for Arc/Arg3.1 is induced by synaptic activity and localizes selectively near active synapses based on signals generated by NMDA receptors. Other mRNAs also move into dendrites in response to external stimuli, such as β-actin mRNA. For export from the nucleus, actin mRNA associates with ZBP1 and later with 40S subunit. The complex is bound by a motor protein and is transported to the target location (neurite extension) along the cytoskeleton. Eventually ZBP1 is phosphorylated by Src in order for translation to be initiated. In developing neurons, mRNAs are also transported into growing axons and especially growth cones. Many mRNAs are marked with so-called "zip codes", which target their transport to a specific location. mRNAs can also transfer between mammalian cells through structures called tunneling nanotubes. Translation Because prokaryotic mRNA does not need to be processed or transported, translation by the ribosome can begin immediately after the end of transcription. Therefore, it can be said that prokaryotic translation is coupled to transcription and occurs co-transcriptionally. In eukaryotic cells the process of translation starts with the information stored in the nucleotide sequence of DNA. This is first transformed into mRNA, then transfer RNA (tRNA) specifies which three-nucleotide codon from the genetic code corresponds to which amino acid. Eukaryotic mRNA that has been processed and transported to the cytoplasm (i.e., mature mRNA) can then be translated by ribosomes. Translation may occur at ribosomes free in the cytoplasm, or targeted to the endoplasmic reticulum by the signal recognition particle. Therefore, unlike in prokaryotes, eukaryotic translation is not directly coupled to transcription. In some contexts, protein abundance can increase even when mRNA abundance decreases, because translation efficiency and protein turnover are regulated independently of transcript levels; this has been reported for mRNA and protein levels of EEF1A1 in breast cancer. == Structure ==

, 5' UTR, coding region, 3' UTR, and poly(A) tail. Coding regions Coding regions are composed of codons, which are decoded and translated into proteins by the ribosome; in eukaryotes usually into one and in prokaryotes usually into several. Coding regions begin with the start codon and end with a stop codon. In general, the start codon is an AUG triplet and the stop codon is UAG ("amber"), UAA ("ochre"), or UGA ("opal"). In addition to being protein-coding, portions of coding regions may serve as regulatory sequences in the pre-mRNA as exonic splicing enhancers or exonic splicing silencers. Untranslated regions Untranslated regions (UTRs) are sections of the mRNA before the start codon and after the stop codon that are not translated, termed the five prime untranslated region (5' UTR) and three prime untranslated region (3' UTR), respectively. These regions are transcribed with the coding region and thus are exonic as they are present in the mature mRNA. The stability of mRNAs may be controlled by the 5' UTR and/or 3' UTR due to varying affinity for RNA degrading enzymes called ribonucleases and for ancillary proteins that can promote or inhibit RNA degradation. One class of mRNA element, the riboswitches, directly bind small molecules, changing their fold to modify levels of transcription or translation. In these cases, the mRNA regulates itself. Poly(A) tail The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the 3' end of the pre-mRNA. This tail promotes export from the nucleus and translation, and protects the mRNA from degradation. On the other hand, polycistronic mRNA carries several open reading frames (ORFs), each of which is translated into a polypeptide. These polypeptides usually have a related function (they often are the subunits composing a final complex protein) and their coding sequence is grouped and regulated together in a regulatory region, containing a promoter and an operator. Most of the mRNA found in bacteria and archaea is polycistronic, Dicistronic or bicistronic mRNA encodes only two proteins. mRNA circularization In eukaryotes mRNA molecules form circular structures due to an interaction between the eIF4E and poly(A)-binding protein, which both bind to eIF4G, forming an mRNA-protein-mRNA bridge. Circularization is thought to promote cycling of ribosomes on the mRNA leading to time-efficient translation, and may also function to ensure only intact mRNA are translated (partially degraded mRNA characteristically have no m7G cap, or no poly-A tail). Other mechanisms for circularization exist, particularly in viruses. In several RNA viruses, long-distance RNA interactions and/or protein-mediated bridges between the 5' and 3' ends can promote translation and genome replication. RNA virus genomes (the + strands of which are translated as mRNA) are also commonly circularized. ==Degradation==

Different mRNAs within the same cell have distinct lifetimes (stabilities). In bacterial cells, individual mRNAs can survive from seconds to more than an hour. However, the lifetime averages between 1 and 3 minutes, making bacterial mRNA much less stable than eukaryotic mRNA. In mammalian cells, mRNA lifetimes range from several minutes to days. The greater the stability of an mRNA the more protein may be produced from that mRNA. Removal of two of the phosphates leaves a 5' monophosphate, causing the message to be destroyed by the exonuclease RNase J, which degrades 5' to 3'. Eukaryotic mRNA turnover Inside eukaryotic cells, there is a balance between the processes of translation and mRNA decay. Messages that are being actively translated are typically associated with cap-binding and translation initiation factors and with poly(A)-binding protein, which can antagonize deadenylation and decapping and help protect mRNA ends from decay machinery. The poly(A) tail of the mRNA is shortened by specialized exonucleases that are targeted to specific messenger RNAs by a combination of cis-regulatory sequences on the RNA and trans-acting RNA-binding proteins. AU-rich element decay The presence of AU-rich elements in some mammalian mRNAs tends to destabilize those transcripts through the action of cellular proteins that bind these sequences and stimulate poly(A) tail removal. and the decapping complex. Rapid mRNA degradation via AU-rich elements is a critical mechanism for preventing the overproduction of potent cytokines such as tumor necrosis factor (TNF) and granulocyte-macrophage colony stimulating factor (GM-CSF). AU-rich elements also regulate the biosynthesis of proto-oncogenic transcription factors like c-Jun and c-Fos. Nonsense-mediated decay Eukaryotic messages are subject to surveillance by nonsense-mediated decay (NMD), which checks for the presence of premature stop codons (nonsense codons) in the message. These can arise via incomplete splicing, V(D)J recombination in the adaptive immune system, mutations in DNA, transcription errors, leaky scanning by the ribosome causing a frame shift, and other causes. Detection of a premature stop codon triggers mRNA degradation by 5' decapping, 3' poly(A) tail removal, or endonucleolytic cleavage. Small interfering RNA (siRNA) In metazoans, small interfering RNAs (siRNAs) processed by Dicer are incorporated into a complex known as the RNA-induced silencing complex or RISC. This complex contains an endonuclease that cleaves perfectly complementary messages to which the siRNA binds. The resulting mRNA fragments are then destroyed by exonucleases. siRNA is commonly used in laboratories to block the function of genes in cell culture. It is thought to be part of the innate immune system as a defense against double-stranded RNA viruses. MicroRNA (miRNA) MicroRNAs (miRNAs) are small RNAs that typically are partially complementary to sequences in metazoan messenger RNAs. Binding of a miRNA to a message can repress translation of that message and accelerate poly(A) tail removal, thereby hastening mRNA degradation. The mechanism of action of miRNAs is the subject of active research. Other decay mechanisms There are other ways by which messages can be degraded, including non-stop decay and silencing by Piwi-interacting RNA (piRNA), among others. == Applications ==

The administration of a nucleoside-modified messenger RNA sequence can cause a cell to make a protein, which in turn could directly treat a disease or could function as a vaccine; more indirectly the protein could drive an endogenous stem cell to differentiate in a desired way. The primary challenges of RNA therapy center on delivering the RNA to the appropriate cells. Challenges include the fact that naked RNA sequences naturally degrade after preparation; they may trigger the body's immune system to attack them as an invader; and they are impermeable to the cell membrane. In the 1990s, mRNA vaccines for personalized cancer were developed, typically using unmodified mRNA. mRNA based therapies continue to be investigated as a method of treatment or therapy for both cancer as well as auto-immune, metabolic, and respiratory inflammatory diseases. Gene editing therapies such as CRISPR may also benefit from using mRNA to induce cells to make the desired Cas protein. The 2023 Nobel Prize in Physiology or Medicine was awarded to Katalin Karikó and Drew Weissman for the development of effective mRNA vaccines against COVID-19. New approaches to modulate RNA levels as a therapeutics include the use of antisense oligonucleotides, including for neurodevelopment diseases associated with high mortality. == History ==

Several molecular biology studies during the early 1950s suggested that RNA played a role in protein synthesis, though the specific role remained unclear. Arthur Pardee also found similar RNA accumulation in 1954. In 1953, Alfred Hershey, June Dixon, and Martha Chase studied E. coli infected with bacteriophage T2 and reported that the bacterium's own DNA decreased while the phage's DNA built up inside the infected cells (including DNA containing 5-hydroxymethylcytosine). In hindsight, this has been discussed as part of the chain of observations that led to the concept of mRNA, and it was not recognized at the time as such. The idea of mRNA was first conceived by Sydney Brenner and Francis Crick on 15 April 1960 at King's College, Cambridge, while François Jacob was telling them about a recent experiment conducted by Arthur Pardee, himself, and Monod (the so-called PaJaMo experiment, which did not prove mRNA existed but suggested the possibility of its existence). With Crick's encouragement, Brenner and Jacob immediately set out to test this new hypothesis, and they contacted Matthew Meselson at the California Institute of Technology for assistance. During the summer of 1960, Brenner, Jacob, and Meselson conducted an experiment in Meselson's laboratory at Caltech which was the first to prove the existence of mRNA. That fall, Jacob and Monod coined the name "messenger RNA" and developed the first theoretical framework to explain its function. In February 1961, James Watson revealed that his Harvard-based research group had been right behind them with a series of experiments whose results pointed in roughly the same direction. Brenner and the others agreed to Watson's request to delay publication of their research findings. As a result, the Brenner and Watson articles were published simultaneously in the same issue of Nature in May 1961, while that same month, Jacob and Monod published their theoretical framework for mRNA in the Journal of Molecular Biology. == See also ==