Structural Biochemistry/Nucleic Acid/RNA/Messenger RNA (mRNA)

Messenger ribonucleic acid (mRNA) is the blueprint of protein reproduction. Transcribed from deoxyribonucleic acid (DNA), mRNA transfers genetic information from the cell nucleus into the protein-producing ribosomes located in the cytoplasm. Similar to DNA, the genetic information is encoded in four nucleotides that are arranged in codons, or triplets of nucleotide bases. Each codon corresponds to a specific amino acid, and the sequence of codons ends with a codon that has a stop signal. The protein synthesis process requires transfer RNA (tRNA) and ribosomal RNA (rRNA). mRNA makes up only about 5% of the different types of RNA found in both Prokaryotic and Eukaryotic cells.



Transcription
During transcription, an RNA strand is copied by an enzyme, RNA polymerase. RNA is then synthesized in the 5' to 3' direction, as is also done in DNA replication. The template of the two DNA strands is the one in which the RNA is synthesized. RNA polymerase binds to the 3' end and replicates via phosphodiester bonds.

The obvious difference between DNA and mRNA in this stage is in the uracil (U) that is present in RNA instead of thymine (T) in DNA.

The RNA first transcribed from the DNA is known as pre-messenger RNA (pre-mRNA) since the exact copy of the DNA region contains both introns and exons. Messenger RNA contains only exons. Introns are removed via splicing by spliceosomes, which recognize intronic sequences based on a GU beginning, a long pyrimidine chain, and an AG ending. Only exons remains in mRNA mainly because it contains useful genetic information for translation - producing a protein. Introns, however, do not provide useful genetic information.

caps and PolyA tails are added as modification to protect the active ends of mRNA after transcription and before translation.

Pre-mRNA
In eukaryotes, the product of transcription of a protein-coding gene is pre-mRNA which requires processing to generate functional mRNA. Several processing reactions occur.

5'processing: capping
Very soon after it has been synthesized by RNA polymerase II, the 5' end of the primary RNA transcript, pre-mRNA, is modified by the addition of a 5' cap(a process known as capping). This process involves the addition of 7-methylguanosine(m 7 G) to the 5'end. To achieve this, the terminal 5' phosphate is first removed by a phosphatase. Guanosyl transferase then catalyzed a reaction whereby the resulting diphosphate 5' end attacks the α phosphorus atom of a GTP molecule to add a G residue in an unusual 5'5' triphosphate link. The G residue is then methylated by a methyl transferase adding a methyl group to the N-7 position of the guanine ring, using S-adenosyl methionine as methyl donor. The ribose of the adjacent nucleotide (nucleotide 2 in the RNA chain) or the riboses of both nucleotides 2 and 3 may also be methylated to give cap 1 or cap 2 structures respectively. In these cases. the methyl groups are added to the 2'-OH groups of the ribose sugars.

The cap protects the 5' end of the primary transcript against attack by ribonucleases that have specificity for 3'5' phosphodiester bonds and so cannot hydrolyze the 5'5' bond in the cap structure. In addition, the cap plays a role in the initiation step of protein synthesis in eukaryotes. Only RNA transcripts from eukaryotic protein-coding genes become capped; prokaryotic mRNA and eukaryotic rRNA and tRNAs are uncapped.

Splicing
RNA splicing is a key step in RNA processing because it precisely remove the intron sequences and join the ends of neighboring exons to produce a functional mRNA molecule. The exon-intron boundaries are marked by specific sequences. In most cases, at the 5' boundary between the exon and the intron(the 5' splice site), the intron starts with the sequence GU and at the 3'exon-intron boundary (the 3' splice site) the intron ends with the sequence AG. Each of these two sequences lies within a longer consensus sequence. A polypyrimidine tract (a conserved stretch of about 11 pyrimidines) lies upstream of the AG at the 3' splice site. A key signal sequence is the branchpoint sequence which is located about 20-50 nt upstream of the 3' splice site. In vertebrates this sequence is 5'-CURAY-3' where R=purine and Y=pyrimidine (in yeast this sequence is 5'-UACUAAC-3'). RNA splicing occurs in two steps. In the first step, the 2'-OH of the A residue at the branch site attacks the 3'5' phosphodiester bond at the 5' splice site causing that bond to break and the 5' end of the intron to loop round and form an unusual 2'5' bond with the A residue in the branchpoint sequence. Because this A residue already has 3'5' bonds with its neighbors in the RNA chain, the intron becomes branched at this point to form what is known as a lariat intermediate (named as such since it resembles a cowboy's lasso). The new 3'-OH end of exon 1 now attacks the phosphodiester bond at the 3' splice site causing the two exons to join and release the intron, still as a lariat. In each of the two splicing reacitons, one phophate-ester bond is exchanged for another (i.e. these are two transesterification reactions). Since the number of phosphate-ester bond is unchanged, no ATP is consumed.

3' processing:cleavage and polyadenylation
A majority of eukaryotic pre-mRNAs undergo polyadenylation which involves cleavage of the RNA at its 3' end and the addition of about 200A residues to form a poly(A)tail. The cleavage and polyadenylation reactions require the existence of a polyadenylation signal sequence (5'-AAUAAA-3') located near the 3' end of the pre-mRNA followed by a sequence 5'-YA-3' (where Y=a pyrimidine), often 5'-CA-3', in the next 11-20 nt. A GU-rich sequence (or U-rich sequence) is also usually present further downstream. After these sequence elements have been synthesized, two multisubunit proteins called CPSF (cleavage and polyadenylation specificity factor) and CStF (cleavage stimulation factor F) aretransferred from the CTD of RNA polymerase II to the RNA molecule and bind to the sequence elements. A protein complex is formed which includes additional cleavage factors and an enzyme called poly(A) polymerase (PAP). This complex cleaves the RNA between the AAUAAA sequence and the GU-rich sequence. Poly(A) polymerase then adds about 200A residues to the new 3' end of the RNA molecule using ATP as precursor. As it is made, the poly(A) tail protects the 3' end of the final mRNA against ribonuclease digestion and hence stabilizes the mRNA. In addition, it increases the efficiency of translation of the mRNA. However, some mRNAs, notably histone pre-mRNAs, lack a poly(A) tail. Nevertheless, histone pre-mRNA is still subject to 3' processing. It is cleaved near the 3' end by a protein complex that recognizes specific signals, one of which is a stem-loop structure, to generate the 3'end of the mature mRNA molecule.

The primary RNA transcript that continues to be synthesized includes both coding(exon) and noncoding(intron) regions. The latter need to be removed and the exon

Transport
In Eukaryotic cells, following synthesis, mRNA typically goes through a series of modifications before being exported to the cytoplasm for translation. These modifications include a 5’ guanine capping and a polyadenylation at the 3' end. This strand of Adenine residues (anywhere from 80-250) is called the Poly-A tail and is needed for the export, protection, translation, and stability of the mRNA. Splicing, the process in which introns are removed and exons are joined, also occurs before exportation.

After all the proper modifications have been carried out, the mature mRNAs are ready to be exported through the nuclear pore into the cytoplasm. Nuclear pores are the channels between the nucleus and cytoplasm, and is a selective barrier that allow macromolecule transportation. Alternate splicing patterns of introns allows the same gene to express in a slightly different way in mRNA creating a different, but similar protein. In order for the mature mRNAs to be carried out, first, the formation of the messenger ribonucleoprotein (mRNPs) export complex with RNA binding proteins and transport factors (carriers) must occur since Mex67-Mtr2 heterodimer, the principal mRNA carrier, binds loosely to bulk mRNA.

Nuclear Transport


Nuclear export is a pathway unique to eukaryotic cells because the nuclear and cytoplasmic compartments within the eukaryotic cells enables spatial separation of the two processes, transcription and translation. The separation between the two processes allows for multiple steps in between for further modification and gene expression regulation, which becomes vital for physiological responses to extra- and intracellular signals.

mRNA nuclear export can be simplified to three stages:


 * 1) the pre-mRNA is transcribed in the nucleus, the site of mRNA synthesis, processing, and packing into mRNP (messenger ribonucleoprotein) complexes (as briefly described earlier)
 * 2) the mRNP molecules are targeted to and translocated through the nuclear pore complexes (NPC) of the nuclear envelope
 * 3) the mRNPs are released into the cytoplasm for translation to occur. Each of these stages involves numerous protein factors and other molecules that need to be recruited to carry out processes.

Formation of mRNP in yeasts:

 * 1) In the nucleus, transcription is mediated through RNA polymerase II. This is followed by modifications like the addition of the 5’ cap, splicing, and 3’ processing. The TREX complex is recruited during these processes and coordinates many of the next steps.
 * 2) The 3’ end processing is necessary because it generates the poly-A-tail which is crucial for the mRNA to be exported. This process requires the factors Rna14, Rna15 and Pcf11. Nab 2 is added onto the poly-A-tail mRNA then recruits Yra1 and Sub 2 during this time. When mRNA is in contact with Pcf11, Yra1 is transferred to the TREX subunit Sub2. (Yra1-Pcf11 binding is an important early step). Yra1 is necessary
 * 3) The MEx67-Mtr2 heterodimer is drafted.
 * 4) mRNPs can now be remodeled by tha DEAD-box helicase Sub2
 * 5) Yra1 dissociates itself from mRNP before export, along with the TREX complex.
 * 6) mRNP is drawn to the nucleus side of the NPC transport channel, where weak interactions arise with FG nucleoporins (proteins that perforate the nuclear pore). To increase the efficiency of export, several mechanisms exist to concentrate mRNAs at the nucleus side of the NPC. Eg: several actively transcribing genes like GAL1 are concentrated at the NPC.
 * 7) mRNP goes through the NPC transport channel, to the cytoplasmic side, where is once again goes through remodeling to prevent from going back into the nucleus.

Cofactors Involved


The NPCs itself have very essential proteins that facilitate mRNA nuclear export. Within the NPC, there is a conical, basket-like feature that protrudes into the nucleus called the nuclear basket. It contains proteins like Nup 60 Nup2, and Mlp2. The cytoplasm similarly has proteins that are cofactors to the export process (Nup1259, Dbp5, Gle1). There are several other key proteins and components of mRNA export that will not be discussed, but they the references for this page will provide much more insight on the specific functions of these export factors.

Here is a short summary of the principle export factors for yeast and metazoans:


 * Mex67-Mtr2 (yeast) and Nxf1-nxt1 (metazoan): facilitate bulk mRNA export through NPCs
 * Yra1 (yeast) and ALY (metazoan): Adaptor linking Mex67-Mtr2 to mRNA molecule
 * Sub2 (yeast) and UAP56 (metazoan): DEAD-box helicase involved in assembly of export-competent mRNPs
 * Nab2 (yeast): Binds the poly (A) til of mRNA to Mlp1 and regulates length of the 3' poly (A) tail
 * Mlp1 (yeast) and (TPR): Nuclear basket protein that binds to Nab2
 * TREX (both yeast and metazoan): The complex involved in coordinating and regulating transcription
 * TREX-2 (both): directs actively expressing genes to NPCs
 * Gle1 and Gdf1 (yeast) and GLE (metazoan): enhances Dbp5 activity
 * Nup159 (yeast) and NUP214 (metazoan): cytoplasmic NPC protein that binds to Dbp5

Recruitment
Recruiting these factors is an essential step for the trafficking and quality control of the export. Most molecules that need to be transported from the nucleus into the cytoplasm involve karyopherin-mediated receptors, like small mRNA export. Its transport direction is based on the gradient of the GTP-bound state of the small GTPase Ran, making the mRNA export process uncharacteristic of normal protein export such as tRNA. Bulk mRNA is exported using Mex67-Mtr2, a non-karyopherin-mediated receptor, via the Nxfl pathway. The Mex67-Mtr2 molecule is recruited to the mRNP using the TREX component. Furthermore, recent works in vertebres shows that the binding of the Yra1 homologue, ALY, to mRNA is stimulated by the presence of the ATP bound form of the Sub2 homologue UAP56. This binding increases the ATPase activity of UAP56. Moreover, Nxf1 binds mRNA associated ALY, forming a ternary complex, and the RNA-binding affinity of Nxf1 is increased in the presence of ALY. Taken together, the events result in an mRNP with bount export receptors. But it is unclear how many receptors must bind a single mRNA for efficient export to occur.

Bulk mRNA Export Pathway


The Nxfl pathway involves a small set of transcripts that are exported via karyopherin Crm1, a protein that also mediates the export of incompletely spliced mRNA from HIV viruses. Therefore, if an mRNA molecule is not properly processed and spliced of its introns, it can be kept in the nucleus to degrade since it is recognized as a viral mRNA molecule. When the mRNP and mRNA are properly processed and have recruited all the necessary receptors and cofactors, it is considered export ready (export competent). The export-competent mRNP is then targeted only to the NPC using its recruited export receptor. The export receptor carries the mRNP to the NPC where it stays and interacts with the NPC proteins to allow recognition. The interactions can be nicely summarized in the figure below.

Bulk Release into Cytoplasm
The directionality of the bulk mRNA release is determined by another mechanism since it does depend on the RanGTP gradient for small mRNA export. It is determined by the function of two important export factors, Dbp5 and Gle1. The Dbp5 protein binds to the NPC cytoplasmic face by interacting with the NPC protein Nup214 As the mRNP comes closer to the cytoplasmic side of the NPC, it interacts with Dbp5 and Gle1. The binding and interactions between mRNP and the two proteins causes a conformational change and activates the removal of a set of proteins from the mRNP. It physically and spatially changes the mRNP making it suitable to be exported out of the NPC into the cytoplasm. These removed proteins are recycled and brought back into the nucleus where it goes through another cycle of mRNA export. In addition, as the mRNP enters the cytoplasm, specific cytoplasmic mRNA-binding proteins are incorporated. These specific links to translation further show the inherent connections between steps in gene expression.

Translational Significance
Since mRNA export is essential for proper gene expression, this process must be properly conducted. Incorrect steps in this export can lead to errors in transcription, and consequently translation. For example, errors in recruiting export factors can lead to incorrect mRNA production, and if the transcript is not recognized by nuclear surveillance the mRNA may be kept inside the nucleus and degraded by exosomes and various other enzymes. Errors in mRNA export can also be linked to many human diseases and developmental issues. Incorrect mRNA export are connected to perturbations that yield mutations in gene encoding export proteins or mRNA-binding proteins as well as mutations in genes that result in the inhibition of correct export of their own mRNA transcripts. Extreme cases also include the decreased regulation or hijacking of endogenous mRNA export complexes by viruses, which enables specific viral genes to hybridize with the mRNA transcript and be expressed in the organism. But with the vast knowledge of the mRNA export process, these malfunctions can be better understood and more easily preventable, and it may be possible to address many issues of diseases and gain a complete understanding of the way cellular function is generated at the simplest level: molecularly.

Translation
In prokaryotes, because the mRNA does not need to be modified or transported, it can be translated by the ribosomes right after transcription.



In eukaryotes, however, mRNA can only be translated after it has been modified and transported to the cytoplasm (the mature mRNAs). mRNA is translated into proteins on the ribosomes located on the endoplasmic reticulum. Translation starts by the ribosomes binding to a site on the 5' side. The ribosome moves along the mRNA until it comes across the start codon AUG. When this binding occurs, the ribosome is joined by an initiator tRNA that carries a formylmethionine (fMet) group that recognizes the start codon. Next, an aminoacyl-tRNA that can base pair with the next codon appears and joins the ribosome complex. Along with the aminoacyl-tRNA is the elongation factor EF-Tu (in bacteria) and a source of energy (usually GTP). The fMet (in bacteria) or Met group covalently bonds to the incoming amino acid of the aminoacyl-tRNA. The initiator tRNA is then released and the ribosome shifts one codon toward the 3' end. A new aminoacyl-tRNA arrives and the amino acid of this aminoacyl-tRNA binds to the previous amino acid. This process continues until the ribosome reaches a stop codon (UAA, UAG, or UGA). The newly bound amino acids are the translated mRNA into a protein. The ribosomal complex containing the tRNA splits back up into its separate parts, re-assembling when new mRNA needs to be translated into protein.

The elongation process "terminates" when a stop codon reaches the A site of the ribosome. Incoming tRNA, which carries the subsequent amino acid, will not be accepted by the ribosome at the A site. The A site will then be specific to a protein called the release factor. The release factor will hydrolyze the bond of the tRNA to the polypeptide in the P site, thus releasing the polypeptide chain. The two ribosomal subunits, release factor, and mRNA then come apart to signify the end of the termination process.


 * Stop Codon - A stop codon implies a sequence of three nitrogenase bases in the mRNA that signifies the termination of polypeptide elongation, or translation. The amino acid sequence is then released from the mRNA template to form its final 3D conformation.



Editing
An mRNA can be changed its nucleotide composition in some instances. This process is called editing. In human, the apolipoprotein mRNA is one of the cases. This editing mRNA takes place in some tissues, but not all of them. In this edition, the mRNA's codon is given an early stop, therefore, it will produce a shorter protein when going to the translation process.

Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies.

Degradating
The message transported through mRNA after a certain amount of time will be degraded and be deleted. This process is called degradation. The cell can easily and quickly changed the protein production in case of any changing needs due to the lifetime of the mRNA. The lifetime of different types of mRNA can be different.The life span of mRNA molecules in the cytoplasm is an important key in determining the pattern of protein synthesis within a cell. Prokaryotic mRNA molecules often are degraded by enzymes within a few minutes of their synthesis and this is one reason as to why prokaryotes can vary their patterns of protein synthesis so quickly in response to changes in their environment. Eukaryotic mRNA, on the other hand, typically survives for hours, days, or for some instances, weeks. One example of multicellular mRNA is hemoglobin polypeptides which, in the process of developing red blood cells which are unusually stable, these long-lived mRNAs are translated repeatedly in the cell. Research done on yeasts suggest that a common pathway for mRNA degradation begins with the enzymatic shortening of the poly-A tail which helps trigger the action of enzymes that remove the 5’ cap. This removal of the 5’cap end is crucial as it is regulated by particular nucleotide sequences in the mRNA. Once the cap is removed, nuclease enzymes can then move in and rapidly chew up the mRNA. This process of mRNA degradation relies on deadenylation. The shortening of poly-A tail is initiated by deadenylase and afterward, mRNA is either fully degraded or stored in the case of certain cells.

Another mechanism that blocks expression of specific mRNA molecules known as MicroRNA (miRNA) or miRNAs have also become of interest. They are formed from longer RNA precursors that fold back on themselves, forming a long, double-stranded hairpin structure held together by hydrogen bonds. These small singled stranded RNA molecules can bind to complementary sequences in mRNA molecules and an enzyme, called the Dicer, can then cut the double-stranded RNA molecules into short fragments. One of the two strands is degraded and then the other stand, often the miRNA associates with a large protein complex and which allows the complex to bind to any mRNA molecule with a complementary sequence to either degrade or block translation of mRNAs.

Scientists also observed that gene expression inhibited by RNA molecules was possible. This was observed when they noticed that injecting double stranded RNA molecules into a cell somehow turned off a gene with the same sequence. Scientists called this phenomenon RNA interference or Interference RNA (RNAi). It was later discovered that this interference was due to small interfering RNAs (siRNAs) which are RNAs of similar size and function as miRNAs. Researched showed that the cellular machinery for making siRNAs was the same mechanism for creating miRNAs in the cell. The mechanisms by which these small RNAs function are also the same. Because the cellular RNAi pathway can lead to the destruction of RNA sequences complementary to themselves, it is believed that they originally acted as a natural defense against infection by RNA viruses.