mRNA (Messenger RNA) is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template and carries genetic information that can guide protein synthesis .
Using genes in the cell as a template, after transcribing to generate mRNA according to the principle of base complementary pairing, the mRNA contains the base sequence corresponding to some functional fragments in the DNA molecule as a direct template for protein biosynthesis. Although mRNA only accounts for 2% to 5% of the total RNA of a cell, it is the most diverse and has a very active metabolism. It is the type of RNA with the shortest half-life. It is decomposed within minutes to hours after synthesis.
Introduction
mRNA----Messenger ribonucleic acid
Messenger RNA is the direct template that directs protein biosynthesis. mRNA accounts for 2% to 5% of the total amount of RNA in a cell. There are many types, and their molecular sizes vary greatly.
Messenger RNA (mRNA) is a large class of RNA molecules that transfer genetic information from DNA to the ribosome , where it serves as a template for protein synthesis and determines the amino acid sequence of the peptide chain of gene expression protein products . RNA polymerase transcribes the primary transcript mRNA (called pre-mRNA) into processed mature mRNA, which is translated into protein.
As in DNA, mRNA genetic information is also stored in nucleotide sequences, which are arranged into codons consisting of three base pairs each. Each codon encodes a specific amino acid, with the exception of the stop codon because it terminates protein synthesis.
The process of translating codons into amino acids requires two other types of RNA: transfer RNA (tRNA) and ribosomal RNA (rRNA). tRNA mediates the recognition of codons and provides the corresponding amino acids. rRNA is the core component of ribosomal protein manufacturing machinery.
The existence of mRNA was first proposed by Jacques Monod and François Jacob, and then discovered by Jacob, Sydney Brenner and Matthew Meselson at the California Institute of Technology in 1961.
Prokaryotic and eukaryotic mRNA have different characteristics
Prokaryotic mRNA often exists in the form of polycistrons . Eukaryotic mRNA generally exists in the form of monocistronics .
The transcription and translation of prokaryotic mRNA are generally coupled, and the pre-mRNA transcribed by eukaryotes needs to be post-transcriptionally processed , and processed into mature mRNA and protein combined to generate a message body before starting to work.
The half-life of prokaryotic mRNA is very short, usually a few minutes, and the longest is only a few hours ( except for RNA in RNA phages ). The half-life of eukaryotic mRNA is longer, for example, mRNA in embryos can reach several days.
The structural characteristics of prokaryotic and eukaryotic mRNA are also different. Eukaryotic mRNA has a 5'cap and 3'poly A tail. Prokaryotes do not have such a head-to-tail structure.
Monocistronic and polycistronic mRNA
The mRNA whose translation product is only a single protein chain (polypeptide) is called monocistronic mRNA. Most eukaryotic mRNAs are monocistronic mRNAs. Polycistronic mRNA carries several open reading frames (ORF), and each open reading frame can be translated into a polypeptide. These polypeptides usually have similar functions and usually constitute different subunits of the final complex protein. The DNA fragments corresponding to these polypeptide chains are located in the same transcription unit and share the same pair of start and end points. Most mRNAs in bacteria and archaea are polycistronic .
Circularization of mRNA
In eukaryotes, due to the interaction between eIF4E and poly(A) binding protein, mRNA molecules form a ring structure. Both binding proteins bind to eIF4G to form an mRNA-protein-mRNA bridge. Cyclization promotes the circulation of ribosomes on mRNA, improves translation efficiency, and ensures that only intact mRNA is translated.
Synthesis and processing
The synthesis of mRNA molecules begins with transcription and ends with degradation. Before being translated, eukaryotic mRNA molecules usually require a large amount of processing and transport, while prokaryotic mRNA molecules do not. The eukaryotic mRNA molecule and the surrounding proteins are called messenger RNP.
Transcription
Transcription refers to the process of synthesizing RNA from DNA. During transcription, RNA polymerase copies the DNA of a gene into mRNA as needed. This process is similar in eukaryotes and prokaryotes.
Obviously different from prokaryotes, eukaryotic RNA polymerase binds to mRNA processing enzymes during the transcription process. Therefore, eukaryotic mRNA processing can proceed quickly after transcription begins. The short-lived unprocessed or partially processed transcription product is called pre-mRNA or pre-mRNA; once processed, it is called mature mRNA.
Eukaryotic pre-mRNA processing
The processing of mRNA varies greatly among eukaryotes, bacteria, and archaea. In essence, non-eukaryotic mRNA is mature during transcription and does not require processing except in rare cases. However, eukaryotic pre-mRNA requires extensive processing.
5'end cap: The 5'cap (also known as RNA cap, RNA 7-methylguanosine cap or RNA m7G cap) is a modified guanine nucleotide that is added to the new The "front" of the eukaryotic mRNA produced is the 5'end. The 5'cap is composed of terminal 7-methylguanosine residues, which are connected to the first transcribed nucleotide through a 5'-5'-triphosphate bond. Its existence is essential for the recognition of ribosomes and the protection of mRNA.
3'-end tailing : refers to the covalent attachment of the polyadenylic acid moiety to the mRNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3'end. The Poly A tail and the protein bound to it help protect mRNA from degradation by exonuclease. The 3'-end tailing is also important for transcription termination, export of mRNA from the nucleus and translation. The mRNA in prokaryotes is often tailed by the 3'end, but the poly(A) tail at this time promotes rather than prevents the degradation of the mRNA by exonuclease.
mRNA transport
Another difference between eukaryotes and prokaryotes is the transport of mRNA. Since eukaryotic transcription and translation are carried out in different organelles, eukaryotic mRNA must be exported from the nucleus to the cytoplasm. This process may be regulated by different signal pathways. Mature mRNA is recognized by its processing modification, and is exported to the cytoplasm through the nuclear pore after binding to the cap-binding proteins CBP20 and CBP80 and the transcription/export complex (TREX).
Translation of mRNA
Because prokaryotic mRNA does not need to be processed or transported, the translation of prokaryotic mRNA in the ribosome can begin immediately after the end of transcription. Therefore, it can be said that the translation and transcription of mRNA in prokaryotes are coupled.
The translation of eukaryotic mRNA (i.e. mature mRNA) that has been processed and transported to the cytoplasm occurs in the ribosome floating freely in the cytoplasm, or in the endoplasmic reticulum to which a signal recognition particle is directed. Therefore, unlike prokaryotes, mRNA translation in eukaryotes is not directly coupled with transcription. In some cases, it may even happen that a decrease in mRNA levels is accompanied by an increase in protein levels.
Degradation
Different mRNAs in the same cell have different lifespans (stability). In bacterial cells, a single mRNA can survive for several seconds to more than an hour, but the average life span is 1 to 3 minutes. Therefore, the stability of bacterial mRNA is much lower than that of eukaryotic mRNA. The life span of mammalian cell mRNA ranges from a few minutes to a few days. The higher the stability of the mRNA, the more protein is produced from the mRNA. The limited lifespan of mRNA allows cells to rapidly change protein synthesis in response to their changing needs. There are many mechanisms that can cause degradation of mRNA.
Degradation of prokaryotic mRNA
The degradation of prokaryotic mRNA is the result of the combined action of different ribonucleases including endonuclease, 3'exonuclease and 5'exonuclease. In some cases, small RNA molecules (sRNAs) with a length of tens to hundreds of nucleotides can promote the degradation of specific mRNA by RNase III by base pairing with complementary sequences.
Degradation of eukaryotic mRNA
There is a balance between eukaryotic translation and mRNA decay. The mRNA being translated is bound by ribosomes, eukaryotic initiation factors eIF-4E and eIF-4G, and poly(A) binding protein, and cannot contact the exosomal complex, and the mRNA is protected. The poly(A) tail of the mRNA is shortened by a specific exonuclease, which is targeted to a specific mRNA through a combination of a cis-regulatory sequence on the RNA and a trans-acting RNA binding protein. The removal of the Poly(A) tail destroys the circular loop structure of the mRNA and reduces the stability of the cap-binding complex, resulting in mRNA being degraded by the exosome or uncapping complex. In this way, the inactive mRNA can be quickly degraded, while the actively translated mRNA is not affected.
Small interfering RNA
In metazoans, the small interfering RNA (siRNA) produced by Dicer is integrated into the RNA-induced silencing complex (RISC). The complex contains endonuclease, which cuts the completely complementary mRNA bound to siRNA, and the resulting fragment is then degraded by exonuclease. siRNA is commonly used to block gene function in laboratory cell culture. SiRNA is considered to be part of the virus innate immune system and can be used for defense against double-stranded RNA viruses.
MicroRNA
MicroRNA (miRNA) is a small RNA that is usually partially complementary to the sequence in the mRNA of metazoans. The combination of miRNA and mRNA can inhibit the translation of the mRNA and accelerate the removal of the poly(A) tail, thereby accelerating mRNA degradation.
Extraction, separation and purification
The most significant structural feature of eukaryotic mRNA molecules is the 5'cap structure (m7G) and the 3'Poly(A) tail. There is a Poly(A) tail composed of 20-30 adenylate at the 3'end of most mammalian cell mRNA, usually represented by Poly(A+).
This structure provides a very convenient selectable marker for the extraction of eukaryotic mRNA. This is the theoretical basis for the separation and purification of mRNA by oligo (dT) cellulose or oligo (U) agarose affinity chromatography.
There are many methods for mRNA separation, among which oligo(dT)-cellulose column chromatography is the most effective and has become a routine method. This method takes advantage of the feature that the 3'end of the mRNA contains Poly(A+).
When the RNA flows through the oligo(dT) cellulose column, under the action of the high salt buffer, the mRNA is specifically bound to the column. The mRNA is eluted at the concentration of salt or in the case of low salt solution and distilled water. After two dT fiber columns, higher purity mRNA can be obtained. Oligo (dT) cellulose column for purification of mRNA.