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This article includes two parts:

• Messenger RNA (mRNA) basics that follows
  
• Messenger RNA (mRNA) splicing
  

The "life cycle" of an mRNA in a eukaryotic cell. RNA is transcribed in the nucleus; once completely processed, it is transported to the cytoplasm and translated by the ribosome. At the end of its life, the mRNA is degraded.

Messenger Ribonucleic Acid (mRNA) is RNA that encodes and carries information from DNA during transcription to sites of protein synthesis to undergo translation in order to yield a gene product.

mRNA "life cycle"

The brief life of a 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. Eukaryotic mRNA molecules often require extensive processing and transport, while prokaryotic molecules do not.

Transcription

Main article: Transcription (genetics)

During transcription, RNA polymerase makes a copy of a gene from the DNA to mRNA as needed. This process is similar in eukaryotes and prokaryotes. One notable difference, however, is that eukaryotic RNA polymerase associates with mRNA processing enzymes during transcription so that processing can proceed quickly after the start of transcription. The short-lived, unprocessed or partially processed, product is termed pre-mRNA; once completely processed, it is termed mature mRNA.

Eukaryotic pre-mRNA processing

Main article: Post transcriptional modification

Processing of mRNA differs greatly between eukaryotes and prokaryotes. Prokaryotic mRNA is essentially mature upon transcription and requires no processing, except in rare cases. Eukaryotic pre-mRNA, however, requires extensive processing.

Splicing

Main article: Splicing (genetics)

Splicing is the process by which pre-mRNA is modified to remove certain stretches of non-coding sequences called introns; the stretches that remain include protein-coding sequences and are called exons. Sometimes pre-mRNA messages may be spliced in several different ways, allowing a single gene to encode multiple proteins. This process is called alternative splicing. Splicing is usually performed by an RNA-protein complex called the spliceosome, but some RNA molecules are also capable of catalyzing their own splicing (see ribozymes).

5' cap addition

Main article: 5' cap

The 5' cap is modified guanine nucleotide is added to the "front" (5' end) of the pre-mRNA using a 5',5-Triphosphate linkage. This modification is critical for recognition and proper attachment of mRNA to the ribosome, as well as protection from 5' exonucleases. It may also be important for other essential processes, such as splicing and transport.

Polyadenylation

Main article: Polyadenylation

Polyadenylation is the covalent linkage of a polyadenylyl moiety to a messenger RNA molecule. In eukaryotic organisms, polyadenylation is the mechanism by which most messenger RNA (mRNA) molecules are terminated at their 3' ends. The poly(A) tail aids in mRNA stability by protecting it from exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Some prokaryotic mRNAs also are polyadenylated, although the poly(A) tail's function is different from that in eukaryotes.

Polyadenylation occurs during and immediately after transcription of DNA into RNA. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, 80 to 250 adenosine residues are added to the free 3' end at the cleavage site. This reaction is catalyzed by polyadenylate polymerase.l

Editing

In some instances, an mRNA will be edited, changing the nucleotide composition of that mRNA. An example in humans is the apolipoprotein B mRNA, which is edited in some tissues, but not others. The editing creates an early stop codon, which upon translation, produces a shorter protein.

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. Mature mRNAs are recognized by their processed modifications and then exported through the nuclear pore.

Translation

Main article: Translation (genetics)

Because prokaryotic mRNA does not need to be processed or transported, translation by the ribosome can begin immediately after the start of transcription. Therefore, it can be said that prokaryotic translation is coupled to transcription and occurs co-transcriptionally.

Eukaryotic mRNA that has been processed and transported to the cytoplasm (i.e. mature mRNA) can then be translated by the ribosome. Translation may occur at ribosomes free-floating in the cytoplasm, or directed to the endoplasmic reticulum by the signal recognition particle. Therefore, unlike prokaryotes, eukaryotic translation is not directly coupled to transcription. l

Degradation

After a certain amount of time, the message is degraded into its component nucleotides, usually with the assistance of RNases. The limited longevity of mRNA enables a cell to alter protein synthesis rapidly in response to its changing needs.

Different mRNAs within the same cell have distinct lifetimes. In bacterial cells, individual mRNAs can survive from seconds to more than an hour; 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 transcript. The presence of AUUUA motifs in some species of mRNA tends to destabilize the transcript through the actions of intracellular binding proteins.

mRNA structure

The structure of a mature eukaryotic mRNA. A fully processed mRNA includes a 5' cap, 5' UTR, coding region, 3' UTR, and poly(A) tail.

5' cap

Main article: 5' cap

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 which 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.

First, the triphosphate at the 5' end of the newly synthesized RNA is cleaved. The enzyme phosphohydrolase cleaves the gamma phosphodiester bonds while leaving the alpha and beta phosphates. Second, the enzyme guanylyltransferase transfers a guanine and its alpha phosphate onto the beta phosphate of the 5' end of the mRNA producing a 5'-5'-triphosphate linkage. Third, the nitrogen-7 (N-7) position of the newly added guanine is methylated (guaninemethylation) by the enzyme guanine-7-methyltransferase. Finally, 2'-O-methyltransferase methylates the 2' position of the ribose sugar. This methyl group provides extra stability to the RNA due to the protection from phosphoester cleavage by nucleophilic attack of the neighbor hydrogen. After the 5' end has been capped, it is released from the cap-synthesizing complex and is subsequently bound by a cap-binding complex associated with RNA polymerase.

Coding regions

Coding regions are composed of codons, which are decoded and translated into protein by the ribosome. Coding regions begin with the start codon and end with the one of three possible stop codons. In addition to protein-coding, portions of coding regions may also serve as regulatory sequences as exonic splicing enhancers or exonic splicing silencers. Dicistronic is the term used to describe an mRNA that encodes for two proteins, usually with a non-coding region in the middle called the intergenic region. It is most common in viral genomes.

Monocistronic versus Polycistronic mRNA

An mRNA molecule is said to be monocistronic when it contains the genetic information to translate only a single protein. This is the case for most of the eukaryotic mRNAs^[1]. On the other hand, polycistronic mRNA carries the information of several proteins, which are translated into single proteins. Most of the mRNA found in prokaryotes is polycistronic^[1].

Untranslated regions

Main articles: 5' UTR and 3' UTR

Untranslated regions (UTRs) are sections of the RNA 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 as part of the same transcript as the coding region. Several roles in gene expression have been attributed to the untranslated regions, including mRNA stability, mRNA localization, and translational efficiency. The ability of a UTR to perform these functions depends on the sequence of the UTR and can differ between mRNAs.

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.

Cytoplasmic localization of mRNA is thought to be a function of the 3' UTR. Proteins that are needed in a particular region of the cell can actually be translated there; in such a case, the 3' UTR may contain sequences that allow the transcript to be localized to this region for translation.

Translational efficiency, and even inhibition of translation altogether, can be mediated by UTRs. Proteins that bind to either the 3' or 5' UTR may affect translation by interfering with the ribosome's ability to bind to the mRNA.

Some of the elements contained in untranslated regions form a characteristic secondary structure when transcribed into RNA. These structural mRNA elements are involved in regulating the mRNA. Some, such as the SECIS element, are targets for proteins to bind. 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.

3' poly(A) tail

Main article: Polyadenylation

The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the "tail" or 3' end of the pre-mRNA through the action of an enzyme, polyadenylate polymerase. The poly(A) tail is added on to the transcripts that contain a specific sequence, the AAUAAA signal. The importance of the AAUAAA signal is demonstrated by a mutation in the human alpha 2-globin gene which mutates the original sequence AATAAA into AATAAG, which can lead to hemoglobin deficiencies.^[2]

Anti-sense mRNA

During transcription, double stranded DNA produces mRNA from the sense strand; the other, complementary, strand of DNA is termed anti-sense. Anti-sense mRNA is an RNA complementary in sequence to one or more mRNAs. In some organisms, the presence of an anti-sense mRNA can inhibit gene expression by base-pairing with the specific mRNAs. In biochemical research, this effect has been used to study gene function, by simply shutting down the studied gene by adding its anti-sense mRNA transcript. Such studies have been done on the worm Caenorhabditis elegans and the bacterium Escherichia coli. This plays a part in RNA interference.

See also

• Non-coding RNA

References

1. ^ ^a b Kozak, M. (March 1983). "Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles." (PDF). Microbiological Reviews 47 (1): 1-45. PubMed. Retrieved on 2006-08-12.
2. ^ Higgs DR, Goodbourn SE, Lamb J, Clegg JB, Weatherall DJ, Proudfoot NJ. (1983). "α-thalassaemia caused by a polyadenylation signal mutation". Nature 306 (5941): 398–400. PMID 6646217 DOI:10.1038/306398a0.

External links

• Life of mRNA Flash animation

In genetics, splicing is a modification of genetic information after transcription, in which introns are removed and exons are joined. Splicing prepares precursor messenger RNA in eukaryotes to produce mature messenger RNA. This mature messenger RNA is then prepared to undergo translation as part of protein synthesis to produce proteins. Splicing occurs by a series of biochemical reactions between RNA nucleotides, which are catalyzed by proteins, RNA, or both.

Simple illustration of exons and introns in pre-mRNA. The mature mRNA is formed by splicing.

Splicing pathways

Several methods of RNA splicing occur in nature. The type of splicing depends on the structure of the spliced intron and the catalysts required for splicing to occur. Regardless of which pathway is used, the excised introns are discarded.

Spliceosomal

Spliceosomal introns often reside in eukaryotic protein-coding genes. Within the intron, a 3' splice site, 5' splice site, and branch site are required for splicing. Splicing is catalyzed by the spliceosome which is a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs, pronounced "snurps"). The RNA components of snRNPs interact with the intron and may be involved in catalysis. Two types of spliceosomes have been identified (the major and minor) which contain different snRNPs.

• Major

The major spliceosome splices introns containing GU at the 5' splice site and AG at the 3' splice site. It is composed of the U1, U2, U4, U5, and U6 snRNPs.

U1- binds 5' splice site U2- binds the branch U4- inhibits U6, lost to activate spliceosome U5 - binds U1 and U2 to create lariat U6 - When, activated, displaces U1 and binds U2. U2-U6 forms active catalytic complex

• Minor

The minor spliceosome is very similar to the major spliceosome, however it splices rare introns with different splice site sequences. Here, the 3' and 5' splice sites are AU and AC, respectively. While the minor and major spliceosomes contain the same U5 snRNP, the minor spliceosome has different, but functionally analogous snRNPs for U1, U2, U4, and U6, which are respectively called U11, U12, U4atac, and U6atac. ^[1]

• Trans-splicing

Trans-splicing is a form of splicing that joins two exons that are not within the same RNA transcript.

Self-splicing

Self-splicing occurs for rare introns that form a ribozyme, performing the functions of the spliceosome by RNA alone. There are two kinds of self-splicing introns, Group I, and II. Group I and II introns perform splicing similar to the spliceosome without requiring any protein. This similarity suggests that Group II and III introns may be evolutionarily related to the spliceosome. Self-splicing may also be very ancient, and may have existed in an RNA world that was present before protein. Although the two splicing mechanisms described below do not require any proteins to occur, 5 additional RNA molecules and over 50 proteins are used and hydrolyzes many ATP molecules. The splicing mechanisms use ATP in order to accurately splice mRNA's. If the cell were to not use any ATP's, the process would be highly inacurrate and many mistakes would occur. Two transesterfications characterize the mechanism in which group I introns are sliced: 1) 3'OH of a free guanine nucleoside (or one located in the intron) or a nucleotide cofactor (GMP, GDP, GTP) attacks phosphate at the 5' splice site. 2) 3'OH of the 5'OH becomes a nucleophile and the second transesterfication results in the joining of the two introns. The mechanism in which group II introns are spliced (two transesterfication reaction like group I introns) is as follows: 1)The 2'OH of a specific adenosine in the intron attacks the 5' splice site, thereby forming the lariat 2) The 3'OH of the 5' exon triggers the second transesterfication at the 3' splice site thereby joining the exons together.

tRNA splicing

tRNA (also tRNA-like) splicing is another rare form of splicing that usually occurs in tRNA. The splicing reaction involves a different biochemistry than the spliceomsomal and self-splicing pathways. Ribonucleases cleave the RNA and ligases join the exons together. This form of splicing does also not require any RNA components for catalysis.

Evolution

Splicing occurs in all the kingdoms or domains of life, however, the extent and types of splicing can be very different between the major divisions. Eukaryotes splice many protein-coding messenger RNAs and some non-coding RNAs. Prokaryotes, on the other hand, splice rarely, but mostly non-coding RNAs. Another important difference between these two groups of organisms is that prokaryotes completely lack the spliceosomal pathway.

Because spliceosomal introns are not conserved in all species, there is debate concerning when spliceosomal splicing evolved. Two models have been proposed: the intron late and intron early models (see intron evolution).

Biochemical mechanism

Diagram illustrating the two-step biochemistry of splicing

Spliceosomal splicing and self-splicing involves a two-step biochemical process. Both steps involve transesterification reactions that occur between RNA nucleotides. tRNA splicing, however, is an exception and does not occur by transesterification.

Spliceosomal and self-splicing transesterification reactions occur in a specific order. First, a specific branch-point nucleotide within the intron reacts with the first nucleotide of the intron, forming an intron lariat. Second, the last nucleotide of the first exon reacts with the first nucleotide of the second exon, joining the exons and releasing the intron lariat.

Computer programs to find splicing sites in genomic DNA sequences

Alternative splicing

Main article: Alternative splicing

In many cases, the splicing process can create many unique proteins by variations in the splicing of the same messenger RNA. This phenomenon is called alternative splicing.

Experimental manipulation of splicing

Splicing events can be experimentally altered^{[2], [3]} by binding steric-blocking antisense oligos such as Morpholinos or PNAs to snRNP binding sites, to the branchpoint nucleotide that closes the lariat, or to splice-regulatory element binding sites^[4].

Splicing errors

Mutations in the introns or exons can prevent splicing and thus may prevent protein biosynthesis.

Common errors:

• Mutation of a splice site resulting in loss of function of that site. Results in exposure of a premature stop codon, loss of an exon, or inclusion of an intron.
• Mutation of a splice site reducing specificity. May result in variation in the splice location, causing insertion or deletion of amino acids, or most likely, a loss of the reading frame.
• Transposition of a splice site, resulting in inclusion or exclusion of more DNA than expected. Results in longer or shorter human-goat hybrid exons.

References

1. Patel, A.A. and J.A. Steitz, Splicing Double: Insights from the Second Spliceosome. Nature Reviews Molecular Cell Biology, 2003. 4(12): p. 960.
2. Draper BW, Morcos PA, Kimmel CB. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown. Genesis. 2001 Jul;30(3):154-6.
3. Sazani P, Kang SH, Maier MA, Wei C, Dillman J, Summerton J, Manoharan M, Kole R. Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res. 2001 Oct 1;29(19):3965-74.
4. Bruno IG, Jin W, Cote GJ. Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements. Hum Mol Genet. 2004 Oct 15;13(20):2409-20. Epub 2004 Aug 27.

See also

• cDNA
• Exon
• Intron
• Primary transcript
• Spliceosome
• Minor spliceosome