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12. Beskrive (kortfattet) følgende for mRNA: splicing (fjernelse af introns), forekomst af alternativ og ukkorekt splicing (og hvordan alternativ splicing udvider repertoiret af genprodukter, mens inkorrekt splicing kan medføre sygdom), 5'-capping og 3'-polyadenylering. Angive kodende og ikke kodende sekvenser

 

Perhaps the most extensively modified transcription product in eukaryotes is the one of RNA polymerase II – mRNA. The immediate product of the RNA polymerase II is referred to as primary transcript or pre-mRNA.

 Pre-mRNA is modified before it leaves the nucleus. The enzymes that participate in this modification are recruited by RNA polymerase during the transcription process. RNA polymerase II contains a C-terminal domain that is extensively phosphorylated when the nascent RNA is about 25 nucleotides long. The phosphorylated C-domain recruits the capping enzyme, splicing and polyadenylation complexes to the transcript. This means that all the modifications are made during transcription. mRNA gets a cap on the 5’-end, a tail of ca. 250 adenylate residues on the 3’-OH and all the introns are spliced out.

 

5´-Capping
 Stryer, s. 798; fig. 28.24
Devlin, s.225

 
 As the transcription complex moves along the DNA, the capping enzyme complex modifies the 5`-end of the nascent RNA. The 5`-end of the DNA has a triphosphate-end which releases a phosphate by hydrolysis. The diphosphate-end then attacks the a phosphate atom of a GTP to form a very unusual 5´-5´triphospahte linkage. This distinctive terminus is called a cap. 

The N-7 terminus of the guanine is additionally methylated (cap 0). The adjacent riboses can also be methylated (cap 1 and 2). The latter methylation occurs by adding -CH3 on the 2'-OH group of ribose.

Function of caps:

 

Splicing
 Stryer, s. 799-801, fig. 28.29
Devlin, s.226-5, fig. 5.14

Splicing – the process in which introns (intervening sequences) in the pre-mRNA are excised and the exons (expressing sequences) are linked together. Spicing must be exquisitely sensitive; a one nucleotide slippage point in a splice site would shift the entire reading frame and gives an entirely different and therefore non-functional protein.

Three sequences are necessary for the splicing process:

 

The splicing principle is the following:

 

  1. The 2’-OH group of the A-residue of the branch site attacks the 5’-splice point on the beginning of the intron, and cleaves the phosphodiester bond between the upstream exon and the GU sequence of the intron.
     

  2. A 2’-5’ phosphodiester bond is formed between the A-residue and the GU of the intron by transesterification, meaning that the phosphodiester bond that used to join the end of the exon and the beginning of the intron now joins the beginning of the intron and its branch site.
     

  3. This leaves the 3’-OH group of the upstream exon free. This group attacks the phosphodiester bond between the intron (the AG-sequence) and the first nucleotide of the downstream exon (3’-splice point).
     

  4. The two exons are joined by a transesterification reaction, while the intron is released with its 5’-splicing point bound to the branch site and a free 3’-splice point.

Thus the splicing process consists of two transesterifications and uses no energy since the number of phosphodiester bonds stays the same.

 

Incorrect splicing
Devlin, s.227, fig. 5.16
Devlin, s.223; s.224 - cc.5.4

 

Mutations in the splicing points cause human diseases. A mutation in the invariant sequences on the beginning and in the end of an intron means that the correct splice junction can not be recognised. This leads to shifting of the reading frame, and an incorrect and non-functional protein.

Fx. if there is a mutation in the intron-start sequence, GU, then the splicing won’t happen because the exon-intron junction can not be recognized. The upstream exon is going to be longer, meaning the protein is going to have extra amino acids and probably be non-functional.

If there is a mutation in the intron stop sequence, AG, the splicing won’t happen here, so the downstream exon is going to be removed until a accidental AG sequence, where the upstream exons is going to be connected. The mRNA is shorter; the protein is missing amino acids.

It is also possible that a stop codon in the mRNA is encountered and the protein is terminated sooner.

Mutations that interfere with introns removal are a major cause of human diseases, fx. b-talasemia.

 

 

Alternative splicing
 Stryer, s.803, fig. 28.33
Devlin, s.227, fig. 5.17

 

Alternative splicing – inclusion of different exons in the mature mRNA. There is only one pre-mRNA, but after the alternative splicing and the translation of the mature mRNA, distinct forms of proteins for specific tissues or developmental stages can be produced.

It is a widespread mechanism for generating protein diversity.

fx.
 A typical example is tropomyosin proteins which are found in muscles. Each type of muscle has its own tropomyosin protein, even though a single gene is transcribed into primary transcript in all cells. Each cell type processes the primary transcript in a characteristic fashion.

 Recent evidence suggests that 30% of human genes are alternatively spliced.  

 

Polyadenylation
 Stryer, s. 798
Devlin, s. 226, fig.5.15

 

Pre-mRNA is also modified at the 3-end. Most eukaryotic mRNAs contain a polyadenylate - poly (A) tail at the end, added after transcription has finished. 

The enzyme that creates this tail is called poly (A) polymerase. This is the principle:

 

The role of polyadenylation:

 

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