lecture8_RNA SPLICING AND PROCESSING (2015)

Apunte Inglés
Universidad Universidad Internacional de Cataluña (UIC)
Grado Medicina - 1º curso
Asignatura Biologia Molecular
Año del apunte 2015
Páginas 7
Fecha de subida 28/03/2016
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Molecular biology – lecture 8 RNA SPLICING AND PROCESSING 1. INTRUDUCTION The protein coding sequences of eukaryotic genes are typically interrupted by noncoding intervening sequences called introns. This means that our genes are broken up into small pieces of coding sequences called exons interspersed with much longer introns; thus, the coding portion of an eukaryotic gene is often only a small fraction of the length of a gene. Both type of sequences are transcribed into RNA but the intron sequence are removed from the newly synthesized RNA through the process of RNA splicing From the transcription unit an hnRNA is produced, which is the molecule that is going to produce the mRNA after introns are removed. Hn = heterologous nuclear RNA. Heterologous because is no homogeneous and nuclear because it’s in the nucleus. This type of RNA only exists in the nucleus. This hnRNA will be modified in the 3’ and 5’ ends. The most important thing is that introns are removed in the nucleus, so the complete mRNA is already synthesized in the nucleus. Once the mRNA is produced, it’s exported into proteins thanks to the ribosomes.
2. SPLICING REACTION The splicing reaction cuts the intronexon junction on the left and on the right, removing th intron and connecting both exons having on single molecule.
This reaction has to be very accurate because the wrong elimination of an intron can cause the addition of extra amino acids or wrong amino acids to the protein. The elimination or addition of a base changes alters the code for amino acids from the modification point onwards.
Molecular biology – lecture 8 The splicing machinery must recognize three sequences of the hnRNA: 1. The 5’ splice site. It’s also called the donor because it donates the first bond to make the lariat 2. The 3’ splice site. It’s also called the acceptor because it accepts the last bond in the splicing reaction 3. The branch point in the intron sequence. Thi sequence initiates the base of the excised lariat.
As expected, each site has a consensus nucleotide sequence that is similar between introns.
Most of our introns start with GU and end up with AG. In addition to the ends, there is another important sequence in the intron: is the branching site. This site is on the middle of the intron and its sequence can be variable, although the consensus sequence is YNYYRAR (Y means pyramid, R means purine and N is any base).
Process of splicing: 1. In the phosphodiester bond ate the 5’ splice is broken and a new bond is made between the 2’OH of adenine of the branch point and the 5’ end of the intron, crating a loop called lariat 2. The free 3’ end of the exon brakes the junction at the 3’ splice site, which releases the lariat and makes a new phosphodiester bond between both exons 3. SLICEOSOME The machine that makes these reactions is called spliceosome. It contains specialized RNA molecules that recognize the nucleotide sequences that specify where splicing is to occur and also participate in the chemistry of splicing. This specialized RNAs are known as snRNAs (small nuclear RNAs) and from a complex with at least 40 protein subunits to from the spliceosome.
Molecular biology – lecture 8 So we can define the spliceosome as a large assembly of RNA and protein molecules that performs pre-mRNA splicing in the cell.
The sn RNA job is recognize the donor, the acceptor and the branch sequences.
By sequence complementary, the complex recognizes sequences on the target RNA. Of these interactions, the two that are essential are the G and U interactions on the donor site.
This interaction is essential for spliceosome to start splicing.
The splicing process is synchronized with transport of the RNA to the cytoplasm. The spliceosome after doing its job leaves on the junction a small group of proteins that will be recognized by another complex of proteins that will finally drive the mRNA to the cytoplasm. This ensures that the RNA is only exported before splicing occurred, the protein that would be produced afterwards would not be functional.
4. DIFFERENTIAL SPLICING Splicing doesn’t follow the same pattern all the time at a particular RNA. Splicing can produce different types of RNA from the same precursor. By inhibiting one of the exons, you can obtain lots of different combinations and so you can have lots of RNA. With 10 exons you could obtain more than 1000 different proteins from a single gene.
Molecular biology – lecture 8 Example β-thalassemia: Sometimes the splicing problems cause slightly modified proteins. This kind of genetic variations don’t cause specific symptoms such as a specific pathologies. Normally from one allele you have to correct protein and on the other is a slightly change that will affect a particular function.
Globin is the protein that forms hemoglobin in our blood cells. This protein has a very important function in our body.
Thalassemia is a group of illnesses that go from very grave symptoms to very slight ones. It can have very different grades of malfunction. The globin gene has 3 exons and two introns (one of them is very long).
Mutations create different problems in splicing. In the case B of the diagram, in addition to the normal branch site the mutation creates another acceptor site in the intron. This causes two different RNAs: one normal and one that will include more amino acids. This can cause difficulties for the hemoglobin to carry oxygen from lungs to muscles.
In case C, the normal donor site is mutated and the spliceosome will use similar sequences that are around to perform the splicing reaction. This is called “activation of cryptic splice sites”.
Minor changes in the sequences of a gene can affect strongly the sequence of a protein. The more we look strange pathologies, the more they seem to be related to splicing process.
Molecular biology – lecture 8 5. THE IMPORTANCE OF INTRONS IN OUR GENES We’ve already seen that introns offer some advantages to our organisms allowing the production of different proteins from the same genes.
In nature we can find some splicing reactions that do not depend on proteins, they don’t need the spliceosome. If we look at how this reactions work, we see that what RNA does by folding is bringing together the donor and the acceptor site. Just a folding of the intron itself brings the two sites together. In addition, the branching site will be just behind the junction of the acceptor and the donor, bringing together all the sites involved in splicing. This suggests that introns were present very early in evolution. These introns that are able to fold are only found in some bacteria nowadays.
On the other hand, when we look at those proteins that have been present during the whole evolution we see that the position of introns is usually conserved. The position in which the protein is stopped by introns is very similar in completely different organisms. This suggests that the position of the introns was there before the evolution of species, appearing in the primitive cell (progenitor). We may imagine a primitive intron that contaminated all the genes but could also eliminate itself from the RNA.
In some organisms, introns were removed. Bacteria and unicellular eukaryotes don’t have introns. This is because their genome was reduced because of the competition between species in limitating nutritional environments, while this didn’t happen in multicellular organisms.
Molecular biology – lecture 8 If we look at the protein fold, there are regions of the sequences that always fold in the same way, in many proteins what we see in that there’s a relation between introns and particular domains with particular shapes. We see that between the specific structures /domains and the exons there is a correlation. Also, if we compare different proteins that are not related we see that there are regions that have specific shape and that all proteins share. From this we may conclude that most of our proteins are produced by shuffling their domains. This shuffling could have been produced by non-homologous recombination mechanisms that were facilitated by the presence of introns. Shuffling protein domains is probably the most likely explanation for protein recombination in our cells.
As introns interrupt the genes, they would increase the chance to having a recombination event. The recombination process could take place in many places in a single intron, thus helping exon shuffling. If there were no introns, the chances of recombination right at the boundaries of two domains would be very small. The more space we have to mix domains, the higher the chance to mix them.
6. ADDITIONAL MODIFICATIONS OF THE RNA - Edition: some RNAs suffer changes in their sequences that affect the encoded protein. An enzyme that is found in certain parts of our organism makes this.
Usually the bases that are modified are present in structures that are recognized by enzymes in our DNA.
This is what we call edition.
Molecular biology – lecture 8 - Modifications of the 5’ end: o When the 5’ end links to another 5’ end, it forms the phosphodiester link, which can be broken.
o Also the nucleotides can be modified by methylation in the group that makes the phosphodiester link. By this methyl groups, the phosphodiester groups can be broken.
Both modifications protect RNAs from enzymes that degrade the RNAs as exonucleases.
- Modifications of the 3’ end: in the 3’ end we also find a structure that protects the RNA called polyadenine tail. This is a structure that may have hundreds of adenines, added by a specific enzyme. This enzyme also recruits some proteins that bind it to have RNA ready for translation ...