Lecture 8 (2015)Apunte Inglés
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Ingrid Guarro Marzoa
Lecture 8-RNA splicing and processing:
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 a eukaryotic gene is often
only a small fraction of the length of the gene.
Both type of sequences are transcribed into RNA but the intron sequences are removed from the newly synthesized RNA through the process of RNA splicing.
From de 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 not 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 from the nucleus to the cytoplasm. Here the mRNA is going to be translated into proteins thanks to the ribosomes.
• Splicing reaction: The splicing reaction cuts the intron-exon junction on the left and on the right, removing the intron and connecting both exons having one 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.
The splicing machinery must recognize three sequences of the hnRNA: 1. The 5’ splice site. It is also called the donor because it donates the first bond to make the lariat 2. The 3’ splice site. It is also called the acceptor because it accepts the last bond in the splicing reaction.
3. The branch point in the intron sequence. This sequence initiates the base of the excised lariat.
2014/2015 Molecular biology Ingrid Guarro Marzoa 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 pyrimidine, R means purine, and N is any base).
Process of splicing: 1. The phosphodiester bond at the 5’ splice site is broken and a new bond is made between the 2’OH of the adenine of the branch point and the 5’ end of the intron, creating a loop called lariat.
2. The free 3’ end of the exon breaks the junction at the 3’ splice site, which releases the lariat and makes a new phosphodiester bond between both exons.
2014/2015 • Molecular biology Ingrid Guarro Marzoa Spliceosome: 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. These specialized RNAs are known as snRNAs (small nuclear RNAs) and form a complex with at least 40 protein subunits to form the Spliceosome.
So we can define the Spliceosome as a large assembly of RNA and protein molecules that performs pre-mRNA splicing in the cell.
The snRNA job is to 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 the 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 to the cytoplasm after splicing. In the hypothetical case that RNA was exported before splicing occurred, the protein that would be produced afterwards would not be functional.
• Differential splicing: Splicing doesn’t follow the same pattern all the time at a particular RNA.
Splicing can produce different type 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 1.000 different proteins from a single gene.
ExampleàBeta-Thalassemia: Sometimes the splicing problems cause slightly modified proteins. This kind of genetic variations don’t cause specific symptoms such as in specific pathologies. Normally from one allele you have the correct protein and on the other there is a slightly change that will slightly affect a particular function.
2014/2015 Molecular biology Ingrid Guarro Marzoa 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 many 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 sequence of a gene can affect strongly the sequence of a protein. The more we look at strange pathologies, the more they seem to be related to splicing process.
• 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.
2014/2015 Molecular biology Ingrid Guarro Marzoa 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 early in very 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 positions 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 limiting nutritional environments, while this didn’t happen in multicellular organisms.
If we look at the way proteins fold, there are regions of the sequences that always fold in the same way. In many proteins what we see is that there’s a relation between introns and particular domains with particular shapes. We see that between the specific structures/domain and the exons there is a correlation. Also, if we compare different proteins that are not related we see 2014/2015 Molecular biology Ingrid Guarro Marzoa that there are regions that have a 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 diversity of our cells.
As introns interrupt the genes, they would increase the chance of 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 chances to mix them.
• Additional modifications of the RNA: o 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 RNA. This is what we call edition.
o Modifications of the 5’ end: -When the 5’ end links to another 5’ end, it forms a phosphotriester link, which can’t be broken.
-Also the nucleotides can be modified by methylation in the group that makes the phosphodiester link. By this methyl groups, the phpsphodiester link can’t be broken.
Both modifications protect RNAs from enzymes that degrade the RNAs as exonucleases.
o Modifications of the 3’ end: In the 3’ end we also find a structure that protects the mRNA 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.