Post-translational modification (L1) Flashcards
Why are splice sites common in the sequence?
Splice donor and acceptor sites are only 2 bases long so are very frequent. Other sequences and secondary structure of the RNA affects the choice of splice site - so the RNA isn’t spliced at every site.
What is meant by optional spicing?
When in some cells there are introns/exons left in - changes gene expression
What is meant by mutually exclusive splicing?
When 1 of 2 exons in retained in mRNA after splicing but not both. Creates isoforms of mRNAs
What is meant by internal splice sites?
When there is a splice site in the middle of an intron or exon - to keep half of it. Creates isoforms of mRNA
What are cryptic splice sites?
When some cells favour one site but can use other ones as well.
Why are proteins used in splicing?
Proteins that bind round splice sites can influence splicing. Splicing is a simple process and is not very accurate so cells influence it and make it more controlled using proteins. E.g. Descam is an example of a mutually exclusively spliced mRNA. It can be spliced in 38000 different ways (one of the most complex) by choosing differently combinations of groups of exons. A similar process is used to make antibodies, how you can make so many different variants from only a few genes.
How can alternative splicing be regulated? give an example.
Sex determination in Drosophila is one of the best-understood examples of this. Three genes are key regulators of male and female differentiation - Sxl, tra, dsx. In males (with 1 X chromosome) The transcripts for sxl and tra are spliced to give inactive versions of the protein. Dsx transcripts give rise to a male-specific repressor protein, which represses transcription from genes required for female development. Females have 2 X chromosomes, which causes a small amount of functional Sxl proteins to be made (alternative promoter) Sxl represses splicing of itself and Tra by blocking binding of U2AF. This feeds back on the Sxl transcripts to make more of itself and also bind to tra transcripts. This results in the female isoform of Dsx (which has 30 AAs on the end of it) Also makes more positive tra due to changes in its carboxy terminus - this represses the male gene and also allows for female development.
Give an example of where polyadenylation site regulation gives rise to functionally different proteins.
The site of polyadenylation on an mRNA can be regulated (addition of a polyA tail in different places) B lymphocytes produce 2 antibody isoforms - 1 that’s membrane-bound and 1 that can be secreted. This is because the antibody gene has 2 possible positions for cleavage and polyadenylation. Initially, B lymphocytes want to produce antibodies on the membrane, so when they’re cleaved they can be secreted. When the cell produces the long transcript, the first stop codon is spliced out. This results in the translation of a transmembrane domain. When the cell switches to the short transcript, the slice acceptor is lost and the first stop codon is not lost. This results in the antibody being secreted. When the short transcript is cleaved, there’s no longer any hydrophobic domain on the protein (because there’s a stop codon now in the exon) so it can leave the membrane. Alternative endings allow the gene to produce 2 different isoforms which have different functions.
Explain how HIV uses alternative splicing
HIV has a small genome that is integrated into the host genome. After integration, the entire genome is transcribed in one piece, so they make large RNA which contain many proteins. Alternative splicing allows for many different protein products to be made. However, the full-length RNA is needed to make new virions, but unspliced RNAs can’t leave the nucleus. So, to get around this the viruses produce a transporter called Rev which goes back into the nucleus, binds to introns (to protect them from splicing) and escorts RNA to outside the nucleus. Rev levels distinguish between 2 phases of infections
What targets an mRNA to a certain part of the cell?
Signals in the untranslated region (UTR). The UTR is on the 3’ end and often forms stem loops which can be recognised by proteins (form due to complementary base pairs like in tRNA) (Intermolecular base pairings). Subcellular localisation of RNA and RNA binding proteins gives rise to localised translocation.
Give an example of when mRNAs have control elements in their 5’ and 3’ UTRs
Ferritin is a protein that stores iron in the cell thus reducing the available Fe. Transferrin receptor imports iron into the cell and therefore has the opposite action, increasing Fe. When there is low iron in the cytoplasm, no ferritin is made because aconitase binds to the stem-loop int he 5’ UTR of ferritin mRNA and blocks translation
AT the same time, aconitase also binds to the stem loops of the 3’ UTR of transferrin mRNA and blocks its degradation, leading to the overall increase of iron in the cell. When levels of iron are high, aconitase binds to the iron through a conformational change. Aconitase then releases mRNAs which provide a rapid and strong regulation. AT the same time, because aconitase is bound to iron, it can’t bind to transferrin or ferritin, so the ferritin is translated (increasing the store of Fe and reducing the amount in the cytosol, and transferrin is degraded so no ore iron can be imported.
Explain the global regulation of translation by EIF2 and EIF-2B
Eif-2/GTP (eukaryotic initiation factor) binds to Met tRNA to start ribosome scanning (start codon)/ eif-2B is required for the dissociation of GDP from eif-2. If the cell is entering G0 or infected by a virus or lacking nutrition, it turns down global translation by phosphorylating eIF-2. This causes eIF-2B to bind to eIF-2 very tightly, blocking its recycling. IRES allows more than one gene to be present on an mRNA. Internal ribosome entry sites (IRES) are stem-loops in RNA that can initiate the formation of the ribosome-independent of the cap/PolyA initiation complex.eiF-4G is required for the IRES-based initiation and binds to the IRES stem-loop. IRESs are often found in viral transcripts. Viruses favour translation of their transcripts by cleaving eIF-4E, but still binds to IRES. Also, during apoptosis eIF-4G is similarly cleaved. Certain genes required during cell death utilize IRES and they continue to be translated.
Explain how RNA stability contributes to the regulation of fo translation.
RNA stability is a common method of regulating translation. The half-life of different mRNAs varies greatly from many hours to a few minutes. polyA tails start at about 200 in length, but an exonuclease chews them down to 30nt at which point, they are decapped and degraded. However, some mRNAs are re-adenylated in the cytoplasm to activate them or to extend their half-life. Often, factors that promote translation block degradation. DAN competes with eIF-4E for binding to the cap.
