Lecture 16 Flashcards
What is meant by the term isoform
An isoform is a protein variant that differs based on posttranscriptional modifications
What process is used to create different protein isoforms
Alternative splicing
What regions of the immature mRNA transcript are removed during splicing
Introns
Splicing means that different proteins can be created from the same gene, T or F
T
All eukaryotic genes contain introns and exons, T or F
F – yeast do not contain introns
What components of genes allow for alternative splicing to occur
Optional introns and exons, mutually exclusive exons and internal splice sites
40% of the Drosophila genome is alternatively spliced. What percentage of human genes are also spliced
0.75
Other than splice donor and acceptor sites within gene transcripts, what other features of the mRNA allows for alternative splicing
Other sequences contained within the mRNA and the secondary structure also affects the choice of splice sites
How is splicing regulated
By RNA binding proteins
Give an example of a gene that undergoes alternative splicing
Dscam in Drosophila is a very large gene that produces a massive mRNA transcript containing 100 exons. The final mature mRNA will contain one exon from 12 A exons, 48 B exons, 33 C exons and 2 D exons, creating 38,000 splice variants of the dscam gene product. This is indicative of its role in the Drosophila nervous system
What are the three key genes in determining sex in Drosophila and what are their roles
Sex lethal (sxl), a RNA binding protein and splicing repressor, transformer (tra), a RNA binding protein that acts as a splicing activator and, doublesex (dsx) a transcription factor.
How are male Drosophila determined using alternative splicing and the interactions between the three sex determining genes
Male Drosophila have one X chromosome and this acts as the default pathway for sex determination in fruit flies. The transcripts for sxl and tra are spliced to give rise to inactive proteins. The dsx transcript is also spliced but this gives rise to a male specific transcription factor that acts as a transcriptional repressor of female-specific genes.
How are female Drosophila determined using alternative splicing and the interactions between the three sex determining genes
Female Drosophila have two X chromosomes and a sex chromosome to autosome ratio of 1. The presence of two X chromosomes results in the transient activation of an alternative sxl promoter sequence which leads to the production of the sxl transcript which is then spliced and translated to form a splicing repressor. The sxl protein produced binds to other sxl transcripts and represses splicing by blocking binding of U2AF. This feeds back to result in more production of functional sxl transcripts. The sxl protein also binds to the tra transcripts causing an alternative splice that produces a functional tra protein after translation. The functional tra protein is a splicing activator and causes splicing of the dsx transcript. Splicing of the dsx transcript produces the female dsx transcript which is translated to the female dsx isoform. The female dsx protein is a transcriptional repressor of male specific genes.
Give an example of how polyadenylation can act as a regulation of gene expression
The site of polyadenylation within the mRNA can be regulated. B lymphocytes for example can produce two different isoforms of an antibody. The antibody gene for a specific antigen has two possible positions for cleavage and polyadenylation. This determines whether the antibody is to be secreted or to remain membrane-bound. To produce the membrane-bound antibody, the cell produces the long transcript of the antibody. In this case the first stop codon within the antibody mRNA transcript is spliced out. This results in the translation of the transmembrane domain. Once the protein is secreted it remains tethered to the membrane via the transmembrane domain. For an antibody to be secreted the short transcript is produced which results in the loss of a splice acceptor site. Thus, no splicing of the transcript occurs and the first stop codon isn’t lost. This results in a termination of translation at the first stop codon, prior to the transmembrane domain region. This means that when the antibody is secreted it isn’t tethered to the membrane by a transmembrane domain.
What is meant by the term leaky scanning
Sometimes the first AUG codon can be missed by the ribosome
Sequences around the start codon help to initiate translation, T or F
T
What is meant by the Kozak sequence
The Kozak sequence is the optimal translation initiation sequence that contains the start codon and ideal bases adjacent.
Recall the Kozak sequence
ACCAUGG
How can leaky scanning lead to the production of different protein products
If the sequence is less than optimal the ribosome can miss the first start codon and begin at the second or third AUG. These proteins will all be produced in the same reading frame, differing only by the sequence in the N-terminus
High levels of what translation cofactor increase the probability that the first start codon will be recognised
eIF-4F
How does the HIV virus make use of regulated nuclear transport to influence gene expression
After integration of the HIV genome into the host cell, the whole genome is transcribed as one piece of mRNA. Alternative splicing allows for the many different protein products to be made. Although the full-length mRNA is needed to make new virions the unspliced mRNA containing the entire virus genome cannot leave the nucleus. One of the proteins encoded by the virus genome is the rev protein. During the early stages of infection when only the alternatively spliced viral mRNA can leave the nucleus, the rev transcript moves through the nuclear pore and is translated. The rev protein then interacts with the nuclear pore in late stage infection to allow the exit from the nucleus of the unspliced mRNA.
Signals within which regions of mRNA transcripts target them to particular parts of the cell
3’ and 5’ untranslated regions
How are 3’UTRs recognised by cellular proteins which lead to the sequestration of RNA in one part of the cell
Intermolecular base pairing within the 3’UTRs form stem loops which are recognised by cellular proteins
What is the role of ferritin in the regulation iron availability
Ferritin is a protein that stores iron inside the cell leading to a decrease in Fe availability
What is the role of transferrin in the regulation iron availability
Transferrin is a receptor that imports iron into the cell leading to an increase in available Fe
How does aconitase interact with ferritin and transferring mRNA
Aconitase binds to stem loops in the 5’UTR of ferritin mRNA and in the 3’UTR of transferrin mRNA
Aconitase can also bind to iron itself, T or F
T
Explain the role of aconitase in increasing Fe availability when iron levels are low
Aconitase binds to stem loops in the 5’UTR of the ferritin mRNA and blocks translation by physically blocking the ribosome from moving along the transcript. Aconitase also binds to stem loops in the 3’ UTR of transferrin mRNA and blocks its degradation. Binding of aconitase stabilises the transferrin mRNA thus increasing transferrin synthesis. Decrease ferritin translation and increase transferrin stability and synthesis results in an increase in intracellular [Fe]
Explain the role of aconitase in decreasing Fe availability when iron levels are high
Aconitase binds to iron itself in the cytoplasm which causes a change in its conformation. A change in aconistase comformation causes it to dissociate from the stem loops in the 5’UTR of the ferritin mRNA and the 3’UTR of the transferrin mRNA. The now unstable transferrin mRNA transcript is then degraded quickly and the ribosome is now free to move along and translate the ferritin mRNA. This leads to a decrease in Fe availability
What co-factor of the translation machinery is required for all mRNA translation
eIF-2
When a cell is in the G0/quiescent phase of the cell cycle, globally, translation is turned down. How is this achieved
Phosphorylation of eIF-2 causes its tight binding to eIF-2B. eIF-2B is usually required as a guanine nucleotide exchange factor but tight binding of eIF-2B to the phosphorylated eIF-2 prevents it’s recycling by GTP displacing the bound GDP. By preventing guanine nucleotide exchange, eIF-2 is prevented from initiating translation.
eIF-2 with its bound GTP binds to Met-tRNA to start ribosome scanning, T or F
T
What are IRES sequences and how can they influence gene expression
Internal ribosome entry sequences are stem loops contained within RNA that can initiation formation of the ribosome independent of the Cap/PolyA initiation complex by mimicking it.
IRES translation initiation is an effective way of encoding a second reading frame within RNA, T or F
F – this is not a very effective method with less than 10% of second ORF translation
Which translation initiation cofactor is required to bind to the IRES stem loop and initiate translation independent of the 5’-Cap and PolyA tail
eIF-4G
Where are IRES sequences commonly found
IRES sequences are often found in viral transcripts
How can viruses use IRES sequences to promote translation of their transcripts
Viruses favour translation of their transcripts by cleaving eIF-4G in a way that prevents it from binding to eIF-4E but can still bind to IRES stem loops
What is the significance of eIF-4G cleavage in apoptosis
eIF-4G is cleaved during apoptosis to prevent its binding to eIF-4E and hence prevent any more translation
RNA stability is defined by the half-life of the different mRNAs and varies greatly, T or F
T
How does the polyA tail change over time and act as a clock to determine mRNA age
The polyA tail usually starts at around 200 residues in length but as time goes by this is gradually degraded by exonucleases. Once it reaches 30 nucleotides in length it is de-capped and degraded. Hence the number of adenines in the PolyA tail can determine the age of the mRNA transcript
How can mRNA half-lives be extended
Re-adenylation of the polyA tail
Often factors that promote translation also promote mRNA degradation to prevent overexpression, T or F
F – de-adenylating nuclease (DAN) is responsible for de-adenylation of polyA tails and competes with eIF-4E binding to the mRNA cap.
