Lecture 9 Tutorial

Question 1

A gene (depicted below) has two different poly(A) sites. What could be consequences when one or the other poly(A) site is used and two different mRNA isoforms are produced?

Answer 1

different protein amount
different stability
different proteins may bind to different isoforms
different miRNAs may bind to different isofoorms
short isoform prevents the production of long isoform

Question 1

You want to generate a mutant of the initiation factor EIF4E that is not able to bind to the mRNA cap anymore. Which amino acid residues within EIF4E would you mutate and with which amino acids would you replace them? Do you think that an organism
expressing only this mutant EIF4E is viable? Please justify your answers briefly.

Answer 1

TRP 102, TRP56, mutated into non - aromatic residues
Probably not viable since Cap binding is essential for translation and translation is essential for survival

Question 2

In your project you generated two reporter cell lines, with which you can measure the intracellular iron levels. You used two different combinations of the iron responsive element (IRE) and a green fluorescent protein (GFP). After integrating the reporters into your cells, you want to test if they behave as expected.
B) What effects would you expect if you knocked out both iron regulatory proteins (IRP1 and IRP2) in
these cell lines?

Answer 2

The cell line with the reporter IRE-GFP shows green fluorescence when iron levels are high

The cell line with the reporter GFP-(IRE)5 shows green fluorescence when iron levels are low

B: The cell line with the reporter IRE-GFP shows green fluorescence under all conditions (iron high and low)

The cell line with the reporter GFP-(IRE)5 does not show green fluorescence (independent of iron levels)

Question 3

Which sequences in exons assist in splicing? Which proteins bind to them and what is their function? What domains are characteristic of these proteins and what are they needed for?

Answer 3

These sequences are called Exonic Splicing Enhancers (ESE); ESEs are bound by SR proteins, which activate splicing and play a role in the regulation of alternative splicing; They contain at least one RNA-binding domain (RRM) and an RS-domain. The RRM is required to bind to the ESE sequence, the RS domain mediates the communication/interaction with the spliceosome.