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Lecture 21

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 October 26 2012 Lecture 21 Dr. Schock There are different ways RNAs can be transported to different regions of the cells (we looked at some of them last time) An experiment in 1994 which gave clues about some factors which may regulate RNAs in the cytoplasm. • They transcribed RNA and then injected it into a Xenopus oocyte and assayed the resulting protein to determine its function. • The xenopus oocyte is a very large cell with a large nucleus making it a very good target for this experiment, because injecting things into this cell is very easy. What they noticed was that the amount of protein produced depends on the type of RNA injected, i.e. the template where the RNA came from. • When they used cDNA as a template, which produced fully spliced mRNA, some protein is transcribed (+) In another experiment, they used genomic DNA of the same gene as a template, which produced primary transcript mRNA, surprisingly, a lot of protein was produced (++++) What could account for this difference? • One reason could be that the mRNA from the cDNA template is not properly exported • Genomic RNA must be spliced and modified in the nucleus before it is exported, but once it is in the cytoplasm, the two RNAs will be pretty much identical • The act of splicing out these introns leave behind proteins which part of the exon-junction complex; they can affect how well an RNA is translated later on. • What actually happened in this experiment was in fact the missing proteins of the exon-junction complex, but they did not know this when the experiment was carried out. The experiment tried to check the export of the cDNA- derived mRNA from the nucleus. To do this, they used two time point: time 0 represents time of injection and time 4 represents 4 hours after injection. • At each time point, they took the cells are fractionated them, separating the nucleus and cytoplasm, and then used a radioactively labelled antisense probe to the gene (CaT) to mark the location and amount of these mRNAs. • Indeed, at time 0, you can see that the mRNA was predominantly located in the nucleus because it has not had a chance to be exported • At time 4, we can see almost all of the mRNA had been exported in the cytoplasm. The control for this system was using the U6 snRNA, which is a nuclear RNA which is never supposed to be exported (it is part of the splicing machinery). Therefore at times 0 and 4, we can see it is always located in the nucleus. • The major thing that they have to control for is the proper fractionation of the cytoplasm and nucleus, which is not always done properly. th BIOL 300 October 26 2012 Lecture 21 Dr. Schock • Usually, the nucleus is denser and upon centrifugation, will sediment first allowing separation from the lighter nucleus. However, from obtaining the correct results for the U6 snRNA, we can see that this was not a problem in this experiment. • Therefore, this procedure allowed them to determine that the export of the RNA is not a factor in why the genomic RNA was translated more than the cDNA-derived mRNA After splicing, there are a few proteins left over bound to the exon known as the exon-junction complex, which stay on the mRNA until the ribosome comes onto the RNA and displaces them during translation. • These are not proteins of the spliceosome itself, and they also must have and NES and an NLS in order for them to be shuttled between the cytoplasm and nucleus. The EJC (exon junction complex) is made of a heterotetramer which is made of 4 proteins: • eIF4AII anchors the complex to RNA (i.e. an RNA binding protein) in an ATP- dependant manner. • 2 proteins, Y14 and Magoh inhibit ATP hydrolysis by eIF4AIII which locks the complex onto mRNA (by preventing the RNA hydrolysis on eIF4AII) • MLN51 increases eIF4AII’s affinity for mRNA Other associated factors include: • Barentz, which we will discuss later on • REF, a nuclear export factor which binds NXF and NXT proteins There are 3 things that the EJC regulates: • Enhanced translation by binding of the EJC; this could be because some factors in the EJC may be able to recruit translation factors. • The localization of mRNA may be regulated
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