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Lecture 20

Lecture 20

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 October 24 2012 Lecture 20 Dr. Shock We have discussed so far various components which are needed for export of mRNAs from the nucleus: • You need exon junction complex proteins to be recognized by NXFI and NXTI in order to the fully spliced mRNA to be exported. • Mpl3 is another proteins which needs to be de- phosphorylated during splicing in order to for the mRNA to be processed. We will begin to talk about mechanisms which will be able retain improperly spliced mRNAs, or primary transcripts, from reaching the cytoplasm. • Thalassemia is a disease involving a deficiency in globin gene products because the gene is improperly spliced and cannot leave the nucleus. These processes have been most studied in yeast, and involve two proteins: Mlp1 and Mlp2, which recognize improperly spliced mRNAs and keep them in the nucleus until they are degraded. • The pre-mRNA in this diagram is the black curvy line, and a leftover intron due to improper splicing is marked in red. • Mlp1 is able to recognize U1 snRNPs which are located on the 5’ splice site of this leftover intron, as well as branch point binding proteins, which would also not be present in a completely spliced mRNA. • The Mlp1/2 complex is able to bind to a Nup on the nuclear pore, assisted by some other factors which sit in the nuclear membrane, and prevent export of the mRNA. Much of these protein factors associated with the Mlp1/2 complex are poorly categorized and we don’t really know exactly what they’re doing. • Therefore, the Mlp1/2 complex can be seen as a “gatekeeper” which monitors mRNAs as they try and leave the nucleus. These factors were identified through a screening experiment which was based on an artificial gene containing a synthetic intron with coding potential (i.e. encodes amino acids in frame), within the lac-z gene which encodes beta-galactosidase (commonly used in screens because its activity is easily detected through simple chemical reactions which turn the yeast colonies blue) • This means that it is necessary to keep the intron INSIDE the final mRNA in order to produce a functional lac-z mRNA which can be translated. Without it, the final protein would be truncated and have no function. • In the wild-type yeast with this artificial gene transfected into it, the intron will be spliced out, creating mRNA which can be exported into the cytoplasm. However, there is no functional beta- galactosidase produced because the protein is truncated (it’s missing the intron) and therefore the colonies don’t turn blue when treated. Another option for the wild-type is that the intron is not spliced out, and therefore the intron is retained. 1 th BIOL 300 October 24 2012 Lecture 20 Dr. Shock • The mRNA could be correct, but there still would not be any beta-galactosidase produced because the mRNA would be stuck in the nucleus and it would never be translated. The only way to get beta-galactosidase in these colonies is to try and knock out any necessary component of the mRNA retention machinery. • This way, in the odd event that the mRNA would not be spliced properly and the synthetic intron was kept in the sequence, there would be nothing to stop the mRNA from being exported, and only then would you have production of beta-galactosidase. The necessary factors which identified through random mutagenesis, in which a chemical mutagen was introduced to induce mutation into the yeast genome. These yeast colonies could be plated and observed; any blue colonies would then be identified to have a necessary mRNA retention factor knocked out. • Further work using staining with antibodies could identify the localization of these factors, but many of the factor’s functions are still unknown. Another mechanism of mRNA transport regulation occurs in the HIV virus, which is an RNA virus The life cycle of HIV: • The complete virus enters the cell; the RNA is enclosed by a capsid as well as an envelope; the envelope fuses with a host membrane, and the capsid is released into the host cell cytoplasm • The capsid degraded and the RNA is released into the host cytoplasm. A reverse transcriptase contained within the viral capsid is also released, which can reverse transcribe the viral RNA into cDNA which can integrate itself into the host genome • Reverse transcription is actually a very similar enzyme to the ones used in a lot of biological experiments • A second protein, packaged in the capsid, is able to bind the cDNA and the importin protein in the host cell, allowing the viral cDNA to be shuttled into the nucleus (via Ran-GTP-importin mediated import) • Most viruses don’t have this mechanism, they are only able to enter the nucleus during mitosis (the same may be true for transfection of cells in experiments); this does not really affect out cells are they are mostly quiescent (not dividing) • The viral DNA, once incorporated into the host genome, can use host factors to transcribe various RNAs, including various mRNAs for proteins needed by the virus (capsid proteins, etc.) as well as the viral RNA genome. Along with transcription of the RNA genome to be packaged into a new viral cell, the viral cDNA is also able to transcribe (using the host machinery) some mRNAs for various proteins:
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