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Lecture 22

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 October 29 2012 Lecture 22 Dr. Shock Normally, a pre-mRNA has a 5’ URT, a start codon, stop codons, and a 3’ UTR. Sometimes, because of errors in transcription, mutations could cause the introduction of premature stop codons, known as a premature termination sequence (PTC). • The result of these mutations are production of truncated proteins which have altered or no function; these must be dealt with through the nonsense-mediated decay pathway. • Somehow, the cell is able to recognize and degrade these mRNAs which contain a premature stop codon. • How does the cellular machinery differentiate between the different stop codons (remember, a stop codon is simply a sequence of three nucleotides)? • The main difference with the premature stop codon is the intron located downstream; the normal stop codon would not have a downstream intron. When pre-mRNA with the premature stop codon is spliced, there is an exon junction complex down downstream of it (as a result of the splicing). • The presence of this EJC, which is not normally downstream of a stop codon, is able to signal to the cell that something’s wrong. • Normally, this mRNA would be exported normally because there is no way for the cell to prevent this, but once in the cytosol, the ribosome, when translating, will stall at this stop codon (this is normal), and somehow the presence of this EJC along with the stalling of the ribosome is recognized as bad and is degraded. • In a normal transcript, the stop codon would be located in the last exon (which is normally large because of the length of the 3’ UTR) This diagram can be separated into two halves; the top right section represents vertebrates and the lower- left section is for invertebrates. While they are slightly different, they share many important similarities, including the use of proteins called UPF1, 2 and 3 (some of which form part of the EJC) In vertebrates: • In the nucleus, the mRNA is spliced and the EJC is formed, containing many proteins, including UPF3, which is important for NMD. • The mRNA is exported at which point the ribosome begins to translate. When the ribosome encounters the stop codon in a non-terminal exon, it will stall at close proximity to the EJC, allowing the two components to interact. 1 th BIOL 300 October 29 2012 Lecture 22 Dr. Shock • In a normal mRNA, the stalling would not occur because there would be no stop codon at this position, and the ribosome would be able to break up the EJC and continue translation. The presence of a stop codon allows for the stalling, which in turn allows for the interaction between the ribosome and the EJC. • This interaction triggers recruitment of eRF1 and 3, which cause the ribosome to dissociate from the mRNA, as well as UPF1. • The EJC will then be able to recruit another protein, UPF2; UPF1 and 2 interact with one another to form the surveillance complex (which triggers degradation of the mRNA they are formed on) • In a normal mRNA, eRF1 and 3 (and this the rest of the cascade) would only be recruited one the ribosome has finished translation, at which point there wouldn’t be any more EJCs and the surveillance complex would not be formed. In invertebrates: • The same proteins are used by invertebrates but you DO NOT need the EJC; the function of the EJC is thought to be taken over by a poly A binding protein PABPCI. • In a normal transcript, the ribosome will translate all the way to the correct stop codon, allowing for interaction with PAPBCI causing the normal release of the ribosome without effecting the mRNA. • With a premature stop codon, the ribosome is not able to interact with PABPCI when it stalls, and allows interactions with proteins including UPF1, 2 and 3 causing formation of the surveillance complex and degradation of the mRNA. • This can be thought of as a series of affinities of the ribosome for UPF1 and PAPBCI; the ribosome will preferentially bind to PABPCI and trigger proper release; in the absence of PABPCI, the ribosome will less preferentially bind to UPF1 triggering NMD Once the mRNA is in the cytosol, it must be translated; once again, there are many different methods of regulating translation • Initiation of translation begins with a capped mRNA 2 th BIOL 300 October 29 2012 Lecture 22 Dr. Shock recruiting eIF4, which then recruits other proteins to form the cap-binding complex • Once this complex is assembled, it can recruit the small subunit of the ribosome along with the initiator Met- tRNA. This complex will scan along the mRNA until it finds the start codon (AUG) • Once the start codon is found, the large ribosomal subunit enters the translation can begin. • An important determining factor Some important determining factors are proteins which are able to bind to the cap binding complex. • Transnationally dormant mRNAs are found predominantly in egg cells, whose functional components normally come from maternal mRNA; they must be translationally dormant before they are fertilized. In the case of xenopus, this is made sure of by a protein called Maskin. • The CPE (a poly A element) of an mRNA in an egg cell binds a protein called CPEB which in turn binds Maskin, which binds eIF4E which prevents formation of the capping complex. • Upon fertilization, the cell will become translationally active through some kind of hormonal signal which induced phosphorylation of CPEB. Phosphorylated CPEB can no longer bind Maskin. • This first allows entrance of proteins like CPSF and PAP which are able to extend the length of the poly A tail (which is not done in dormant cells), allowing binding of poly A binding proteins like PABPII, which binds eIF4G to help form the cap binding complex which will initiate translation. • As soon as Maskin can no longer bind eIF4E, eIF4G can come in and bind eIF4E and help form the pre- initiation complex; there are many regulatory mechanisms which block or enhance this eIF4E-G interaction. • Also note that the mRNA usually takes on a circular shape by linking the 3’ and 5’ ends, thanks for the arr
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