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Lecture 6

BIO230 lecture 6 notes.docx

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University of Toronto St. George
Darrel Desveaux

BIO230 lecture 6 notes - Quality control of mRNA in the cytosol IN EUKARYOTES:  Only about 1/20 mRNA molecules make it out of the nucleus. Some mRNAs are incompletely processed or damaged when they come out to the cytosol. The cell needs to prevent these mRNAs from being translated because they can produce aberrant proteins that have toxic effects on cells (they can be sticky and either induce or interfere cellular processes that can hurt the cell)  First let’s look at translation because a lot of these quality control mechanisms are tightly coupled with translation:  tRNA molecules match amino acids to specific codons in the mRNA; codons are 3 nucleotides. The process of translating those 3 nucleotides into specific amino acids is called the genetic code. 3 codons do not encode for amino acids: they indicate a stop of translation. There is also one codon for methionine which serves as a start codon for translation.  This translation occurs on large complexes called ribosomes. Ribosomes are a mixture of proteins and RNA; almost 50 different proteins and RNA molecules make it up. The ribosome is divided into a large and small subunit that come together on the mRNA for protein synthesis. There are 3 sites in the ribosome, A site (amino acid site) where the tRNA with the free amino acid goes, the P site (peptide site) where the tRNA with the peptide associated with it stays, and the E site (exit site) where the tRNA will leave the ribosome.  A specific tRNA with a specific amino acid attached to it comes in, matches to a codon at the A site, and transfers the amino acid onto the growing peptide chain to produce a peptide bond (note that the amino acid is added to the C-terminus of the growing polypeptide chain  that is why protein synthesis occurs from the N- terminus to the C-terminus from the 5’ end to the 3’ end of the mRNA).  Recall that initiation of translation requires eukaryotic initiation factors that need to come together for the ribosome to start translating. These initiation factors bind to the 5’ cap and the poly-A binding proteins that are bound to the poly-A tail of the mRNA. There are 2 specific eukaryotic initiation factors that bind: EIF4E (displaces the cap binding complex that is bound to the 5’ cap) and EIF4G (it binds to the poly-A binding proteins that bind to the poly-A tail. This binding is a sign that the mRNA has been processed properly and that the mRNA molecule is intact; damaged mRNA won’t have both the 5’ cap and the poly-A tail. After this, there is the recruitment of the small ribosomal complex, which eventually initiates translation at the first start codon, AUG.  There are some exceptions where this first codon will be skipped and the second one will be used  this is called leaky scanning  The exon junction complex (EJC) also stimulates translation; it has a critical function in an important quality control mechanism called nonsense-mediated mRNA decay. It is based on the recognition of premature stop codons in the mRNA molecules, and these premature stop codons are called nonsense mutations because it shifts the protein and you get a stop codon before translation should actually stop. This often occurs if there is improper splicing; it shifts the frame of the mRNA molecule and this introduces a stop codon where it doesn’t belong.  Normal situation: As the mRNA molecule leaves the nucleus, a ribosome will bind to it as it emerges from the nuclear pore and do a “test round” of translation. As it is doing so, it is translating the mRNA and it removes/displaces the EJCs. In this normal situation, the stop codon is in the last exon and once the ribosome reaches it, there are no more EJCs on that mRNA molecule because they have all been displaced. The mRNA will be released in the cytosol and the protein will be translated from that mRNA molecule.  Abnormal splicing: there is a premature stop codon and it shifts the frame of the mRNA so that the ribosome will encounter a stop codon before it is actually supposed to. The ribosome will again bind it, go through the “test round”, but as it is moving along and displacing EJCs, when it reaches the premature stop codon, there will still be EJCs present in the mRNA molecule. This presence of EJCs serves as a sign to the cell to degrade that mRNA molecule. This degradation is assisted by Upf proteins that bind to the EJC and degrade that mRNA molecule.  This is actually a very important process in eukaryotes and is likely to have contributed to the evolution of novel genes in eukaryotic organisms by allowing the selection of DNA rearrangements/ alternative splice patterns that produce full length proteins while also protecting the organism by premature stop codons that may occur during these rearrangements.  In the immune system, there is a lot of rearrangement going on to produce all of the antibodies involved in recognizing a diverse array of pathogens. This means that many times the mRNA molecule produced from the rearrangements will encode premature stop codons. In these cells, the decay will protect the cell from many mRNA transcripts that encode premature stop codons.  About 1/3 of inherited genetic diseases are caused by premature stop codons and the cells can degrade the aberrant mRNA to allow functional protein to accumulate. - Quality control of mRNA IN PROKARYOTES:  This is for incomplete or broken mRNAs.  If the ribosome comes to an incomplete or broken mRNA, it will stall on that molecule. When a ribosome stalls on a prokaryotic mRNA molecule, a special type of RNA will bind to the A-site, and this is called a tmRNA. It carries an alanine amino acid with it and as its name implies, it acts as both a tRNA and an mRNA.  Once the tmRNA binds to the ribosome, the broken mRNA will be released from the ribosome.  The tmRNA will transfer that alanine onto the growing polypeptide chain; this is an aberrant protein because the original mRNA is damaged. This transferring of the alanine makes the tmRNA behave as a tRNA. NOTE: there is no codon encoding for this alanine; the tmRNA doesn’t require a codon on the corresponding mRNA to do this.  The tmRNA encodes for 10 amino acids, thereby acting as an mRNA. These 10 amino acids will be added onto the growing polypeptide chain to create an 11-amino acid tag (alanine + 10 amino acids encoded by the 10 codons of the tmRNA). This serves as a signal to proteases to degrade the whole protein. This 11-amino acid sequence that makes up this tag varies for each bacteria cell but they all signal the proteases to come and degrade the protein. - mRNA stability:  In prokaryotes, mRNA molecules are very short-lived – less than a minute! In prokaryotes, there are very effective exonucleases that rapidly degrade mRNA molecules. Exonucleases degrade nucleotides/molecules from the ends and they are differentiated from endonucleases that degrade from the inside of the molecule.  In eukaryotes, the mRNAs are more stable and can live for a maximum of 10 hours, with the average being less than 30 minutes (in terms of stability).  There are 2 main mechanisms to degrade mRNA molecules in eukaryotes and they both involve the gradual poly-A shortening. Recall that a properly processed mRNA molecule has a poly-A tail at the 3’ end; once that poly-A tail is produced, it starts to slowly degrade in the cytosol. There are about 200 A nucleotides that are added in the poly-A tail and this tail is slowly being shortened by an exonuclease found in the cytosol called deadenylase. This exonuclease acts as timer for the mRNA lifetime.  T
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