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23 RNA Processing 2012.docx

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Western University
Biochemistry 2280A
Chris Brandl

23- Formation of mRNAs: RNA Processing Eukaryotic Cells  DNA in the nucleus is somewhat constraint like a rope fixed to certain spots. When polymerase binds and unwinds the DNA, then the transcription bubble moves downstream and starts to wrap super coils in front which prevent movement of DNA. Enzymes in the cell have to remove those super coils by cutting the DNA and allowing it to unravel.  Going from RNA produced in the nucleus to a mRNA that can be translated in the cytoplasm, in Eukaryotic cells.  Primary transcripts becomes and mRNA for export out of the nucleus after translation Primary transcript becomes a mRNA for export out of the nucleus and translation • 5’ capping with 7-methyl guanosine • 3’ polyadenylation • Splicing 3 processing events. These processes are concerted so they are all happening at the same time as transcription and each of these require a specific enzyme or enzyme complexes 5” Capping - upon synthesis the first base is a triphosphate - to this is added a 7-mG Importance - Marks the 5’end of the mRNA as intact. - Required for mRNA export and for translation (the latter because it is recognized by the ribosome) Fig. 7-16 7-methyl guanosine is put on the 5’ end of mRNA. It has this unique 5’-5’ linkage. A bunch of enzymes are required to put this on the end of the RNA. Those enzymes actually bind RNA polymerase, which may happen as soon as the 5’ end of the mRNA is made. This is important because it’s a sign that the RNA is intact. If the translational/export machinery sees an RNA with a 5’ cap, it knows that this is the authentic 5’ end of the RNA. An RNA that somehow has gotten degraded in the cell will not have a cap on it. Only RNAs that are meant to be translated will have a 5’ cap. So tRNA and rRNA do not have the cap. That 5’ cap is essential for RNA export and later for translation. When the RNA is made, the first nucleotide added is a triphosphate, to that is added a 7- methylguanosine (at the 5’ end) added very soon after the 5’ end of the messenger is created. It is through a unique 5’-5’ linkage. Importance of 5’ Cap - Marks the 5’ end of the mRNA as being intact - Required for mRNA export from the nucleus - Required for export of the RNA out of the nucleus and for translation of the mRNA into protein (translational initiation) - And RNA that isnt capped wont be translated or exported. 3’ Polyadenylation There’s a signal downstream form the stop signal (AAUAAA), which is a signal for the polyadenylation machinery (which brings in an endonuclease and recruited to that is a poly A polymerase that adds a poly A tail to the end of the message. The RNA that is being transcribed is actually longer, the polymerase is still going.) to come in and clip the RNA about 30 bases downstream from that signal sequence. And adds along poly-A-tail along the 3’ end of RNA. That poly A tail can be about 300 bases long. An intact mRNA then must have a 5’ cap and a 3’ poly-A-tail. • Helps protect the 3’ end of the mRNA from degradation • Indicates that the 3’ end of the mRNA is intact and therefore is important for export out of the nucleus and for translation • There are enzymes in the cell called 3’ prime exonuclueases that start degrading RNA form the 3’ end, with the poly A tail it helps protect the 3’ end of the RNA from degradation. The longer the poly-A-tail, the more stable the message is. • The poly-A-tail also defines an intact 3’ end of the mRNA, to allow cell to distinguish between authentic and not authentic mRNA. If you have an mRNA with a 5’ cap and a 3’ tail, then the export machinery knows that that is an authentic mRNA. Having either one of these is not sufficient, the mRNA must have both, with a few exception mRNAs. • There are a few RNA in the cell that are not polyadenylated. • - Has a 5’ cap and 3’ poly a tail to be considered intact. In Eukaryotic cells protein encoding sequences are interrupted by one or more noncoding sequences called introns Fig. 7-17 At the top is a bacterial gene with promoter, coding region continuous- non interrupted. No introns in Proks. In Euks, we have introns (interrupted) and exons (exons for expressed). Splicing in euks, protein-encoding sequences are interrupted by one or more non coding sequences- the gene is interrupted. In mamalian cells, all sequences have introns.  Exons = expressed  Introns = interruptions  The introns are “spliced” out of the primary transcript to form the mature mRNA that will be translated Fig. 7-18 Shown at the top is human beta-globin gene- very simple gene with 3 exons. In contrast, the human factor VIII gene has 26 exons but most of the gene is introns (25 introns). Much more introns than exons. So to get mRNA from this, there’s going to be a lot of splicing. 2000 nucleotide pairs- most of it is introns it not clear why there’s so many introns structure, but they are a major component of this gene itself. - Some of the introns do code for things, there is stuff going on in there but it just isnt making the proteins but there is information in there. Splicing – discovery When people were looking initially at genes, they had the RNA that could be purified from the cell, and the DNA from the genome. They did hybridization from the RNA and DNA from the genome. They got this RNA-DNA hybrid with all of these big loops shown here. These loops were the introns. That’s how they discovered introns. An EM pic demonstrating the level at which our genes are interrupted with introns. Why is splicing important? Differential Splicing: A single RNA (primary transcript) can be spliced in different ways to create related but distinct (mRNAs) proteins. This is one aspect of splicing we know about. Cell can splice an mRNA in multiple ways, depending on what introns are spliced out of the gene and what exons included in the mRNA. A reason is differential splicing: because of intron/ exon structure, a single thing can be spliced in different ways to make different proteins/ functions. Differential Splicing often is tissue specific Fig. 7-21 The torpomyosin gene is expressed in many different tissues. Depending on the type of tissue, different exons are put together in the mRNA. The mRNA transcripts are a lot in common but there some differences in the exons that makes up each type of tissue. Without the splicing we would have had to duplicate the gene many times to get the same level of complexity, so splicing substitutes for a whole bunch of gene duplication. Differential splicing allows diversity without taking up too much genome space. There specific proteins that help determine how the genes get spliced. The splicing pattern for any RNA is usually tissue specific. - The tropomyosin gene can be spliced in different ways- and it varies from tissue to tissue. - Smooth muscle does it a different way - Fibre blasts look like striated muscle mixed with smooth muscle. Splicing Requires Specific Sequences in the RNA There are 3 key sequences in the RNA required for splicing. Shown is a primary transcript with 5’ and 3’ end. Blue = exons, yellow = introns. Shown is the consensus sequence for each of the 3 key sequences, the sequences can be slightly different in different RNAs which may also effect the efficiency of splicing. 1. 5’ splice junction: this sequence is crucial for splicing to occur. Located between the exon and the intron- a concensus sequence requires for that. 2. 3’ splice junction: this sequence is crucial for splicing to occur- some is very high conserved- others is a little bit looser. 3
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