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BIO240 Lecture 22

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Jennifer Harris

Lecture 10: Processing of RNA to produce mature mRNA Lecture Outline 1) RNA processing 2) RNA world Readings Alberts, Ch 6, pp.349- 366, 402-408 - From Chang’s we are mostly interested in the paradigm shift of views regarding RNA and the likelihood of a completely RNA world (in the beginning) Which statement concerning DNA replication or transcription prokaryotes is correct? a) Single-stranded DNA binding proteins require ATP in order to stabilize single stranded DNA during replication (False because it does not require ATP) b) The lagging strand requires two different polymerase for its synthesis whereas the leading strand requires only one (False because in prokaryotes there is only one type of polymerase required) c) The helicase powers the movement of the primosome during replication (True because it powers the movement using ATP and conformational changes) d) The RNA polymerase initiates transcription on the start codon on the template strand (False - doesn’t initiate transcription at the start codon, that is where translation is initiated not transcription) e) Primase is subunit of polymerase (False) - In eucaryotes, the structure of genes are far more complex than prokaryotes, have introns and exons, modifications that happen. - These introns get spliced out & exons get spliced together – that transcript is then capped – all of this happens simultaneously. Splicing also happens & poly-adenylation signal. - mRNA is then exported (all of these things are happening in nucleus – all of this initial transcription as well as processing of primary mRNA transcript) into cytoplasm – tightly regulated process. Final part is the addition of the adenosines (poly A tail) polyadenylation Why would we go to all this trouble synthesizing all this shitty RNA and then cutting it out (making a long dress and cutting pieces out) Next slide explains why  RNA processing - This is one of the realities that makes gene prediction very hard. - Alternative splicing means that in the genome, you can have a gene with many exons & introns interspersed throughout the code. Depending on the tissue/cell type that is processing the primary RNA transcript, you can get different mature transcripts resulting. - In all of these cases, in all of these different cell types, initial transcription happens so theoretical primary RNA transcript doesn’t actually exist because they are processed as they are synthesized but theoretical primary RNA transcript would be exactly the same in all these different cell types. - During the splicing, some exons are discriminated and spliced out of mature transcript, creating a final mature mRNA transcript that doesn’t include all exons. These different mature transcripts that contain different exons, will code for slightly different proteins (same gene in genome, slightly different proteins in different cell types) – thought to be related to slightly different functions in different cell types – 1 way of generating needed variation in protein structure & function that is tailored to what cell needs it to do. - Alternatives splicing mechanisms are not necessarily errors – may be intended outcome however mistakes can also happen – so complex that in fact it is fairly often that mistakes can happen. When you look at the mature RNAs you can find different combinations of exons that have been used The AA structure then are similar, not completely different since they have identical regions but not exactly the same Whole families originate from this type of splicing From one primary transcript you get different variations Same type of cell could have variants or variations between cells in term of splicing Sometimes splicing errors can be found in disease situations - In normal genes, these types of errors would be grossly elevated if not for the double checking in the spliceosome with complex RNA-RNA rearrangements where 1 set of RNAs check & another set of RNAs check & then that region is identified as appropriate splice site. - Errors still happen but they happen at a lower rate. - Different cases of exon splicing mistakes are depicted in the slide. - Can have case that you have single nucleotide change that destroys 3’ splice site – one common problem is that it can’t find 3’ splice site so it keeps gong & then eventually finds other 3’ Resembles true splice site (but not under splice site so this entire thing is spliced out including exon (important exons may be skipped over because the splice site was normal conditions intended to be the splice site not recognized and something important could be removed – similar enough that in unusual circumstances, under certain conditions, are recognized & used completely). For gene that is not normally alternatively spliced as true splice sites) where this exon is absolutely critical for function of protein this would kill the function of the enzyme. - Other things that can happen: single nucleotide changes can destroy normal splice sites that activate cryptic splice sites – can’t find splice site there, instead 1 is activated there – this part of intron is actually read as protein coding which could have serious consequences for protein. - Some nucleotide changes can create new splice sites: can cause new exons to be incorporated – additional exon sequence (additional protein coding sequence). Sometimes there are mistaken start areas and the mature mRNA could end up with intron areas in the transcript which could contain a bunch of useless stuff. Beta globin splicing shown in slide – It is part of hemoglobin Normal splicing is on the left – Form a normal messenger RNA Phalacemia is problem with hemoglobin, known to have defects in splicing Part of genetic condition is that they have mutations with defects in splicing of mRNA A mutation at the end of an exon might make splicing miss integral region and cause the entire exon to get removed along with introns Sometimes there is a mistake sequence inserted due to exon like regions mutated into the introns sequence Cryptic splice sites that can come up too, they are splice sites that may resemble the splice site that under certain conditions they are recognized and used as true splice sites - The entire splicesome complex is to help ensure fidelity – if we didn’t want fidelity & speed we wouldn’t need splicesomes at all (for the removal of introns). - 2 factor in ensuring splicing fidelity is fact that the splicing mechanisms happens while mRNA transcript is being made so there is always a 5’ splice site that is recognized & a 3’ that is recognized. There is an order by which things happen which helps to ensure fidelity. - 3 rdmajor factor (least well understood) is related to exon definition hypothesis: the top graph show that exons in terms of length across vastly different animals (worms  humans) tends to be fairly standard (majority is 150 nucleotides in length Co-transcriptionally (as RNA polymerase is approx). going along, splicing is happening at same - Intron length is much more variable across different species – time) can see that humans have on average much longer intron lengths than either worms or flies do – this is related to idea that you will know approximately when you should be seeing another splice site – important to get exons correct. Lots of different proteins that associate with mRNA as its transcribed & help to make available splice sites or cover up splice sites. Ex: Intron sequences usually complexed with particular type of ribonuclear protein which tends to hide cryptic splice sites in introns whereas exon sequences have different proteins that associate with them. This all helps to ensure that the correct splice sites are being used – this all happens co-transcriptionally, the demarcation of exons. If you look at the average length of exons and you also look at average lengths of introns you find graphed relationship Percent of exons with certain lengths Protein coding regions really are very short and interspersed by variable and large introns A lot (especially in humans), you can get up to 30 000 nucleotide introns Exon lengths are more common / most conserved
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