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Lecture 18

Lecture 18

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 October 19 2012 Lecture 18 Dr. Shock In addition to splicing, there are some more exotic types of RNA modifications, one of these is RNA editing, which involves chemical changes individual nucleotides to the primary transcript (pre-mRNA) There are two types of changes: • Cytidine can be changed to a uracil through cytidine de-aminase which removes the amino group on cytidine to produce the uracil. • Adenine can also be de-aminated through another protein known as ADAR, to produce another nucleotide called inosine, which is similar to guanine. What are the consequences of these two reactions? • If this change is within the coding sequence, the codon, and maybe the amino acid which is produced, could be changed. • You could also introduce a stop codon in the coding sequence, truncating the resulting protein. • Regardless of its location in the gene, it could affect splicing in some way by either inserting itself into an ESS, ESE, ISS, ISE, as well as splice sites or the branch point region. • It could also affect stability of the mRNA, as well as export of the mRNA into the cytoplasm, and even the localization of the mRNA. We can see from the pre-mRNA, there are various sites of modification depending on the concentration of various de-aminase proteins. • The different modifications could result in different proteins with different functions; normally, not all transcripts are modified, meaning you can get a mix of edited and unedited proteins. • If the modification occurs at a splicing related area, it can result in alternative splicing of the protein • Editing of regulatory RNAs like miRNAs can have functions as well. How was RNA editing discovered, and how do we identify edited sites? • We can compare cDNA to genomic DNA on a large scale; T (cDNA) – C (genomic DNA) mismatches when hybridizing two sequences could hint that this is a possible modification sites. • This could also be done directly with the RNA, but it is much harder and more technical, so cDNA is much more commonly used. 1 th BIOL 300 October 19 2012 Lecture 18 Dr. Shock There are some problems with this procedure: • Very commonly, these mismatches between cDNA and genomic DNA could simply be errors in the sequencing machine. Alternatively, it could be an error made by RNA polymerase rather than a post-transcriptional chemical change. • Also, RNA editing of certain genes can be specific to certain cell types; for example, if you take a random gene for which you know the sequence, and you sequence multiple cDNAs of this gene, and you only find a mismatch in 10% of them. • This could be because of a sequencing error, RNA Pol error, or simply because the RNA isn’t edited in 90% of these sample cDNAs because they come from a cell type which does not carry out RNA editing of this gene. • Some of these problems are solved now using deep sequencing, in which extremely large samples of cDNAs are sequenced 100s of times to reduce the amount of error dramatically so that you can essentially rule out sequencing errors as a possible reason for a mismatch. • Deep sequencing in humans showed a lot of editing in Alu repeat sequences, which are transposons (i.e. not encoded into proteins) that don’t have a clear function. No one knows the purpose of this editing. • Apart from this, there are not many examples of RNA editing which have been researched and understood in detail The first example of RNA editing is in the liver and intestines in the apolipoprotein B gene (apoB) involved in cholesterol uptake • In the intestines, CU editing introduces a stop codon in the sequence which creates a truncated protein known as ApoB-48, in comparison to the liver, where no editing occurs and an ApoB-100 protein is formed. • Since editing is not 100% efficient, in the intestines, about 90% is the truncated version ApoB-48, while in the liver, there is very little editing, forming only about 1% of the truncated protein. • The cytidine de-aminase protein for this specific reaction is known as APOBEC1 which recognizes a short sequence surrounding this specific CAA region, which allows it to de-aminate only this C and not other Cs in the gene (and other genes) • Note that all RNA-editing proteins are RNA binding proteins, in which the RNA binding domain recognizes a 26 base pair sequence. • Knowing this, you can now narrow down your search for mismatches to those only containing certain known 26 base pair sequences which bind RNA binding proteins; this will allow for much more confident predictions than the mismatch found is in fact due to RNA editing. 2 th BIOL 300 October 19 2012 Lecture 18
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