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Gene Silencing Treatments - Days of Future Past.

floofer

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Many of you will have seen the new treatment trialled for porphyria - a condition which is a result of a large buildup of porphyrins. Heam is a protein that is a constituent of haemoglobin, a notable component of our red blood cells that utilises an iron ion to carry oxygen. Heam is composed of an iron ion and porphyrin components. In the porphyria there is an imbalance of the porphyrin constituents due to a mutation in the enzymes converting the various porphyrins to needed types. What we get is proper Heam biogenesis, but due to the imbalance, insufficient. This treatment corrects that imbalance by inhibiting the translation through RNAi - a process which has been around since the 90s, and used to be very popular until CRISPR/Cas9 was discovered. 

 

RNAi is an evolutionary ancient mechanism for genome defence in many organism, RNA uses when they replicate will go through a strand of dsRNA. RNAi functions to silence viruses and rogue genetic elements that make dsRNA intermediates. Anything that forms dsRNA, e.g. transposons will affect this process. RNAi uses the invading dsRNA as a template to process an enzyme to chop up the RNA. This means RNAi will silence transposons and will have dosage-dependant silencing

 

It was noted that plants silencing genes via RNAi  produced short RNAs. The RNA were 20-25nt and matches the gene being silenced to the mRNA transcribed. Their small size, compared with rRNA and mRNA, led to them being undiscovered until recently. These can either be miRNA (Micro-RNA) or siRNA (Silencing RNA). As Heam biogenesis is an essential process, miRNA regulation of mRNA is necessary. 

 

miRNA assists the process of RNAi to regulate transcription factors. miRNA are 19-25nt ssRNAs. Genome encoded on most multi-cellular organisms. The miRNA genes account for 0.5-1% of the predicted genes in those organisms, they are the miRNA genes. These genes are evolutionary conserved and developmentally regulated - they have an important role for RNAi in endogenous gene regulation. miRNA will silence genes at the stage of protein synthesis (they are translational inhibitors) by binding to the 3’ UTR. MicroRNA will turn on Translation proteins to get developmental processes turned on. There is fine tuning of how many mRNA gets translated into protein by miRNA.

 

miRNA’s are produced by post-transcriptional processing of precursors. Precursors form a stem-loop structure and may be polycistronic. Drosha enzyme, in the nucleus will chop off the stem looped formed in the miRNA process - processes the pri-miRNA into a 70nt pre-miRNA. The pre-miRNA is transported out of the nucleus to be acted upon by Dicer, which then chops the pre-miRNA to form miRNA.

 

miRNAs base-pair with partially complementary sequence in mRNA, while siRNA’s bind with perfect complementarity. miRNAs function to block synthesis, in this pathway the mRNA is not degraded. They inhibit translation but transcription is not impaired. There is no transcriptional inhibition via conventional miRNA pathway. The degree of translational inhibition depends upon how many miRNA are bound to the target mRNA, there is logical suppression of message of protein.

 

miRNAs typically bind at the 3’ UTR through partially complementary regions. mRNAs will have many binding sites for miRNAs in the 3’ UTR. Several different miRNAs will target the same mRNA - the more bind, the greater the level of inhibition, and allows more to bind. Base-pairing with the 7-8 nt near the 5’ terminus of the miRNA is essential. This region has perfect complementarity and there are regions which do not.

 

Essentially:

  1. The gene encoding for miRNA transcribed a 17nt polycystonic Pri-miRNA
  2. The Pri-miRNA gets cleaved into a hairpin pre-miRNA molecule by Drosha
  3. Dicer cleaves the pre-miRNA into a mature miRNA molecule
  4. The mature miRNA is assembled into a miRNP complex, which targets to bind to the 3’ end to block translation. It will bind logistically, so greater binding involves greater inhibition.

 

 

 



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