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Gene Silencing Antisense RNA and RNA Interference How antisense RNA works • During protein synthesis a single strand of mRNA is produced by transcribing the DNA code inside the nucleus • This mRNA contains a “sense” code; it is sent to the ribosome to facilitate making a specific protein How antisense RNA works • If we want to stop that protein from being made we can disable the sense strand of RNA • RNA can become double stranded • What is needed is an “antisense” strand of RNA to bind with the “sense” strand How antisense RNA works • To get the antisense strand we can insert a gene into the DNA that codes for the antisense chain of RNA • When this gene is read an antisense RNA chain is produced • The antisense chain binds with the sense chain disabling it • No protein is produced – the gene is silenced Applications of this process • Scientists used this process to silence a gene in the tomato that produced ethylene and caused the tomato to ripen • Scientists used this process to silence a gene in the potato that produced either amylose or amylopectin so that these two starches did not have to be separated later Modeling this process • We will modify our protein synthesis model activity to role play how this process works • Two additional roles will need to be added: – We will need another mRNA molecule to read the antisense gene – We will need another mRNA polymerase to prepare the antisense chain and deliver it to the sense chain to stop it from entering the ribosome for translation • We will not need the amino acid beads or the tRNAs because no proteins will be made How RNA interference works • mRNA is found in the cytoplasm as a single stranded molecule • If any double stranded RNA is found in the cytoplasm it is attacked by an enzyme called dicer How RNA interference works • Dicer cuts the double stranded RNA into small fragments • The fragments then separate into a sense and antisense strand. The fragment is called siRNA How RNA interference works • The antisense strand combines with another protein (argonaute) - this combo molecule is called RISC (RNA induced silencing complex) • With the aid of this second protein the antisense chain can combine with the mRNA on its way to the ribosome • The mRNA is disabled and the gene is silenced How RNA interference works Double stranded RNA + Dicer = siRNA siRNA + Argonaute = RISC RISC degrades mRNA No Protein Applications of this process • Scientists are studying how to use RNAi technology to remove caffeine from coffee • Scientists used this process to save the papaya from a virus • Scientists used this process to enable soybeans to make a healthier oil Animation Let’s watch how this works in the following animations • RNAi animation (http://www.youtube.com/watch?v=cK-OGB1_ELE) • RNAi animation second link (http://blogs.nature.com/news/2011/12/videoanimation-rna-interference.html) • There are other methods of gene silencing being studied, like the micro RNA described in the animation.