Interfering with RNA: Part 2, Dicer and the Argonauts

RNAi

RNA interference, through the RISC pathway

A couple of weeks ago I wrote a post explaining the process of gene silencing, or RNA interference. I wanted to expand on that further today by diving in a bit more into the molecular details of exactly how this happens inside a cell.

• As I explained earlier, the Central Dogma of Molecular Biology is DNA –> RNA –> Protein. This means the DNA blueprint makes a sequence-specific copy of RNA, which in turn acts as a blueprint for a specific sequence of amino acids which make up a protein. So if you could somehow destroy the RNA blueprint (known as the messenger RNA, or mRNA) for a particular protein, this would prevent the protein from being made – no blueprint, no protein. This is what RNA interference, or gene silencing is.

• How does this work within the cell? How can introducing a sequence-specific double stranded RNA molecule (i.e. two RNA strands wound up together exactly like a double-helix DNA molecule) into a worm destroy the mRNA blueprint? It’s a beautifully intricate, complicated process involving many different proteins and many different steps. The key point to remember is that the double stranded RNA is sequence specific (i.e. the order of the nucleotides ATCG etc), meaning it will target a specific mRNA blueprint to prevent a specific protein from being made.

• First, the double stranded RNA molecule is ‘diced up’ by a protein imaginatively named Dicer. Dicer chops up the long double-stranded RNA molecule into smaller pieces of RNA, again imaginatively named small interfering RNAs, or siRNAs. These are 21-26 nucleotides in length (in comparison, a typical double-stranded RNA molecule could run into several hundred nucleotides!).

• Next, these siRNAs go on to form a RNA-induced silencing complex, or RISC. The RISC consists of a single stranded siRNA and a group of proteins collectively known as the Argonaute Proteins.

• The Argonaute Proteins act to ‘hold’ the siRNA from either end within the RISC, allowing the middle of the siRNA to base pair with the target mRNA (the sequence specific blueprint). The Argonaute Proteins are key in mediating target mRNA sensing, subsequent recognition and complementary base pairing between the RISC complex and the target mRNA.

• Once the target mRNA has base paired with the siRNA within the RISC, the mRNA is ‘snipped’ by one of the Argonatue proteins within the complex. The snipped bits of the mRNA are then degraded and recycled within the cell. So think of the Argonaute Proteins as the ‘seek and destroy’ part of the RISC. To give you an idea of how complex this process is, so far over twenty seven different Argonate Proteins have been identified in the worm C. elegans!

• It is thought that RNA interference evolved to protect against viral infections, as many viruses are double stranded RNA viruses. RNA interference in the context of microRNAs are extremely important in development, and there are dire consequences when things go wrong – but that’s another post entirely!

• Needless to say, RNA interference is a tightly regulated process, with multiple checkpoints within the cell to ensure that an important necessary mRNA blueprint doesn’t ‘accidentally’ get destroyed. Understanding this process at the molecular level provides insight into how we can use it to silence various genes to elucidate their functions.

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