Structural Biochemistry/Nucleic Acid/RNA/RNA Interference/RISC (RNA-induced silencing complex)

Main Component: Argonaute
The main component of RISC is the argonaute (Ago) proteins. These proteins will associate the RNAs. The Ago family can be divided into the Argo subfamily and the Piwi sub family. siRNAs and miRNAs bind to the Argo subfamily and piRNAs bind to the Piwi subfamily. In mammals, each of the four Ago subfamily proteins (AGO1-4) can repress translation but only AGO2 can cleave the RNA and result in RNA interference (RNAi)

Two Steps in RISC Assembly: RISC Loading and Unwinding
siRNA and miRNA come from double stranded RNA that has been chopped up by the RNase III enzymes, Drosha and Dicer. The resulting RNA are call RNA duplexes. There are two models of when RNA unwinds when binding to Ago proteins. The ‘helicase model’ propose that the RNAs were separated into single stranded RNA first then incorporated into the Ago proteins. The other model is the ‘duplex-loading model’ which states that the double stranded RNA binds to the Ago proteins then dissociated within the protein. Recent studies show that the ‘duplex-loading model’ may be the model for when RNA unwinds. Therefore, RISC assembly can be divided into 2 steps: small RNA duplex is bound to Ago protein, the double stranded RNA dissociates into two single stranded RNA. RNA duplex bound to Ago protein is called pre-RISC while Ago protein with single stranded RNA is called mature RISC.

Since the double stranded RNA will unwind into two single stranded RNA one of these strands must be discarded. The discarded RNA strand is called the passenger strand and the other strand is called the guide strand. The strand with a less stable 5’ end will serve as the guide strand while the other strand is discarded.

RISC loading machinery
Ago proteins need the help of RISC-loading machinery to bind to RNA. RISC-loading machinery is composed of Dicer-2 (DCR-2) and R2D2 for Drosophila Ago2. R2D2 binds to the more stable end of RNA while Dcr-2 binds to the more stable end. Although Dcr-2 can both dice up RNA and load RNA into Ago proteins, studies has shown that the siRNA duplexes must dissociate from Dcr-2 after dicing then rebind to the Dcr-2-R2D2 dimer according to its stability. Human only has one type of Dicer, human Dicer and its partner protein TRBP (TAR binding protein) helps load RNA into AGO2-RISC. However, studies have shown that Dicer is only needed when loading into fly Ago2. It is not needed when loading RNA complexes for other Ago proteins. It seems that there are two pathways of RISC loading, a Dicer dependent pathway and a Dicer independent pathway.

Small RNA sorting
siRNA duplexes usually have perfectly complementary sequences so that all the bases are lined up. However, miRNA-miRNA* complexes usually have central mismatches. In flies, the Dcr-R2D2 likes to bind to the perfectly complementary siRNA like complexes but doesn’t like RNA strands with mismatches. On the other hand, Ago1 likes to bind to sequences that has central mismatch around nucleotide 8-11.

Another guild loading in the right orientation is the identity of the nucleotide in the 5’ end of the guild strand. In flies, Ago1 favors U while Ago2 favors C. For plants, the orientation of the strands relies heavily on the identity of the nucleotide as well. Arabidosis AGO1 prefers U, AGO2 and AGO 4 prefer A and AGO 5 prefers C. The MID and PIWI domain of Arabidosis Ago proteins confer recognition of the nucleotide at the 5’ end. However, mammalian Ago proteins only prefer perfectly complementary siRNA like complexes and disfavor RNA with non-central mismatches. However, if the RNA only has central mismatches, Ago protein will incorporate it without any difficulties as well. Also, human Ago protein does not have a preference for the 5’ end nucleotide. Therefore, human Ago proteins do not have a strict small DNA sorting system.

Slicer-dependent unwinding
A pre-RISC loaded with double stranded RNA is very similar to a mature RISC that is bounded to a target mRNA. Therefore, the passenger RNA is like the target RNA for the guide strand. In slicer-dependent unwinding, the passenger strand is discarded just like how a target mRNA would be discarded. This type of unwinding only occurs in siRNA like complexes that has highly complementary strands.

Slicer-independent unwinding
Human AGO1, 3, and 4 does not have any slicer activity, therefore, it cannot use slicer-dependent unwinding. Also, if the RNA strands have mismatches, the slicers would not unwind the two strands. Therefore, another pathway was proposed as the slicer-independent unwinding. In this type of unwinding, the mismatched RNA will actually accelerate the unwinding process and it is essential for this type of unwinding. Therefore, scientists dub this the ‘mirror-image’ process of target recognition. It is basically the opposite of when the guild strand anneals to the target strand.