Structural Biochemistry/Making RISC's

Making RISC’s

MicroRNA's, Piwii-interacting RNA's, and small interfering RNA's are unique in the world of RNA catalysts because they cannot perform any designated functions on their own. In order for small RNA's to function, they must first make RISC's. RISC stands for RNA-induced Silencing Complex. RISC's play an important role in regulating a multitude of biological processes by interfering with gene expression. Analyzing the assembly of these effector complexes can help us gain a better understanding of how small RNA's such as siRNA silence specific sequences. The assembly of RISC's has puzzled the scientific community because the final product contains single stranded RNA, while its precursors contain double-stranded RNA.

Argonaute

The central protein of an RISC is the Argonaute, abbreviated Ago. The term Argonaute encompasses a family of proteins that act as catalyst in RISC's. The specific function that the small non-coding RNA will perform is determined, in part, by which Ago protein it is associated with. There are two primary classes of Ago proteins. One class binds to miRNA's and siRNA's while the other primarily binds to PiRNA's. Argonaute proteins share the ability to prevent translation. However, they differ in how they interfere with the production of polypeptides. For example, in humans, the AGO2 protein uses a cleaver to create RNAi. Whereas in flies, the AGO1 protein works with miRNA to regulate gene expression.

The Assembly of RISC’s

Although Ago proteins are central to the formation of RISC's, the mere binding of a small noncoding RNA to its complementary protein will not result in a complete RISC. Research has shown that RISC assembly is the result of a highly regulated mechanism. This mechanistic pathway results in the processing of small RNA until the desired RISC is produced. The assembly of an RISC can be broken down into two primary steps, loading and unwinding. In the first step, the noncoding RNA is “loaded” onto its corresponding Ago protein. In the second step, the double stranded small RNA is separated inside of the Ago protein. This is the “unwinding” step which results in a single stranded RISC molecule.

ATP Powers RISC Assembly

Researchers have found that ATP is needed to load miRNA onto the Ago protein but it is not required to unwind the complex within the protein. These results have been confirmed for both drosophila and humans. Upon closer examination of Ago protein complexes, it was found that these proteins lack any domains that could be used to bind ATP. Scientists hypothesize that the ATP is consumed by machinery in the process of non-coding RNA loading.