MicroRNAs (miRNAs) are small RNA molecules generated in the cell to control the generation of proteins. Like siRNA, miRNAs are single stranded RNA molecules around 22 nucleotides in length that are bound by proteins called Argonaute (Ago) proteins. These Ago proteins are also able to bind to messenger RNA (mRNA), the RNA molecules that are the blueprints for making a specific protein. Bringing the miRNA into close contact with the mRNA, and if there is base pair matching between the miRNA and mRNA, they will bind to each other. This generates a small portion of double stranded RNA that either blocks reading of the mRNA to generate protein, or results in destruction of the mRNA molecule, thereby inhibiting protein synthesis. This is akin to brining the perfect Tetris piece into alignment with the last gap in the row: it will slot into the space and make the row disappear.
In this paper, the authors wanted to determine how miRNA molecules are guided to the proper binding location on an mRNA by Ago proteins, and what the minimal match between the miRNA and mRNA is needed to cause protein synthesis to be blocked. They tested this by fluorescently labeling both the miRNA molecule and a target mRNA molecule that had been immobilized on a coverslip. The miRNA were added to purified Ago proteins, with the complexes then being added to the mRNA. If the researchers saw the fluorescent signal, which occurred due to the FRET principle, it meant the miRNA and mRNA were in contact, indicating binding.
The authors found that the miRNA could bind to the mRNA when the nucleotides numbered 2 through 4 of the miRNA match a portion of the mRNA. If multiples sites existed on the mRNA that the miRNA nucleotides 2 through 4 match, the Ago/miRNA complex would jump between the multiple sites. Once there is a match at one of the sites on the mRNA to the miRNA nucleotides 2 through 8, the miRNA would irreversibly bind, and would then lead to inhibition of translation. This study therefore succeeded in showing the minimal requirements for miRNA binding, leading to inhibition of protein synthesis, helping to show the method how cells properly employ miRNA, which was previously unknown.
Stanley D. Chandradoss, Nicole T. Schirle, Malwina Szczepaniak, Ian J. MacRae, and Chirlmin Joo. 2015. A dynamic search process underlies microRNA targeting. Cell, 162: 96-107.