Small interference; significant interpretation

To discover what something does, often we remove it and observe what changes. This is especially true in biology. Jinquan observed that under certain conditions, cells start producing a lot of a protein called ATF4. Proteins in cells are frequently linked in pathways, wherein one protein turns on another protein, which turns on another, and so on, until the final protein actually carries out the cells’ responses to a stimulus. Jinquan wants to figure out if ATF4 turns on several other proteins in a pathway. If she can make some cells stop producing ATF4 while allowing other cells to produce it, and then show (using western blotting, discussed previously) that the cells without ATF4 are producing less of those other proteins than the cells that are still producing ATF4, then she has evidence that ATF4 is part of that pathway. But how to turn off ATF4 protein?

RNA interference is one method we use to stop making a protein. Normally, to make a protein, a cell first uses the DNA segment encoding the protein (a gene) to create a piece of RNA, a molecule similar to DNA, in a process called transcription. This type of RNA, which codes for a protein, is messenger RNA (mRNA). The cell then uses a large group of molecules known as a ribosome to read the mRNA and make a protein from its code of bases, a process called translation.

DNA → mRNA → protein

Eukaryotic cells (cells with nuclei, such as those of all macroscopic organisms) have evolved a way to prevent the translation of a specific protein by destroying the mRNA encoding it: RNA interference. RNA interference is useful in the lab, where we can use it to dramatically reduce the amount in which a cell produces a specific protein, and, by observing the differences between cells with and without the protein, infer the protein’s function. We can induce RNA interference by adding, or transfecting, another type of RNA called small interfering RNA (siRNA) into cells. siRNA, like DNA, is a double-stranded helix, but, unlike DNA, is automatically chopped up into small fragments by a protein called Dicer. Then, a collection of proteins known as an RNA-induced silencing complex binds to each fragment, discards one strand of siRNA, and displays the strand it retains. Just like two complementary DNA strands in a double helix bind together, mRNAs that have sequences that match the siRNA sequences will bind to the siRNA. A protein called Slicer (or Argonaute), part of the RNA-induced silencing complex, then cuts up the mRNA, preventing it from being translated into a protein. RNA-induced silencing complexes can work over and over, degrading many mRNAs, and shutting down the production of the protein that the mRNA encodes. For an in-depth video on RNA interference, follow this link.

Mechanism_of_RNA_interference

 

Simone Mocellin and Maurizio Provenzano: Mechanism of RNA interference. Wikimedia Commons. Here, the double-stranded siRNA (abbreviated dsRNA) is chopped by Dicer to form siRNA fragments, which associate with the RNA-induced silencing complex (RISC) and cleave mRNA (blue) into fragments, preventing translation.

Earlier this week, I watched Jinquan transfect cells with siRNA that specifically targets the mRNA that produces a protein called ATF4. A day before the transfection, Jinquan had cultured the cells she wanted to transfect in 4 plastic wells.

Two wells would be transfected with siRNA for ATF4. To prepare this siRNA, Jinquan added in one tube 8 microliters (µL) of siRNA for ATF4 to 200 µL of a special siRNA transfection medium containing solutes that facilitate the transfection. Next, to 200 µL of siRNA transfection medium in a second tube, she added 10 µL of siRNA transfection reagent, which binds to the siRNA and helps to carry it into the cells. She pipetted the dissolved siRNA into the transfection reagent tube and waited 30 minutes while the two bound together.

(8 µL siRNA + 200 µL medium) + (10 µL transfection reagent + 200 µL medium) =  ~ 400 µL

To understand what the lack of ATF4 does, cells without ATF4 must be contrasted with cells with ATF4—the negative control cells. To make sure that any differences between the negative controls and the transfected cells arose from differences in ATF4 levels, and not from the process of transfection itself or the addition of the transfection reagent, Jinquan also mixed 10 µL of transfection reagent with 400 µL of transfection medium.

10 µL transfection reagent + 400 µL medium = ~ 400 µL

Using a small vacuum pipette, Jinquan sucked out the liquid medium in which the cells were growing (the cells remained attached to the bottom of the plastic wells), and she washed them with transfection medium, which she removed. After adding 800 µL more of transfection medium, she pipetted the approximately 200 µL of siRNA with transfection reagent into 2 wells and 200 µL of transfection reagent alone into the other 2 wells, drop by drop, each drop in a different place. She gently rocked the wells to distribute the siRNA, then incubated them for 6 hours to let the wonders of transfection and RNA interference occur.

Now, a few days after the transfection, Jinquan is making western blots of proteins extracted from the cells. If she finds that ATF4 is much lower in transfected cells, then the transfection worked. What she is curious about, though, is whether the other proteins she suspects are part of the ATF4 pathway are much lower in transfected cells. If they are, her results will be evidence that ATF4 is upstream of the other proteins in the same pathway. Slowly, using RNA interference, the pathways of cellular proteins can be elucidated, which can lead to novel drugs to inhibit proteins in cancer pathways or to turn on more salubrious proteins.

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