Day 13 Afternoon Lecture Notes

Steve Williams, Smith College

June 18, 2004

RNA interference (RNAi)

The expression of a given gene can be down-regulated by eliminating its cognate mRNA. An RNA should be blocked by insertion of its antisense strand into the cell as dsRNAs are degraded. RNA interference is similar to gene knockout but is much cheaper. It's a fast, inexpensive way to do "gene silencing." RNA interference uses a "phenocopy" of a mutation.

There are many methods of performing RNA interference. For example, feed C. elegans nematode worms some E. coli that are expressing the interfering RNA. Alternatively microinject the interfering RNA, induce absorption via electroporation, or simply put the worms in a solution containing the RNA.

Surprisingly a recent paper (A. Fine, Nature 391 (1998)) showed that dsRNAs have a stronger interfering effect than antisense RNAs. dsRNAs are most effective because they copy themselves and move from cell to cell like a retrovirus. RNA interference has also been shown to work in Drosophila, mammals and plants.

Organisms use naturally occurring "microRNAs" as post-transcriptional method of regulating gene expression. Methods of destroying RNAs are important since otherwise transcripts can hang around for days in eukaryotes. "Short interfering" SI-RNAs are 20-25 nt in length and work best in mammals. Longer interfering sequences are called RNAis. Small RNA fragments found in gels were always thought to be degraded RNAs but they can also be microRNAs!

Postulated in vivo roles for RNAis:

• block retroviruses;
• transposon (like LINE1) suppression;
• gene regulation via microRNAs.

Exogenous (artificially inserted) dsRNA binds to dicer RNAse III. Dicer cuts it into SI-RNAs of 21 nt length in mammals. 21 nt fragments bind into RISCs (RNA interfering short complexes). RISCs cuts mRNAs and gene expression is turned off. All of this occurs in the cytoplasm. dsRNA works as a silencer because it binds the RISC complex. Antisense RNA only works by binding to mRNA directly. It's unknown if retroviruses have any method to block dicer.

To turn off a particular gene using dsRNA, clone the appropriate cDNA into a plasmid. Use the T3 and T7 promoters to make two ssRNAs in pBluescript and then mix them together.