- Define microRNA and siRNA.
- Briefly describe the mechanisms by which RNAi interference affects gene expression.
- Predict the effect of a miRNA or siRNA that is fully or partially complementary to an mRNA.
MicroRNAs (miRNAs) are small (20-25 nucleotide) RNAs that are encoded by genomic loci. MicroRNAs were first discovered when scientists tried to identify the gene responsible for a heterochronic mutant (lin-4) C. elegans in the early 1990s. The mutant had been identified nearly two decades prior as one in which the normal pattern of larval molting did not stop, instead adult worms continued to repeat larval patterns, hence the name meaning cell lineage-defective. The mutation turned out to be in a non-protein-coding region that produced a small RNA with partial complementarity to seven sites in the 3' UTR of the lin-14 RNA (Lee et al, 1993). Previous studies had shown that lin-4 was a negative regulator of lin-14, so this result revealed that the mechanism involved an RNA acting by repressing the another RNA! Further work in C. elegans revealed that another gene, let-7, also encodes a microRNA that regulates multiple target RNAs via RNA-RNA interactions.
This work has led to the discovery of many more microRNAs in many different organisms. The current estimate of number of miRNAs in the human genome is about 2,300 (Alles et al., 2019).
Why are small RNAs important?
The discovery of two non-coding RNAs in C. elegans turned out to be just the beginning of our understanding of the prevalence and activity of small RNAs. Two other C. elegans biologists, Andrew Fire and Craig Mello, were studying another aspect of RNA activity at the same time. They were following up on some research that had used antisense RNAs to inhibit the expression of certain genes.
Antisense RNA: the theory
Remember that only one strand of a gene is transcribed into RNA.
The mRNA contains the codons needed to make the correct protein so it makes SENSE, but it was transcribed using the ANTISENSE strand as a template.
The ANTISENSE RNA does not usually exist in cells, but if you provide it, it will be complementary to the mRNA.
The theory was that this would prevent the translation of the RNA.
The experiments of Fire and Mello revealed that injecting double-stranded RNA (dsRNA = sense RNA + antisense RNA) cause a more potent response than injecting antisense alone. They also demonstrated that the injected dsRNA caused a decrease in mRNA levels, not just protein levels that was predicted by the antisense RNA idea. The phenomenon of a small RNA that targets a cellular mRNA for degradation is known as RNA interference, and these small RNAs are known as siRNAs (small interfering RNAs). These typically differ from microRNAs in that they have perfect complementarity to their target genes and cause their destruction.
The timely discovery of microRNAs and RNA interference by dsRNA revealed some shared mechanisms of action. Both microRNAs produced by transcription and siRNAs provided experimentally are cleaved into smaller (20-25 nt) fragments by the enzyme Dicer. The short pieces then are loaded to a RISC (RNA induced silencing complex), which contains a single-stranded small RNA while surveilling the cell for complementary sequences. When perfect (100%) complementarity is found, the target RNAs are cleaved; when imperfect complementarity is found, the outcome is often translation repression, although other effects are possible.
Fire, A., Xu, S., Montgomery, M. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998). https://doi.org/10.1038/35888
Julia Alles, Tobias Fehlmann, Ulrike Fischer, Christina Backes, Valentina Galata, Marie Minet, Martin Hart, Masood Abu-Halima, Friedrich A Grässer, Hans-Peter Lenhof, Andreas Keller, Eckart Meese, An estimate of the total number of true human miRNAs, Nucleic Acids Research, Volume 47, Issue 7, 23 April 2019, Pages 3353–3364, https://doi.org/10.1093/nar/gkz097
Vella, M.C. and Slack, F.J. C. elegans microRNAs (September 21, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.26.1, http://www.wormbook.org.