It’s time for a new edition of my biochemistry textbook

Jon Karpilow, PhD

My biochemistry book, a classic 2^nd edition of Lehninger from 1978, sits on the top shelf at home and tells me that RNA belongs to three functional groups – messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA). In 1978, mRNA was the most abundant of these three, and was known to serve a critical function in transferring the genetic information from DNA into protein.  This process required mRNA’s two non-coding sidekicks, rRNA and tRNA. Aside from these three, the world of RNA was somewhat predictable.

A decade later, RNA casts off this dated reputation. In addition to its classical role in transmission and translation of genetic information, it was shown to embody new capabilities. We now know that RNA can replicate, catalyze reactions, and even evolve in a test tube. So broad are these newly discovered functions, that some have suggested that RNA is the most abundant biopolymer in both quantity and diversity of function and a possible contender for the “first molecule of life” award.[1,2]

The diversity of RNA functions was considerably expanded with the recent discovery of RNA interference (RNAi), a post transcriptional gene regulatory phenomenon mediated by small non-coding RNAs called microRNAs (miRNAs).[3,4] First discovered in plants and C. elegans, RNAi has since been demonstrated to serve a vital role in virtually all eukaryotes.[5] In fact, miRNAs are so prevalent yet so seemingly invisible, that they have been coined the “dark matter” of the cell.[6] How do they work? miRNAs appear to function by targeting the 3’-untranslated regions of dozens of mRNAs for translation attenuation. The synchronized modulation of these proteins defines a regulatory program that controls cellular fate and homeostasis in lower eukaryotes[7,8,9] and most certainly provides a similar function in higher eukaryotes. As the regulatory role of RNAi becomes clearer, so has an understanding of the molecular machinery that mediates this process. Critical among the cellular components that drive RNAi is the highly conserved multi-protein complex known as the RNA-induced silencing complex (RISC). It is the interaction of the small regulatory RNA intermediates with RISC and the target mRNA that largely defines the silencing outcome – specifically, partial complementarity of the miRNA towards its target effects translational repression while perfect complementarity leads to target cleavage.  

In mammalian systems, it is possible to appropriate this highly conserved cellular machinery to induce the reduction of specific transcripts.  Since the first demonstration of target specific silencing,[10] this reverse genetic tool has been broadly implemented by the scientific community and used in basic research to define gene function and in drug discovery platforms to validate targets for drug development programs. By transfecting or transducing the short interfering RNA (siRNA) which represents the active intermediate of RNAi into the cell, one can effectively eliminate expression of the gene.  siRNAs are typically 19-23mer double-stranded oligonucleotides designed to have perfect complementarity to the gene target and can be generated by a variety of enzymatic, expression-based or chemical methods.  By far, chemically synthesized siRNAs are the most commonly used method for cell culture-based assays. However, expression from viral-based vectors is also gaining in popularity as a complementary tool that offers the possibility of tissue specific or sustained knockdown. It is important to note that vector expression does have its shortcomings. Recent studies by Grimm and associates at Stanford demonstrated that expression of shRNAs from vector-based constructs can over-saturate the cellular pathways dedicated to transporting the natural RNAi substrates, miRNA.[11] Thus additional studies and designs are needed to minimize the effect of DNA-based RNAi on endogenous miRNA gene regulatory mechanisms.

One of the primary challenges facing RNAi as an emerging technology was that for any targeted gene, thousands of candidate siRNAs exist.  Some of these duplexes are functional and capable of >90% gene knockdown, but others are not. The development of rational design principles and algorithms that can predict functionality was a significant step in making the technology a robust tool.[12]  Today, with sequence information for any well annotated gene, it is possible to rationally design a functional silencing reagent. This approach has been so successful that collections of synthetic silencing reagents that target complete genomes are now in widespread use. Large scale screening efforts using RNAi are currently underway in both academic and industrial research centers around the globe and RNAi technology has already contributed significantly to our understanding of gene function.[13]  Similar work is beginning with expressed shRNAs; however design strategies for shRNAs are still evolving.

Because we are appropriating such an important cellular pathway, it comes as no surprise that experimental artifacts commonly referred to as non-specific or “off-target” events have been observed, resulting in an unintended down regulation of non-targeted genes. Many of these activities can be eliminated by careful experimental design and the inclusion of siRNA controls (positive, negative and non-functional controls). More recently, a leap in our understanding of off-targeting has come from analysis of the sequence dependence of the event. Analysis of the miRNA pathway revealed that the seed region, positions 2-7 of the antisense strand, plays a critical role in targeting the 3’-UTRs of their regulated transcripts.  Eloquent studies by a number of labs have now demonstrated that off-targeting by siRNAs occurs by a similar mechanism, thus tying the two pathways together.[14] How do you eliminate off-targets and the (false positive) phenotypes that they generate? A joint study between Dharmacon and Rosetta Inpharmatics recently identified a chemical modification pattern that dramatically reduces off-target events.[15] In addition, the R&D group at Dharmacon has added an important dimension to a silencing strategy that relies on the concept of introducing not just one potent siRNA but a mixture of four rationally designed siRNAs to effect reliable, potent and specific silencing (e.g. siGENOME collection of SMARTpool silencing reagents from Dharmacon).  By combining a more fundamental understanding of the pathway, sophisticated bioinformatics and advanced siRNA chemistries, RNAi will become indispensable as a tool for discovery biology, drug development and ultimately in the therapeutic arena.

While the intense efforts in the field of RNAi have quickly opened a new world of research, recent studies hint that we are looking at just the tip of the iceberg. Current work by Lau and colleagues has identified a second class of small, regulatory RNAs (called piwi-interacting RNAs, or piRNA) that are slightly longer than mature miRNAs, but equally broad in diversity.[16] Similarly, a group of investigators have recently discovered a new mechanism where by components of the RNAi pathway return to the nucleus and regulate expression by targeting the chromatin for modification.[17] In future issues of m360, we will focus on these and other observations as the field develops to address new discoveries, technological challenges, and novel methods of implementing RNAi with complementary technologies. We hope this forum serves as site to educate, inform and open the door to novel applications.

 

References:

1. Vaughn, M. W., and R. Martienssen. "It's a Small RNA World, After All." Science 309.5740 (2005): 1525-26.
2. Waldrop, M. M. "Did life really start out in an RNA world?" Science 246.4935 (1989): 1248-9.
3. Lim, L. P., et al. "Vertebrate microRNA genes." Science 299.5612 (2003): 1540.
4. Bartel, D. "MicroRNAs: genomics, biogenesis, mechanism, and function." Cell 116.2 (2004): 281-97.
5. Fire, A., et al. "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans." Nature 391.6669 (1998): 806-11.
6. Riddihough, G. "In the Forests of RNA Dark Matter." Science 309.5740 (2005): 1507.
7. Lee, R., R. Feinbaum, and V. Ambros. "The C. elegans heterochronic gene lin-4 encodes small RNAs with complementarity to lin-14." Cell 75.5 (1993): 843-54.
8. ---. "A short history of a short RNA." Cell 116.2 Suppl (2004): S89-92, 1 p following S96.
9. Karp, X., and V. Ambros. "Developmental biology. Encountering microRNAs in cell fate signaling." Science 310.5752 (2005): 1288-9.
10. Elbashir, S. M., et al. "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells." Nature 411.6836 (2001): 494-8.
11. Grimm, D., et al. "Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways." Nature 441.7092 (2006): 537-41.
12. Reynolds, A., et al. "Rational siRNA design for RNA interference." Nature Biotechnology 22.3 (2004): 326-30.
13. Sorkina, T., et al. "RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis." J Neurosci 26.31 (2006): 8195-205.
14. Birmingham, A., et al. "3' UTR seed matches, but not overall identity, are associated with RNAi off-targets." Nat Methods 3.3 (2006): 199-204.
15. Jackson, A. L., et al. "Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing." Rna  (2006).
16. Lau, N. C., et al. "Characterization of the piRNA RNA complex from rat testes." Science 313.5785 (2006): 363-7.
17. Verdel, A., et al. "RNAi-mediated targeting of heterochromatin by the RITS complex." Science 303.5658 (2004): 672-6.