www.nature.com/collections/antisense December 2019 Antisense RNA Produced by: With support from: Nature Structural & Molecular Biology, Nature Reviews Molecular Cell Biology and Nature Communications MILESTONES IN ANTISENSE RNA RESEARCH 1970s Antisense cellular RNA and synthetic oligonucleotides inhibit mRNA translation (MILESTONE 1) 1990 RNA-mediated post-transcriptional gene silencing (PTGS) in petunia (MILESTONE 2) 1993 microRNAs emerge as novel post-transcriptional regulators of gene expression (MILESTONE 3) 1998 Double-stranded RNA mediates sequence-specific gene silencing in animals (MILESTONE 4) FDA approval of the first antisense oligonucleotide drug (MILESTONE 5) 1999 Small RNAs trigger different types of PTGS in plants (MILESTONE 6) 2000 Elucidation of the molecular mechanism of RNA interference (MILESTONE 7) 2001 First demonstration that small interfering RNAs trigger gene-specific silencing in mammals (MILESTONE 8) Discovery of PIWI-interacting RNAs (piRNAs) (MILESTONE 9) 2002 Advent of plasmid-based gene silencing and large-scale RNA interference screens (MILESTONE 10) 2005 Targeted gene silencing in vivo by antibody-mediated siRNA delivery (MILESTONE 11) 2012 Gene editing by CRISPR–Cas9, from bacteria to humans (MILESTONE 12) 2014 Chemical optimization enables targeted delivery of siRNA drugs to the liver (MILESTONE 13) 2016 FDA approval of an antisense oligonucleotide drug to treat spinal muscular atrophy (MILESTONE 14) 2018 First RNAi-based therapeutic agent approved by the FDA (MILESTONE 15) S4 | DECEMBER 2019 www.nature.com/collections/antisense MILESTONES ‘Waltz of the polypeptides’ by Mara G. Haseltine, Cold Spring Harbor Laboratory, NY, USA. Credit: Ivanka Kamenova / Springer Nature Limited MILESTONE 1 First signs of antisense RNA activity The first reports of antisense RNA activity in to sequestering the inhibitory RNA through replication and oncogenic transformation eukaryotes were published when the efforts base-pairing. The ideas put forward by of the cells. The chemically modified oligo- to understand the mechanism of mRNA Ochoa — that small regulatory RNAs exist as nucleotide performed better, likely because the translation were well underway. At that time, double stranded structures and are generated modifications conferred resistance to nucleases cell-free in vitro translation systems were by endogenous RNase enzymes — have been in the culture medium. widely used to probe the function of different revisited in subsequent decades, as the mech- The most likely target processes of the translation co-factors. These minimal systems, anisms for processing of small interfering complementary oligonucleotide were the cir- comprised of a cell extract and a template RNAs and microRNAs were uncovered. cularisation of provirus DNA or the translation mRNA, proved instrumental in the initial Whereas the above reports described the of viral mRNA. Although the first possibility characterisation of cellular RNAs that could activity of cellular antisense RNAs, in 1977, was not ruled out, in a companion paper control mRNA translation through antisense Paterson et al. demonstrated that exogenous published in the same issue of Proceedings of recognition of their targets. plasmid DNA fragments complementary to the National Academy of Sciences, Stephenson Working in such a cell-free system, in an mRNA could also inhibit its translation in and Zamecnik demonstrated that the delivered 1975, Stuart Heywood and colleagues demon- vitro. A year later, Paul Zamecnik and Mary oligo inhibits translation of viral mRNA strated that short ribonucleotide sequences Stephenson reported the first synthetic DNA through sequence-specific hybridisation. purified from chicken muscle messenger ribo- oligonucleotide delivered to cells, capable of Our knowledge of the mechanisms nucleoprotein and polysome fractions could inhibiting viral replication and oncogenic by which endogenous RNAs regulate key control translation of the mRNA encoding transformation caused by Rous sarcoma virus. processes in cell function and in devel- myosin. They called the RNAs ‘translation The 35S RNA of this retrovirus had recently opment has since grown exponentially. control RNAs’ (tcRNAs) and proposed that been found to contain a 20-nucleotide repeat Yet, these early experiments, using highly tcRNAs act by binding to their mRNA targets sequence in its 5ʹ and 3ʹ ends. The authors purified com ponents, provided the first in a sequence-specific manner. In follow-up synthesized 13-nucleotide-long DNA oligo- hints that cells produce small RNAs with work a decade later, Heywood demonstrated nucleotides complementary to part of the viral the capacity to affect translation, and that that one of these tcRNAs, tcRNA102, repeat sequence, which was hypothesized to be the complemen tarity of these RNAs to their recognises a sequence in the 5ʹ untranslated important for viral replication, and tested their target is important for their function. The region of chicken myosin mRNA, albeit with effects on the growth of chicken embryonic experiments by Zamecnik, Stephenson and imperfect homology. fibroblasts infected with Rous sarcoma virus. others laid the foundation of the field of anti- In the years immediately following Two synthetic oligonucleotide variants were sense RNA therapeutics and resulted in the Heywood’s original tcRNA discovery, several tested: one with free 3ʹ and 5ʹ termini, and one founding of Hybridon, the first biotechnology groups isolated similar small RNA species with chemical modifications. Strikingly, addi- company dedicated to developing synthetic from different organisms and demonstrated tion of either oligonucleotide to the cell culture oligonucleo tides for therapeutic purposes. that these short RNAs could modulate trans- medium at the time of infection inhibited viral Ivanka Kamenova, Associate Editor, Nature Protocols lation. Notably, in 1977, Severo Ochoa and colleagues purified two distinct short RNAs MILESTONE STUDIES Bester, A. J. et al. Two classes of translational control RNA: their role in the regulation of protein synthesis. Proc. Natl Acad. Sci. USA 72, 1523–1527 (1975) | Lee-Huang, S. et al. Eucaryotic oligonucleotides affecting mRNA translation. Arch. Biochem. from a small crustacean, Artemia salina, and Biophys. 180, 276–287 (1977) | Paterson, B. M. et al. Structural gene identification and mapping by DNA–mRNA hybrid-arrested cell- showed that these RNAs exerted activating free translation. Proc. Natl Acad. Sci. USA 74, 4370–4374 (1977) | Zamecnik, P. C. et al. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl Acad. Sci. USA 72, 280–284 (1978) | Stephenson, M. L. et al. Inhibition and inhibitory effects on A. salina mRNAs. of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl Acad. Sci. USA 75, 285–288 (1978). The activator RNA is complementary to the FURTHER READING Heywood, S. M. tcRNA as a naturally occurring antisense RNA in eukaryotes. Nucleic Acids Res. 14, 6771–6772 inhibitory RNA, and thus its stimulatory (1986) | Zamecnik, P. C. From protein synthesis to genetic insertion. Annu. Rev. Biochem. 74, 1–28 (2005) | Kresge, N. et al. The Discovery of tRNA by Paul C. Zamecnik. J. Biol. Chem. 280, e37 (2005). effect on translation was proposed to be due NATURE MILESTONES | ANTISENSE RNA DECEMBER 2019 | S5 MILESTONES MILESTONE 2 Patterns of co‑suppression in plants If cuttings were taken from the branches, the new individuals would have the same flower pattern as the …experiments branch from which they were cut. on pigmentation Back-crossing the transformed in petunias did plants to parental lines allowed not simply have the researchers to show that the unexpected altered pigmentation only occurred when the transgene was present results, and active. RNase protection assays but results using radiolabelled RNA probes completely antisense to the gene showed that opposite to messenger RNA levels for both what had been endogenous and transgenic CHS in transgenic plants were reduced or anticipated, essentially abolished. arguably It would have been very easy for marking the Jorgensen and his team to dismiss beginning of these results as a failed experiment, the field of RNA not least because a similar approach interference attempted by a group at the University of Amsterdam had not produced the dramatic phenotypes seen by the Californians. However, discussions between the two groups Credit: blickwinkel / Alamy Stock Photo Stock Alamy / blickwinkel Credit: identified subtle differences in grow- ing conditions. Increasing the light It is often said that truly novel discov- by increasing the number of copies levels resulted in the Dutch petunias eries arise when experiments do not of the gene encoding the enzyme confirming Jorgensen’s observations. go as planned. This is certainly true chalcone synthase (CHS). CHS catal- These experiments showed clearly of one of the earliest observations of yses the condensation of one mole- that co-suppression of gene expres- an RNA-mediated gene silencing cule of 4-coumaroyl-CoA and three sion was dependent on high-copy- mechanism by Richard Jorgensen molecules of malonyl-CoA to form number transcription of a transgene and colleagues at the DNA Plant naringenin chalcone, from which a homologous to a native gene, but was Technology Corporation in Oakland, wide variety of flavonoid pigments not affected by the proximity of those California. Their experiments on are synthesised. genes in the plant genome. pigmentation