RNA Silencing in Surgical Disease

REVIEW ARTICLE RNA Silencing in Surgical Disease

Jyoti Arya, MD; John Y. Cha, MD; Anirban Banerjee, PhD; Alden H. Harken, MD

ach of our cells contains a complete copy of the genes required to create a whole organ- ism. Thus, it was possible to clone Dolly from a single sheep epidermal cell, and we should be able to reconstruct a dinosaur from the genetic material buried in the mud of “Jurassic Park.” However, it is hard to believe that by adding water to the $5 worth of chemicals in aE human, it is possible to create a human being. Although 30000 instructions, or genes, sounds like a lot, the creation of a hand or a heart or a mind is still a daunting task. Our current genetic code is a lot like the King James Bible—both have evolved. The travails of Moses or Noah were initially “transcribed” by clerics into huge books safely stored in cathedral vaults. With frightening frequency, these imaginative priests “improved” the story as they wrote. The testaments were then “translated” into many languages to enhance access by many peoples. These translations were found, and the data enjoyed further incremental evolution. In eukaryotes, the functional DNA rarely leaves the safety of the nuclear cathedral vault. Traditionally, we have envisioned the Gideon messengers as transcrib- ing reliable molecular copies and distributing them out to motel rooms. While chromosomes are made of double-stranded DNA comprising millions of nucleotide pairs, single-strand messenger RNA (mRNA) carries only single verses, requiring a nucleotide code of only 50 to several thousand nucleotides. Ribosomal RNA synthesizes protein from the mRNA script, while transfer RNA serves an adaptor role in converting the message to protein.1

Two decades ago, RNA assumed exalted sta- serves as antiviral protection in a fashion tus when catalytic RNAs, or ribozymes, were that intuitively might be harnessed for thera- recognized as capable of promoting reac- peutic benefit. The purposes of this article tions similar to protein enzymes. The re- are (1) to delineate the rapidly expanding alization that RNA could also store infor- subtypes of RNA that appear critical to mation spawned the concept that RNA, as healthy cellular function; (2) to explore a simpler molecule than DNA, was a logi- RNA silencing as a highly conserved method cal earlier step in the “origin of life.” As of cellular regulatory protection; (3) to ex- brighter light was shone on RNA, a rapidly amine mechanisms by which ncRNA pro- expanding group of noncoding RNAs tects the genome from external viral at- (ncRNAs) has surfaced. These short- tack; and (4) to present posttranscriptional segment, or micro-RNAs, contain only sev- RNA interference or silencing as a therapy eral dozen nucleotides. In creating func- for medical and surgical disease. tional cells and organs, it is clear that coordinating the expression of gene- CLASSICAL DNA-DRIVEN orchestrated protein synthesis requires PROTEIN SYNTHESIS highly sophisticated temporal and spatial tuning. Micro-ncRNAs appear to exert con- RNA is formed from the same nucleotides siderable posttranscriptional control of mo- as DNA (adenine, cytosine, guanine, and lecular-level activities within a cell. It seems thymine) except, in RNA, uracil pairs with that this regulatory function of ncRNA also adenine as thymine does in DNA. Under the control of promoters and enhancers, seg- From the Departments of Surgery, University of California–San Francisco, East Bay, ments of chromosomal DNA are isolated Oakland, and University of Colorado Hospital, Denver. for copy into relatively short segments of

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 WHAT DO ncRNAS DO? Synthetic RNA RNA Virus Until recently, we could comfortably assume that any change in phenotype or cellular function was driven directly by the genome’s control of protein structure. It is now clear that ncRNAs exhibit both posttranscriptional and posttranslational regulation of protein function, structure, and brane em location. Estimates of the number of ncRNAs in Escheri- siRNA ell M Dicer C + RISC chia coli range up to 200, and several thousand may be op- erative in man. Criteria used in searching for ncRNAs in- ATP clude (1) large gaps between identified protein coding genes, (2) extended stretches of introns conserved among spe- RISC cies, and (3) orphan (unlinked) promoter or terminator RdRP Target mRNA sequences. It is also clearly possible that ncRNAs are pro- duced by the opposite DNA strand of protein-coding genes.2 In its simplest fashion, ncRNAs work by direct base- pairing with RNA or DNA molecules. In this manner, the ncRNAs can bind to the target nucleotide sequence (simi- Endonuclease lar to a blocking antibody) and clog transcription or trans- lation. Some ncRNAs appear to masquerade as promot- mRNA Degradation ers or ribosomal start sequences and can fool the polypeptide synthesis machinery into producing the de- Synthetic “therapeutic” RNA or viral RNA enters a cell. This double-stranded sired protein. Noncoding RNAs can also participate as sig- RNA is recognized and processed by the dicer enzyme into small interfering nal recognition sites in large RNA-protein complexes.4 RNAs (siRNAs). The duplex siRNAs are unraveled by the RNA-induced silencing complex (RISC) into single antisense strands. The RISC complex is activated by adenosine triphosphate (ATP), and the antisense RNA strands HOW DOES ncRNA PROTECT THE CELL? are amplified by RNA-directed RNA polymerase (RdRP). The short antisense strands then bind and “clog” the target messenger RNA (mRNA), marking The critical first step in any successful military coup is to this mRNA for degradation by an endonuclease. With this strategy, virtually any human gene (ie, an activated oncogene) or virus (ie, hepatitis or human take over the central government and the radio and tele- immunodeficiency virus 1) should by accessible to silencing. vision stations. A virus successfully incorporating its genetic material into the genome of a cell is analogous to a foreign agent infiltrating the politburo. Human cells are an mRNA. The transcribed mRNA is the complement of the attractive target for invasion by viruses and other trans- gene to be expressed. After the mRNA is disengaged from posable elements. Plasterk5 estimates that as much as 45% the DNA, the coding regions (exons) are retained, and the of the human genome consists of remnants of prior viral/ apparent noncoding introns are discarded. The resulting transposon infiltration. In warding off a viral attack, the hu- mRNA travels out to the cytoplasmic ribosomes. The short- man cell faces the standard array of immunological prob- segment mRNA is translated into protein within the cy- lems apparent in the spectrum from transplantation toplasmic ribosome. Typically, there is a 3-nucleotide code (accepting “good” nonself) to surgical oncology (rejecting for each of the 20 amino acids. Thus, ribosomal polypep- “bad” self). Thus, the cell must (1) recognize the viral/ tide synthesis begins by reading the start nucleotide codon, transposon invader as nonself and (2) amplify the defen- proceeding through the triple nucleotide amino acid iden- sive response specific to the invader. Eukaryotic cells (like tifying codons, and finishing at the stop codon with a com- ours) appear to have evolved a posttranscriptional gene si- plete protein. Messenger RNA is quite unstable, and 80% lencing strategy that is analogous to “clonal selection” in of it is degraded before it is ever transcribed into protein. the vertebrate immune system. If the genetic message (mRNA) is so unstable, the op- All functional flavors of RNA are relatively short single portunity for posttranscriptional regulation by modifica- strands. Thus, the cell’s warning alarm of nuclear attack is tion and stabilization of the mRNA should be huge—and sounded by double-stranded RNAs (dsRNAs). When an in- it is. Recently, a family of RNA that is not classified as mRNA, filtrating transposon/virus provokes production of dsRNA, transfer RNA, or ribosomal RNA, has been identified.2 This its cover is blown. There are several methods (all hypo- was initially termed other RNA. It is now termed ncRNA. thetical) by which an exogenous nucleotide sequence might Some are short, consisting of 21- to 25-nucleotide seg- promote dsRNA constructs: (1) inserted transposons might ments, and are sometimes referred to as micro-RNA. Other contain a terminal inverted repeat that would trigger du- larger ncRNA appear to influence protein structure and plicate complementary RNA transcription; (2) identical vi- function by regulating transcription, RNA processing, RNA rus/transposons might incorporate into multiple random modification, and RNA stability.2 Following protein syn- locations on the genome with the resultant production of thesis, ncRNAs also provide posttranslational regulation duplicate RNA strands; and (3) “good” self genes may share by influencing protein stability and translocation.3 Intu- some ZIP code or Social Security number motif on their itively, ncRNA and, presumably, nonfunctioning RNA mRNA, identifying the mRNA message as self.5 Whatever should not be conserved through evolution. In this in- the mechanism, cells recognize dsRNA as a death warrant. stance, our intuition is correct. These small bits of ncRNA A successful cell must heed the dsRNA warning and use it seem to do a lot. as a code for self-catharsis.

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 As indicated, dsRNAs contain both sense and anti- RNA SILENCING AS THERAPY sense fragments of the genetic material (viral/transposon) that needs to be silenced. Small bits of the dsRNA are suf- Although it sounds fantastic, multiple groups now state that ficient to bind and clog the target mRNA. It is now clear virtually any gene can be disrupted in cultured human cells.8,9 that these warhead fragments, or small interfering RNAs The problem—likely solvable—is to extrapolate this strat- (siRNAs), are still double stranded and are cleaved from egy to the intact patient. In that small ncRNAs are ubiqui- longer dsRNAs by an adenosine triphosphate–dependent tous processors of gene expression, it is up to us to harness ribonuclease, appropriately named “dicer.” The cell per- the cell’s regulatory machinery. Novina and colleagues10 forms a security check at this point. A 5Ј phosphate is re- have successfully used ncRNA to inhibit events in the life quired on the target complementary strand of siRNA for it cycle of human immunodeficiency virus (HIV) 1 both prior to perform its war on terrorism.6 After the siRNA is con- to and after HIV infection. Double-stranded RNA seg- firmed as targeting the appropriate transposon/gene, it is ments (ncRNA) were introduced into human cells, target- transferred to the RNA-induced silencing complex (RISC), ing mRNA for the HIV1 CD4 cellular receptor (prior to in- which peels off the antisense strand of the siRNA. This an- fection) or the viral structural Gag protein (postinfection). tisense strand of siRNA is now armed and ready to attack This group persuasively espoused the therapeutic poten- and clog the mRNA to be silenced. Chemically synthetic tial of ncRNA for HIV1 and other viral infections. Korneev siRNAs patterned after dicer products have triggered si- and coworkers7 seem to have overcome the animal ncRNA lencing of genes in animals7 and cultured human cells.8 delivery problem in rodents. These investigators injected whole animals with small-segment, nc-dsRNAs, targeting siRNA AMPLIFICATION neuronal nitric oxide synthase. By decreasing nitric oxide synthesis in the brain, feeding was reduced. Hannon11 has Clearly, the unique RNA-dependent RNA polymerase that championed the concept of “RNA therapeutics.” This labo- amplifies the workhorse siRNA probe is the key to this an- ratory has successfully delivered ncRNA with high effi- cient immune process. DNA transcription is not im- ciency, using replication-deficient viruses and has har- peded, but as soon as the suspect mRNA emerges into the nessed the exquisite specificity of RNA silencing, making cytosol, the expanding number of siRNA copies gener- it possible to single out and silence the cancer-causing ac- ated there destroys it. Just as Guttenberg’s printing press tivated oncogene without affecting the healthy allele.11 Simi- mushroomed the availability of scriptural knowledge, the larly, once the genetic mutation that provokes the risk of amplification of stabilized siRNA fragments (that have suc- cardiac arrhythmias has been identified,12 RNA silencing cessfully found an existing mRNA in the RISC) ensures of this potentially fatal gene will soon be feasible. Noncod- that soon, all copies of threatening mRNA will be quenched. ing RNA can now control the biological spectrum from vi- Thus, only a few nanograms of siRNA (21 nucleotides long) ral receptor docking to animal behavior. These observa- will completely silence a gene in a day or 2. This ampli- tions are presented with the conviction that surgeons should fication of a few RNA molecules harkens back several hun- be tuned into the information superhighway—we must drive dred million years, when RNA-based life preceded the ar- the steamroller, and not be part of the road. rival of DNA. Unfortunately—but predictably—several Accepted for publication March 23, 2002. viruses have evolved strategies to counterattack the com- This research was supported in part by grants P50 ponents of the silencing/surveillance system. GM49222 and T32 GM8315 from the National Institutes of Health, Bethesda, Md. SURGICAL GENE KNOCKOUT Corresponding author and reprints: Jyoti Arya, MD, Uni- OWNER’S MANUAL versity of Colorado, Department of Surgery, Box C311, 4200 ENinthAve,Denver,CO80262(e-mail:[email protected]). 1. Identify the target gene, such as the tumor necrosis factor gene (in a patient with an acute myocardial REFERENCES infarction) or the toll-like receptor-4 gene (in a patient with gram-negative bacteremia) or the inducible nitric 1. Riddihough G. The other RNA world. Science. 2002;296:1259. oxide synthase gene (in a patient with sepsis). 2. Storz G. An expanding universe of non-coding RNA’s. Science. 2002;296:1260. 3. Keenan RJ, Freymann DM, Stroud RM, Walter P. The signal recognition particle. 2. Go to PubMed and find the gene nucleotide code Annu Rev Biochem. 2001;70:755-775. and corresponding mRNA sequence. 4. Batey RT, Rambo RP, Lucast L, Rha B, Doudna JA. Crystal structure of the ri- bonucleoprotein core of the signal recognition particle. Science. 2000;287:1232- 3. You will find a nucleotide sequence (which for hu- 1239. man tumor necrosis factor is a 1581-item sequence), within 5. Plasterk RHA. RNA silencing. Science. 2002;296:1263. which you must locate 21 sequential nucleotides that be- 6. Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 2001;107:309-321. gin with AA. With this template, you can construct an an- 7. Korneev SA, Kemenes I, Straub V, et al. Suppression of nitric oxide (NO)- tisense or short-segment, mRNA-“clogging” strand. dependent behavior by double-stranded RNA-mediated silencing of a neuronal 4. Copy this sequence and feed it into Ambion’s siRNA NO synthase gene. J Neurosci. 2002;22:RC227. 8. Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specific inhibition of gene ex- target finder and designer tool engine (www.ambion.com pression by small double-stranded RNA’s in invertebrate and vertebrate sys- /techlib/misc/siRNA_finder.html). tems. Proc Natl Acad Sci U S A. 2001;98:9742-9747. 9. Zamore PD. Ancient pathways programmed by small RNA’s. Science. 2002;296: 5. Order your nucleotide sequence at $15 a pop for 1265-1269. each nucleotide (21ϫ$15=$315). 10. Novina CD, Murray MF, Dykxhoorn DM, et al. siRNA-directed inhibition of HIV-1 6. You may now inject/transfect your designer siRNA infection. Nat Med. 2002;8:681. 11. Hannon GJ. RNA interference. Nature. 2002;418:244-251. or ncRNA into a cell, tissue, or intact patient. 12. Marx J. Gene mutation may boost risk of heart arrhythmias. Science. 2002;297: 7. Do not try this at home. 1252.

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