RNA Activation ﬂew in the Face of at Dallas, Was Convinced She Had Messed Would Be Even More Unbelievable Than What Everyone’S Perceived Wisdom Regarding Something Up

MODIFIED FROM ORIGINAL PHOTO. © 2009, THE ANN ARBOR NEWS. ALL RIGHTS RESERVED. REPRINTED WITH PERMISSION. fter getting the tion by as much as 90%.1 Interestingly, the other genes could also switch on gene data back from the findings confirmed work by Kevin Morris, mkZgl\kbimbhg'Ebd^<hk^rZg]CZghpldb% very first experi- now at the Scripps Research Institute in EbZg]IeZ\^p^k^^qi^\mbg`bgZ\mboZmbhg% ment at her new EZCheeZ%<Zeb_'%pahaZ]in[ebla^]ma^Öklm ghmma^hiihlbm^'ÊP^p^k^lnkikbl^]maZm job, Rosalyn Ram, evidence of this phenomenon in human we didn’t observe gene silencing by the a lab technician cells a year earlier. ]hn[e^&lmkZg]^]KG:%ËlZrlEb'ÊBglm^Z]% at the University ;nm<hk^rZg]CZghpldbZelhghmb\^] we observed strong gene activation.” of Texas Southwestern Medical Center something in their data that, if correct, RNA activation flew in the face of at Dallas, was convinced she had messed would be even more unbelievable than what everyone’s perceived wisdom regarding something up. The results were decidedly Ram saw on her first day at the bench. A few RNA-based regulation. RNA was thought Êp^bk]%Ëla^k^\Zeel'A^keZ[a^Z]l%ma^ h_<hk^rZg]CZghpldbÍll^^fbgèrÊbgZ\- to silence genes only by cutting up mRNA husband-and-wife research duo David tive” RNAs that did not reduce gene expres- via the RNAi pathway, not at the point <hk^rZg];^maZgrCZghpldb%aZ]Zek^Z]r sion reproducibly enhanced transcription of gene transcription and definitely not shown that synthetic DNA molecules with by around 25% to 50%. Such relatively through activation. As such, many of the protein-like backbones, known as peptide small changes weren’t enough to say defin- big names in the RNA world dismissed the nucleic acids, could block gene transcrip- findings out of hand. tion. And as a long shot, in October 2004 ÊBmpZllhaZk]mh`^mmablphkdin[- they had tasked the new lab tech with ebla^]%ËlZrlCZghpldb'ÊBmpZlebd^Zibm\a- trying to do the same with small RNA fork coming out. The RNAi community molecules, fully expecting it not to work. “RNA activation was so hostile.” ;nm bm ]b] phkd3 Ebd^ ma^ iîmb]^ . went against the N nucleic acids, the RNAs targeted to the oth groups had trouble pub- O same promoter also silenced gene expres- grain of everything lishing their papers. Corey MISSI lbhgZmmaêôêh_mkZgl\kbimbhg'ÊPa^g Zg] CZghpldbÍl phkd pZl PER [Ram] saw the silencing, she thought we knew about rejected by Science before it was published she had done something wrong,” says in Nature Chemical Biology bg CZgnZkr WITH CZghpldb'ÊLa^]b]gÍmpZgmmhlahpf^ma^ how small RNAs 2007,3pabeÊbZg]IeZ\^[Zmme^]_hkmph ED data because she thought it was supposed years and faced four rejections before INT regulate gene to be a negative result.” they finally published their paper in the The data just didn’t make sense: Sin- expression.” Proceedings of the National Academy of ED. REPR 2 V gle-stranded peptide nucleic acids bind Sciences in November 2006. So, when ER S directly to unwound DNA at the transcrip- —John Rossi <hk^r Zg] CZghpldbÍl iZi^k \Zf^ hnm E R tion start site, and double-stranded RNAs two months later showing the same basic were thought only to target messenger h[l^koZmbhg%ÊbmpZldbg]h_Zkêb^_%ËEb RIGHTS KG:!fKG:"mhikô^gmmkZgleZmbhgÉZ lZrl'ÊBm_êm`hh]maZmlhf^hg^êl^Zelh LL . A well characterized process known as RNA itively that the researchers had observed saw a phenomenon that was similar to WS interference, or RNAi. So how could they `^g^Z\mboZmbhg%Ê[nmbmieZgm^]ma^l^^]l ours,” adds Place. both be causing the same effect? With in our minds that maybe activation could However, neither group offered a mablhg^Ög]bg`%ÊZeemahl^mabg`lmaZmrhn be occurring,” says Corey. To investigate the plausible mechanism for how RNA acti- ARBOR NE thought you could predict just flew out the trend further, the researchers switched to a oZmbhgfbàmh\\nk'ÊB]b]gÍmmabgdmaZm NN pbg]hp%ËCZghpldblZrl' cell line with much lower background activ- ma^ ôb]^g\^ maZm TEb Zg] IeZ\^V aZ] E A H ÊBmmhhdfhgmal[^_hk^p^\hgobg\^] ity levels of their gene of interest, the human really supported the mechanism they were ourselves that the results were real,” says progesterone receptor, and the data became kîhkmbg`%ËlZrlChagKhllb%Zfhe^\neZk 2009, T Corey. But eventually they did. They tested glaringly obvious: The same RNAs that did geneticist at City of Hope Comprehensive © several different double-stranded RNAs— not inhibit transcription could now trigger Cancer Center in Duarte, Calif., who was O. T O each 21 nucleotides long, just like standard, as much as 10- to 20-fold increases in gene Zkôb^p^k_hkmaîZi^k'ÊBmp^gmZ`Zbglm H P small interfering RNAs used in RNAi. All expression. The activation effect was real. the grain of everything we knew about of these RNAs perfectly matched regions Before they could publish the findings, how small RNAs regulate gene expression. IGINAL of the DNA promoter but had little to no however, another lab published results The data were clear, but were they looking OR 2 M overlap with the gene’s mRNA, to ensure showing essentially the same effect. A at something indirect? We wanted more the RNAs were acting on transcription, m^Zfe^][rEhg`&<a^gÈbZg]Kh[^km experiments to validate the mechanism ED FRO not translation. Ultimately, in September Place at the University of California, San Zg]ma^rTEbZg]IeZ\^Vgô^k]b]ma^f'Ë I F I +)).<hk^rZg] CZghpldb lahp^] maZm Francisco, discovered that small RNAs Nonreviewers alike were unconvinced. MOD introduced RNAs could inhibit transcrip- targeting the DNA promoters of three ÊBaZo^ZaZk]mbf^mh^qieZbgma^Ög]bg`l% Ω May 2009 THE SCIENTIST 35 RNA and what mechanism can explain it,” says Argonaute proteins—known members the same direction as mRNA transcription Thomas Tuschl, an RNA expert at New of the RNAi pathway—help facilitiate ZlpêeZlbgma^hiihlbm^ÉhkÊZgmbl^gl^ËÉ York’s Rockefeller University, in an email. RNA-RNA interactions. Thus, the new- orientation. These ubiquitous noncoding ÊBml^^fllhb]bhlrg\kZmb\Zg]li^\bZeZm found connection between transcrip- RNAs probably served some purpose, the moment,” adds Timothy Nilsen, an tional regulation and Argonaute proteins perhaps even in RNA activation, the sci- RNAi researcher at Case Western Reserve indicated that RNA activation’s target entists reasoned. Ngbo^klbmrbg<eôêZg]%Habh'ÊBmÍlcnlmghm was naturally occurring RNA, not DNA, Using reverse transcription–PCR, a generalizable phenomenon like RNAi. as was the case for peptide nucleic acids <hk^rÍl `kZ] lmn]^gm CZ\h[ L\apZkms With RNAi, you know it’s going to work.” !Zg]pab\ama^k^l^Zk\a^klaZ]gZboêr scanned for noncoding RNAs around The concerns are valid. Introducing assumed would be true of activating the progesterone receptor promoter. He nucleic acids into cells is a notoriously KG:l%mhh"'Fhkkblma^gm^lm^]mablb]^Z couldn’t find any RNA coded in the direc- artifact-prone experiment, so the observed directly and confirmed that RNAs were, tion of transcription, but did discover effects could easily be due to the intro- indeed, interacting with each other.4 three antisense transcripts spanning duced RNA interacting with nontarget :ee mh`^ma^k% Êbm ^lmZ[ebla^] ma^ the promoter region. At first, however, molecules, such as unintended proteins link between transcriptional and post- Schwartz didn’t believe his own results. or RNA that in turn cause the activation, transcriptional gene silencing,” says L\apZkmsÍl Ê_bklm r^Zk `kZ]nZm^ \hnkl^ the skeptics argued. CZghpldb' FZr[^% ma^r k^Zlhg^]% mabl perspective of transcription” led him to Another part of the problem was that believe that there wasn’t going to be any unlike RNAi, in which the introduced RNA transcripts in the promoter region, RNA always perfectly matches the mRNA he says, so he thought he was just seeing targets, RNA activation did not seem to genomic DNA contamination. For two follow any predictable set of rules. The “One can be picky p^^dl%a^mkb^]Êô^krpab\apZrËmh`^m researchers constructed the activating about the mecha- rid of the bands in his PCR before he RNAs to match stretches of the promoter convinced himself otherwise. In the end, DNA, but couldn’t devise a reliable design nism, but it’s early Schwartz showed that activating RNAs, scheme: Single-base differences in the together with Argonaute, bound to these RNA’s target sequence could turn an acti- days. What’s clear antisense transcripts in the vicinity of the vator into a repressor and vice versa, they is that RNA activa- promoter. This RNA-protein complex found. What’s more, the most effective then acted as a scaffold to recruit and activator RNAs for some genes matched tion exists.” redirect other protein modifiers to either DNA sequences right at the transcrip- crank up or slow down transcription.5 tional start site, but for others the target —John Mattick ÊRhnk^ZeeraZo^mhmabgdh_ma^lîkh- site was way upstream. moters as very dynamic,” explains Corey. The RNA community adopted a wait- Ê@^g^lZkîhbl^]mh^bma^k[^mnkg^]hghk Zg]&l^^Zmmbmn]^'ÊPa^gô^klhf^\hg- off.” In his view, antisense transcripts act troversial findings like these ones emerge, wasn’t such a new and implausible phe- as the regulators of this delicate balance. I will not touch the topic and I wait for nomenon after all. But the question still So by introducing RNAs that interacted follow-up by the labs that brought the remained: How could their introduced with these RNA gatekeepers, his team topic up,” says Tuschl.

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