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Identification of Factors Involved in Target RNA-Directed Microrna

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Identification of Factors Involved in Target RNA-Directed Microrna Published online 24 January 2016 Nucleic Acids Research, 2016, Vol. 44, No. 6 2873–2887 doi: 10.1093/nar/gkw040 Identification of factors involved in target RNA-directed microRNA degradation Gabrielle Haas†,SemihCetin†,Melanie´ Messmer, Beatrice´ Chane-Woon-Ming, Olivier Terenzi, Johana Chicher, Lauriane Kuhn, Philippe Hammann and Sebastien´ Pfeffer* Architecture and Reactivity of RNA, Institut de biologie moleculaire´ et cellulaire du CNRS, Universite´ de Strasbourg, 15 rue Rene´ Descartes, 67084 Strasbourg, France Downloaded from https://academic.oup.com/nar/article-abstract/44/6/2873/2499455 by guest on 01 February 2019 Received March 09, 2015; Revised January 12, 2016; Accepted January 13, 2016 ABSTRACT Consequently, their synthesis and turnover must be tightly controlled. Briefly, miRNAs are transcribed in the nucleus The mechanism by which micro (mi)RNAs control as a primary transcript (pri-miRNA) containing a hairpin their target gene expression is now well understood. structure, which is recognized and cleaved by the RNase III It is however less clear how the level of miRNAs them- Drosha. This cleavage generates a precursor miRNA (pre- selves is regulated. Under specific conditions, abun- miRNA) which will be exported to the cytoplasm, where a dant and highly complementary target RNA can trig- second cleavage by the RNase III Dicer gives rise to a ≈22 ger miRNA degradation by a mechanism involving nucleotides (nt) miRNA duplex (1). One strand of the du- nucleotide addition and exonucleolytic degradation. plex (guide strand) is loaded in the RISC (RNA-Induced One such mechanism has been previously observed Silencing Complex) and becomes the active miRNA, while to occur naturally during viral infection. To date, the the second strand (passenger strand) is often degraded. molecular details of this phenomenon are not known. miRNA biogenesis is regulated both transcriptionally and post-transcriptionally by different mechanisms control- We report here that both the degree of complemen- / ling the level of pri-miRNA transcription, the activity or tarity and the ratio of miRNA target abundance are the accessibility of Drosha and/or Dicer or the stability of crucial for the efficient decay of the small RNA. Us- the pre-miRNA (1). One of the best described example of ing a proteomic approach based on the transfection miRNA biogenesis regulation involves the LIN28 protein, of biotinylated antimiRNA oligonucleotides, we set which negatively impacts the synthesis of Let-7 miRNA (6– to identify the factors involved in target-mediated 11). LIN28 reduces the cleavage activity of both Drosha miRNA degradation. Among the retrieved proteins, and Dicer, at respectively the pri-Let-7 and the pre-Let- we identified members of the RNA-induced silencing 7levels(6–8). LIN28 also recruits the Terminal-Uridylyl- complex, but also RNA modifying and degradation Transferases TUT4/TUT7 (9,10), which uridylate pre-Let- enzymes. We further validate and characterize the 7 leading to its subsequent degradation by the exonuclease importance of one of these, the Perlman Syndrome DIS3L2 (11). More recently, TUT4 and TUT7 were also de- scribed to have a more widespread role in the control of pre- 3 -5 exonuclease DIS3L2. We show that this protein miRNA degradation via a mechanism involving the RNA interacts with Argonaute 2 and functionally validate exosome (12). its role in target-directed miRNA degradation both Mature miRNAs, which represent the active end prod- by artificial targets and in the context of mouse cy- ucts of this biogenesis were long thought to be very sta- tomegalovirus infection. ble molecules with half-lives ranging from hours to days (13,14). But recently, several examples showed that they INTRODUCTION are also subjected to active regulation. In this case, mod- ifications of the small RNA play essential roles to influ- Among the various classes of small regulatory RNAs, miR- ence its stability or function. For example, miR-122 mono- NAs represent one of the most studied in mammals. They adenylation by GLD-2 (TUT2) stabilizes this miRNA in act as guides to recruit Argonaute proteins (Ago) to target mammals (15). At the opposite, miR-26a is no longer func- mRNAs, resulting in translation inhibition and reduced sta- tional as a consequence of its uridylation by ZCCHC11 bility (1). These tiny regulators are involved in a wide variety (TUT4) (16). In addition, accelerated miRNA turnover has of biological processes (2,3), and their aberrant expression been reported. This is especially true for biological situ- can be the cause of genetic diseases and/or cancers (4,5). *To whom correspondence should be addressed. Tel: +33 3 88 41 70 60; Fax: +33 3 88 60 22 18; Email: [email protected] †These authors contributed equally to the paper as first authors. C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2874 Nucleic Acids Research, 2016, Vol. 44, No. 6 ations that require rapid changes in gene expression (i.e. AGO2 was re-amplified from the pIRESneo-FLAG/HA- cell cycle, light-dark transitions) (17–19), or during viral in- AGO2 vector (Addgene). The vector expressing GFP-MBP fections (20–22). Moreover, the presence of a highly com- was a gift from E. Izaurralde. To generate GFP fusion pro- plementary target can induce miRNA degradation via a teins, TUT1 (BglII/EcoRI), DIS3L2 (BglII/BamHI), mechanism involving tailing (3 addition of non-templated mTUT1 (BglII/EcoRI), mDIS3L2 (EcoRI/KpnI), nucleotides) followed by trimming of the miRNA (13,23). TUT2 (EcoRI/BamHI), TUT4 (XhoI/SmaI) and TUT7 From now on, we will refer to this phenomenon as target (XhoI/BamHI) were each cloned in the pEGFP-C2 expres- RNA-directed miRNA degradation (TDMD) according to sion vector (Clontech) using the restriction sites indicated a recent report from the Grosshans laboratory (24). in parentheses. For HA-tagged proteins, the pcDNA3.1(+) In several organisms, small RNA species are usu- vector (Invitrogen) was first modified by mutagenesis to ally protected from degradation by addition of a 2’O- insert the coding sequence of the HA-tag 5 of the MCS cas- methyl group at their 3 extremity by the methyltrans- sette. Then, AGO2 (EcoRI/NotI), mAGO2 (EcoRI/NotI), Downloaded from https://academic.oup.com/nar/article-abstract/44/6/2873/2499455 by guest on 01 February 2019 ferase HEN1 (23,25). In hen-1 mutant plants and flies, TUT1 (EcoRI/NotI), mTUT1 (EcoRI/NotI), DIS3L2 the lack of a 2’O-methylated 3 terminal residue results in (BamHI/NotI) and mDIS3L2 (EcoRI/NotI) were in- 3 uridylation/adenylation and subsequent 3 to 5 degra- serted between the indicated restriction sites. The catalytic dation of small interfering (si)RNAs (and plant miR- mutants of DIS3L2 (D391N, D392N) and mDIS3L2 NAs) (23,26). As opposed to siRNAs or plant miR- (D389N) were generated by mutagenesis. For the luciferase NAs, Drosophila and mammals miRNAs are not 3 pro- reporters, m169–3 UTR or SH fragment was cloned in the tected and usually present only a partial complementarity pcDNA3.1 vector using EcoRV restriction site. Mutation with their target RNAs. This explains why a near-perfect in the seed region was then done by mutagenesis. To miRNA/target complementarity (involving an extensive concatamerize the SH region, SH fragments were amplified pairing of the 3 region of the miRNA) coupled to a high with the insertion of different cloning sites, then treated for abundance of the target seems to trigger miRNA degrada- multiple ligation before being re-amplified and cloned as tion rather than mRNA regulation (23). Therefore, TDMD described above. Finally, the F-Luc coding sequence was could represent a potential and powerful spatiotemporal inserted in pcDNA-m169 versions using NheI/KpnI. All way to regulate mature miRNA accumulation. Accordingly, the sequences of the cloning and mutagenesis primers are TDMD has been observed in vivo in the context of mouse available in Supplementary Table S2. cytomegalovirus (MCMV) infection. Indeed, MCMV ex- presses an abundant viral transcript (m169), which local- Cell culture and transfection izes to the cytoplasm and induces degradation of the cellu- lar miR-27a and b (20,21). To date, the molecular details of HeLa cells, Hepa 1.6 cells and HEK293 cells were cultured the process and the cellular enzymes involved in this mech- in Dulbecco’s modified Eagle’s medium (DMEM) supple- anism are unknown. mented with 10% (v/v) fetal calf serum at 37◦C in a hu- In this study, we developed a biochemical approach midified 5% CO2 atmosphere. For co-immunoprecipitation, that allowed us to induce TDMD in cell lines and to un- HeLa and Hepa cells were transfected with a mixture con- cover the identity of the cellular factors responsible for taining plasmids expressing HA-tagged and GFP-tagged the target-induced degradation of mature miRNAs. We re- protein using the Turbofect reagent (Thermo Scientific). trieved known components of the RISC as well as several For RNA-immunoprecipitation, HeLa cells (15 × 106 cells putative candidates for the miRNA modification and degra- in 15-cm plates) were transfected with 60 ␮g of plasmid dation. Among those, we identified the Terminal-Uridylyl- expressing GFP-tagged protein. The day after, cells were Transferase TUT1 and the 3-5 exoribonuclease DIS3L2. divided and re-seeded in two 15-cm plates. After 10 h, We confirmed that these two proteins interact with Arg- cells were retransfected with either control antimiRNA di- onaute 2 and together in an RNA-dependent manner.
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