Mapping RNA interactome oin viv) technology to massively reveal RNA–RNA interactions from unperturbed cells. We mapped tens of thousands of endogenous RNA–RNA interactions from mouse embryonic stem cells and brain. We validated seven interactions by RNA antisense purification and one interaction using single-molecule RNA–FISH. The experimentally derived RNA interactome is a scale-free network, which is not expected from currently perceived promiscuity in RNA–RNA interactions. Base pairing is observed at the interacting regions between long RNAs, including transposon transcripts, suggesting a class of regulatory sequences acting in trans. In addition, MARIO data reveal thousands of intra-molecule interactions, providing in vivo data on high-order RNA structures.The pervasive transcription of our genome presents a possibility of revealing new genomic functions by investigating RNA interactions. Current methods for mapping RNA–RNA interactions have to rely on an ‘anchor' protein or RNA and often require molecular perturbations. Here we present the MARIO  technology that can massively reveal RNA-RNA interactions and RNA structure from unperturbed cells. Inferring from the notion that regulatory RNAs ‘promiscuously' interact with 300–1,000 target RNAs112121The MARIO technology maps RNA–RNA interactions in a massive scale. MARIO can identify protein-assisted between-molecule and within-molecule RNA interactions. The MARIO identified RNA–RNA interactome is composed of tens of thousands of interactions, which involve mRNA, long intergenic noncoding RNA (lincRNA), small nucleolar RNA (snoRNA), small nuclear RNA, tRNA, miRNA, transposon RNA, pseudogene RNA, antisense RNA and novel transcripts. The MARIO identified RNA–RNA interactome is a scale-free network. Long non-coding RNA including lincRNA, transposon RNA and pseudogene RNA are observed to interact with mRNA. Sequence complementation is observed in interactions between transposon (LINE and LTR) RNA and mRNA, as well as in mRNA–mRNA, mRNA–peudogeneRNA, lincRNA–mRNA, miRNA–mRNA and LINE–miRNA interactions. MARIO data also provide spatial-proximity information related to RNA folding in three-dimentional space.in vivo. In this procedure, RNA molecules are cross-linked with their bound proteins and then ligated to a biotinylated RNA linker such that proximal RNA molecules co-bound by the same protein form a chimeric RNA in the form of RNA1–Linker–RNA2. These linker-containing chimeric RNAs are isolated using streptavidin-coated magnetic beads and subjected to paired-end sequencing . After cross-linking and cell lysis, the lysates from the two cell lines were immediately mixed before any subsequent steps. The mixture was subjected to the rest of the experimental procedure and resulted in a sequenced library (Fly-Mm). The proportion of RNA pairs mapped to two species is in the range of 2.5–6.8%, depending on whether the Drosophila genome and the mouse genome were assembled into a pan genome16in silico simulations (We carried out two independent MARIO assays on mouse embryonic stem (ES) cells with minor technical differences , which w2324ulations .http://mariotools.ucsd.edu). It also provides visualization tools for both the RNA–RNA interactome and the proximal sites within each RNA (A suite of bioinformatics tools was created (MARIO tools) to analyse and visualize MARIO data. MARIO tools automated the analysis steps, including removing PCR duplicates, splitting multiplexed samples, identifying the linker sequence, splitting junction reads, calling interacting RNAs, performing statistical assessments, categorizing RNA interaction types, calling interacting sites and analysing RNA structure  . For example, an interaction between the 3′ untranslated region of Trim25 mRNA and Snora1 snoRNA was supported by multiple paired-end reads in ES-1 and ES-2 samples, but was not detected in MEF , followed by ES indirect, MEF, and then brain tissue . The inton test) . The intd in MEF . We did The ES-1 and ES-2 libraries were merged to infer the RNA–RNA interactome in ES cells. This dataset included 4.54 million non-duplicated paired-end reads that were unambiguously split into two RNA fragments with both fragments uniquely mapping to the genome (mm9). We identified tens of thousands of inter-RNA interactions  . As expeMalat1 lincRNA and Slc2a3 mRNA in vivo by two-colour single-molecule RNA fluorescence in situ hybridization (smRNA–FISH)Actb mRNAs in the cytoplasm  and controls =446, P-value <10−20, χ2-test).We used two methods to validate selected interactions in the MARIO identified interactome. These two methods were selected because they do not perturb the cells or change RNA expression levels. First, we examined co-localization between ytoplasm . We desid Slc2a3  and labed Slc2a3 . We noteectively . We callectively . Sixteenf Slc2a3 . In the P-value<0.0003, χ2-test). This suggests a strong overlap of Malat1 targets in MARIO and Malat1 RIA-seq. Next, we asked whether Tfrc RIA could reversely identify Malat1 by Tfrc RIA-seq , suggesting that antisense purification of Tfrc could reversely pull down Malat1. In addition, three out of four other Tfrc-interacting RNAs identified by MARIO exhibited 1.4- to 13.6-fold increases . Taken together, seven additional MARIO-identified interactions were validated by RIA-seq.To test interactions at a larger scale, we carried out RNA interactome analysis and sequencing (RIA-seq) RIA-seq . The TfrThe experimentally derived RNA interactome offered an opportunity to test a fundamental physical property. Most known biological networks are hierarchical networks21The MARIO-derived RNA–RNA interactome is scale free . In otheA number of the interacting RNAs exhibited overlapping MARIO reads , suggest40P-values<2.4−18, test by random shuffling), but was not observed in LTR–pseudogeneRNA interactions ractions . This daIf these RNA–RNA interactions are sequence specificSnora73 transcripts  and 1,000 U ml−1 of LIF (Millipore). The cells were maintained in an incubator at 37 °C and 5% CO2.Undifferentiated mouse E14 ES cells (gift from Huck-Hui Ng) were cultured under feeder-free conditions. ES cells were seeded on gelatin-coated dishes and were cultured in DMEM medium (GIBCO) supplemented with 15% fetal bovine serum , 0.055 mM 2-mercaptoethanol (Sigma), 2 mM Glutamax (GIBCO), 0.1 mM MEM non-essential amino acid (GIBCO), 5,000 U ml−1 penicillin/streptomycin (GIBCO). MEFs were also maintained in an incubator at 37 °C and 5% CO2.MEFs  were cultivated in 15-cm dishes in DMEM (GIBCO) supplemented with 15% FBS (Gemini Gemcell), 0.055 mM 2-mercaptoethanol (Sigma), 2 mM Glutamax (GIBCO), 0.1 mM MEM non-essential amino acid (GIBCO), 5,000 U mlDrosophila S2 cells (Invitrogen) were maintained in 15-cm plates in Schneider's Drosophila Medium (GIBCO) supplemented with 10% heat-inactivated FBS (Gemini Gemcell) and 5 ml 1:100 penicillin/streptomycin (GIBCO) in an incubator at 28 °C without CO2.−2 in an ultraviolet cross-linker (254 nm) with gentle swirling between each irradiation. Cross-linked, powdered tissue was immediately lysed and subjected to MARIO procedure as described.Mice handling was approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. Adult female (C57BL/6J background) was killed by cervical dislocation and the whole brain was immediately collected, rinsed with ice-cold PBS three times and snap frozen. Frozen, whole mouse brain tissue was ground into fine powder in liquid nitrogen using a mortar and pestle. The tissue powder was quickly transferred into a Petri dish on a bed of dry ice and irradiated on dry ice three times at 400 mJ cmin vivo in an unbiased manner without genetically or transiently introducing exogenous molecules; (ii) allow stringent removal of non-physiologic associations that form after cell lysisMARIO was designed to: (i) capture interacting RNAs Drosophila S2 were irradiated three times with UV-C (254 nm) at 400 mJ cm−2 in ice-cold PBS on ice. Cells were harvested by scraping and pelleted by centrifugation at 1,000 g for 5 min at 4 °C. Cell pellets were snap-frozen in liquid nitrogen and stored at −80 °C.Ultraviolet irradiation was used to form covalent bonds between photoreactive nucleotide bases and amino acids. Ultraviolet irradiation generates highly reactive, short-lived states of the nucleotide bases within the RNA, inducing covalent bond formation only with amino acids at their contact points without additional elements that might cause conformational perturbationin vivo dual cross-linking method with previously validated parameters5556g for 5 min at 4 °C, snap-frozen in liquid nitrogen and stored at −80 °C.We generated a MARIO library (ES indirect) in which we cross-linked protein–protein complexes as well. This was to capture the RNA that were brought together by protein interactions. We applied an 8 cross-linked cells stored at −80 °C were thawed on ice and resuspended in ∼3 volumes of lysis buffer ). Lysis was performed on ice for 20 min. Cell debris and insoluble chromatin were removed by centrifugation at 20,000 g for 10 min at 4 °C. The supernatant was collected and treated with TURBO DNase (Invitrogen) at concentration of 10 μl TURBO DNase per ml lysate for 20 min at 37 °C. RNAs were digested into ∼1,000- to 2,000-nt (ES-1) or ∼1,000-nt (ES-2) fragments by adding 10 μl of 1:100 diluted RNase I (NEB) per ml of lysate and incubating at 37 °C for 3 min. Following RNase I treatment, the lysate was immediately transferred to ice for at least 5 min. Both RNase I and sonication-based fragmentation leave 5′-OH and 3′-P ends, incompatible with RNA ligation, which suppress undesirable RNA ligations. To stop DNase digestion, we added EDTA (Ambion) to a 25-mM final concentration and incubated the mixture at 4 °C for 15 min with rotation. The fragmented dual cross-linked (ES indirect) lysate was prepared as follows: after the lysis on ice for 20 min, the suspension was directly subjected to fragmentation by sonication (Covaris E220) under the following settings: 20 min with 5% duty cycle, 140 Watts peak incident power and 200 cycles per burst at 4 °C.Approximately 6 × 108 E14 mES cells and 3 × 108Drosophila S2 cells were lysed separately and then mixed before protein biotinylation.For cross-species experiment (Fly-Mm), ∼3 × 10−1) EZlink Iodoacetyl-PEG2-Biotin (IPB) (Pierce Protein Research Products) and rotating the mixture in the dark for 90 min at room temperature. We quenched the biotinylation reaction by adding dithiothreitol (DTT) to a 5-mM concentration and incubating at room temperature for 15 min. To neutralize SDS, Triton X-100 (Sigma) was added to a 2% final concentration and incubated at 37 °C for 15 min. The lysate sample was dialysed in a 20-kDa cutoff Slide-A-Lyzer Dialysis Cassette (Pierce Protein Research Products) at room temperature in 2 l of dialysis buffer  to remove excess biotin. The dialysis buffer was changed at least thrice, once every 2 h. Following dialysis, the lysate was transferred to a 15-ml tube.To dissociate loosely bound proteins, 500 mM NaCl final concentration was added and the solution was incubated at 4 °C for 10 min with rotation. To further dissociate protein complexes and non-cross-linked RNAs, and halt the activities of RNase I, we added SDS to a 0.3% final concentration and incubated the mixture with shaking at 750 r.p.m. for 15 min at 65 °C. After letting the solution mixture cool down to room temperature, we biotinylated the cysteine residues by adding to the lysate 1:5 volume of 25 mM (13.56 mg ml2 surface area). The advantages of immobilization on a solid surface include: (i) reduction of random intermolecular ligations between non-cross-linked oligonucleotidesThe protein–RNA complexes were immobilized at low bead-surface density on streptavidin-coated beads ; two times with each buffer, with a rotation for 5 min at 4 °C during the second wash.Eight hundred microlitres of MyOne T1 beads were washed thrice with PBST (PBS with 0.1% Tween-20), resuspended in 800 μl of the same buffer and transferred into the biotinylated lysate. The bead-lysate suspension was rotated at room temperature for 45 min. During this incubation, we prepared 200 μl of neutralized 25 mM IPB by adding equal molarity of DTT and incubating at room temperature for at least 30 min. The beads were immobilized using a magnetic stand and most of the supernatant was aspirated out, leaving behind 4 ml of the supernatant. The beads were resuspended in the leftover solution followed by the addition of 200 μl of neutralized IPB. IPB was used to saturate excess of unbound streptavidin after immobilization, which may interfere with subsequent step that involves biotin-tagged RNA linker. To remove the undesired RNAs non-covalently attached to proteins or via nonspecific protein–protein interactions59Next, a biotin-tagged RNA linker (5′- rCrUrArG/iBiodT/rArGrCrCrCrArUrGrCrArArUrG rCrGrArGrGrA -3′) was attached to the RNA's 5′-end. The biotin-tagged linker serves as a selection marker to enrich for the ligated RNAs; it also delineates a clear boundary to unambiguously split any sequencing read that covered a ligation junction. The 5′-end of the RNA linker was temporarily ‘blocked' from ligation, to avoid linker circularization or concatenation. This was achieved by synthesizing the linker with a 5′-OH group, which is incompatible with ligation but can be ‘re-activated' by phosphorylation. However, RNase I leaves 5′-OH end, which is incompatible for linker ligation; thus, we first phosphorylated the 5′-end with PNK, 3′-phosphatase minus (NEB). We did not use the wild-type T4 PNK due to its additional 3′-phosphatase activities, which modifies the 3′-ends of RNAs from 3′-P into 3′-OH, making them susceptible to self-ligation.−1 T4 PNK (3′-phosphatase minus) (NEB), 2 μl of RNAsin Plus (Promega)) and incubating for 1 h at 37 °C with intermittent shaking at 1,200 r.p.m. for 5 s every 2 min. The beads were washed with wash buffer I, II, III and PNK, two times with each buffer, with rotation for 5 min at 4 °C in the second wash. The ice-cold washes were used to eliminate any leftover PNK, which may phosphorylate the RNA linker, inducing it to be potentially ligated to the 3′-end of RNAs. After wash buffer was removed, the biotin-tagged RNA linker was ligated to RNA 5′-ends by adding 160 μl RNA ligation reaction mixture, which contained 2 μl RNAsin Plus (Promega), 16 μl of 10 mM ATP, 16 μl of 10 × RNA ligase buffer, 16 μl of 1 mg ml−1 BSA, 30 μl of 20 μM biotin-labelled linker, 64 μl of 50% PEG8000 (NEB), 16 μl of 10 U μl−1 T4 RNA ligase 1 (NEB). Ligation was carried out at 16 °C overnight with intermittent shaking at 1,200 r.p.m. for 15 s every 2 min. BSA was added to enhance the activities of T4 RNA ligase and prevent bead aggregation. Polyethylene glycol (PEG) was used to enhance intermolecular ligation by increasing the concentrations of the donor and the acceptor endsThis was achieved by removing wash buffer and subsequently resuspending the beads in 100 μl of PNK reaction mixture , 5 μl of 10 U μl−1 T4 PNK (3′-phosphatase minus) (NEB), 2 μl of RNAsin Plus (Promega) and incubated for 20 min at 37 °C with intermittent shaking at 1,200 r.p.m. for 5 s every 2 min. The beads were washed once with PNK wash buffer and the 5′-end of the biotin-labelled linker was phosphorylated in 100 μl of PNK reaction mixture  (NEB), 2 μl of RNAsin Plus (Promega)) for 1 h at 37 °C with intermittent shaking. Following phosphosrylation, the beads were wash twice in PNK wash buffer and proximity ligation was then performed under extremely diluted conditions in a 15-ml total volume reaction , 25 μl of 1 M DTT, 2.25 ml of 100% dimethyl sulfoxide, 0.75 ml of 10 U μl−1 T4 RNA ligase 1 (NEB)), to minimize inter-complex ligations. The proximity ligation was carried out at 16 °C overnight with continuous rotation.Next, the beads were washed twice with ice-cold wash buffer II, once with ice-cold wash buffer III and PNK wash buffer. To prepare for proximity ligation, we first dephosphorylated the RNA 3′-end using the 3′-phosphatase activities of T4 PNK, leaving a 3′-hydroxyl group−1 Proteinase (NEB) and incubated at 55 °C for 2 h. We topped up the mixture with RNase-free water to the final volume of 400 μl. RNAs were extracted in 400 μl of phenol:chloroform:isoamyl alcohol  (Ambion) and incubation at 37 °C for 20 min with shaking at 1,000 r.p.m. The mixture was transferred into a 2-ml MaXtract high-density phase lock gel tube (Qiagen) and centrifuged at 16,000 g for 5 min at room temperature. Residual phenol was removed by adding 400 μl of chloroform to the same MaXtract tube and centrifugation at 16,000g for 5 min at room temperature. Following centrifugation, the aqueous phase was transferred into a new tube and RNAs were precipitated by adding 1:9 volume of 3 M sodium acetate pH 5.2, 1.5 μl of glycoblue (Ambion) together with 1 ml of 1:1 ethanol:isopropanol and incubating at −20 °C overnight. We pelleted the precipitated RNA by centrifugation at 21,000 g for 30 min at 4 °C. After discarding the supernatant, the pellet was washed twice with 80% ethanol and air dried until ethanol completely evaporated. The purified RNAs at this stage were a mixture of RNAs without linkers (RNA1 or RNA2), RNAs ligated with linkers but not proximity ligated with other RNAs (5′-linker–RNA2) and the desirable chimeric constructs in the form of 5′-RNA1–linker–RNA2. RNA1 can be depleted by selection of the biotin-tagged linker. We therefore depleted the non-informative 5′-linker–RNA2 as well in the next reaction with T7 exonuclease.The following day, ligation was stopped by adding EDTA to a final concentration of 25 mM and rotating for 15 min at 4 °C, to prevent inter-molecular ligation from happening as the beads were collected on the wall of the tube. The beads were washed once in PBST. We next eluted protein–RNA complexes from streptavidin beads twice in 100 μl of Elution Buffer ) by heating to 95 °C for 5 min. The resulting solutions were combined, mixed with 50 μl of 800 U mlThis was based on the RNase H activity of T7 exonuclease, which not only removes 5′-mononucleotides from duplex DNA but also exert exonucleolytic activity on the RNA strand from a RNA–DNA hybrid−1) to 25 °C. The annealed mixture was then mixed with 8 μl of 10 U μl−1 T7 exonuclease (NEB), 4 μl of 1 mg ml−1 BSA and incubated at 37 °C for 1 h. We removed the DNA oligonucleotides and any contaminating genomic DNA using TURBO DNase rigorous treatment: 44 μl of RNase-free water, 10 μl of 10 × TURBO DNase buffer and 6 μl of TURBO DNase (Invitrogen) was added, and the resulting mixture was incubated at 37 °C for 1 h. DNase-treated RNA was purified by phenol:chloroform extraction and ethanol precipitation as described above.The RNA pellet was resuspended in 17 μl of RNase-free water, 4 μl of 10 × NEBuffer4 and 7 μl of 100 μM cDNA oligo. Annealing was performed by denaturing at 95 °C for 2 min and then slowly ramping down the temperature  and ethanol precipitated as described above.−1 T4 RNA ligase 2, truncated KQ (NEB). The reaction was incubated overnight at 16 °C.Next, the RNAs were ligated with a 3′-RT adapter (5′- /5rApp/AGATCGGAAGAGCGGTTCAG/3ddC/ -3′) that served as a primer for RT reaction. Following ethanol precipitation, the RNA pellet was resuspended in 20 μl of ligation reaction mixture: 1 μl RNAsin Plus (Promega), 2 μl of 10 × RNA ligase buffer, 7 μl of 20 μM pre-adenylated L3-App adapter, 8 μl of 50% PEG8000 (NEB), 2 μl of 200 U μlGgatcCTGAACCGCTCTTCCGATCT -3′. According to this scheme, the first read of every sequencing read pairs contains a barcode that takes the configuration of NNNNXXXXNN (reverse complement of that from the RT primer), where the Ns are a random 6-nt barcode for removing PCR duplicates195051Following ligation, RNA was purified by 2 × SPRISelect beads (Beckman Coulter Genomics) and eluted in RNase-free water. The following RT reaction is described for 2 μg of RNA and was scaled up accordingly for higher amount of RNAs. For each experiment or replicate, a different RT primer containing individual experimental barcode sequence was used. Each RT primer has the form of 5′- /5Phos/NNXXXXNNNNAGATCGGAAGAGCGTCGT−1 T4 gene 32 protein (NEB) were added. The resulting mixture was incubated at 50 °C for 2 min before adding reverse transcriptase enzyme to minimize mispriming. Next, we added 2 μl of 200 U μl−1 Superscript III reverse transcriptase (Invitrogen) to the solution. The RT reaction mixture was then incubated at 50 °C for 45 min, 55 °C for 20 min followed by 4 °C hold. Here, the heat inactivation of reverse transcriptase enzyme was omitted, to preserve the RNA–cDNA hybrids.For cDNA synthesis, 9 μl of RNA was mixed with 1 μl 10 mM dNTPs and 1 μl of 50 μM RT primer. The mixture was heated at 65 °C for 5 min and snap-cooled in ice for at least 2 min. Four microlitres of 5 × First-Strand buffer (Invitrogen), 1 μl DTT, 0.1 M, 1 μl RNasin Plus and 1 μl of 10 mg mlStreptavidin–biotin affinity purification was used to enrich for chimeric RNA–DNA hybrids. This pull-down was carried out after the second RNA fragmentation and RT, to allow a substantial fraction of the sequencing read pairs to cover the RNA–linker or linker–RNA junctions, in one end of the read pair.−1 RNase H (NEB)) for 1 h at 37 °C. The beads were collected on the tube wall using a magnetic concentrator and the supernatant was collected in a new tube for subsequent manipulations. We inactivated RNase H by heating at 70 °C for 20 min. cDNA was purified by 2.2 × SPRISelect beads (Beckman Coulter Genomics) (v/v).Specifically, 50 μl of Myone C1 beads (Invitrogen) was prepared by washing twice with 1 × Tween B&W buffer  and once with 1 × B&W buffer . The beads were then resuspended with 100 μl of 2 × B&W buffer . The RT mixture was topped up with RNase-free water to the final volume of 100 μl before combining with 100 μl C1 bead suspension and incubated at RT for 30 min with rotation. The beads were reclaimed and washed thrice with 1 × B&W buffer before transferring into a new tube, followed by washing once with TE buffer pH 8.0. Next, the cDNA strand was released from streptavidin beads by completely digesting the RNA strand in 50 μl RNase H elution mixture ) and incubated for 2 h at 60 °C. CircLigase II was inactivated by incubating the reaction at 80 °C for 10 min.cDNA was circularized by CircLigase II (Epicentre). Briefly, cDNA was eluted from SPRISelect beads in 20 μl CircLigase reaction mixture  was added into the CircLigase II reaction. Annealing was carried out by heating to 95 °C for 2 min, followed by 71 cycles of 20 s each, starting from 95 °C and decreasing the temperature by 1 °C after every cycle down to 25 °C and holding at 25 °C. Six microlitres of FastDigest BamHI (Fermentas) was added and incubated at 37 °C for 30 min. Re-linearized cDNA was purified by 2 × SPRISelect beads (Beckman Coulter Genomics) (v/v) and eluted in nuclease-free water.A cDNA oligo was annealed to the RT primer, generating a short double-stranded region suitable for BamHI restriction. This strategy also prevents BamHI activities on other endogenous BamHI restriction sites. Next, BamHI were applied, creating linear cDNAs with adapters at both 5′- and 3′-ends to prime subsequent PCR amplification. Next, oligo annealing mixture , 5 μl 20 μM Cut_oligo  with small number of cycles (six cycles). We found that the final libraries were less prone to be contaminated with undesirable smaller size fragments  by doing size selection at this stage.Six cycles of PCR were performed in a 40-μl reaction, which contained 20 μl of NEBNext High-Fidelity 2 × PCR Master Mix (NEB), 0.625 μM of each DP5/DP3 primer using the following temperatures: 1 cycle of initial denaturation at 98 °C for 30 s; 6 cycles of amplification with 98 °C for 10 s, 65 °C for 30 s, 72 °C for 30 s; followed by final extension at 72 °C for 5 min; and hold at 4 °C. The PCR product was purified by 1.8 × SPRISelect beads (v/v) and size-selected using E-gel EX 2% Agarose gels (Invitrogen). The DNA fragments between 150 bp and 350 were excised from the gel and purified using MinElute gel extraction kit (Qiagen).−1 DSN enzyme (Axxora) was added, mixed and incubated at 68 °C for 25 more minutes. We stopped the reaction by adding 40 μl of 2 × DSN stop solution (Axxora) to the reaction mix tube, mixing well and transferred the tube to ice. The reaction mixture was then purified using 1.8 × SPRISelect beads.. To reduce rRNA cDNAs from ES-1 and ES-indirect library, we also pre-amplified ss-cDNA using the truncated PCR primer DP5/DP3. However, the PCR cycle number was increased until we could obtain 80–100 ng of cDNA after purification by 1.8 × SPRISelect beads (Beckman Coulter Genomics) (v/v). We skipped the size selection by agarose gel, as this would largely reduce the amount of DNA. The eluted DNA from SPRISelect beads was mixed with 4.5 μl hybridization buffer  and sterile water (if necessary) to a final volume of 18 μl. The resulting mixture was denatured at 98 °C for 2 min and re-annealed at 68 °C for 5 h on a thermal cycler. Although the reaction mix tube was still in the thermal cycler, we added 20 μl of 68 °C-preheated 2 × DSN buffer (Axxora) to the reaction mix, mixed well by pipetting up and down ten times and incubated the reaction for 10 min at 68 °C. Two microlitres of 1 U μlWe performed PCR amplification of DNA produced from previous steps using full-length PCR primer PE 1.0 and 2.0 (Illumina). The number of PCR cycles was carefully titrated by running pilot PCRs with small aliquots of DNA to avoid overamplification. We purified the PCR products by 1.8 × SPRISelect beads (v/v) and size-selected fragments between 250 and 550 . Final libraries were quantified by Qubit (Invitrogen) and quantitative PCR, quality-checked by Bioanalyzer (Agilent Technologies) and submitted for paired-end sequencing on Illumina HiSeq platform.The custom-designed RNA and DNA oligonucleotides used in the procedure are as follows:Biotinylated RNA linker (RNase-free HPLC-purified from IDT):5′- rCrUrA rG/iBiodT/rA rGrCrC rCrArU rGrCrA rArUrG rCrGrA rGrGrA -3′cDNA strand with RNA linker (RNase-free HPLC-purified from Sigma):5′- T*C*G*C*ATTGCATGGGCTACTAGCAT -3′Pre-adenylated RT adapter (RNase-free HPLC-purified from IDT):5′- /5rApp/AGATCGGAAGAGCGGTTCAG/3ddC/ -3′RT primers (adapted from ref. RT Primer for the ES-1 sample:GgatcCTGAACCGCTCTTCCGATCT -3′5′- /5Phos/NNAGGTNNNAGATCGGAAGAGCGTCGTRT Primer for the ES-2 and MEF samples (sequenced on different lanes):GgatcCTGAACCGCTCTTCCGATCT -3′5′- /5Phos/NNCGCCNNNNAGATCGGAAGAGCGTCGTRT Primer for the ES-indirect sample:GgatcCTGAACCGCTCTTCCGATCT -3′5′- /5Phos/NNCATTNNNNAGATCGGAAGAGCGTCGTCut_oligo (HPLC-purified from IDT):GGATCCACGACGCTCTTCAAAA/3InvdT/ -3′5′- GTTCABamHI restriction site is highlightedTruncated PCR Forward Primer DP5 (HPLC-purified from IDT):5′- CACGACGCTCTTCCGATCT -3′Truncated PCR Reverse Primer DP3 (HPLC-purified from IDT):5′- CTGAACCGCTCTTCCGATCT -3′Illumina PE PCR Forward Primer 1.0 (PAGE-purified from Sigma):5′- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT -3′Illumina PE PCR Reverse Primer 2.0 (PAGE-purified from Sigma):5′- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT -3′Oligonucleotides were synthesized with a biotin attached to their 5′-end (IDT). Labelling was achieved by incubation of oligonucleotides and dyes  coupled with streptavidin at room temperature for 30 min at a ratio of 0.5 μM of oligonucleotides per 1 μM of dye.D-lysine  and laminin . Following incubation for 2 h, cells were washed in nuclease-free PBS and permeabilized with methanol at −20 °C. smRNA-FISH experiments were conducted using a modified version of an established protocolES cells were seeded on glass-bottom micro-chamber  previously coated with poly-z axis.Wide-field fluorescence imaging was conducted in an Olympus IX83 inverted microscope equipped with appropriate cubes for the dyes used , Chroma x–y–z centre of mass.Following imaging, raw image stacks were processed in ImageJhttp://mariotools.ucsd.edu. The pipeline takes pair-end sequencing reads as input  theas input ; and (3)as input . The majThe binding energies between two RNA interaction sites were calculated by the DuplexFold programme from RNAstructure version 5.6 ref. . The basFor every read pair in the RNA1–Linker–RNA2 category (output of Step 4 from MARIO tools), we obtained the PhyloP conservation scoresStarting from the RNA1–Linker–RNA2 type of read pairs (output of ‘Step 6. Selection and extraction of desired RNA–RNA interactions and reverse transcription' from MARIO tools), we applied the following filters to identify the pair-end reads generated from self-interacting RNAs. We removed those read pairs mapped to two different genes. If a read pair mapped to the same gene, we also removed those pairs that: (1) did not contain any fraction of the linker sequence; (2) the forward and the reverse reads mapped to opposite strands within 2,000 bp; (3) the read mapped to plus strand has smaller coordinates than the read mapped to minus strand in the genome within the pair. This step minimizes the inclusion of any intact (continuous) RNA fragment in the structural analysis.Structural information of the RNAs with known or generally accepted structures was downloaded from fRNAdb database v3.4 in DOT format (graph description language). We drew figures from the DOT files using the command line version of VARNA Applet version 3.9. For the RNAs without structural information in fRNAdb, we predicted their secondary structures based on the sequence using the ‘Fold' programme in RNAstructure version 5.6GSE61489.Data that support the findings of this study have been deposited in Gene Expression Omnibus with the accession number Accession codes: Data that support the findings of this study have been deposited in Gene Expression Omnibus with the accession number GSE61489.How to cite this article: Nguyen, T. C. et al. Mapping RNA–RNA interactome and RNA structure in vivo by MARIO. Nat. Commun. 7:12023 doi: 10.1038/ncomms12023 (2016).Supplementary Figures 1-28, Supplementary Tables 1-8, Supplementary Notes 1-5 and Supplementary References


By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA–ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3′UTR. Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem–loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation. Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3′ untranslated region (3′UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3′UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3′UTR, along with its  Requiem (REQ), also known as DPF2, ubi-d4 or BAF45D, was initially identified as an essential gene for apoptosis following IL-3 deprivation in myeloid cells and was named for its role in cell death . REQ is Since the overexpression of G8 appeared to mediate globin switching, we speculated that 3′UTR-mediated translational control could also play a role in the regulation of REQ gene expression. Indeed, when REQ was initially identified as a proapoptotic factor by screening for phenotypes induced by antisense cDNA expression , a partiDrosophila embryogenesis and in mammalian neurons (Alus)  length, the presence of a cytoplasmic polyadenylation element (CPE), specific RNA binding elements and microRNAs (miRNAs)  harboring the firefly luciferase gene or with pRLuc containing the escribed ,25. CommTo generate stable K562 cell lines expressing the Tet-on regulator, 10 μg of the linearized Tet-on regulator plasmid, p172-1 (a kind gift from Dr Manfred Gössen), was electroporated into K562 cells at conditions of 50 μF, 500 volts x 2. Positive cells were selected in the presence of G418 (200 μg/ml). After 2 weeks of sib-selection in 96-well plates, selected clones were assessed for the presence of p172-1 by monitoring the reporter activity after transient transfection of either pTRE-Luc (Clontech) or pTRE-EGFP (enhanced green fluorescence protein) into the cells.Total cellular RNA was prepared using Trizol (Invitrogen) according to the manufacturer's protocol. To eliminate possible contamination by exogenous or endogenous DNA, the RNA was treated with RQ DNase I (Promega) for 30 min at 37°C before continuing with the RNA extraction protocol. The quantity and quality of the isolated RNA were confirmed using UV spectroscopy  and electrophoresis on a 1% agarose gel, respectively. For RT-PCR, cDNA was synthesized using random hexamers (Invitrogen); reverse-transcribed cDNA was then amplified by the PCR using various sets of exon-specific primers. To amplify cDNA, sqPCR was performed using GoTaq polymerase (Promega). Primers used to PCR-amplify EGFP and hHPRT RNA were 5′-CAC CCT GGT GAA CCG CAT CG-3′ (sense) and 5′-GCT GCA CGC TGC CGT CCT CG-3′ (antisense) and 5′-GCT GGT GAA AAG GAC CCC A-3′ (sense) and 5′-AGC TCT ACT AAG CAG ATG GC-3′ (antisense), respectively. The pFLuc and pRLuc mRNAs were amplified by RT-qPCR using a CFX96 system (Bio-Rad) and Fast SYBR Green Master Mix (Applied Biosystems). cDNA synthesized from HeLa cells transfected with RLuc and FLuc mRNA was amplified using the primer pairs 5′-ATA TTG AGC CAG TAG CGC GG-3′ (sense) and 5′-GCC AAA CAA GCA CCC CAA TC-3′ (antisense) and 5′-AAT GTC CGT TCG GTT GGC-3′ (sense) and 5′-GTA GGC TGC GAA ATG CCC-3′ (antisense), respectively. The primers used to amplify endogenous REQ and GAPDH mRNA were 5′-TTT GTG ATG ACT GCG ATC GG-3′ (sense) and 5′-GGT GAG GGT GAT GTA AGC AG-3′ (antisense) and 5′-CAA GAT CAT CAG CAA TGC C-3′ (sense) and 5′-CTG TGG TCA TGA GTC CTT CC-3′ (antisense). C-Jun and IL-7R mRNA were amplified as previously described .In vitro transcribed [α-32P]-labeled RNA (100 000–500 000 cpm) was heated at 95°C for 5 min and subsequently allowed to cool to room temperature for 30 min. Protein extracts were incubated with the RNA probe in a buffer containing 30-mM Tris (pH 7.4), 3-mM MgCl2, 70-mM KCl and 1.5-mM Dithiothreitol (DTT). Then, 10 μg of protein was incubated with heparin sulfate (5 mg/ml) in a total volume of 15 μl. The reaction was carried out at room temperature for 30 min. To discriminate heterogeneous secondary-structured RNA probes from the specific RNA–protein complexes and to estimate the molecular weights of the RNA–protein complexes using UV-induced crosslinking, RNase T1 and RNase A were added at final concentrations of 3000 U/ml and 10 μg/ml, respectively, and the mixture was incubated for another 15 min at room temperature. Samples were resolved on a 5% polyacrylamide gel containing 5% acrylamide-bisacrylamide (29:1) and 0.05% NP-40. For supershift assays, antibodies specific for STAU1 (Abcam) were added to the reaction.rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Minimum free energy and partition function were set as the fold algorithms and basic options, and interactive RNA secondary structure plot and RNA secondary structure plot with reliability annotation were set as the output options.The optimal secondary structures for wild-type and truncated RNAs were predicted using the RNAfold web server  and anti-PLCγ (Santa Cruz Biotechnology). Antibodies were diluted 1:200–1:1500 in 5% non-fat dry milk in 0.1% Tween 20/TBS and then incubated overnight at 4°C with rocking. Immunoreactive bands were then visualized using ECL reagents (Thermo).Proteins in cell lysates were resolved by electrophoresis on 6–10% polyacrylamide gels and subsequently transferred to PVDF membranes (Millipore). The following antibodies were used: anti-EGFP (Clontech), anti-β-actin (Santa Cruz), anti-GST (Sigma), anti-UPF1 , anti-UPF2 (Santa Cruz Biotechnology), anti-calnexin (Santa Cruz Biotechnology), anti-REQ (in-house manufactured anti-rabbit peptide antibodies raised against the peptide Ac-LGEFPVSNSRARC-NHRNA immunoprecipitation (IP) was performed as previously described , with soAn antisense sequence of the REQ 3′UTR was previously reported to rescue the FDCP-1 cell line from apoptosis , correspin silico screen for conserved miRNAs predicted to target the REQ PBS (see below for definition) using various miRNA-matching algorithms (http://www.targetscan.org and http://www.microrna.org). Through these analyses, we could not identify any putative miRNAs that targeted the PBS (data not shown), suggesting that downregulation of translation may not result from miRNA-mediated mRNA turnover. To further study the mechanism of mRNA turnover mediated by the REQ 3′UTR and to determine which region of the 3′UTR is bound to cellular components, we performed RNA EMSAs using radiolabeled RNA probes consisting of a series of G8-deletion mutants. For this analysis, we generated various deletion mutants of the G8 region; each deletion segment was then subcloned into either pGEM3 or pT7Blue, vectors suitable for the in vitro synthesis of RNA probes  were initially screened with an [α-32P]-labeled G8-RNA ligand to detect specific RNA–protein interactions  or a nonspecific control siRNA Figure  and B. WTo determine whether STAU1 binding to the REQ 3′UTR leads to SMD, the levels of endogenous REQ mRNA in UPF1- and STAU1-knockdown cells were quantified by RT-qPCR. In these experiments, siRNA-mediated knockdown of either UPF1 or STAU1 upregulated endogenous REQ mRNA by ∼1.5- and 2-fold, respectively Figure . Two knoTo further investigate the effect of SMD on the REQ 3′UTR, we generated reporter constructs harboring various REQ 3′UTR variants downstream of the firefly luciferase gene, thus replacing the SV40 late poly(A) signal sequences Figure . HeLa ceThese observations led us to test whether the PBS core itself was necessary for SMD. To this end, we inserted various PBS core variants immediately after the stop codon in pFLuc and then cotransfected each variant with pRLuc into HeLa cells. The levels of FLuc mRNA were then quantified by RT-qPCR and normalized to the levels of RLuc mRNA Figure . These etrans-acting factor STAU1, has the ability to induce REQ mRNA decay. We presented evidence that G8, a region of the REQ 3′UTR, is involved in the downregulation of REQ protein, concomitantly with the downregulation of REQ mRNA, by using a Tet-regulated transactivation system  to the REQ 3′UTR. When the predicted stem–loop structure in the mouse PBS core was partially disrupted, the binding activity of these trans-acting factors was greatly reduced or eliminated (Figures In this study, we showed that the 3′UTR of REQ, along with its  Figures . And theRecently, post-transcriptional regulation, especially when it is mediated by the mRNA 3′UTR, has been identified as a major mechanism of gene regulation that contributes to cell fate decisions during differentiation and development. At the same time, through studies that have employed RNA EMSAs, the functions of the 3′UTRs of many viral genomes have been elucidated . The majDrosophila STAU was reported to preferentially bind to stem structures in long 3′UTRs (g 3′UTRs . Recent g 3′UTRs . Althougg 3′UTRs . Indeed,g 3′UTRs , suggestg 3′UTRs ,42. Howetrans-acting factors that normally bind to the REQ 3′UTR, thereby increasing the level of REQ protein and directly or indirectly driving β-globin expression in γ-globin-expressing K562 cells. In addition, we have shown in the present study that REQ is subject to SMD (Figures Several points should be emphasized from the perspective of REQ-mediated globin-switching. Although G8 was originally cloned as a putative globin-switching factor ,11, the  Figures , keratin Figures  and adip Figures , it will Figures . Thus, iSupplementary Data are available at NAR Online.


This study analyzed the impact of area residential property values, an objective measure of socioeconomic status (SES), on self-rated health (SRH) in Seattle, Washington and Paris, France. This study brings forth a valuable comparison of SRH between cities that have contrasting urban forms, population compositions, residential segregation, food systems and transportation modes. The SOS (Seattle Obesity Study) was based on a representative sample of 1394 adult residents of Seattle and King County in the United States. The RECORD Study  was based on 7131 adult residents of Paris and its suburbs in France. Socio-demographics, SRH and body weights were obtained from telephone surveys (SOS) and in-person interviews (RECORD). All home addresses were geocoded using ArcGIS 9.3.1 . Residential property values were obtained from tax records (Seattle) and from real estate sales (Paris). Binary logistic regression models were used to test the associations among demographic and SES variables and SRH. Higher area property values significantly associated with better SRH, adjusting for age, gender, individual education, incomes, and BMI. The associations were significant for both cities. A one-unit increase in body mass index (BMI) was more detrimental to SRH in Seattle than in Paris. In both cities, higher area residential property values were related to a significantly lower obesity risk and better SRH. Ranked residential property values can be useful for health and weight studies, including those involving social inequalities and cross-country comparisons. •We studied the impact of area property values on health in Seattle and Paris.•Higher area property values associated with better SRH in both cities•Ranked area property values can be useful for health and weight studies.•BMI was more detrimental to SRH in Seattle than in Paris. In geneThis paper makes three distinct contributions. First, disparities in SRH can also be observed at the microscale. While it is instructive to compare social disparities across countries, it may also be useful to compare SRH across cities that differ in urban form, population composition, residential segregation, food systems and transportation modes. The question is how the socioeconomic determinants of health affect SRH across cities and their neighborhoods. The present analyses compare Paris and Seattle.Second, past educational attainment and current income may not capture the multiple facets of SES . Given tThird, this research adds to the growing literature of studies examining the interaction between urban-form-related SES variables, functional limitations and SRH. Obesity rates, determined by body mass indexes, are linked to social inequalities and obesity prevalence is higher in the US than in France , with 3222.1The Seattle Obesity Study (SOS), conducted in 2008–2009, was a population-based survey of 2001 adult residents of King County, WA . Detailewww.record-study.org), conducted in 2007–2008, was based on 7290 eligible persons attending 2-hour preventive checkups conducted by the Centre d'Investigations Préventives et Cliniques in four of its health centers located in Paris, Argenteuil, Trappes, and Mantes-la-Jolie . Annual household income was divided into tertiles based on the most recent census information in the two cities and the underlying distribution of the data. Questions about marital status and household size were comparable in the SOS and the RECORD Study. The dichotomous variable “living alone or not” was based on reported household size in the SOS and on a question regarding cohabitation status in the RECORD Study.Participant heights and body weights were obtained through telephone self-report in the SOS and measured by a nurse in the RECORD Study using a wall-mounted stadiometer and calibrated scales . Body ma2.3Procedures for geocoding of home addresses were specific to each country but each resulted in spatial coordinates that were used to calculate network distances. In the SOS, home addresses were geocoded to the centroid of the home parcel using the 2008 King County Assessor parcel data, by means of standard methods in ArcGIS 9.3.1 . Addresses that failed the automatic geocoding (60% of the samples were geocoded based on 100% match score) were manually matched using a digital map environment, augmented by online resources such as GoogleMaps, QuestDEX and Yelp. Each home point was checked for plausibility and accuracy. In the RECORD Study, home addresses in 2007–2008 were geocoded using the Geoloc software of the National Institute of Statistics and Economic Studies. Research assistants rectified all incorrect or incomplete addresses with the participants by telephone. Additional data were obtained from local authorities. Spatial coordinates and block group codes were identified for 100% of the sample.2.4Property values were handled differently in the SOS and in the RECORD Study. The residential property values for Seattle were based on county tax assessor data for individual parcels, whereas the data for Paris were based on prices of properties sold over a given period of time.In the SOS, assessed residential property values were obtained from the 2008 King County tax assessor parcel database. Property values are determined by the combined value of both land and improvements (buildings and other structures), and based on recent local sales data. For home parcels with multiple residential units , assessed value per unit was calculated as the sum of a parcel's land and improvement values divided by the number of residential units on the parcel. Tax assessment in Washington State aims to estimate the full market value of a given property (King County Department of Assessments). The primary variable of interest was the mean assessed property value per residential unit in the 833 m radius buffer around the each respondent's home.In the RECORD Study, residential property values were assessed on the basis of notary data obtained from Paris-Notaries related to the price at which properties  were sold. Mean value of dwellings sold within a French IRIS (similar size to a US block) between 2003 and 2007 was computed (after accounting for variations of price levels between years) and captured with a 500 m radius buffer around each respondent's home. The mean score was categorized by quartiles.2.5Separate binary logistic regressions were conducted to examine the impact of demographic, socioeconomic, average area property value, and BMI on SRH. In detail, Model 1 only included demographic and socioeconomic variables. In Model 2, average area property value was added. Model 3 included all the variables in Model 2 and added three categories of BMI . An alternative model also considered BMI as a continuous variable (results not shown). All analyses were conducted using Stata 10.0.33.1RECORD Study participants were predominantly male (65%), married (70%), and > 45 years old (65%). Only two out of five persons (38%) were college educated. With an average BMI of 25.47, the obesity rate was 12%, which was equal to the national estimate for France . “Poor oLiving in the city of Seattle, as opposed to the suburbs, was associated with higher education, living alone (39% vs. 27%) and with lower obesity rates (18% vs. 24%). Living in the city of Paris, as opposed to the suburbs, was associated with higher education, living alone (37% vs. 27%), and with lower obesity rates (9% vs. 14%).3.2Binary logistic regression analysis examined the associations between SRH, demographic and SES variables, including residential property values . Model 1The impact of SES factors on SRH was greater in Seattle than in Paris for the three SES variables, even after mutual adjustment. In Seattle, residents living in the top quartile of property values were 59% less likely to report a fair/poor health status compared to residents living the bottom quartile of property values. In Paris, the corresponding reduction was only 23% (Model 3). In Seattle, individuals having a college degree or higher were 59% less likely to report a poor health status compared to samples with high school or less education. In Paris, the corresponding reduction was 42%. Similarly, the effect of household income was also stronger in Seattle than in Paris.Living within the city limits was not related to SRH in either of the cities. There was some evidence that the relationship between gender and SRH was in the opposite direction in Seattle and in Paris. While men tended to more often have a fair/poor vs. good/excellent SRH in Seattle, they less often reported a fair/poor SRH than women in Paris.Being obese was negatively correlated with SRH; however, the detrimental obesity effect was much greater in Seattle than in Paris (Model 3). Being overweight was negatively correlated with SRH in Paris but was not related to SRH in Seattle. Alternative models also tested the impacts of BMI as a continuous variable on SRH. The results showed that a one unit BMI increase in Seattle would increase the likelihood of reporting a poor/fair health status by 9% (95% CI: 6%–12%). In Paris, the corresponding increase was only of 5% (95% CI: 3%–7%). We also added a quadratic term for BMI, but the 95% CI of the coefficient overlapped 0 in both cities, and this quadratic term was dropped from the final model reported in 4The SOS and the RECORD Study are among the first studies to examine interactions between SES variables, SRH, and obesity rates. In both Seattle and Paris, SRH was strongly linked to SES variables, both reported and objective. Remarkably, regression models provided very comparable findings for both cities, suggesting that the social mechanisms underlying SRH may be the same. In both cities, low area property values, low educational attainment and low incomes had significant impacts on SRH.This comparative study also showed that the impact of socioeconomic disparities on SRH was larger in Seattle than in Paris . Significantly, obesity was both higher and also appeared to be more detrimental to SRH in Seattle than in Paris. In Europe, obesity among women was shown to be more strongly and more consistently related to education, rather than to occupation or incomes . In the The present approach to measuring SES was based on property values within an area or neighborhood buffer zone around each respondent's residential address, which required reconciling different methodologies. In the US, property values are available from tax rolls that are in the public domain. In Seattle, property values are derived from recent sales because by law, property assessment must equate sales value. In France, comparable data were obtained from tax files and notaries, based on recent sales. Property values, whether from tax rolls or real estate sales, can complement self-reports of education and incomes in the US  and the The use of the novel methodology requires some new distinctions of what constitutes individual SES as opposed to area SES. There has been some debate as to whether SES derived from administrative unit areas was more or less important than SES derived from individual neighborhoods . PampaloWith respect to neighborhood influences on health, Cummins and colleagues found that in the UK, fair to very bad SRH was significantly associated with poor neighborhood-level physical residential environment and lower access to private transport . FurtherThe links between obesity and SRH require some discussion. Delpierre et al. have reported that functional limitations in the US were associated more strongly with poor SRH in the most educated men than in the least educated, as compared to France . This fiThe relation between obesity and SRH was found to be markedly stronger in Seattle than in Paris where the population is leaner. A hypothesis was that it may be due to the much higher percentage of obese population in Seattle than in Paris (21% vs. 12%). A separate model coding BMI as a continuous variable showed that a one unit increase in BMI was associated with a larger deterioration of SRH in Seattle than in Paris. Thus, this finding further suggests that there may also be differences between the two cities in the perception of how a high body mass index is detrimental to health.Both studies had limitations. The SOS sample, based on a random landline-telephone survey (standard BRFSS procedure) was older, better educated and had a higher proportion of females than the background population. Heights and body weights were based on self-reported data, which may have resulted in some discrepancies due to underreporting body weights. A recent publication focused on a comparable Seattle-based cohort also showed a good correspondence between self-reported and measured heights and weights .The Seattle sample might exclude some low SES subjects who do not have landline telephones. Conversely, the Paris sample was employment-based, with an over-representation of males, and people with higher education attainment and coming from more affluent and lower-density neighborhoods. Furthermore, because of limitations in data availability, the authors could not include the same built environment or spatial variables in both models.Both studies were cross-sectional, limiting our ability to draw causal inference from the observed associations. Nonetheless, the present Seattle–Paris comparisons are valuable given the different socioeconomic, behavioral and environmental context of the two cities. The relative influences of income, education, and property values on SRH can provide additional insight into the contextual socioeconomic determinants of SRH.The authors declare that there are no conflicts of interest.


C. elegans genome encodes many RBPs conserved throughout evolution. The rich analytic tools in molecular genetics and simple neural anatomy of C. elegans offer advantages to define functions of genes in vivo at the level of a single cell. Notably, the discovery of microRNAs has had transformative effects to the understanding of neuronal development, circuit plasticity, and neurological diseases. Here we review recent studies unraveling diverse roles of RBPs in the development, function, and plasticity of C. elegans nervous system. We first summarize the general technologies for studying RBPs in C. elegans. We then focus on the roles of several RBPs that control gene- and cell-type specific production of neuronal transcripts.Neurons have evolved to employ many factors involved in the regulation of RNA processing due to their complex cellular compartments. RNA binding proteins (RBPs) are key regulators in transcription, translation, and RNA degradation. Increasing studies have shown that regulatory RNA processing is critical for the establishment, functionality, and maintenance of neural circuits. Recent advances in high-throughput transcriptomics have rapidly expanded our knowledge of the landscape of RNA regulation, but also raised the challenge for mechanistic dissection of the specific roles of RBPs in complex tissues such as the nervous system. The  C. elegans' genome encodes approximately 500 RBPs, defined by having an RNA binding domain such as the RNA recognition motif (RRM) and K Homology Domain (KH)  and C. elegans (Fem-3)  and small RNAs.Precise regulation of RNA, including mRNAs and small RNAs, is essential for controlling gene expression in a spatial and temporal manner. Research has identified critical roles of RBPs in neuronal development and synaptic transmission, which when disrupted through mutations, can cause neurological diseases. The 3) Table  (MacNico3) Table . StudiesC. elegans, comprehensive transcriptome analyses for several developmental stages by the modEncode Consortium have provided valuable information for validating gene structures as well as revealing many new alternative splice junctions  in mRNA, as well as many previously unannotated 3′UTRs . miRNAs, as part of the miRISC, guide the miRISC to specific mRNA targets. Two key components of the miRISC are the Argonautes C. elegans adult hermaphrodite has 302 neurons and the major tissues are non-neuronal cells, a technical hindrance in defining transcriptomes of neurons is that neuronal mRNAs are often poorly represented in samples prepared using whole animals. Thus, a clever method, developed by Kudlow et al. allows tissue-specific CLIP in the intestine and muscle by using specific promoters tagged to the protein of interest . One hypothesis for the mechanism of SMA is the loss of the survival motor neuron 1 protein (SMN1), which can assemble pre-mRNA splicing machinery, resulting in abnormal mRNA splicing in neurons  mutations genetically enhance less severe loss of function mutations of unc-52, which, through alternative splicing, produces multiple isoforms of perlecan, an extracellular matrix heparan sulfate proteoglycan (HSPG) conserved to humans  shows lethality at the 2-fold embryo state and appears paralyzed but certain alleles of unc-52 are viable and fertile , these viable alleles of unc-52 mimic the lethal and paralyzed phenotype  results in differential expression of specific isoforms of unc-52 by RT-PCR and immunofluorescence compared to wild type, supporting the notion MEC-8 may act as a splicing factor of unc-52 mRNA in the epidermis  involved in mechanosensation UGUUGUG shared among these targets and confirmed UNC-75 could bind this sequence, which is similar to other studies that identified G/U-rich motifs for the CELF proteins  suppressed the behavioral phenotype of this unc-32 allele by allowing expression of the other isoforms of unc-32 in the nervous system to compensate for the loss of exon 4b isoform. Thus, these data provide functional evidence for unc-75's regulation of this specific exon in the nervous system  mutants as they showed decreased fluorescence intensity of the glr-1 reporter and a reduction in a response to a nose-touch assay, a behavioral test dependent on the AVE interneuron  consists of RBPs, named after the discovery of the gene in rpm-1, display an abnormal synaptic phenotype consisting of fewer and disorganized synapses , ess-2, related to the mammalian ES2/DGCR14 protein, was found to regulate dlk-1 mRNA when splicing junction was altered. ES2/DGRC14 is a component of the splicesome complex  mutants, by themselves, do not show obvious effects on splicing, but reduce the levels of dlk-1 mRNA expression in a mutant in which the splice site in the third intron of dlk-1 was mutated. ess-2(lf) was also found to act similarly on an allele of dpy-10, a cuticle collagen, which changes a splice acceptor site leading to unspliced and spliced transcripts  endocytosis, such as synaptojanin and endophilin. Consistent with a model that the abnormal neurotransmission may result from problems in endocytosis, Wang and colleagues found through electron microscopy decreased number of SVs, altered distribution of SVs away from the active zone, and vesicle accumulations in the presynaptic terminal  mutants displayed abnormal synapses and ectopic axon branches , which implies specific roles for certain members of this machinery in the nervous system  mutants indicating fbf-1 promoted translation of the reporter through egl-4's 3′UTR  in the msi-1(lf)   mutants displayed precocious synaptic remodeling   transcription factor family shown to inhibit dauer formation  was paired with the GW182 homolog, ain-1(lf), suggesting microRNA involvement. However, the individual mutants of the microRNAs and unc-3(lf) could not completely recapitulate the percentage of dauer formation observed in unc-3; ain-1, suggesting that multiple microRNAs contribute to the dauer phenotype  by inhibiting neurotransmitter release  inhibits adult axon regeneration  and found to be necessary for axon regeneration 's phenotype, suggesting rpm-1 negatively regulated cebp-1 through its 3′UTR. Second, using a photoconvertible protein tagged to cebp-1's 3′UTR, they showed upregulation of protein synthesis in the distal axon 2 h after axotomy. When examining a similar reporter tagged to a control 3′UTR this increase in fluorescence in the distal axon was absent. Taken together, these data suggest upregulation of cebp-1 through its 3′UTR after injury is important for the regenerative response, illustrating a function for the 3′UTR in the adult nervous system. Though so far there are few studies of RBPs and microRNAs in C. elegans in axon regeneration, they all implicate roles for RBPs and microRNAs in controlling local responses to injury and regeneration.In PLM mechanosensory neurons, Yan et al. reported the 3′UTR of a transcription factor important for axon regeneration (Yan et al., C. elegans has helped advance the field by providing genetic tools to dissect RNA pathways, specifically in the nervous system. Since many RBPs are conserved, their function and mechanism of regulation in C. elegans may likely be the same in humans. Additionally, in vivo studies in C. elegans have displayed the importance of RBPs in mRNA splicing, mRNA 3′-end processing and microRNA pathway, and their resulting effects on synaptic plasticity, synapse formation and axon regeneration. These studies have allowed single-neuron and global dissection of these interactions to highlight the complexity of the nervous system. Future studies of mRNA in the nervous system of C. elegans will continue to build a deeper understanding of the complex regulations by RBPs.The complexity of the nervous system poses many questions about how it develops, grows and maintains its connections. Studying RNA regulation will identify key mechanisms that can address these questions. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Labeling RNA is of utmost interest, particularly in living cells, and thus RNA imaging is an emerging field. There are numerous methods relying on different concepts ranging from hybridization-based probes, over RNA-binding proteins to chemo-enzymatic modification of RNA. These methods have different benefits and limitations. This review aims to outline the current state-of-the-art techniques and point out their benefits and limitations. At that time, the only method by which RNA could be visualized wasin situ hybridization (ISH)2. Since then, the options for RNA detection have expanded greatly. Labeling RNA—particularly mRNA—is of utmost interest, as mRNA localization has been shown to be important in a range of situations. For example, in a developingDrosophila oocyte, asymmetrically localized mRNA produces abicoid protein gradient through localized translation, which specifies the anterior-posterior polarity of the developing larva3. In neurons, localization of mRNA is also particularly important, and localized mRNA leads to multiple rounds of translation at the synapse and activity-dependent changes4. Additionally, since the number of genes transcribed was found to surpass the amount of protein-coding genes, interest in non-coding RNAs has increased5. Thus, imaging microRNAs or small interfering RNAs (siRNAs) is also of interest6. Furthermore, defects in mRNA localization play an important role in some diseases such as fragile X syndrome7, and non-coding RNAs have been shown to be important in diseases such as cancers8.The localization of mRNA in the cell has been a topic of interest since the 1980s, when protein localization was linked to localized mRNA translationin situ hybridization (FISH) can distinguish between RNA molecules that differ in only a single base9. FISH is highly sequence-specific, and individual RNA strands may be detected when combined with various amplification procedures in fixed cells10. A variety of derivatives of ISH that reduce background signal have been developed, most notably molecular beacons  makes FIT probes an attractive improvement of ISH. FIT probes have been expanded to contain dyes that emit in the blue and green ranges17 and have been successfully used for mRNA visualization in cells andDrosophila embryos18.An alternative approach to increase the specific fluorescent signal upon binding is to use forced intercalation (FIT) probes . FIT proin vivo hybridization (FIVH)21. In particular, 2′-O-methylated oligonucleotides exhibit faster hybridization kinetics, increased melting temperatures, enhanced binding specificity, improved nuclease stability, and the ability to bind structured molecules—properties beneficial for FIVH probes20. They were used to detect a variety of RNA types, such as snRNAs, rRNA, and poly(A) RNA20. Nevertheless, these probes need to be introduced into the cells and thus FIVH requires transient permeabilization of cells.Hybridization-based RNA detection is an excellent tool for use in fixed samples and can be used in living cells and organisms when strong turn-on effects are achieved . However, probes based on modified nucleic acids or derivatives thereof are neither cell-permeable nor can they be produced by the cell itself. Furthermore, hybridization has to occur in regions of the target RNA free of secondary structure, and hybridization conditions are typically not optimized for the cellular milieu. Recently, probes and conditions have started to be developed for use in live cells; this approach is termed fluorescencein vitro selection22. Recently, an aptamer termed “Spinach” was selected that folds to allow binding of a small-molecule fluorophore that fluoresces only upon binding the RNA aptamer  dynamics of mRNA could be monitored32.An alternative method to mark RNA is to incorporate visualizable moieties directly into the RNA. A convenient way to achieve marking RNA without introducing a large moiety that may interfere with RNA function is to incorporate a small chemical group that may be further reacted by using click chemistry to attach to a fluorophore. There are a number of different click reactions, the most prominent being the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC). Here, an azide reacts with an alkyne in the presence of Cu(I) as a catalyst. CuAAC is rapid and extremely selective; however, Cu(I) at millimolar concentrations is toxic to cells and thus this approach is limited to fixed cell samples. Jao and Salic succeeded in incorporating ethynyl groups into total RNA by feeding cells with the uridine analog 5-ethynyluridine (EU), which is converted to the respective triphosphate inside the cell 31. Usinet al. synthesized an azido-modified UTP analog that can be used in the bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC)34. This allowed the click reaction to proceed in live cells; however, this UTP analog had to be transfected into the cells as its uridine precursor was no longer cell-permeable or was not a good substrate for the ribonucleoside salvage pathway  analog can be made from cell-permeable and stable methionine analogs by a variant of the methionine adenosyltransferase (MAT), which is responsible for AdoMet synthesis37.A downside of incorporating modified nucleotides during transcription or poly(A) tail addition is that different subtypes of RNA cannot be distinguished. A possible method by which to obtain specific labeling of different subtypes is to attach chemical groups used for click reactions post-synthetically by using RNA-modifying enzymes. Subtypes of RNA may also be labeled by taking advantage of certain structures or modifications in an RNA type. For example, the 5′ cap of mRNA may be specifically labeled by using an engineered methyltransferase that is only active on the mRNA cap 35–37. in vitro by using a box C/D methyltransferase-guide RNA complex and the respective propargyl-bearing analog of the cosubstrate AdoMet38. Liet al. developed an RNA labeling system in which an RNA of interest was extended by a tRNA-derived sequence and an enzyme that specifically modifies this sequence  was introduced into a cell  system . This co46. Like many RNA-binding proteins, pumilio is modularly composed of domains that can be engineered to alter the specific RNA sequence bound49. Pumilio is of particular interest as it can target RNA directly without the need to introduce an RNA tag into the target RNA.Another RNA-binding protein worth mentioning is pumilio. Pumilio is a member of the RNA-binding protein family PUF51. The potential drawback of high background fluorescence due to unbound protein may be countered by using a split GFP, which fluoresces only upon dimerization (53.An advantage of using RNA-binding proteins to visualize RNA is that two individual RNA sequences may be targeted by separate RNA-binding proteins, thus allowing the imaging of the association of two RNA molecules of interestization 52,53.et al. described a method based on trans-splicing that results in the expression of luciferase in cells of a living organism microinjected with an exogenous RNA that was processed to pre-mRNA54. Soet al., employing a similar approach, developed an engineered ribozyme, which fuses a reporter gene to a specific gene of interest55. The authors were able to detect p53 in a whole organism and on a cellular level. Despite theoretical expansion potential56, the approach taken by the Gambhir lab has not been significantly developed since the publication of the original study, leaving it with the limitation that only exogenous RNA can be visualized. Similarly, the work of Soet al. has not been further developed.Two approaches have been developed by which a pre-mRNA may be spliced into a functional form, which allows the expression of a reporter protein. This enables tissue-specific localization of an mRNA of interest, although the resolution at a subcellular level is lost. Bhaumik57. RNA imaging at the whole organism level is important to determine the tissue expression pattern of a specific transcript. RNA labeling has seen extensive use in imaging of infection by RNA viruses (e.g.,59.Imaging of RNA is of interest at the level of both single cells and the whole organism. Labeling RNA in a single cell can show the localization of a specific transcript, which may have important biological consequenceses e.g.,. AnotherIn summary, RNA may be visualized by a variety of methods. RNA may be seen via hybridization of a reporter molecule, most commonly through FISH or variations thereof. Alternatively, RNA-binding proteins that bind specific sequences may mark an RNA molecule of interest, or an RNA aptamer that fluoresces upon binding of a fluorophore may be incorporated into the target molecule. RNA may be sequence- or subtype-specifically labeled by using click chemistry. Challenges facing the field of RNA imaging are the cell permeability of dyes used and the low abundance of target RNA. Furthermore, no method of RNA labeling is yet able to yield quantitative data on its target RNA. However, with continued development, RNA imaging will continue to provide important biological insights.


O-MTases (Rlml/RlmN), spOUT MTases and the NSun2 MTases are dissected for the insight they provide on the transition from an RNA world to our present RNA/DNA/protein world.RNA, the earliest genetic and catalytic molecule, has a relatively delicate and labile chemical structure, when compared to DNA. It is prone to be damaged by alkali, heat, nucleases, or stress conditions. One mechanism to protect RNA or DNA from damage is through site-specific methylation. Here, we propose that RNA methylation began prior to DNA methylation in the early forms of life evolving on Earth. In this article, the biochemical properties of some RNA methyltransferases (MTases), such as 2′- Figure 1). Nature’s selection of contemporary bases in RNA might have been driven by modifications like N-glycosyl bonds that made the bases more resistant to hydrolysis  as a cofactor. In fact, folate is required for synthesis of SAM and as a cofactor to speed up the methylation reaction  since its precursors are derived from adenine (the first base likely to have been synthesized in the prebiotic soup), methionine (the first amino acid added during translation) and ribose (the first sugar made in nucleic acid anabolism).The prebiotic appearance of RNA has received considerable attention during the past decade and this event has been succinctly summarized by reaction . PerhapsO-methyl intermediate has already been pointed in earlier literature  or RNA complexes is indeed the primordial manifestation of the methyl transfer reaction and is the most likely mechanistic platform for the de novo creation of deoxyribose sugar  on Earth before the appearance of genetic code or ribonucleotide reductase. In fact, 2′-O-methylation or the insertion of an oligodeoxynucleotide (short piece of DNA) in some ribozymes leads to enhanced activity  and the other is through C/D box ribonucleoproteins  has been reported to methylate both rRNA and tRNA at the second aromatic carbon of adenosine .The two known family of MTases involved in methylation of rRNA, as well as tRNA, are spoUT family and RlmN enzymes. The spoUT enzymes employ a minimal domain to carry out the methylation reaction, though the exact binding and catalytic mechanisms remain elusive . spoUT gine m2A; . RlmN caNSun2 and Dnmt2 are, at this time, the only known 5-methylcytidine (m5C) RNA MTases in higher eukaryotes; tRNA is the confirmed target substrate for both enzymes . RecentlBase flipping is a biological process in which a specific nitrogenous base from the stacked region turns around 180° at the catalytic site of MTase without disturbing the remainder of the nucleic acid backbone . In the Bacterial genomes evolve rapidly through mutation, rearrangement and horizontal gene transfer . Phages in vitro are capable of self-sustained replication  forms rather than racemic mixtures. Much remains to be understood about the exact nature of early Earth conditions, the constituents of the primordial mixture, the appearance of RNA and DNA, the introduction of chirality in shaping life and how methylation could be one of the mechanisms to conserve chirality in the biomolecules .All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MicroRNAs act by post-transcriptionally regulating the gene expression of 30%–60% of mammalian genomes. MicroRNAs are key regulators in all cellular processes, though the mechanism by which the cell activates or represses microRNA-mediated translational regulation is poorly understood. In this review, we discuss the RNA binding protein Fragile X Mental Retardation Protein (FMRP) and its role in microRNA-mediated translational regulation. Historically, FMRP is known to function as a translational suppressor. However, emerging data suggests that FMRP has both an agonistic and antagonistic role in regulating microRNA-mediated translational suppression. This bi-functional role is dependent on FMRP’s interaction with the RNA helicase Moloney leukemia virus 10 (MOV10), which modifies the structural landscape of bound mRNA, therefore facilitating or inhibiting its association with the RNA-Induced Silencing Complex. FMR1 gene [FMR1 coding region. Two identified missense mutations are the arginine to glutamine mutation (R138Q) in the Nuclear Localization Sequence (NLS) [The clinical consequence of a loss in functional Fragile X Mental Retardation Protein (FMRP) is known as Fragile X Syndrome (FXS), the most common inherited form of intellectual disability ,2. AlongMR1 gene . Howeveration R13Q in the ce (NLS) ,10 as wece (NLS) . Such inThe evidence that FMRP is a translational regulator has long been known, but the mechanisms for this regulation are poorly understood. Two factors that may influence FMRP’s role in the microRNA (miRNA) pathway are the structural landscape of the 3’ Untranslated Region (3′ UTR) and FMRP’s interaction with other RNA-binding proteins (RBPs). Here we discuss the RNA G-Quadruplex (GQ) and FMRP’s interaction with the RNA helicase Moloney leukemia virus 10 (MOV10). The bi-functional role of FMRP to suppress or de-repress its target transcripts through the miRNA pathway may be, in part, through modulating RNA secondary structure through its interaction with MOV10.in vitro. Although not specifically identified in any cellular RNAs, exogenously introduced kissing complex was able to compete FMRP off of polyribosomes [Though expressed in many tissue types, FMRP is highly expressed in the testes and the brain, with elevated expression in the cortex and hippocampus ,13. FMRPibosomes , suggestibosomes . Howeveribosomes . The thiibosomes ,21,22. Dibosomes ,24,25, Fibosomes . More reibosomes . Thus, aAnother KH domain was recently discovered and appropriately named the KH0 domain, as it resides N-terminal to the first KH domain (KH1) ,10. Alth3K79 through its agenet domains and modulated the DNA repair response [FMRP contains two N-terminal in-tandem agenet domains, Agenet1 and Agenet2, thought to associate with trimethylated lysine . Other fresponse . A loss response . Evidencresponse  is a domresponse ,34.Though mostly cytoplasmic, FMRP contains both nuclear localization and export sequences and has been shown to shuttle into the nucleus to bind target mRNAs, eventually transporting back into the cytoplasm with its bound mRNA through the nuclear RNA export factor 1 (Tap/NXF1) . Once trXenopus oocytes and in rabbit reticulolysate [Drosophila ortholog of FMRP, as was much of the initial work implicating FMRP in the RNA-induced silencing complex (RISC) pathway, discussed in more detail below. Drosophila has one fragile X family member, whereas mammals have three , which are 60% identical [Drosophila ortholog likely give insight into the function of all of the fragile X family members. Like FMRP, loss of expression of the Drosophila ortholog result in viability but impaired behavior [FMRP was identified as associating with the 60S large ribosomal subunit in 1996, although the functional consequences of that association were unknown . The firlolysate . Later wlolysate . In a lalolysate . Recent lolysate . Howeverdentical . Thus, ebehavior .An alternative mechanism in which FMRP suppresses the translation of its bound mRNAs is by modulating the interactions required for translation initiation. Cytoplasmic FMRP Interaction Protein (CYFIP1) directly interacts with FMRP, with its bound mRNA, along with the eukaryotic initiation factor 4E (eIF4E), sequestering it into an RNP and effectively suppressing cap dependent translation initiation . TranslaDrosophila ortholog of FMRP was found to be associated with components of RISC, and regulated mRNA expression through an unknown mechanism [The first evidence of FMRP’s role in the RNA interference (RNAi) pathway came from studies in which the echanism ,52. In hechanism  or supprechanism  expressiechanism . Subsequechanism ,56. FMRPechanism ,58. Alonechanism . FMRP waechanism  and thisechanism . Soon afechanism . PSD-95,echanism . Phosphoechanism . Howeverechanism ,63. MOV1echanism .The 3′ UTRs of RNA contain excessive secondary structure and are heavily bound by RBPs , which cThere is evidence that proximal binding of RBPs to MRE sites may act to block MREs, restricting the accessibility of RISC on target mRNAs and therefore up-regulating the expression of these genes. For example, dead end 1 (Dnd1) competitively binds to 3′ UTR MRE sites in human cell lines and zebra fish germ cells, prohibiting miRNAs from binding, counteracting translational suppression of target mRNA . The CodRNA-binding proteins have also been shown to have both an agonizing or antagonizing function in the miRNA-mediated translational regulation of their bound mRNAs. The polypyrimidine tract binding protein (PTB) functions to antagonize miRNA-mediated translational regulation by competing with miRNA targets in the 3′ UTRs of a subset of mRNAs. In this case, partial overlap of the MRE sequence to that of the PTB binding site causes direct competition for this region, decreasing RISC’s ability to bind. Contrary to competing for binding sites with the miRNA, higher concentrations of PTB also facilitate miRNA targeting by modifying secondary structure to reveal or expose MREs . In thisAnother RBP exhibiting both agonistic and antagonistic functions in miRNA regulation based on RBP binding and secondary structure is human antigen R (HuR). HuR facilitates miRNA binding in the 3′ UTR of the c-Myc mRNA, by either recruiting Let-7a or by changing the local structure proximal to the MRE , therebyRNA secondary structure itself has a large effect on the efficacy of miRNA function . The modIn the 5′ UTR of RNA, GQs have been shown to suppress translation by blocking translation initiation ,81. Within vitro [RNA helicases function to remodel and unwind RNA and RNA-protein complexes (RNP), and play a role in many different processes. Because secondary structure plays such a significant role in miRNA functional efficiency, it stands to reason that resolving secondary structure is paramount for miRNA-mediated translational regulation. Examples of how RNA helicases function to unwind GQs and enhance or suppress a cellular process is observed by work involving the DEAH-box RNA helicases RNA associated with AU-rich element (RHAU) and DEAH Box Helicase 9 (DHX9). RHAU’s function to unwind GQs  has beenin vitro .MOV10 is an RNA helicase that binds single stranded RNA proximal to the stop codon and translocates along the 3′ UTR in a 5′ to 3′ direction . Like maMOV10 was first identified as being associated with RISC when it was found to be required for miRNA reporter cleavage and co-localized with AGO2 in P-bodies . A studyAnalysis of CLIP-seq data for MOV10  AGO2 7070 and FMIn conclusion, RNA binding proteins and the structural landscape of the 3′ UTR of mRNA play an important role in miRNA translational regulation. It is becoming more apparent that secondary structure not only adds a level of complexity to the dynamic process of miRNA regulation, but the emerging data also supports that the dualistic functions of RBPs can create a multitude of new and exciting methods by which RNA is post transcriptionally regulated. There is still much more work to be done to gain a clearer understanding of how these components work together to cooperatively and/or competitively drive gene expression via the microRNA pathway.


The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation. The La protein LARP3) is a cancer-associated RNA-binding protein –6 initia is a canHuman La protein has three RNA-binding surfaces: the N-terminal located La motif (LAM), the RNA Recognition Motif 1 (RRM1) and the non-canonical RNA Recognition Motif 2 (RRM2) located in the C-terminal extension characteristic for mammalian La protein . The RNAcis-splicing and strand-annealing/dissociation assays in vitro, bacteria and yeast  phosphorylation site ,59–63. IWe have shown recently that the La protein is overexpressed in solid tumors, supports proliferation and mobility of cancer cells and established that La promotes IRES-mediated translation of cyclin D1 (CCND1) in HeLa cells ,4. The r32P]-CTP (Cytidine triphosphate) labeled, were synthesized using the MEGAshortscript High Yield Transcription Kit (Ambion) according to the manufacturer's instructions. For the transcription of internally labeled RNAs, reactions were assembled in the following order: 2 μl T7 10x reaction buffer, 8 μl of 75 mM T7 ATP-GTP-UTP Mix (18.75 mM each), 1.5 mM cold CTP, 40 μCi [α-32P]-CTP, 100 nM of DNA template containing a T7 promoter, 2 μl T7 enzyme mix and nuclease-free water ad final volume of 20 μl. For DNA templates <75 nucleotides, 150 nM of template DNA was used for the in vitro transcription. The reactions were incubated for 3.5 h at 37°C and subsequently treated with 1 μl TURBO DNase for 15 min at 37°C. Non-radioactive RNA transcripts were synthesized by using a similar reaction, but using a mix of 100 mM T7 NTP Mix  instead of unlabeled and [32P]-labeled CTP.RNA probes, non-radioactive as well as radioactive -labeled D1-FL RNA was added. A reaction with only the RNA transcript and no protein served as negative control. The samples were equilibrated for 10 min at room temperature before 2.5 μl RNA loading buffer (5% glycerol (v/v), 0.25% (w/v) bromphenol blue in 1x TBE buffer) was added and then samples were subjected to electrophoresis for 10 min at 240 V followed by 60 min at 100 V. The gel was removed from the glass plates, placed on Whatman filter paper, dried and exposed to a storage phosphor screen. The phosphor screen was scanned using a Storm PhosphorImager. The La–RNA complex (La–RNP) formation was quantified using the ImageQuant TL software and plotted as a function of the La concentration in Prism 5. The dissociation constant was determined using the one-site hyperbolic binding fit in Prism 5.Several recombinant La mutants were tested for their ability to form complexes with the 5′-UTR of CCND1 mRNA. The binding affinity of recombinant La to the 5′-UTR was determined by titrating La against the RNA. A 6% native TBE (Tris-Borate-EDTA) polyacrylamide gel was prepared in a Mini-PROTEAN Tetra Handcast System, and was pre-run for 35 min at 240 V in 1x TBE buffer . The RNA was re-annealed for 10 min at 85°C in 1x RNA-binding buffer  Nonidet P-40) and slowly cooled down to 37°C in a thermomixer. The binding reaction was prepared in a total volume of 20 μl in 1x RNA-binding buffer. Increasing concentrations of recombinant LaWT protein: 40, 80, 160, 320, 640 and 960 nM were placed in a 96-well microliter assay plate and 10 nM of re-annealed [KD) was determined in Prism 5 after plotting the La–RNA complex formation as a function of the La protein concentration.The binding affinity of wild-type (WT) recombinant La protein to the D1-AUG RNA oligoribonucleotide, was determined by native electrophoretic mobility shift assay (EMSA) as described above. Increasing concentrations of recombinant His-tagged La protein of 10, 60, 200, 400, 600, 800 nM, 1 M, 1.6 and 3 μM were used. The La–RNA complex (La–RNP) formation was quantified using the ImageQuant TL software. The dissociation constant , 250 (25×) and 500 nM (50×) in order to challenge the binding. The specificity of the assay was tested by utilizing the cold transcripts of the radiolabeled D1-FL RNA or the unlabeled D1-AUG RNA oligoribonucleotide as competitor RNAs.Incubating the protein of interest with a highly specific antibody leads to a much larger complex when bound to the RNA (super-shift). Three hundred nanogram of recombinant La protein was incubated for 1 h at 4°C with 5 μl of the mouse monoclonal anti-La SW5 antibody in 10 mM Tris/HCl pH 7.4 and 20 units RNasin (Promega) in a total reaction volume of 10 μl. A parallel reaction with 5 μl of the isotype control mouse IgG2α,κ antibody (Dako) was performed. The antibody pre-incubated La protein was subsequently used for EMSA as described.2, 0.5 mM EDTA, 0.5% (v/v) IGEPAL® CA-630), heated for 10 min at 80°C and slowly cooled down to 37°C. 5 mg/ml (w/v) bovine gamma globulin was added to a final amount of 100 μg/ml. The 1.0 μM recombinant La protein stock solutions in FP assay buffer were prepared, and the La protein and its mutants were placed at increasing concentrations (1–300 nM) in black 384-well plates. Ten nanomolar of re-annealed RNA was added to the proteins and FP assay buffer was added to a total volume of 100 μl per reaction. Hundred microliter of the FP assay buffer was used as blank sample. To determine the background polarization, 10 nM free 5′-FAM-labed D1-AUG RNA in FP assay buffer was used as control. The POLARstar Omega plate reader (BMG Labtech) was utilized to measure FP after 5 min incubation at room temperature. The assay was done in quadruplicates for each protein concentration, to reduce the standard deviation. A 5x RNA–protein master mix of 500 μl was prepared and 100 μl was added per well. The background polarization was subtracted from the blank corrected data. La–RNA complex formation was determined as a change in millipolarization (ΔmP) and plotted as a function of the La concentration in Prism 5. The dissociation constant, KD, was determined by the one-site (hyperbola) non-linear fit algorithm in Prism 5.An alternative quantitative method to monitor RNA–protein interactions is based on the alteration in tumbling properties of a fluorescent ligand upon binding to a larger molecule. The RNA of interest, D1-AUG, was synthesized and 5′-end labeled with the fluorophore 6-carboxyfluorescin (6-FAM) by Integrated DNA Technologies, Inc. The lyophilized RNA was resuspended in 1x siRNA (Dharmacon) buffer to a stock concentration of 100 μM. The 5′-FAM-labed D1-AUG RNA stock solution was diluted to 10 μM in fluorescence polarization (FP) assay buffer  and heat up for 10 min at 80°C and subsequently place on ice. Recombinant La protein was added to the wells containing dialysis buffer  to reach a final volume of 20 μl. Subsequently, 20 μl MB-D1-AUG (on ice) was added to three wells containing La or just 20 μl dialysis buffer (Blank). The 96-well plate was placed into the pre-heated (37°C) micro-plate reader and fluorescence light emission was measure at indicated time.All reagents, dilutions, proteins, the 96-well plate was kept on ice. RNA chaperone assays (RCA) were performed in a final volume of 40 μl in a black 96-well plate. The molecular beacon MB-D1-AUG was diluted to 50 nM in RCA buffer . After incubation for 3 h at 30°C reactions were stopped by adding SDS-loading buffer, boiled and resolved on a 12.5% SDS–PAGE. Gels were dried under vacuum and analyzed by autoradiography.Recombinant LaWT or LaT389A (600 ng of each) were incubated with recombinant AKT-1  in a 20 μl reaction mixture containing 20 mM,Tris/HCl, pH7.4, 50 mM KCl, 5 mM MgClg at 4°C, washed three times with 200 μl RCA buffer and eluted according manufacturer's instructions. The volume was adjusted to 40 and 20 μl were analyzed in the RCD assay as described above.To prepare AKT-1 phosphorylated LaWT protein for the RCA, 1.2 μg recombinant La protein was phosphorylates in 20 μl volume over night at 30°C in reaction buffer described above containing 2.0 μg AKT-1. After incubation, 200 μl RCA buffer was added and the reaction was transferred into VWR centrifuge units, spun for 5 min at 14 000 x 2 atmosphere in complete advanced dulbecco's modified eagle's medium containing 2 mM L-glutamine (Life Technology) plus 10% fetal bovine serum (Gibco). For transfection 4 × 105 HEK 293 cells were plated per well of a 6-well plate. The next day cells were transfected with plasmid DNA encoding gfp-tagged LaWT, LaΔRCD, LaT389A using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufactures instructions. Two days later cells were collected and replated into 96-well plates (4 × 104 cells/well). The following morning cells were transfected with 150 ng of capped and polyadenylated bi-cistronic RNA with CCND1 IRES or without an IRES (empty) using the TransiT® mRNA kit (Mirus). Seven hours later cells were harvested and luciferase expression was determined using the Dual-Luciferase Reporter Assay (Promega). Renilla and firefly luciferase activity is blotted as percent, in which renilla and firefly luciferase activity measured for the empty bicistronic RNA was set as 100%.HEK 293 cells were purchased from ATCC (American Type Culture Collection), expanded and aliquots were stored in liquid nitrogen. Cells were cultured at 37°C in a humidified 5% CO32P-UTP-labeled in vitro transcript referred to as D1-FL -mediated mRNA translation . Now we L Figure  increasiin vitro transcripts spanning the 5′-half of the 5′-UTR (part A (nts −209 to −110)) and the 3′-half of the 5′-UTR . Those unlabeled transcripts were used in competitive EMSAs to map the La-binding site . La-WT and La-Δ1  bound D11 to 99 . In cont11 to 99 ) or La-Δ11 to 99 )  to GGVGGG (LaΔRCD1) and FQGKKTKF (aa 357 to 364) to VQGGGTGV  are important for La's ability to support the synthesis of various precursor RNA molecules , suggestin vitro transcribed bicistronic CCND1 IRES reporter RNA, transfected the RNA into HEK 293 cells expressing gfp-tagged LaWT or LaΔRCD  and measured renilla and firefly luciferase activity after 7 h. Interestingly, these experiments demonstrate that LaWT not only stimulates CCND1 IRES-mediated firefly expression, but also cap-dependent renilla luciferase expression , and that the RCD of La contributes to the helix-destabilization of D1-AUG RNA. Now we plan to demonstrate that La mutants with impaired RNA chaperone activity are defective in supporting CCND1 IRES-mediated translation in cell-based assays. To test this notion we generated capped and polyadenylated n Figure . In contin vitro phosphorylation of LaWT and LaT389A by recombinant AKT-1. As shown, recombinant AKT-1 was able to phosphorylate LaWT, but not LaT389A  or a phosphomimetic mutation (LaT389E) affects the helix destabilization activity of La. We purified three independent sets of recombinant La proteins  and measured their helix destabilization activity. We did not observed a significant difference in helix destabilization activity between LaWT, LaT389A or LaT389E , suggesting that we cannot replicate the results we observed by AKT-1 phosphorylation by mutating T389 to glutamic acid.Recently, a genome wide study identified threonine 389 (T389) as AKT phosphorylation site located upstream of the newly identified RCD in La , Figure . To valiA Figure , which dD Figure . Hence, D Figure . The datIt has been shown earlier that AKT activity represses CCND1 IRES-mediated mRNA translation ,67. To tin vitro by AKT-1 and that phosphorylation at T389 impairs the RNA chaperone activity of La protein. Furthermore, we demonstrate that overexpression of LaT389A mutant impairs specifically CCND1 IRES-mediated translation in cells.In summary, we show that La can be phosphorylated Herein we define for the RNA-binding protein, La, a novel RCD. We further demonstrate that the RNA chaperone activity of La is required to facilitate CCND1 IRES-mediated mRNA translation in cells and can be regulated by a nearby AKT-1 phosphorylation site (T389). These findings suggest a novel interplay between RNA-binding, RNA chaperoning and AKT-1 phosphorylation of the La protein and its regulation of CCND1 IRES-mediated mRNA translation.Human La is suggested to facilitate ,25–30,32drosophila and zebrafish, La orthologs display strong similarities in respect to the presence of arginine, lysine and phenylalanine residues in the two regions (RCD1 and RCD2) that we mutated in the RCD . This suggests that these amino acids play an important role in the cellular function of La proteins in higher eukaryotes. It is also important to note, the RCD1 domain (aa 333 to 340) overlaps with the dimerization domain  suggest to 348, , suggestin vivo, we demonstrate that the same residues important for full RCD activity in vitro are required to stimulate CCND1 IRES-mediated mRNA translation in cell-based luciferase reporter assays  is required for CCND1 IRES-mediated translation in cells. We conclude that the La protein is a modular protein requiring the concerted action of various domains to stimulate IRES-mediated mRNA translation, and that its function can be regulated by AKT signaling. We are convinced that this study provides novel information illuminating the function of the La protein during mRNA translation and its aberrant role in tumorigenesis.Summarizing the data shown herein suggest that the function of La in protein synthesis is not only mediated by its RNA-binding, but significantly, by its RNA chaperone activity, which requires the newly defined RCD located in the C-terminal intrinsic disordered region of La. In addition, we found that AKT-1 phosphorylation impairs the helix destabilization activity of La Supplementary Data are available at NAR Online.