Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1–3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein∶RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway. Fully activated DC are able to induce T-cell mediated immunity and have the capacity to activate even naïve CD4Surface expression of CD83 protein serves as a marker for immune-competent mature DC Since membrane-bound CD83 is strongly upregulated during DC maturation and activation, it became of particular interest to investigate the regulation of CD83 expression in detail on both, the transcriptional and the posttranscriptional level cis-active RNA element in the coding region, termed the posttranscriptional regulatory element (PRE), which is directly recognized by the cellular RNA-binding shuttle protein HuR In metazoans, the vast majority of mRNAs are transported from the nuclear site of transcription to the cytoplasm, the site of protein synthesis, via the NXF1/TAP pathway  protein The human HuR protein is a ubiquitously expressed member of a family of RNA-binding proteins that is related to the In the present work we have characterized the HuR:CD83 mRNA interaction by in vitro binding assays using various HuR and PRE RNA mutant constructs. Furthermore, we analyzed the contribution of the CRM1- and the NXF1/TAP-specific nuclear export pathway to the nucleocytoplasmic transport of selected CD83 mRNA mutants (i.e. PRE mutants) in transfected cell cultures.To assess the binding of HuR to CD83 mRNA, RNA gel retardation experiments were performed. For this, HuR was expressed and purified in the context of a fusion to GST (glutathione S-transferase) and then analyzed in combination with in vitro transcribed radiolabelled CD83-specific RNA probes. In agreement with data presented in a previous study Next, we investigated the specificity of this protein∶RNA interaction. Therefore, RNA competition experiments were performed in which increasing amounts of unlabelled and highly structured heterologous RNAs were added to the CD83 PRE:HuR binding reaction. As shown in HuR belongs to a family of ELAV-like proteins that are characterized by a modular structure composed of several functionally distinct domains To investigate which region of HuR is involved in CD83 mRNA binding, recombinant GST and several GST-HuR fusion proteins were prepared  and subjInspection of the PRE revealed, that this CD83 mRNA-derived sequence does not contain a classical ARE The CD83 PRE is a structured RNA element that potentially forms a stem loop with three apical substructures To detect an association of HuR with CD83 mRNA in vivo, we next performed an immuno-PCR experiment. COS7 cells were transiently transfected with expression vectors encoding either for human CD83 cDNA flanked by the entire homologous 5′- and 3′-untranslated region (UTR) or variants thereof, encoding PRE SubL-deletions. At day two posttransfection, endogenous HuR protein was covalently crosslinked to the CD83 mRNA by addition of formaldehyde to the respective cell culture. Afterwards, HuR was immunoprecipitated using a polyclonal HuR antiserum, bound CD83 mRNA was reverse transcribed and the presence of coimmunoprecipitated CD83 mRNA was determined by specific PCR. By using this experimental set-up, it has been already previously shown that no specific binding could be observed when the CD83 message lacked all three PRE SubL elements It has been previously shown that CD83 expression and the nuclear export of CD83 mRNA is sensitive to leptomycin B (LMB) Since the PRE-deficient CD83 transcript was apparently exported from the nucleus in a CRM1-independent fashion , we now Obviously, the PRE in CD83 mRNA enables nucleocytoplasmic translocation of this mRNA via the CRM1-pathway, whereas deletion of the PRE makes this RNA subject to regulation by the NXF1/TAP export receptor and its associated factors.The CD83 protein serves as surface marker for fully matured DC cis-active CD83 PRE mRNA sequence Lately it was shown that the expression of CD83 is regulated at the level of nucleocytoplasmic transport The RNA-binding HuR protein has been shown to efficiently shuttle between the nuclear and cytoplasmic compartment It has been demonstrated that ARE recognition by HuR is mediated by the protein's two tandemly arranged RRMs (RRM1 and RRM2) cis-active PRE secures nucleocytoplasmic CD83 mRNA translocation via CRM1. One may therefore speculate that this specific export route might provide a significant advantage with respect to efficient protein synthesis when compared to the more general pathway involving NXF1/TAP, particularly under conditions of cellular activation. In fact, this notion is increasingly supported by studies identifying specific cellular transcripts that are translocated from the nucleus to the cytoplasm by CRM1. These include the transcript coding for interferon-α1, c-Fos, Cyclin D1, cyclooxygenase-2, HLA-A and, most notably, HuR itself cis-active element has been recently identified in the coding region of interferon-α1 mRNA that mediates, although in an HuR-independent manner, the aforementioned CRM1-dependent nuclear export of this message The RNA binding and immuno-PCR experiments performed in the present study demonstrated that all PRE substructures are required for optimal HuR binding . FurtherGAT TTT TCT→GAC TTC TCA), p3CD83PREΔURE2  and p3CD83PREΔURE1+2 , were constructed using PCR technology. The respective CD83 CDS-derived fragments were ligated between the HindIII and EcoRI sites of the pcDNA3 vector.The plasmids p3CD83-CDS , p3UTR-CD83ΔSubL2 (CD83 CDS nt 543–555 deletion), p3UTR-CD83ΔSubL3 (CD83 CDS nt 561–594 deletion), p3UTR-CD83ΔSubL1–3 (CD83 CDS nt 498–594 deletion), were constructed by ligating the respective PCR-generated CD83 CDS-derived fragments between the HindIII and Asp718 sites of the p3UTR-CD83 vector The expression plasmids for purification of the GST-proteins, pGex-HuR1–244 , pGex-HuR27–93 , pGex-HuR108–174 , pGex-HuR246–317 , pGex-HuR103–244 , pGex-HuR190–328  and pGex-HuR175–245  are based on the parental vector pGex-5×-1 (Pharmacia Biotech).E. coli BL21 and purified from crude lysates by affinity chromatography on glutathione-Sepharose 4B (Pharmacia) as described previously 32P]-labelled CD83-derived probes, MS2 competitor RNA and GST fusion protein as previously described GST fusion proteins were expressed in 32P] UTP (Amersham Pharmacia) using a commercial T7 RNA polymerase-based kit following the manufacturer's instructions (Promega).Labelled and non-labelled RNA was obtained by in vitro transcription with or without [α-6) were transfected with 8 µg DNA of either p3UTR-CD83, p3UTR-CD83ΔSubL1, p3UTR-CD83ΔSubL2 or p3UTR-CD83ΔSubL3 expression vector, respectively, using the DEAE dextran transfection method. Two days after transfection endogenous HuR protein was covalently cross-linked to CD83 mRNA by adding 1% formaldehyde to the medium for 5 minutes. Afterwards, crude extracts were prepared and HuR was immunoprecipitated with a polyclonal HuR antiserum, bound CD83 mRNA was reverse transcribed and the presence of coimmunoprecipitated CD83 mRNA sequences was determined by specific PCR using DNA-oligos described below. Rabbit IgG (instead of HuR antibodies) or water was used for PCR control.COS7 cells  was maintained as previously described 5 cells were lysed on ice for 1 min using 100 µl NP40 buffer . Subsequently, the lysates were cleared by centrifugation at 470× g for 5 min at 4°C. Cytoplasmic RNA was isolated from 80 µl of the supernatant using TRIzol reagent. The nuclei were washed again in 100 µl NP40 buffer to deplete residual cytoplasmic RNA. Afterwards, the nuclear RNA was isolated by using TRIzol reagent. DNAse treatment of the RNA samples was performed. Selected RNA samples were reversely transcribed using the first strand cDNA (AMV) synthesis kit for RT-PCR  according to the manufacturer's instructions. Subsequently, RT products were assayed by PCR. For detection of GAPDH sequences following primers were used: forward, 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′; reverse, 5′-CATGTGGGCCATGAGGTCCACCAC-3′. The amplification profile involved 25 cycles of denaturation at 95°C for 1 minute, primer annealing at 56°C for 1 minute, and primer extension at 72°C for 2 minutes. CD83 mRNAs were detected by using following primer pairs: forward, 5′ GGT GAA GGT GGC TTG CTC CGA AG-3′, and reverse, 5′-GAG CCA GCA GCA GGA CAA TCT CC-3′. The amplification profile involved 25 cycles of denaturation at 95°C for 1 minute, primer annealing at 56°C for 1 minute, and primer extension at 72°C for 1 minute. Cytocrome C oxidase subunit II mRNAs were detected by using following primer pairs: forward, 5′ GCA AGT AGG TCT ACA AGA CG-3′, and reverse, 5′-GTA GTC GGT GTA CTC GTA GG-3′. The amplification profile involved 25 cycles of denaturation at 95°C for 1 minute, primer annealing at 56°C for 1 minute, and primer extension at 72°C for 1 minute. Real time PCR was performed as described previously Total cellular RNAs were isolated according to the manufacturer's protocol by using TRIzol reagent (Invitrogen). For isolation of cytoplasmic and nuclear RNAs, 2×10


Coral taxa colonising the artificial reef were a subset of those on the surrounding natural reef, but occurred in different proportions—Pseudosiderastreatayami, Mycediumelephantotus and Leptastreapurpurea being disproportionately abundant on the artificial reef. Coral cover increased rapidly in the later stages of the study, reaching 2.3 ± 0.7 SE % at 62 months post construction. This study indicates that simple materials of opportunity can provide a suitable substrate for coral recruitment in Dampier Harbour, and that natural colonisation at the study site remains sufficient to initiate a coral community on artificial substrate despite ongoing natural and anthropogenic perturbations.A 0.6 hectare artificial reef of local rock and recycled concrete sleepers was constructed in December 2006 at Parker Point in the industrial port of Dampier, western Australia, with the aim of providing an environmental offset for a nearshore coral community lost to land reclamation. Corals successfully colonised the artificial reef, despite the relatively harsh environmental conditions at the site . Coral settlement to the artificial reef was examined by terracotta tile deployments, and later stages of coral community development were examined by in-situ visual surveys within fixed 25 x 25 cm quadrats on the rock and concrete substrates. Mean coral density on the tiles varied from 113 ± 17 SE to 909 ± 85 SE per m Pilbara coast of northwestern Australia is located between 22° S 114° E and 20° S 119° E, in a semi-arid environment with a range of coastal habitats including rocky shores, sandy shores, mud flats and mangroves [Pilbara coast in the inner Mermaid Sound, a rock-dominated area with a complex coastline and numerous islands , 4 m on lowest astronomical tide (LAT) and 9 m on highest astronomical tide (HAT).The artificial reef was surveyed at six month intervals between August 2007 and February 2012 to determine the level of coral settlement to tiles on the artificial reef and nearby natural reef, and the recruitment and growth of corals on the artificial reef. The intent of the monitoring was to test whether a coral community would develop on the reef to a targeted minimum cover of 10% within 10 years, and to describe coral recruitment and growth processes to help predict coral recovery rates within the broader area of the inner Dampier Harbour.The work described in this article was undertaken in accordance with Permit SD2005/0028 issued on the 22nd of March 2006 by the Australian Government Department of the Environment and Heritage.Water temperature and turbidity were logged at a natural reef site  100 m southeast of the artificial reef during various environmental monitoring programs unrelated to the artificial reef project. The loggers were placed among coral colonies at a depth of 4.5 m MSL. Water temperature was recorded hourly between February 2008 and February 2010 with a TidBiT V2 water temperature data logger, and turbidity was recorded (in nephelometric turbidity units - NTU) at 30 minute intervals between November 2007 and August 2010 with either an SAS  or Wetlaterracotta tiles (11 x 11 x 1 cm) at six subsites across the artificial reef  with stainless steel screws and 10 mm PVC spacers. Besser blocks were used as the attachment medium because the rock substrate of the artificial reef was too hard to drill for direct tile attachment. The three blocks at each subsite were closely spaced and oriented horizontally.Acroporidae, Pocilloporidae, Poritidae and ‘other’, based on reference images in Babcock et al. [ln(x+1). A two-way analysis of variance was undertaken on the artificial reef data, testing the effect of survey and aspect (seaward v. landward) on coral density.On retrieval, tiles were bleached in a 10% NaOH solution for 24 hours, rinsed in fresh water, dried and examined under a dissecting microscope . Corals k et al. . RecruitCoral recruitment was sampled by in-situ visual inspection within fixed 25 x 25 cm quadrats. Before the first survey in August 2007, small concrete lugs were cemented to the substrate as a means of positioning the quadrats consistently in each survey. 162 quadrats were distributed equally amongst the nine subsites shown in ln(x+1) transformation.Two three-way analyses of variance were undertaken on the quadrat data. The first examined the effects of substrate type, substrate orientation and location  on recruit density on the landward side of the reef and the second examined the effect of aspect, substrate orientation and location on recruit density on the rock substrate. It was not possible to combine all four factors in a single analysis because the concrete substrate was restricted to the landward side of the reef . AnalysePatches of coral reef occur to the north, east, and southeast of the artificial reef. Coral cover and taxonomic composition were surveyed in the southeast coral patch, adjacent to the water quality instruments, during environmental monitoring programs unrelated to the artificial reef project. The site is 100m from the artificial reef  at a depEstimates of coral cover and composition at the natural reef site were derived from point count analysis of digital still images along five fixed 10 m x 1 m transects, at 30 images per transect and 30 points per image. Hard coral colonies were identified to genus and counted from the images. Care was taken to ensure corals spanning more than one image were only counted once.The natural reef site underwent marked seasonal temperature variation, with seasonal minima of approximately 19°C in July and maxima of approximately 32°C in February , consistTurbidity at the natural reef site was highly variable. Although the average daily median turbidity over the 31 months of records was only 2.4 NTU, many high turbidity events were recorded, with daily median turbidity often above 10 NTU and occasionally exceeding 50 NTU .Lobophoravariegata) and coralline algae. A ubiquitous layer of fine sediment, bound by turf algae, covered the horizontal surfaces of the artificial reef to a depth of one to six mm. Despite this sediment cover, corals were present on the reef within the first few months after construction, although they remained inconspicuous until the second year. The artificial reef was also colonised by a diverse fish community, initially composed primarily of migrants from surrounding reefs but later including juveniles recruiting directly to the reef [The rock and concrete substrates of the artificial reef proved stable over time, remaining in position despite several cyclones affecting the area over the 2006-2012 study period. Turf algae and barnacles were conspicuous early colonisers, followed by encrusting sponges, colonial and solitary ascidians, macroalgae (primarily 2), with a pulse to 909 per m2 in autumn 2011 followed by a reduction to 164 per m2 in spring 2011. The density of settled corals showed high interannual variability, with greater settlement to tiles on the landward side of the reef than the seaward side, although the magnitude of the difference varied between years , due primarily to aggregations of the most abundant species Pseudosiderastreatayami.Coral abundance in the quadrats was initially low and increased slowly; mean density (± SE) in the 162 quadrats on the artificial reef was 6.0 ± 1.0 per mtruction . In subsPseudosiderastreatayami was the most abundant coral species identified on the artificial reef, comprising 30% of the 243 colonies recorded in the final survey. Other common species included Turbinariamesenterina (14%), Mycediumelephantotus (6.6%) and Leptastreapurpurea (6.2%). Several faviid genera were present in proportions of less than 5% each, including Cyphastrea, Goniastrea, Favia and Favites. Juvenile (attached stage) fungiids constituted 4% of the total. Additional genera recorded in lower numbers  included Acropora, Acanthastrea, Psammocora, and Heteropsammia. Several additional genera were recorded as only one or two individuals.Leptastreapurpurea, with radial extension rates averaging 10-15 mm per year. The largest colony recorded in the final survey was a Lpurpurea.  colony of 152 mm diameter. A few larger colonies were found outside the quadrats, including Leptastrea, Cyphastrea and Porites colonies of more than 200 mm diameter. The growth pattern of these genera was typically flat and spreading; even the largest colonies appeared to be less than 10 mm thick  than horizontal surfaces (1.8 ± 0.8 SE %), despite the lower mean coral density on vertical surfaces.Mean coral cover on the artificial reef rose slowly in the first couple of years but increased rapidly in the latter half of the study, reaching an estimated 2.3 ± 0.7 SE % in the final survey . LeptaFaviidae (57%), Dendrophyllidae, Poritidae and Mussidae. Favites was the most abundant coral genus, comprising 24.3% of the 531 colonies recorded in the final survey (Turbinaria (16%), Favia (10%), Platygyra (9.6%) and Goniastrea (9%). In contrast to their abundance on the artificial reef, Pseudosiderastrea, Leptastrea and Mycedium accounted for only 1.9%, 1.7% and 0.6%, respectively, of recorded colonies on the natural reef transects.The coral community at the natural reef site occurs as a discontinuous veneer over the subtidal hard substrate, with mean live cover of approximately 30-35% between 2006 and 2010, as assessed from belt transects. Common coral families at the site, in order of numerical abundance, were l survey . Other cThe water temperature range at the natural reef site was greater than that reported for almost all reefs elsewhere except those of the Arabian Gulf –23. The The turbidity regime was characterised by moderate background turbidity levels punctuated by intermittent events that increased turbidity to high levels , often for several days. We are uncertain of the extent to which this turbidity regime, and the associated sedimentation, may have affected the health and resilience of corals at our study site. Certainly, a layer of sediment was present on abiotic surfaces during each visit to the site.Erftemeijer et al.  emphasis-2 over a spawning period, which falls within the typical range of 100-1000 recruits m-2 recorded in other studies using terracotta tiles on Indo-Pacific reefs [Coral settlement was recorded in all five deployments, including the single summer deployment in 2011/2012. Settlement rates to tiles deployed on the artificial reef and the natural reef were generally within the range 100-400 per mic reefs –31, althic reefs . TherefoPseudosiderastrea, Faviidae, Turbinaria and Mycedium amongst juvenile corals. Very few of the settling Acroporidae or Pocilloporidae appear to survive to a size which might represent a reproductive coral, either on the artificial reef or the natural reef. This contrasts with some nearshore Queensland reefs with apparently similar environmental settings to our study site, where adult Acropora colonies are abundant [Acroporidae and Pocilloporidae appear better suited to clear water environments further from the mainland, where they often comprise a significant component of the adult community [Although the coarse taxonomy of the tile data limits comparisons with the quadrat data, the abundance of the ‘other’ category on the tiles appears to carry through to the quadrats as a predominance of abundant . In Dampommunity .Coral density in the quadrats was far lower than on the tiles, as expected given the limitations of in-situ counts versus microscope counts, the likely high mortality of newly settled corals –37 and tStatistically significant coral distribution patterns in the quadrat data include differences in coral density between substrates (concrete > rock), aspects (seaward > landward), locations  and surface orientations . As the mechanisms behind these relationships were not investigated it is not possible to link cause and effect from our data. Some speculative influences on the observed distributions are outlined below.The greater density of coral recruits on concrete than rock was a relatively late development, and is not a particularly strong trend, given the variability observed between surveys . If the The effects of aspect and location on coral density —i.e. greater density on the seaward side of the reef and on the central and eastern locations—appear to be due to differential post-settlement survival, because the tile data displayed a greater density to landward than seaward, and no significant effect of location. The greater coral density on horizontal surfaces than vertical surfaces was a consistent pattern throughout the study. We interpret this relationship to result primarily from the pre-emption of space on vertical substrates by encrusting sponges and colonial invertebrates. Other explanations such as restricted light availability on vertical surfaces are feasible but we believe less likely, because coral colonies on the vertical surfaces are, on average, larger than those on the horizontal surfaces, indicating that the vertical surfaces are not inherently poor coral habitat. It seems the few colonies that manage to settle and survive on the vertical surfaces are able to thrive there, perhaps due to the relatively low sedimentation rate. The higher sedimentation rate on the horizontal surfaces is probably detrimental to corals, but may be even more detrimental to filter feeding invertebrates. In this scenario corals on the horizontal surfaces may actually benefit from sedimentation because they appear to withstand it better than their invertebrate competitors .Pseudosiderastreatayami, Mycediumelephantotus and Leptastreapurpurea, and the scarcity of Porites, non-Leptastrea faviids and mussids on the artificial reef relative to the natural reef. We interpret these differences primarily as demographic traits; Ptayami. , Melephantotus.  and Lpurpurea.  appear to be early colonisers whereas Porites, non-Leptastrea faviids and mussids recruit in lower numbers but may survive and grow to become abundant over the longer term.The current coral community composition on the artificial reef differs markedly from that of the nearby natural reef. The most obvious differences are the abundance of It remains to be seen whether coral community composition and diversity on the artificial reef will eventually approach that of the natural reef. In most reported cases artificial reef communities do not closely resemble the adjacent natural communities, even after several decades ,44. HoweLeptastreapurpurea was by far the greatest contributor to coral cover because it was both abundant and fast-growing. In contrast, the slow growth rate and small size of Ptayami.  preclude it from contributing significantly to coral cover on the artificial reef, despite its numerical dominance.Coral cover on the artificial reef increased rapidly in the last two years of the study . This isDrupellacornus and bleaching-related mortality were recorded [The current rate of increase of coral cover on the artificial reef must eventually slow. Physical mechanisms that may slow or reverse the trend include disturbances such as cyclones, high or low water temperature excursions and high turbidity/sedimentation events . Biological mechanisms include disease, predation and competition. Observations on the natural reef suggest that multiple mechanisms operate to constrain coral cover. Cyclone damage was evidenced by displacement or removal of entire colonies, but this affected only a minor proportion of colonies. Partial colony mortality was much more common, occurring across all coral taxa. In some cases it was clearly due to competitive interaction where coral colonies grew into close contact, but it was also common among colonies that were not in close contact. The cause(s) of mortality in these colonies was rarely determined, but a few instances of predation by recorded . We presThe steady increase in coral density and cover on the Parker Point artificial reef to date is an encouraging sign that natural regenerative processes in Dampier Harbour remain adequate to restore lost coral habitat, where substrate and water quality permit, and to generate new coral habitat if that option is appropriate. We do not advocate artificial reef construction as a general solution to anthropogenic coral loss; restoring damaged reefs to their natural state is far preferable and should be attempted wherever possible . However


Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine, which is then recognized as guanosine. To study the role of ADAR proteins in RNA editing and gene regulation, we sequenced and compared the DNA and RNA of human B cells. Then, we followed up the findings experimentally with siRNA knockdown and RNA and protein immunoprecipitations. The results uncovered over 60,000 A-to-G editing sites and several thousand genes whose expression levels are influenced by ADARs. Of these ADAR targets, 90% were identified. Our results also reveal that ADAR regulates transcript stability and gene expression through interaction with HuR (ELAVL1). These findings extend the role of ADAR and show that it cooperates with other RNA-processing proteins to regulate the sequence and expression of transcripts in human cells. APOB and NF1 genes.Molecular studies and, more recently, genome and transcriptome sequencing have uncovered the complexity of RNA processing. From the same DNA templates, events such as RNA editing generate different forms of transcripts. In this study, we focused on ADAR-mediated RNA editing and its interactions with other RNA processing steps to regulate gene expression. In human cells, two classes of proteins are known to be involved in RNA editing: the ADAR and APOBEC families. ADARs, which are expressed in a wide variety of cell types, deaminate adenosine to inosine, which is then recognized by the translation and splicing machineries as guanosine . APOBEC1Recent work has uncovered many more RNA-editing events mediated by ADAR proteins. These findings led to new questions. Most of the A-to-G editing sites were identified by computational analysis of sequence data without experimental validation. Some of the findings were based on a comparison of RNA sequences with reference DNA sequences that were not derived from the same cells. In addition, it has been suggested that ADAR plays a role in other biological processes in an editing-independent manner , but theWe compared DNA and RNA sequences in human B cells from two individuals to identify RNA-DNA sequence differences (RDDs). We validated the findings by RNAi and RNA immunoprecipitation (RNA-IP). The results uncovered ~10,000 known and ~50,000 unknown ADAR-mediated A-to-G editing sites in premature and mature mRNAs and long noncoding RNAs (lncRNAs). We also found that ADAR proteins have an editing-independent effect on gene expression. Our results showed that ADAR1 interacts with HuR (ELAVL1) to regulate transcript stability. Together, these results provided us with a deeper understanding of ADAR proteins in RNA editing and gene regulation.We sequenced the DNA and mRNA from cultured B cells of two individuals using Illumina-based next-generation sequencing (NGS) . We condADAR1 is >20 times higher than that of ADAR2 and ADAR3 (reads per kilobase of transcript per million mapped reads [RPKM] of ADAR1 = 7 compared with RPKM of ADAR2 and ADAR3 < 0.3), suggesting that ADAR1 is the predominant form of ADARs in human B cells. Following gene knockdown with four independent siRNAs and a pool comprising the four siRNAs, ADAR1 was reduced by >50% at mRNA and protein levels  sites decreased by 20% or more in samples from both individuals, whereas only 43 sites of the other 11 types of RDDs decreased by the same extent  and the von Hippel-Lindau (VHL) tumor suppressor, which have been implicated in various cancers. In contrast, the levels of the other types of RDDs did not change or decreased very modestly. This suggests that ADAR1 mediates the majority of A-to-G editing in B cells and does not contribute to the other types of RDDs. In addition, these results show that the FDR of A-to-G editing is no more than 4%.ADAR and RNAi pathways work cooperatively , so the e extent . A smallADAR1 and the editing levels of its many targets remained low throughout the time course  and antiviral defense (p < 10−3).The above data were obtained at one time point. In order to study the kinetics of A-to-G editing, we carried out RNA-seq on the cells at several time points after siRNA transfection. The expression level of e course . For insanalysis , 2009b sADAR2 (ADARB1) followed by nucleic acid sequencing. The ADAR2 mRNA level was reduced by 25%  are mainly targeted by ADAR1; therefore, following ADAR2 knockdown, a compensatory increase in ADAR1 binding or activity would lead to higher editing levels, which would be abolished by the simultaneous silencing of ADAR1 and ADAR2. This hypothesis was confirmed by a decrease in EIF2AK2 editing following double knockdown of ADAR1 and ADAR2  and the base 3′ to A-to-G editing sites is enriched for G .ADAR1 knockdown, 635 genes showed significant changes in gene expression in two individuals , DNA damage response proteins (p < 10−10), and zinc-finger proteins  were editing targets of ADAR1. ADAR1 regulated the expression of zinc-finger proteins regardless of whether they were editing targets or not . Following et genes . For exas or not . For insproteins .ADAR1 knockdown, the expression levels of 67 genes increased and those of 39 genes decreased. Changes in editing levels and gene expression following ADAR1 knockdown were not significantly correlated (r < 0.05). For example, IKZF3, a transcription factor that regulates proliferation and differentiation of B lymphocytes, has 68 A-to-G editing sites. Its expression level increased by 1.3-fold, whereas its editing level decreased by >7-fold following ADAR1 knockdown. In contrast, both the editing and expression levels of CENPN (43 A-to-G editing sites) decreased following ADAR1 knockdown. The positions of edited sites within genes  and the number of edited sites per transcript also did not correlate with changes in expression following ADAR1 knockdown. These results further suggest that ADAR1 can affect gene expression independently of its deamination activity.Another way to investigate the relationship between RNA editing and gene-expression regulation is to study the 106 genes that are both edited and regulated by ADAR1 at the mRNA expression level . Among tADAR2 knockdown, the expression levels of 4,154 transcripts  increased by 2-fold, and those of 872 transcripts (in 734 genes) decreased by 2-fold  and regulates transcript stability and gene expression . We carr2, p < 0.0001) than in control transcripts . Similarly, other HuR-binding sequences (including (U/A) UUUA, (U/C)UUUA, and AUUU(U/C); 2, p < 0.0001), showing that HuR binds to ADAR1 targets in vivo. These common binding targets of HuR and ADAR1 include MCM4, which plays a key role in DNA replication; TMPO, which encodes a nuclear membrane protein; and GSR, which encodes the enzyme glutathione reductase in the antioxidative stress pathway.To examine how ADAR and HuR interact with their RNA targets, we carried out additional analyses. First, we studied the HuR-binding sites in ADAR-bound transcripts. As mentioned above, we found that sequences of transcripts bound by ADARs were enriched for AU-rich elements (AREs), which are HuR-binding sites. Among the 4,279 ADAR-bound transcripts, 4,198 (98%) had at least one and often many HuR-binding sites. There were 172,000 TA(T/A)TTTT sites in ADAR-bound transcripts, significantly more  that we previously identified as shared targets of ADAR and HuR. After incubating these transcripts with B cell lysates, we pulled down the transcripts using their biotin tags and immunoblotted for ADAR and HuR proteins. Our results showed that ADAR and HuR are specifically pulled down on MCM4 and GSR transcripts, confirming concurrent ADAR and HuR binding . For example, the expression levels of MCM4, TMPO, GSR, and CTH transcripts were reduced in both individuals following ADAR1 knockdown, consistent with binding of their transcripts by both HuR and ADAR1 . Correlation plots for MCM4 and TMPO with ADAR1 and HuR are shown in After identifying that ADAR is required for HuR binding to transcripts, we examined the effects of ADAR and HuR on transcript levels. Since HuR regulates gene expression by stabilizing mRNAs , we examnd ADAR1 . These r our lab , we compOur findings suggest that ADAR1 and HuR proteins cooperate to regulate RNA processing through editing and mRNA turnover. These proteins coregulate transcripts by binding to specific sequences and secondary structures that mediate these processing steps.In this study, we uncovered ~60,000 A-to-G RNA editing sites mediated by ADAR1 and ADAR2 proteins in human B cells. We show that ADAR proteins are involved in gene regulation, particularly in regulating RNA stability and processing.ADAR gene knockdowns and ADAR RNA-IP. The same analysis method was used to analyze sequence reads from all samples, and thus the sites in which editing is responsive to gene knockdown, or that are bound specifically to ADAR proteins, cannot be artifacts of computational analyses. In a recent study on RNA editing in Drosophila  and at least ten sequence reads at each site. However, computational analysis alone may not be adequate. To determine a list of high-confidence ADAR targets, we coupled deep sequencing with osophila , RNA-seqThe large number of sites in which RNA sequences differed from the underlying DNA sequences is surprising and requires further attention in genetic studies. Results from this and other studies show that there are likely many thousands of A-to-G editing sites in each individual. Previously, we showed that there are individual differences in the number of RDDs . Here, iRNA transcripts are tethered to regulatory factors, and the combinatorial binding of RBPs to transcripts coordinates different steps of RNA processing . We founIn summary, in this work we studied ADAR-mediated RNA editing and gene-expression regulation. Our findings uncover editing targets, reveal ADARs’ role in mediating RNA editing and regulation of gene expression, and show that the ADAR protein complex coordinates multiple steps in RNA processing. However, they also raise new questions. Our findings suggest that other mechanisms, such as those that mediate non-A-to-G type RDDs, remain to be identified. In addition, the RNA sequence and structural signatures of the regulatory codes for co- and posttranscriptional processing are largely unknown. Elucidating ADAR’s functions will further our understanding of RNA processing and provide insights into human diseases.B cell lines from two individuals in the Centre d’Étude du Polymorphisme Humain database were cultured and genomic DNA and RNA were extracted. DNA-seq and RNA-seq libraries were prepared and sequenced on a HiSeq 2000 instrument (Illumina). DNA-seq and RNA-seq data were aligned to the reference genome (HG18) using CASAVA and GSNAP, respectively. To identify RDDs, we compared each RNA sequence with its corresponding DNA sequence. We required an editing site or RDD site to be covered by a minimum of 10 total DNA-seq and RNA-seq reads, 100% concordance in the DNA sequence, an RDD level ≥ 10%, and an RDD event to be found in both individuals. Potential sites were then filtered using stringent thresholds.ADAR1 and ADAR2. Sequences surrounding RDD sites were PCR amplified using genomic DNA or cDNA as the template, and PCR products were sequenced. The 3′ UTR of ATM was amplified from cDNA of B cells and cloned into TOPO vector (Invitrogen).Cultured B cells were transfected with Accell siRNAs (Thermo Scientific) against For droplet digital PCR, DNA probes specific to the DNA and RNA variants at RDD sites were synthesized and labeled by VIC and FAM, respectively (ABI Biosystems). Emulsion PCR was carried out and quantified on a QuantaLIfe Droplet Reader (Bio-Rad Laboratories).Anti-ADAR1 and anti-HuR RNA-IP was carried out with a Magna RNA-Binding Protein Immunoprecipitation Kit (Millipore). Quantitative PCR and RNA-seq of immunoprecipitated transcripts were carried out. RNA-editing sites that were detected in transcripts pulled down by ADAR1 antibody, but not by negative-control IgG, were identified as ADAR1-specific targets.Transcripts of HuR and ADAR1 targets were synthesized and biotin labeled in vitro, and incubated with whole-cell lysates. RNA-protein complexes were pulled down and analyzed by western blot (Pierce).2, 0.2% SDS. To examine RNA-dependent interactions, whole-cell lysates were diluted to 1 μg/μl, RNase A or RNase V1 was added, and lysates were incubated at room temperature for 15 min. Protein samples were analyzed by western blot.B cell lysates were incubated with anti-ADAR1 or negative-control rabbit IgG at 4°C overnight. The immunocomplex was pulled down using Protein A agarose (Roche), washed, and finally eluted in 20 mM Tris/7.5, 150 mM NaCl, 2.5 mM MgClA detailed description of the materials and methods used in this work is provided in the 12345678


In order to provide gene expression profiles of different cell types, the primary step is to isolate the specific cells of interest via laser capture microdissection (LCM), followed by extraction of good quality total RNA sufficient for quantitative real-time polymerase chain reaction (qPCR) analysis. This LCM-qPCR strategy has allowed numerous gene expression studies on specific cell populations, providing valuable insights into specific cellular changes in diseases. However, such strategy imposed challenges as cells of interests are often available in limited quantities and quality of RNA may be compromised during long periods of time spent on collection of cells and extraction of total RNA; therefore, it is crucial that protocols for sample preparation should be optimised according to different cell populations.We made several modifications to existing protocols to improve the total RNA yield and integrity for downstream qPCR analyses. A modified condensed hematoxylin and eosin (H&E) staining protocol was developed for the identification of rat renal proximal tubular cells (PTCs). It was then determined that a minimal of eight thousands renal PTCs were required to meet the minimal total RNA yield required for downstream qPCR. RNA integrity was assessed using at every progressive step of sample preparation. Therefore, we decided that the shortened H&E staining, together with microdissection should be performed consecutively within twenty minutes for good quality for gene expression analysis. These modified protocols were later applied on six individual rat samples. A panel of twenty rat renal drug transporters and five housekeeping genes showed Ct values below thirty-five, confirming the expression levels of these drug transporters can be detected.We had successfully optimized the protocols to achieve sufficient good quality total RNA from microdissected rat renal PTCs for gene expression profiling via qPCR. This protocol may be suitable for researchers who are interested in employing similar applications for gene expression studies. Gene expression studies of isolated tissues are often confounded by the reality that such tissues are made up of highly heterogeneous cell types. The relative difficulty in obtaining a pure population of cells has often imposed a huge challenge for researchers as manual isolation of cells is often laborious, requires long working hours and complicated procedures . FurtherLCM often requires several upstream procedures such as tissue collection, cryosectioning and staining prior to microdissection. The majority of these preparations are performed at room temperature, leading to possible deleterious effects on the RNA quality by the presence of RNases. Furthermore, microdissected cells often come in small numbers which may not be sufficient for commercialised qPCR kits. Such difficulties may lead to biased expression profiling and missing information, especially for rare transcripts.While detailed reports had been described for specific cell population expression profiling studies, data about RNA quality assurance specifically for cells harvested through LCM are seldom available in the literature -12. It hIn this study, we looked into the collection of rat renal proximal tubular cells (PTCs) using LCM, and also evaluated several aspects during sample preparation to ensure total RNA isolated is of good quality and quantity for downstream qPCR analysis. The optimized protocols were then applied onto a group of six rats for the gene expression profiling of twenty drug transporters and five housekeeping genes.All procedures were performed in an RNase-free environment. RNase decontamination wipes  were used as a cleaning agent for removing RNase for pipettors and countertops, nuclease-free 70% ethanol, and pipette tips with filters were used.Rats were euthanized via carbon dioxide asphyxiation. Both kidneys were removed immediately after death and middle cross-section was sectioned using a sterile blade. Sectioned kidneys were wrapped in aluminium foil and frozen in liquid nitrogen immediately. Frozen kidneys were then transferred to Leica Cryostat CM3050S (Leica Biosystems) at −16°C. Kidneys were then embedded onto block holder using Tissue-Tek® OCT™ Compound . The embedded kidneys were sectioned thrice with 10 μm in thickness. The kidney sections were then gently transferred onto PEN membrane glass slides. The slides containing kidney sections were allowed to dry in cryostat for 2 minutes before histology (H&E) staining.All procedures were performed on wet ice to minimize RNase activity. Table XT™ LCM instrument  modular stage. Illumination contrast and desired magnification objective were adjusted for optimal visualisation. Using simple drawing tools supplied, renal PTCs or desired cells were outlined on monitor screen for microdissection. After dissection, renal PTCs or desired cells that were captured and transferred onto CapSure® HS LCM Caps  were inspected under microscope for reconfirmation.H&E stained slides were placed on ArcturusTotal RNA was isolated immediately after microdissected renal PTCs. PicoPure® RNA Isolation Kit (Applied Biosystems®) was used for the total RNA extraction according to manufacturer’s protocol with several modifications.Briefly, microdissected PTCs on caps and ExtracSure Extraction Device was assembled. This assembly was placed in a HS Alignment Tray and 30 μL of extraction buffer was gently pipetted into the well and the alignment tray was covered with a pre-warmed incubation block. The assembly was then incubated for 30 minutes at 42°C. Microcentrifuge tube with CapSure-ExtracSure assembly was centrifuged at 1000 g for 2 minutes for collection of cell lysate to the bottom of centrifuge tube. The RNA purification column was pre-conditioned by adding 250 μL of conditioning buffer before incubating at ambient temperature 5 minutes. The RNA purification column was then centrifuged at 16, 000 g for 1 minute for removal of conditioning buffer. 30 μL of 70% ethanol was then added to the cell extract and mixture was mixed well by pipetting up and down gently. The cell extract-70% ethanol mixture was transferred to RNA purification column and centrifuged at 100 g for 2 minutes for RNA binding, followed by a quick spin at 16, 000 g for 30 seconds to remove flowthrough. This step was repeated twice to ensure maximal RNA binding to the column membrane. 100 μL washing buffer 1 was pipetted to the same RNA Purification Column and centrifuged for 1 minute at 8, 000 g. The bound RNA was later treated with DNAse I . 10 μL of DNAse I stock solution was added to 30 μL RDD Buffer and mixed well via gently pipetting. This diluted DNAse I-RDD mixture was then added to RNA Purification Column containing bound total RNA and allowed to stand in ambient temperature for 30 minutes. 40 μL Wash Buffer 1 was added to the column and centrifuged at 8, 000 g for 15 seconds to stop the treatment. The DNAse I treated RNA was washed twice with 100 μL of Wash Buffer 2. The purification column was transferred to a new 0.5 mL microcentrifuge tube provided and 11 μL elution buffer was added. The tube assembly was left to incubate at room temperature for 1 minute and centrifuged at 1, 000 g for 1 minute to distribute elution buffer in the column, followed by spinning at 16, 000 g for 1 minute to RNA elution. The eluted RNA was then transferred back to the same purification column and elution was repeated twice to maximize total RNA yield.RIN was measured using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit. All procedures were performed according to manufacturer’s protocol.Total RNA concentrations and purity ratios (260/280 and 260/230) were measured using NanoDrop 2000 UV–vis Spectrophotometer (Thermo Scientific) unless otherwise stated.Total RNA was then converted to complementary DNA (cDNA) using RT2 PreAMP cDNA Synthesis Kit  according to manufacturer’s protocol. Freshly synthesized cDNA then underwent one round of amplification (RT2 PreAMP PCR Mastermix and RT2 PreAMP Pathway Primer Mix – CAPR09572) before qPCR. Using customized PCR array plates from SABiosciences (Plate ID – CAPR09572), preamplified cDNA was added into each well containing specific primers for genes of interest. DNA Engine Opticon-2 (BioRad) was used for qPCR.In order to optimise the protocol for the collection of good quality RNA for qPCR, we expanded the following: (a) suitability of modified H&E staining on the identification of rat renal proximal tubular cells (PTCs); (b) the minimal number of rat renal PTCs required to yield sufficient total RNA for downstream qPCR; (c) the quality of total RNA, (d) modifications made to DNase I treatment and lastly, (e) the application of isolated total RNA on qPCR analyses.In order to maximise the time for collection of renal cells via LCM, we used a shortened H&E staining protocol to minimise the degradation of RNA by surrounding RNases. Although the original H&E protocol of approximately twenty-six minutes was condensed to a five-minute protocol, the modified H&E staining protocol was adequate for the identification of rat renal PTCs against other renal cells. The rat renal PTCs were identified via their unique brush border membranes and were selected in red in Figure There have been a number of reports on the various number of cells required for downstream gene expression analyses. These ranged from single cell studies on traumatic brain injury, ten avian embryo to less than five thousand breast cancer cells -16. HoweAs seen in Figure It was previously reported that stained cryosections should undergo LCM immediately to prevent RNA degradation . AlthougWe also assessed the RIN of RNA at progressive stages of sample preparation. As listed in Table Approximately 4000 renal PTCs which were collected within twenty minutes had a RIN of 6.2. We attempted to collect more renal PTCs within 40 minutes but were unsuccessful as the increased time cased RIN to be degraded to 1.0. Thus, two HS caps of 4000 renal PTCs were collected individually within 20 minutes for a sample and placed on dry ice before pooling together during RNA isolation stage.Using the standard DNase I treatment, we observed a Ct value of 27.10 for genomic DNA, suggesting the potential of genomic DNA contamination. Therefore, modifications were made to the existing DNase I treatment as elaborated under Methods section. The Ct value for genomic DNA was then increased to 40 after the changes made in DNase I treatment.As seen in Figure The fully optimised protocol was then applied on a total of six rats for the collection of renal PTCs via LCM and subsequent total RNA extraction using commercially available kit. Table A panel of twenty drug transporters which were previously reported to be expressed in rat kidneys were studied. Figure One of the greatest challenges when using LCM for qPCR is to collect sufficient RNA of good quality. It was made difficult due to upstream sample preparation procedures which exposed the total RNA to RNases. RNases are found in all cell types and organisms and also commonly detected in typical laboratory settings such as reagents used, environmental exposure and human contact. In order to overcome these issues, we have optimised the progressive steps leading to the collection of total RNA from rat renal PTCs.A histological stain is required for the efficient identification of rat renal PTCs, and at the same time, minimizing RNA degradation. We attempted to condense the time taken for H&E staining from 26 minutes to 5 minutes. It was then observed that the modified H&E staining not only allowed straightforward identification of rat renal PTCs, and also a minimal impact on RIN.We also agreed with publications that LCM should be carried out immediately after H&E staining. We later evaluated the RIN of total RNA collected at the progressive stages of sample preparation. We then observed that RIN of rat renal PTCs collected within 15 minutes had a significant better RNA quality than rat renal PTCs collected within 35 minutes. The RIN of an archived rat kidney was extremely poor at 2.2, recommending the usage of fresh kidney tissues to be studied.As different cell types have different amount of RNA yield, it is important to have a protocol specifically designed for different cell types . We lookDNase I treatment is often crucial in qPCR analyses. It aids in the elimination of genomic DNA contamination which may contribute to a higher gene expression than its true value. We observed that small modifications made to the existing DNase I treatment as recommended by manufacturer aided in removing more genomic DNA from total RNA isolated. We doubled the amount of DNase I units and the incubation period. It was shown that the Ct values for genomic DNA control increased from 27.10 to 40, indicating a significant reduction in genomic DNA contamination.The above optimised protocol for extracting total RNA from microdissected rat renal PTCs showed that the amount of total RNA recovered from the cells depends not only on the cell type or size but also the isolation method and/or treatments used.The above modified techniques were then applied in a total of six Wistar male rats. We were able to collect an average of 8000 rat renal PTCs all six rats. The total RNA concentrations and purity ratios were measured using NanoDrop 2000 and RIN were analysed via RNA 6000 Pico kit. The total RNA extracted from each rat was applied onto qPCR array in the gene expression analysis of twenty drug transporters and five housekeeping genes. The average Ct values of respective genes were below 35 indicating a measurable gene expression level according to manufacturer’s protocol. Therefore, we concluded that the optimised techniques were suitable for a more meaningful qPCR analysis.These techniques may be applicable for scientists looking into similar cell groups, such as rat renal distal tubular cells or glomerulus. However, researchers may require paying attention on the number of required cells for sufficient RNA concentration and the organs or cells of interest which may possess different levels of RNases, thus requiring more attention to the protocols Microarray study is currently a popular choice for the understanding of gene expression of a huge pool of genes simultaneously. There are several studies conducted for the preparation of RNA from microdissected cells for microarray analyses ,19. The In this study, we described a protocol for the isolation of good quality RNA from microdissected rat renal PTCs for the application of qPCR analyses. In specific, we have optimised a few important procedures: 1) modified shortened H&E staining, 2) minimal number of rat renal PTCs required for qPCR analyses, 3) the quality control of total RNA, and 4) modified DNase I treatment for a more efficient removal of genomic DNA contamination. Using this optimized protocol, we are able to achieve sufficient good quality total RNA from microdissected rat renal PTCs for gene expression profiling via qPCR.The authors declare that they have no competing interests.JYY and Dr. LMGL optimized the described total RNA isolation method and DNase I treatment, measured the RNA RIN and purity ratios and performed qPCR gene expression profiling. KR, SMR and VSYL performed the tissue preparations, cryosectionings, LCM and input helpful suggestions in the optimizations of protocols. Professor EJDL is the principle investigator of this study shared invaluable experience and guidance in this study. All authors read and approved the final manuscript.


Systematic manipulation of the loop length between each G-tract revealed that the −1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher −1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating −1 ribosomal frameshifting (−1 FS)  G-quadruplex G4) structures formed by guanine (G)-rich nucleic acid sequences are characterized by their four-stranded G-tracts in combination with multiple stacked G-quartets. As opposed to typical Watson–Crick base pair forming duplexes, G-quartets are constituted by noncanonical Hoogsteen hydrogen bonds between these G bases. Although both RNA and DNA can adopt G4s, structural analysis demonstrates that RNA G4s fold into parallel-stranded conformations independent of nucleotide sequences, the species of cations and the concentration of RNA molecules . DNA G4s structurAs opposed to extensive studies on DNA G4s, our knowledge of RNA G4s, especially their biological consequences, remains limited. Recent advances have demonstrated that RNA G4s are key players in various cellular functions, including telomere homeostasis, pre-mRNA processing (splicing and polyadenylation), mRNA targeting, RNA turnover and translation . Among t−1 ribosomal frameshifting (−1 FS) is a translational recoding mechanism whereby translating ribosomes are forced to move one nucleotide (nt) backward, leading to the decoding of a second open reading frame (ORF) located in the −1 register with respect to the first ORF . Two eleBecause stable RNA G4s in the 5′-UTRs can impede 40S ribosomal subunit scanning, and stable structures are required to stall translating ribosomes to induce −1 FS, we hypothesized that G4 RNAs in the coding region can stall ribosomes, owing to their unusual stability, and thus promote −1 FS. While ribosomal stalling by G4s has recently been demonstrated in a bacterial system , we demo−1 FS was monitored by the pSF208 construct described earlier . Sets ofin vitro translation. The translation mixtures (10 µl) contained 5 nM of mRNA, 4 µl of nuclease-treated rabbit reticulocyte lysate , 0.5 µl of 1 mM amino acids mix (Promega) without methionine, 0.25 µl of 35S-methionine (>1000 Ci (37.0TBq)/mmol, EasyTag, Perkin Elmer), and indicated amounts of PhenDC3 . After transcription, RNA samples were loaded on a 1% agarose gel to determine the quality and quantity. Appropriate dilutions of the transcription mixtures in RNase-free water were directly used for  PhenDC3  or TMPpy PhenDC3 , and wer2 at 37°C on a regular subculturing regime. Cells were transfected with 300 ng of plasmid by 1 µl of Lipofectamine 2000 (Invitrogen) in a 24-well culture plate. Cells were lysed 20–24 h after transfection and luciferase activities were measured by GLOMAX multidetector (Promega) using Dual-Luciferase Reporter Assay Kit (Promega). Frameshifting efficiency was obtained by dividing the ratio Renilla luciferase (RL) over Firefly luciferase (FL) activity of the mutant by the RL/FL ratio of the in-frame control and multiplied by 100.Selected G4 constructs were tested in HEK293T cells using the dual luciferases reporter, pDUAL-HIV(0), as described earlier . These data indicate that the stable RNA G4s, which can interrupt 40S ribosomal subunit scanning are able to act as roadblocks to stimulate frameshifting as well.To investigate the possibility of RNA G4 in inducing −1 FS, we first examined two well-defined suppressive RNA G4s located in the 5′-UTR of NRAS  and Trf23U)4 background (3U)4 has been reported as the most stable G4 structure both at RNA and DNA level 4NA level ,37 and tNA level B, SP5, wNA level B, NC. FSin vivo was related to an in-frame control whose activity was set at 100% . Similar to in vitro experiments, optimal FS was observed for the 6-nt spacer with decreasing FS efficiencies for shorter and longer spacers . In general, FS efficiencies measured in vivo are much lower than those obtained in vitro [see e.g. . For the six constructs with total loop length of 5 nt, the −1 FS efficiency ranges from 2.3 to 3.2% . Constructs with a total loop length of 6 , 7  and 10  nt displayed a decreasing ability in promoting −1 FS. In combination with previous data showing that the thermodynamic stability of RNA G4s is inversely correlated with total loop length 5, we observed less efficient −1 FS by incrementing the number of G3U repeats . These data are in contrast to a previous study applying G4 RNAs in the 5′-UTR as translational suppressors (3U)4 at the right distance from the slippery sequence will decrease, thus actually resulting in a decrease in FS. For obstructing ribosomal scanning, distance is not an issue, and as melting of the first G-stretch still allows formation of a new G4 by downstream G-tracts, scanning inhibition is enhanced by increasing numbers of G-tracts. Moreover, decreasing or increasing the number of G-quartets results in less FS  in agreement with their lower thermodynamic stability 4 construct reaching a 1.4-fold increase at a concentration of 2 µM . Addition of TMPyP4 at 2 µM, although affecting global translation, decreased FS ∼3-fold , while both TMPyP4 and PhenDC3 had no significant effects on frameshifting induced by a 12 base-pair hairpin . These data verify that RNA quadruplex formation is responsible for the observed FS.Next we investigated the effect of G4 binding ligands on FS efficiency. We chose the G4 stabilizing ligand PhenDC3, which has been reported to increase the stability of a variety of DNA  and RNA 3U)4 G4 sequence 4 as stimulator. Although this level is rather modest compared with levels obtained with pseudoknot-stimulated FS, which can reach >40% as in the case of the ‘Infectious Bronchitis virus’ FS pseudoknot 4 has been measured to be only −8.16 kcal/mol 4 sequence was also capable of stimulating +1 FS as well as stop codon RT with an efficiency of 3.0 and 1.5%, respectively. Although these values may seem low, in vitro RT frequencies reported for CTFV are between 3.6 and 6.7% 4 RNA.We have also investigated the effect on −1 FS of several known G4 ligands. The bisquinolinium derivative PhenDC3, which is a general stabilizer of RNA G4s , was fouin vitro translation assays already contains a high level of potassium  but it is conceivable that at even higher concentrations of K+ (>100 mM), FS may be enhanced. In addition to potassium, several proteins like FMRP  . HoweverIn conclusion, RNA G4s are capable of stimulating −1 and +1 FS as well as stop codon RT, thereby expanding the repertoire of RNA structures involved in translational recoding. Whether RNA G4s are present at natural recoding sites remains to be investigated.Supplementary Data are available at NAR Online.Leiden Institute of Chemistry, Leiden University, Leiden, The NetherlandsFunding for open access charge: Conflict of interest statement. None declared.


Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2) with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research.Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs) are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and  RNA-world, a term first used by Gilbert in 1986 [RNA-world hypothesis has implicated a crucial role for RNA in the origin of life. Also in today’s DNA-based life, the function of RNA molecules is not limited to being a messenger for protein synthesis. In fact, only about 1–2% of the RNA present within a human cell is protein-coding, the remainder being non-coding RNA (ncRNA). The vast majority of this ncRNA is ribosomal RNA (rRNA) and transfer RNA (tRNA)—both involved in the process of translation [versus the 20,447 known protein-coding loci (GENCODE v15) with lncRNA numbers likely to increase further in later releases of GENCODE [RNA molecules are best known for their ability to convey genetic information encoded in the DNA into the synthesis of specific proteins. This messenger function makes RNA an essential player in today’s DNA/RNA/protein world. It is commonly believed that our current DNA/RNA/protein world was preceded by a so-called  in 1986 ,3. The Rnslation —as well nslation . To date GENCODE ,15,16,17 GENCODE ,22). ImpFor most mRNAs ample evidence for their protein coding ability exists. Likewise, an ever-growing list of publications proves the involvement of lncRNAs in diverse aspects of gene regulation. Despite this major discrepancy in function, lncRNAs are in many ways very similar to mRNAs. The majority of active lncRNA genes are occupied by the same histone modifications as protein-coding genes, are synthesized by the same RNA polymerase II transcriptional machinery, 5' capped and are often spliced with similar exon/intron lengths ,24. MoreProtein-coding ability—Proof of protein-coding ability can be obtained from experiments such as Western blotting using specific antibodies or via mass spectrometry. For example, in 2012, about one-third of all annotated human protein-coding genes were supported by peptide hits derived from mass spectrometry spectra submitted to PeptideAtlas [i.e., 100 amino acids) [H19, Xist, Mirg, Gtl2, and Kcnq1ot1 all contain ORFs longer than 100 codons, while they do not code for protein [ideAtlas . This stideAtlas . Theoreto acids) . Similaro acids) . Unfortuo acids) . For exa protein . Secondl protein . In fact protein  or ribos protein . Noncano protein . HoweverSequence conservation—Instead of measuring the length of the ORF one could also examine the evolutionary conservation of the ORF. If the ORF of a novel transcript shows homology with other known proteins this indicates that the RNA could function as mRNA, while novel, non-conserved ORFs are likely to occur by chance and often do not function as protein-coding [Xist gene evolved from a protein-coding gene and therefore still shows great overlap with mRNA features and a strong conservation [n-coding . Howevern-coding . A furthervation . Other pervation .LncRNAs versus untranslated regions of mRNAs—Interestingly, a recent study revealed significant similarities between lncRNAs and the 3' untranslated regions (3' UTRs) in protein-coding RNAs in structural features and sequence composition [position . Both lnposition . Moreoveposition . In conclusion, although lncRNAs and mRNAs do differ in their protein-coding ability, the above-mentioned facts about lncRNAs reveal a high degree of similarity between lncRNAs and mRNAs, or parts thereof. LncRNAs have been shown to play critical regulatory roles in diverse cellular processes including chromatin remodeling, transcription, post-transcriptional processing, as well as intracellular trafficking ,49,50. Tcis (on neighboring genes) or in trans (distantly located genes). The protein complexes brought on by the lncRNAs can act as epigenetic repressors and activators, as well as transcription factors [LncRNAs appear to be involved in nearly all aspects of gene regulation, including X-inactivation, imprinting, epigenetic regulation, nuclear and cytoplasmic trafficking, transcription, mRNA splicing and translation . Through factors .HOTTIP lncRNA [Knowledge on how lncRNAs search for selective sites in the genome and how they interact with chromatin or target RNAs is slowly accumulating. LncRNAs can interact with RNA molecules via the formation of complementary hybrids ,59,60. TP lncRNA .cis-regulatory functions of mRNAs are known—mostly residing in the non-coding regulatory elements —and involve the regulation of stability, splicing and translation of the transcript [For more detailed information about the mechanisms of lncRNAs action we refer to excellent reviews by others ,66,67,68anscript ,71,72. Ranscript ,74. Likeanscript ,76,77,78trans-regulatory functions. Below, we describe known and putative trans-regulatory functions of mRNAs and compare them to known lncRNAs with similar functions. Each example is also mentioned in Importantly, mRNAs can also affect other genes or gene products via Satellite III (SATIII) [SATIII is involved in the formation of nuclear stress bodies (nSBs) when cells are subjected to thermal, hypertonic or chemical stresses [SATIII repeats on chromosome 9q11‑12 to a euchromatin state. After transcription, SATIII RNA remains within the locus and recruits serine-arginine rich splicing factor SF2/ASF and several heat shock transcription factors like HSF1 and SAF-B to form nSBs [SATIII was even shown to be sufficient for the formation of nSBs in the absence of a stress trigger [NEAT1). NEAT1 is a 3.7 kb long unspliced, polyadenylated transcript that is localized at the edges of SC35 domains in paraspeckles, which are found in all cells in interphase [NEAT1 was concluded to be essential for the assembly, maintenance and structural integrity of these paraspeckles [LncRNAs can serve as structural scaffolds involved in the formation of nuclear domains. The first described non-coding RNA with a structural role is (SATIII) . SATIII stresses . These corm nSBs . SATIII  trigger . A seconterphase ,126. NEAspeckles ,126,127.de novo formation of both these nuclear components was shown to be induced by histone 2b (H2B) pre-mRNA [VegT, found in Xenopus laevis oocytes [VegT transcript was shown to be an integral part of the cytokeratin cytoskeleton at the vegetal cortex of the oocytes and responsible for the localization of Vg1, Bicaudal-C and Wnt11 mRNAs at this position. Depletion of VegT mRNA therefore resulted in the delocalization of these mRNAs [VegT mRNA [Not only ncRNAs, but also mRNAs have been shown to perform architectural roles for cellular substructures. Two of these nuclear structures are the histone locus bodies (HLBs) and the associated Cajal bodies. The HLBs are known to harbor large amounts of histone pre-mRNA and histone 3'-end processing components , whereaspre-mRNA . In the  oocytes . The Vegse mRNAs . FurtheregT mRNA .(co-)transcriptional control. Here, the recruitment of RNA polymerase II, transcription factors and/or co-factors to gene promoters is facilitated or prevented by long non-coding RNAs. The lncRNA MEG3 activates the p53 tumor suppressor gene and the growth differentiation factor 15 (GDF15) gene by enhancing p53 binding to the GDF15 gene promoter, thereby inhibiting cell proliferation [MEG3 is expressed in many normal human tissues, reduced levels of MEG3 are frequently observed in a variety of cancers and associated with hyper-proliferation [MALAT1, which is frequently upregulated in many cancers and can regulate alternative splicing by modulating the phosphorylation of serine/arginine-rich splicing factors (SRSFs) [MALAT1 altered the localization and activity of these splicing factors, leading to altered splicing patterns for a set of pre-mRNAs [GAS5 contains a hairpin sequence motif, mimicking a DNA binding site of the glucocorticoid receptor, thereby serving as a decoy to release the receptor from the DNA and preventing transcription of metabolic genes [dihydrofolate reductase (DHFR) gene, a lncRNA initiated from the upstream DHFR-minor promoter inhibits the assembly of the pre-initiation complex at the major promoter by forming a stable triple helix complex with promoter sequences, as well as through direct interactions with the general transcription factor IIB (TFIIB) resulting in the silencing of the DHFR gene [A second level of lncRNA-directed regulation is by feration . While Mferation ,132,133. (SRSFs) ,135,136.re-mRNAs . The lncic genes . In caseHFR gene .Steroid Receptor RNA Activator (SRA) transcript was initially identified as a ncRNA that co-activates the Progesterone Receptor [SRA RNA has been confirmed to co-activate many nuclear receptors, including estrogen (α and β), androgen, glucocorticoid, retinoic acid (α), peroxisome proliferator activated receptors (δ and γ), thyroid and vitamin D receptors [SRA RNA can enhance the activity of transcription factors like MyoD and GATA3 [SRA ncRNA functions as a scaffold for nucleoprotein complexes with both positive regulators  nd Pus3p ,139). Wiein SRAP . Conside cancers ,145,146. cancers . C-terminal domain (CTD) of RNAPII, allowing the escape into productive elongation [In vivo, P-TEFb is present in two states: an active P-TEFb form, associated with Brd4 and other factors, and in an inactive ribonucleoprotein from, referred to as 7SK snRNP, containing a 331-nt non-coding RNA known as 7SK snRNA. RNase footprinting and mutagenesis experiments have indicated that 7SK contains a high degree of secondary structure, with stem-loops at both the 5' and 3' ends [HIC mRNA for P-TEFb activation [HIC mRNA binds to and activates P-TEFb by displacing 7SK RNA from inhibitory complex. Analysis of the secondary structure of HIC mRNA 3' terminal region revealed the existence of hairpins resembling similar structures within 7SK RNA [Transcriptional pausing is a well-known phenomenon, where RNA polymerase II (RNAPII) becomes trapped downstream of the transcriptional start site (TSS) and is unable to escape into productive elongation . P-TEFb,ongation . In vivo 3' ends ,149,150. 3' ends ,151,152.tivation . The 3'  7SK RNA . It is s 7SK RNA . PTEN-P1 lncRNA was found to bind the same set of regulatory miRNA sequences that normally target the tumor-suppressor gene PTEN, alleviating the PTEN mRNA repression and allowing its translation into the tumor-suppressor protein PTEN [HULC which may act as an endogenous miRNA sponge that down-regulates a series of miRNAs, including miR-372. Inhibition of miR-372 by HULC led to reduced translational repression of its target gene, PRKACB, which in turn induced phosphorylation of transcription factor CREB [MicroRNAs—a large class of small ncRNAs—have emerged as a critical element in gene regulation by interacting with incompletely complementary sequences in target messenger RNAs ,153,154.ein PTEN ,101. Anotor CREB ,160.Versican (VCAN) mRNA in hepatocellular carcinoma (HCC) and of CD44 mRNA in breast cancer cells [VCAN mRNA in HCC and HepG2 cells sequester miR-133a, miR-199a*, miR-144 and miR-431, thereby increasing the protein levels of amongst others CD34 and fibronectin (FN1), which have similar miRNA binding sites in their 3' UTRs [VCAN increased proliferation, survival, migration, invasion, colony formation, and enhanced endothelial cell growth, but decreased apoptosis [CD44 mRNA is elevated in breast cancer cells and its 3' UTR harbors binding sites for miR-328, miR-512-3p, miR-491 and miR671 [CD44 (3' UTR) levels sequester these miRNAs thereby increasing the protein levels of amongst others COL1α1 and fibronectin 1 (FN1), and enhanced the cell motility, invasion and cell adhesion and metastasis. Similarly, two mRNA transcripts were recently shown to act as miRNA sponges: the 3' UTR regions of er cells ,104. The 3' UTRs . Increaspoptosis . Similarcis, as described above, but they can also be transcribed in trans from separate loci (trans-NATs). Both cis- and trans-NATs can affect gene expression at the level of transcription, maturation, transport, stability and translation [cis- and trans-acting lncRNAs base-pairing with mRNA molecules and affecting its stability or translation have been describe so far [Global transcriptome analyses has provided evidence that a large proportion of the genome can simultaneously produce transcripts from both strands, and that antisense transcripts commonly link “neighboring genes” in complex loci into chains of linked transcriptional units . Accordinslation . Numeroue so far ,165,166.trans-acting lncRNAs, termed half-STAU1-binding site RNAs (½‑sbsRNAs), can activate the decay of specific target mRNAs. Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs upon STAU1 binding to the 3' UTR via double-stranded RNA [½-sbsRNAs mode of action [A recently discovered group of nded RNA . STAU1-bnded RNA . Evidentf action . Whethercis- and trans-acting lncRNAs are capable to pair with mRNA molecules forming double-stranded RNA structures and thus inhibiting mRNA translation [LincRNA-p21 is an example of a trans-acting lncRNA involved in translation inhibition [CTNNB1 and JUNB  base-pair imperfectly with lincRNA-p21 at several places throughout the coding regions and UTRs. The formed lincRNA-p21-mRNA complex further interacts with translation repressors Rck and Fmrp, suggesting that lincRNA-p21 can repress the translation of target mRNAs by operating via multiple mechanisms [cis-acting lncRNAs is antisense mRNA for PU.1 gene [PU.1 transcript and stall translation between initiation and elongation steps [LncRNAs are best known for their roles as regulators of transcription. However, recent studies have shown an important role of long non-coding RNAs in mRNA translation ,108,164.nslation . Alternahibition . The trachanisms . AnotherU.1 gene ,168. Theon steps ,168. Antisense BCMA RNA is transcribed from the same locus as BCMA and has typical mRNA features, e.g., polyadenylation, splicing, Kozak consensus sequence and an open reading frame encoding an experimentally proven 115 amino acid peptide: p12 protein [antisense BCMA inhibits the expression of BCMA protein, while it does not affect the expression level of BCMA mRNA. The inhibition of BCMA expression is obtained through the action of the antisense RNA and not of the p12 protein, although the exact mechanism is not fully understood [Protein-coding antisense mRNA transcripts are also capable to form RNA duplexes with sense mRNA molecules leading to translation inhibition.  protein . Experimderstood . Xenopus laevis transcript BC1. BC1 transcript—expressed in neurons and germ cells—inhibits the assembly of the translation initiation complex [BC1 RNA interacts with eIF4A and PABP and disrupts the functional link between the two factors which is necessary for efficient translation in Xenopus oocytes [BC1 happens via eIF4A and PABP [A ncRNA that acts by affecting the general translation machinery is the  complex . The 3'  oocytes . A near-and PABP .trans-regulators by inhibiting translation through the activation of PKR [P23/TCTP full-length mRNA but not a truncated version thereof, was able to bind and activate PKR, resulting in the inhibition of translation [VEGFA mRNA [IFN-γ mRNA [TPM1 mRNA [TNF-α mRNA [cis effect on the translation level of mRNA itself as well as more general trans effect on the translation level of other transcripts.The ability to inhibit general translation machinery is also identified for several mRNAs. These transcripts mainly act through the interaction of their UTRs with the RNA-dependent protein kinase (PKR). PKR is a serine-threonine protein kinase that is activated by intermolecular autophosphorylation upon binding to RNA molecules. The 3' UTR regions of cytoskeletal muscle mRNAs can act as n of PKR . Specifin of PKR . Once acn of PKR . An impon of PKR . Similarnslation . SeveralGFA mRNA  and IFN-N-γ mRNA , and thePM1 mRNA  and TNF-F-α mRNA . In all p53 [trans-activate a variety of target genes which promote cell cycle arrest, senescence or apoptosis [MDM2 gene. Its protein product is an E3 ubiquitin ligase which promotes polyubiquitination and proteasomal degradation of p53, thereby forming a negative regulatory feedback loop [p53 can interact with the RING domain of MDM2, which prevents the E3 ligase activity and furthermore stimulates translation of the p53 mRNA [p53 mRNA was considered to control the function of MDM2 [p53 mRNA-MDM2 interaction and thereby acts as the switch for MDM2 between being a negative or a positive regulator [Another mRNA with translational control is the tumor suppressor gene p53 . This gep53 ,171. Thep53 . When p5poptosis ,174. Anoack loop ,176,177.p53 mRNA . At firs of MDM2 . Later iegulator .Prostate Cancer-Associated Transcripts, or PCATs) were identified, representing potentially functional lncRNAs associated with prostate cancer. One of these transcripts, called PCAT-1 was selectively upregulated only in prostate cancer and shown to function predominantly as a transcriptional repressor by facilitating trans-regulation of genes preferentially involved in mitosis and cell division, including known tumor suppressor genes, such as BRCA2 [Recently, an example of a regulatory lncRNA in prostate cancer was described, with a proven functionally, but through a yet unknown mechanism of action . In thisas BRCA2 .trans-acting regulators in feedback loops that inhibit cell division and promote differentiation [prohibitin (PHB), an inhibitor of cell proliferation, significantly suppresses the tumorigenic properties and metastatic phenotype of transformed MCF7 cells [ribonucleotide reductase (RNR), a key rate-limiting enzyme in DNA synthesis, significantly suppresses the tumorigenic properties and metastatic phenotype of transformed fibroblasts cells [c-myc P0 transcript suppresses the malignant phenotype of human breast cancer cells with decreased anchorage-independent proliferation, enhanced susceptibility to programmed cell death, and complete loss of the ability to form tumors in the intact animal [trans-regulatory functions, but the exact mechanism of their action is currently still unknown. Also several mRNAs, and more specifically their UTRs, have been reported to function as regulators (riboregulators) that suppress tumor formation but through unknown mechanisms. Results from Rastinejad and Blau suggest that the 3' UTRs of certain differentiation-specific RNAs are ntiation . More reF7 cells . Similarts cells . Also tht animal . For allHOTTIP [Mistral [HOTAIR [HOTAIRM1 [ANRIL [Kcnq1ot1 [Air [Xist [pRNA [HOTAIR can simultaneously bind to both the polycomb repressive complex 2 (PRC2) and the LSD1-CoREST complex using specific domains of the RNA molecule [ANRIL and HOTAIRM1 directly interact with proteins from both PRC1 and PRC2 complexes [Kcnq1ot1 interacts with both the PRC2 and G9a (EHMT2) to lay down the silencing histone marks H3K27me3 and H3K9me2, respectively [MEG3 in particular, which directs PRC2 to the reciprocally imprinted Dlk1 coding gene [et al. only describe results for non-coding intronic RNA sequences [in cis. One of their examples is the SMYD3 intronic RNA, which can bind to EZH2, a component of the PRC2 complex, thereby targeting this repressive complex to the SMYD3 gene. SMYD3 is a SET domain-containing H3K4 methyltransferase with oncogenic properties, which is frequently overexpressed in colorectal, breast and liver cancer [SMYD3 intronic RNA, resulted in reduced tumor growth, and revealed SMYD3 intronic RNA to harbor tumor suppressive abilities [It is well known that many lncRNAs are involved in the regulation of gene expression at the epigenetic level. Approximately 20–30% of all lncRNAs have been shown to be able to physically interact with specific epigenetic enzymes, which control the reversible modification of histone residues and DNA methylation, thereby influencing the activity of genes ,180. UpoHOTTIP  or Mistr[Mistral ) or gene [HOTAIR , HOTAIRMHOTAIRM1 , ANRIL [1 [ANRIL ,57, KcnqKcnq1ot1 , Air [12ot1 [Air , Xist [1ir [Xist  or pRNA st [pRNA ). LncRNAmolecule , while Aomplexes ,57,120. ectively . In receectively ,122,183.equences . They rer cancer ,185. Redbilities . Similarbilities . Finallybilities ,187.HOTAIRM1, ANRIL and KCNQ1OT1  via next-generation sequencing and compared these results to input material . To our KCNQ1OT1 A. IndepeKCNQ1OT1 B. The (rquencing . There, In conclusion, all three studies described above imply a vast level of interaction between proteins of the PRC2 complex and protein-coding RNAs. These results are also in line with recent mRNA-proteome interaction studies where mRNAs appear to interact with regulatory enzymes and proteins. In these large proteome studies hundreds of mRNA binding proteins were identified ,189. As trans-regulatory roles for mRNA. Side-by-side, we have compared lncRNA and mRNA examples with similar regulatory functions. We have shown that mRNAs can frequently be associated with the PRC2 complex components and hypothesize a common guiding role for mRNA molecules. Future experiments need to further substantiate these speculations. Lastly, conclusions from loss-of-function experiments for mRNAs may need to be reinterpreted as the effects may not automatically be solely attributed to the associated protein function, but instead may also be partially due to affected regulatory functions. Again, further experimentation will show the extent of these regulatory roles for coding RNA.From the vast amount of papers it is clear that long non-coding RNA can have a variety of important roles in gene deregulation in cancer. Evidence of similar roles for protein-coding transcripts is now slowly accumulating. Here, we have combined, reviewed and extended the current knowledge of


RNA editing is a process that modifies RNA nucleotides and changes the efficiency and fidelity of the central dogma. Enzymes that catalyze RNA editing are required for life, and defects in RNA editing are associated with many diseases. Recent advances in sequencing have enabled the genome-wide identification of RNA editing sites in mammalian transcriptomes. Here, we demonstrate that canonical RNA editing (A-to-I and C-to-U) occurs in liver, white adipose, and bone tissues of the laboratory mouse, and we show that apparent non-canonical editing  is an artifact of current high-throughput sequencing technology. Further, we report that high-confidence canonical RNA editing sites can cause non-synonymous amino acid changes and are significantly enriched in 3′ UTRs, specifically at microRNA target sites, suggesting both regulatory and functional consequences for RNA editing. The commonly taught, simplest version of the central dogma of molecular biology (DNA to RNA to protein) has been complicated in recent years by the discoveries of alternative splicing, non-coding RNAs, and other transcriptional regulatory mechanisms. RNA editing disrupts the faithful transfer of information from DNA to RNA to protein by altering the sequence of RNA molecules co- or post- transcriptionally, potentially altering translational regulation and leading to a diversified proteome. RNA editing was first discovered in 1986 in trypanosomes, where nucleotide insertions cause reading frame shifts et al. study further demonstrated that at least some editing sites are translated into peptides detectable by mass spectrometry New applications of high-throughput sequencing technology to RNA have expanded the number of characterized A-to-I and C-to-U “canonical” editing sites, and several studies have suggested widespread DNA-RNA differences, including all 10 other possible “non-canonical” base substitutions. However, no enzymes or biochemical processes have been identified that can catalyze these non-canonical RNA edits. Initial computational efforts focused on A-to-I editing and searched through expressed sequence tag (EST) libraries to identify thousands of candidate sites subsequently verified through targeted RNA-seq http://genome.ucsc.edu) in a manner that tolerates a large number of mismatches and the possibility of reads spanning splice junctions. Potential RNA editing sites were identified as bases supported by at least 2 high quality edited reads with an edit ratio  greater than 5% in all three biological replicates  that cuts only the edited version of the sequence. These results agree with the sequencing results in that the cDNA contains both edited and un-edited forms (since cleaved and un-cleaved products are visible), while the gDNA contains only the un-edited (un-cleaved) sequence  and genomic DNA (gDNA) were extracted from two liver or adipose samples of additional C57BL/6J mice. For each site, the surrounding region was amplified by PCR from both gDNA and cDNA, and each was sequenced. All 19 canonical RNA editing sites were validated in both independent samples through Sanger sequencing . Sanger sequencing of several locations within Hjurp showed mixed sequencing peaks in both gDNA and cDNA samples , suggesting that there may be additional polymorphic copies of Hjurp in our genetic background that are not reflected by the reference genome, leading to observed editing in our RNA-seq data. As such, we removed all sites within Hjurp from our final results. A similar phenomenon could occur because of somatic mutations, or genetic drift relative to the reference genome.To validate the strand bias filter, we selected 3 canonical editing sites with a significant strand bias for testing . None of these sites were validated through Sanger sequencing , confirming our assessment that strand biased canonical editing sites are likely artifacts. The remaining non-canonical editing sites that do not exhibit a strand bias also appear to be the product of biases or errors, falling into several categories. First, errors or omissions in the reference genome relative to our samples can lead to apparent non-canonical editing. For example, roughly 50% of our observed non-canonical editing sites without any strand bias are localized to a single gene, Apparent non-canonical editing could also be the result of complementary canonical editing of double stranded substrates or of antisense transcripts. For example, the AID/APOBEC family has been shown to edit C-to-U in DNA sequences, which when repaired can produce a C-to-T edit in the primary strand, and a G-to-A edit in the complementary strand Due to these potential sequencing errors and biases, we focused further analyses on a set of replicated, canonical RNA editing sites. A summary of our overall approach is shown in nd to 8th nucleotides of microRNAs, the key positions for microRNAs to recognize their targets Rpa1 , a gene essential for replication, recombination, and DNA repair, we observed 12 high-confidence A-to-I editing sites, 7 of which localize to targets of microRNA seeds. Six of these 7 sites are within 100 nucleotides, forming a very dense cluster, including a pair of adjacent editing sites within overlapping targets of microRNAs  occur in 3′ UTRs, and of these, they are significantly biased towards target sites of the predicted “seeds” of microRNAs , suggesting that it may be a result of sequencing errors other than the strand bias issue, and that further analysis of high-throughput sequencing errors is warranted.Strand bias explains the majority of non-canonical editing sites in our data, while a smaller minority are likely errors or biases from the reference genome; but the balance between these factors may be different for other analysis settings, sequencing platforms, or organisms. The challenge of incomplete or imperfect reference genomes is somewhat mitigated by our use of homozygous C57BL/6J mice, which closely match the NCBI mouse reference genome. However in other populations or organisms, including human datasets, genetic diversity, heterozygosity, and varying qualities of reference genomes will likely present an even greater challenge. In fact, a recent re-analysis of human B-cell transcriptome data Despite mounting evidence from this and other studies, it is difficult to completely rule out the possibility of rare non-canonical RNA editing events, especially for editing sites that are complementary to canonical editing types. Nevertheless, our results suggest that canonical RNA editing plays both regulatory and functional roles, as editing sites are significantly biased towards 3′UTRs and microRNA target sites, and can cause non-synonymous amino acid changes. Although we have some biochemical knowledge of how the AID/APOBEC and ADAR gene families catalyze canonical editing, many open questions remain regarding the regulation and function of RNA editing, including how sites are targeted for editing and which enzymes catalyze which targets. As costs decrease and technologies improve , future studies of RNA editing will be able to address these outstanding issues.The Institutional ACUC of The Jackson Laboratory approved the use of mice for this study . All animals were treated humanely and in strict accordance with the recommendations provided by the National Institutes of Health.2) on bedding of sterilized white pine shavings under conditions of 12 hours light; 12 hours darkness. All mice had free access to water and rodent chow for the duration of the study.Samples for RNA-seq were obtained from 16 week old female C57BL/6J mice (JR stock #000664), which were purchased from the resource colonies of The Jackson Laboratory . Validation experiments were performed in additional, independent C57BL/6J mice, one male and one female. Mice were maintained in polycarbonate boxes . Briefly, sequencing libraries were constructed by purifying mRNA from total RNA, which was then fragmented and converted to double stranded cDNA using random primers. Overhangs were converted into phosphorylated blunt ends, and adaptors were ligated to the cDNA fragments. An agarose gel was used to remove excess adaptors and isolate fragments roughly 300 bp long, and PCR was used to enrich for adapter-modified fragments. Libraries were prepared and sequenced using a barcoding scheme across multiple sequencing pools to enable the identification of technical artifacts. As such, each sample was sequenced to a depth of approximately 15 million 68 base pair long paired-end reads .RNA and DNA was isolated from livers of two additional, independent C57BL/6J mice. In order to account for potential variation in protocols and reagents, one sample was prepared using a Qiagen kit protocol, and the other using TRIzol (Invitrogen). RNA was digested with DNase I to exclude DNA contamination. Random primers were used for first-strand cDNA synthesis, and Superscript III RT (Invitrogen) was used for reverse transcription. As a control for DNA contamination, DEPC-treated water was used in place of the reverse transcriptase enzyme. PCR was carried out with primers specific for each RNA editing site of interest. Primers were designed to amplify fragments of an appropriate size for sequencing and that could be used on both gDNA, and on potentially spliced cDNA. Amplified PCR products were purified and subjected to Sanger sequencing using standard methodologies. Sites were considered validated if the cDNA sequence at the candidate editing site contained two peaks (both a reference peak and an edited peak), while the corresponding gDNA sequence contained a single reference peak. Sites were considered un-validated if the cDNA and gDNA traces both contained a single reference peak.Serinc1 gene creates a sequence that is recognized by the restriction enzyme BspDI, and the site at chr9:123370996 in the Lars2 gene creates an RsaI recognition site. In both cases, site specific PCR products for cDNA and gDNA were obtained as described above. These products were subjected to restriction enzyme digest or a mock control for 2 hours at 65°C for BspDI and 37°C for RsaI. To insure enzyme integrity, two positive controls for RsaI were amplified by PCR using specific primers and digested with or without RsaI. The resulting fragments were visualized using standard agarose gel electrophoresis.We selected two potential editing sites for validation as their sequences were amenable to validation with RFLP analysis. Specifically, the editing site at chr10:57235791 in the http://genome.ucsc.edu) by splitting each 68 nucleotide read into two smaller portions which were separately aligned using the Bowtie software Our goals for analysis were to identify high-confidence RNA editing sites from inherently noisy high-throughput sequencing of RNA. As such, we analyzed data to filter out poor quality sequences and alignments, but in a manner that tolerates a large number of mismatches in order to account for potential “clusters” of editing sites. First, all reads that do not meet Illumina's chastity filter were removed, and reads that did not contain a perfect barcode sequence were removed, since sequencing errors early in a read are a potential indicator of poor read quality. Second, reads were aligned first to a custom set of all possible exon splice junction sequences based on RefSeq annotations Based on these high-quality aligned reads, we identified potential RNA editing sites as genomic locations with mapped reads containing at least 2 high quality mismatched bases (Phred score >20) and where the edit ratio  is at least 5%. Known SNP locations  and editing ratio (model: EditRatio∼run*tissue). In both cases the run effect did not significantly contribute to variability . As such, downstream analyses pooled technical replicates together for analysis.The genomic locations of RNA editing sites were classified as 3′UTR, coding, intronic, 5′UTR, or intergenic based on RefSeq gene annotations nd to 8th nucleotide) of a microRNA defined by miRBase Editing sites were classified as within a microRNA target site if they fall within the region complementary to the “seed” Click here for additional data file.Figure S2Non-canonical RNA editing candidates with a significant strand bias fail to validate through Sanger sequencing. Results are shown as in Fig S1.(TIF)Click here for additional data file.Figure S3Distribution of average editing ratios for canonical and non-canonical editing sites. A) In bone samples, and B) in liver samples. The x-axis is sorted by editing ratio.(TIF)Click here for additional data file.Figure S4Canonical sites with a strand bias, and a non-canonical site without strand bias fail to validate. A) Sanger sequencing of 3 canonical sites with a significant strand bias. B) Sanger sequencing of 1 non-canonical site without a significant strand bias.(TIF)Click here for additional data file.Figure S5Examples of DNA polymorphisms within the Hjurp gene.(TIF)Click here for additional data file.Figure S6Motifs discovered at or near strand biased sites. A,B) Significant motifs within 50 bp of strand biased sites discovered with MEME (TIF)Click here for additional data file.Figure S7Effect of technical sequencing variation on gene expression and RNA editing. A) Effect sizes determined from fitting an ANOVA model to gene expression levels (measured as RPKMs) with respect to technical replicate (labeled “run”) and tissue analyzed (model: RPKM∼run*tissue). B) Effect sizes based on ANOVA analysis of edit ratios similarly to A (model: EditRatio∼run*tissue). In both cases, the effect size of run is negligible.(TIF)Click here for additional data file.Table S1Summary of reads and RNA editing sites identified from each tissue.(DOCX)Click here for additional data file.Table S2Homologues of RNA editing sites previously validated in human studies present in our samples.(DOCX)Click here for additional data file.Table S3All high confidence canonical editing sites.(XLSX)Click here for additional data file.