It has a plethora of biological effects, appears to be particularly pervasive in humans with respect to other mammals, and is implicated in a number of diverse human pathologies. Here we present the first human inosinome atlas comprising 3,041,422 A-to-I events identified in six tissues from three healthy individuals. Matched directional total-RNA-Seq and whole genome sequence datasets were generated and analysed within a dedicated computational framework, also capable of detecting hyper-edited reads. Inosinome profiles are tissue specific and edited gene sets consistently show enrichment of genes involved in neurological disorders and cancer. Overall frequency of editing also varies, but is strongly correlated with ADAR expression levels. The inosinome database is available at:  Large-scale projects such as ENCODE have shed light on the dynamic nature of eukaryotic transcriptomes, where diverse molecular processes interact to fine-tune gene expressionIn humans, the most prevalent type of RNA editing converts adenosine (A) residues into inosine (I) in double stranded RNAs through the deamination reaction carried out by members of the adenosine deaminase (ADAR) family of enzymes, comprising ADAR1 , ADAR2  and ADAR3 Inosine is commonly interpreted as guanosine by the cellular machinery (and sequencing enzymes) leading to functional consequences including the alteration of codon identity, the creation or elimination of splice sites and the modification of base-pairing interactions within higher-order RNA structures57ad hoc filters to minimize false candidate calls due to sequencing or read mapping errors910119101112High-throughput sequencing platforms and effective computational technologies have facilitated transcriptome-wide studies of RNA editing9Distinct from other large-scale studies1516Strand-specific RNA-Seq reads were subjected to stringent quality trimming and aligned to the reference human genome using the spliced aligner GSNAPThe impact of misalignments was mitigated by applying Blat, a more accurate aligner than fast-mappers like GSNAP, on reads carrying mismatches9The concomitant use of strand oriented RNA-Seq reads and whole genome sequences, in combination with a refined computational methodology, allowed us to detect a very low fraction of apparent non-canonical RNA editing sites  which liWe also extended our screening to C-to-U editing sites, the other type of canonical RNA editing in human through the action of APOBEC1 enzymeWe also applied a recent approach to rescue heavily edited RNA-Seq reads that are generally missed by current methodsOur catalogue of A-to-I editing sites was compared to the DARNED databaseMore than 2 million of the editing events identified here are novel and our resource is the largest single collection of human editing events. Our data further highlight the pervasive nature of A-I editing  and are In agreement with previous studies in humans91216About 2.5% of A-to-I changes were in 3′ untranslated regions (3′UTR) and several (38) of these overlapped with miRNA targets predicted by the TargetScan algorithm . Very feDespite the large number of RNA editing events identified, only 1741 (0.05%) A-to-I changes fell in CDS regions . Of thes27et al.Nonsynonymous events in non-repetitive regions were enriched in brain (85 sites) and lung (62 sites), and included 31 out of 35 mammalian conserved recoding sites described by Pinto DrosophilaCaenorhabditisAcromyrmexThe large number of detected RNA editing sites allowed us to investigate the sequence context flanking A-to-I changes. As reported previously31Further, we noted under-representation of A in regions upstream and downstream editing sites . Such deIn addition, we inspected sequences flanking editing sites detected in hyper edited and non-hyper edited regions . AlthougIn sequences surrounding RNA editing sites detected in Alu elements, repetitive non Alu regions and non repetitive regions , we noteFinally, we calculated the distribution of RNA editing levels and found that the median value was rather low (17%). This is due to the fact that the vast majority of A-to-I events occur in Alu repeats and typically exhibit editing levels lower than 1%To investigate the impact of RNA editing on human transcriptome, we mapped all detected events on Gencode (v19) annotations and discovered that 17,140 loci over 55,496 (31%) underwent RNA editing in their exons and/or introns. Interestingly, most of the detected events (92%) occurred in protein coding genes, modifying 13062 loci out of 20173 annotated genes (65%). The remaining A-to-I events (8%) were distributed in the non-coding RNA fraction (ncRNA) . AlthougSince the vast majority of RNA editing events occur in Alu elements, the number of A-to-I changes per locus should be correlated with the Alu abundance per gene. In examining this relationship, we found a strong correlation of 0.78  . TherefoFurther, we explored the presence of RNA editing in ncRNAs and detected that a notable number  of A-to-I events resided in long-non-coding RNAs (lncRNAs), including primarily antisense RNAs and long-intergenic RNAs (lincRNAs) . Two per36As shown in The total number of distinct events in brain  was not exceptionally high compared with other tissues as, for example, lung  . RNA ediDespite the difference in the number of editing sites per sample, the distribution of RNA editing levels was quite similar across tissues and more evident within each tissue group . The vasWe also investigated the inosinome similarity across human tissues. Cluster analysis based on pairwise comparison of RNA editing levels per sample by the Spearman correlation coefficient, showed well-defined tissue segregation and three major groups involving 1) brain and heart, 2) muscle and 3) lung, liver and kidney . This ob13We also considered the genomic localization of RNA editing events across all six human tissues, finding the same trend as observed for the entire collection of sites. The largest fraction of edited positions per tissue occurred in introns and non-coding regions rich in Alu repeated elements. In addition, RNA editing events occurred mainly in protein-coding genes and the fraction of edited genes was quite similar across tissues but muscle in which only 5,564 protein coding genes out of 20,173 (27%) were modified by RNA editing.The main prerequisite for A-to-I RNA editing is the double-stranded RNA structure, like the one formed during the miRNA maturation process. Therefore, RNA editing is expected to affect miRNA biogenesis, through A-to-I changes in pri- or pre-miRNAs, and miRNA target interaction by A-to-I modifications in mature miRNAs, especially in the recognition site, known as the “seed” region. Previous studies focusing on RNA editing in miRNAs of human brain samples by NGS technologies have demonstrated that A-to-I changes in mature miRNAs are rare39Our screen on mature miRNAs revealed 4 novel A-to-I changes, 3 of which were identified in the brain .In addition, we checked RNA editing changes in the full set of events detected by RNA-Seq data. Although these libraries were not prepared to capture mature miRNAs, a fraction of reads might derive from pri-miRNA transcripts and align to miRNA genes. Indeed, we found 113 A-to-I changes in genes for miRNAs, 20 of which were supported by hyper edited reads . Of thesFinally, we noted that about 50% of detected sites (53) have been already identified in past genome wide screenings and included in the RADAR databaseHierarchical clustering analysis as well as the uneven distribution of A-to-I changes across tissues indicates that RNA editing profiles are strongly tissue dependent. This behaviour may be mainly due to tissue specific regulation of ADAR enzymes and only partially to variable RNA-Seq coverage among samples. To investigate the correspondence between tissue expression profile of ADARs and RNA editing, we initially calculated expression values of ADAR genes from RNA-Seq data. As shown in In the ADAR locus, we noted high expression level of isoform NM_001025107, coding for the short and almost exclusively nuclear p110 ADAR protein, in brain and high expression level of isoform NM_001111, coding for the interferon (IFN)-inducible p150 variant, in lung . This tiExpression values of ADARs were thus strongly correlated to inosinome profiles across tissues as both ADAR expression and RNA editing extent decreased from brain and lung to muscle. Comparing the global level of editing per sample, by summing editing levels over all positions, with the expression of ADARs (excluding ADARB2 since expressed only in brain as shown in −263 against the shuffled distribution) (−35 against the shuffled distribution)  and editing levels per position. As background distribution we used the same dataset in which editing levels were randomly shuffled. Limiting the analysis to sites covered by at least 10 RNA reads, we found a striking and statistically significant positive correlation between ADAR expression and individual editing levels  . The coribution) . Togetheibution)  and, thuAs aforementioned, RNA editing profiles are strongly tissue dependent and previous studies have shown that some A-to-I events are tissue specificInterestingly, we found that brain contained the highest number of tissue specific RNA editing events , 5-fold more sites than in kidney  and 620-fold more sites than in muscle . This reIn addition, we investigated the enrichment of disease-associated genes in each list of tissue specific edited genes. Remarkably, we discovered significant enrichments for diseases typical of the tissue under investigation. For instance, brain specific edited genes were enriched in neurological and neurodegenerative disorders while kidney specific edited genes were enriched in urogenital diseases . TogetheDifferential editing levels of individual sites has been proposed to underlie deregulated A-to-I sites linked to human diseases. Differential editing has been addressed only for a handful of recoding sites, since they are generally considered more informative than editing events residing in other genomic regions and more likely associated to pathological phenotypes. Here, we investigated differential RNA editing across human tissues using RNA-seq data and performing tissue pairwise comparisons of detected events. Since the RNA-Seq coverage is not uniform among independent samples, we selected only positions showing editing evidence in all samples of each tissue group and supported by more than 10 RNA reads. A-to-I changes displaying statistically significant differential editing, after the paired t-test with P-value corrected by Benjamini–Hochberg procedure, were recovered.Overall, we detected 2,636 sites with significant differential editing level across all six tissues . Such siThese findings suggest an active functional role of RNA editing and corroborate our previous evidence of lack of correlation between gene expression and RNA editing levels . We founhttp://srv00.ibbe.cnr.it/editing/, is freely available and can be interrogated by genomic region, gene name and other features as well as the tissue of origin. Query results are shown in sortable and downloadable tables in which the main characteristics of individual RNA editing events are reported. Unlike other collections, we provide RNA-Seq and DNA-Seq coverage per site as well as the RNA editing level.Our work reports the largest collection of RNA editing events in human tissues comprising more than 3 millions of sites. To make available our entire collection as an atlas of RNA editing in human, we loaded all detected A-to-I events in a SQL database. The RNA editing atlas, accessible at High throughput sequencing has dramatically improved our appreciation of complex eukaryotic transcriptomes providing an opportunity to investigate co- and post-transcriptional mechanisms such as alternative splicing and RNA editing at single nucleotide resolution91644RNA editing by A-to-I deamination may be extremely relevant for modulating gene expression in human transcriptomes5Here we produced high throughput strand specific RNA-Seq data from six tissues  from three post-mortem healthy individuals. To accurately call RNA editing events, we resequenced the whole exome and genome of each individual . All datComparing RNA editing profiles across human tissues we discovered an uneven distribution of the number of events, even in the same tissue group, that is mainly due to differences in the RNA-Seq coverage. Despite this observation, some tissues such as brain and lung appeared more edited that others like heart and muscle. In particular, muscle was the tissue with the lowest number of A-to-I changes. Nonetheless, we notably found that RNA editing profiles clustered according to tissue type. This result is particularly relevant for investigations in pathological conditions. Indeed, the direct comparison of inosinomes in different conditions allows the identification of potential anomalies at RNA editing level or the discovery of global editing dysregulation.The clustering of whole editing profiles highlights the tissue specific regulation of RNA editing. We noted that gene expression of ADAR enzymes was strongly correlated with the number of events and the global RNA editing level per tissue. Notably, we showed that ADAR expression was more strictly correlated with editing levels than ADARB1 expression and this finding is perfectly consistent with a recent genomic analysis of ADAR binding in which the majority of CLIP-Seq sites were located in Alu repeatsOur results, therefore, suggest that tissue-specific inosinomes depend on the expression of ADARs and are regulated by specific tissue requests. In addition, RNA editing levels are not associated to gene expression meaning that the increased expression of a given gene does not entail higher editing levels.The pervasive RNA editing landscape in the human transcriptome raises essential and intriguing questions about functional roles of A-to-I changes. Experiments involving ADAR enzymes knock-out in different model organisms have clearly demonstrated the indispensability of RNA editing in survival and in preserving the cellular homeostasis75Our genome wide screening indicates that more than 90% of RNA editing sites resides in known protein coding genes (affecting about 65% of mRNAs) and may profoundly affect transcriptome dynamics with a variety of functional consequences. This makes RNA editing an essential and vital mechanism in human. Employing our large collection of A-to-I modifications, we investigated the indispensability of RNA editing in human, exploring the relationship between inosinome and human diseases. To this end, we downloaded all known genes associated to human disorders from DisGeNET database comprising over 380,000 associations between more than 16,000 genes and 13,000 diseasesIn addition, we investigated how many edited genes were in common with a collection of human essential genes obtained from DEG databaseAnother important aspect emerging from our results is the relationship between RNA editing and human diseases of the nervous central system. To explain this relationship we focused on tissue specific genes and RNA editing events occurring herein. The majority of edited genes are specifically expressed in brain than in other tissues, indicating that RNA editing may play indispensable functional roles in brain. However, edited genes in other tissues deserve also careful attention since they are associated to tissue specific pathologies.In conclusion, our study provide a comprehensive overview of RNA editing in human primary tissues, highlighting inosinome variations and the importance of using matched RNA-Seq and DNA-Seq data from the same individual to accurately profile RNA editing. Our collection is freely available through the web and may be a relevant resource to investigate editing alterations in human disorders. We think that RNA editing deregulation may be a key phenomenon in many diseases and the understanding of mechanisms relating edited genes and disrupted cellular homeostasis may lead to new biomarkers and hopefully to the design of novel biotechnological drugs.Six different post-mortem human snap-frozen tissues  from three unrelated “nondiseased” Caucasian individuals  were obtained from Cureline . Sample IDs and further details are reported in DNA was extracted and purified using the DNeasy Blood & Tissue Kit  according to the manufacturer’s instructions. Each sample was then quantified and qualitatively checked by the NanoDrop 2000c .Total RNA was extracted and purified using the RNeasy Plus Mini Kit , according to the manufacturer’s instructions. RNA quality was assessed by the Agilent Bioanalyzer 2100, obtaining RIN (RNA Integrity Number) values ranging from 5 to 7, that were considered acceptable for RNA derived from post-mortem tissues.Exome capture was performed using the TruSeq Exome Enrichment Kit , according to the manufacturer’s instructions. Briefly, for each tissue, a DNA library, including inserts ranging in size from 200 to 400 bp approximately, was prepared using the TruSeq DNA Sample Prep kit (Illumina). Then, each library was hybridated with biotinylated probes targeting the exonic regions . After two steps of enrichment with the probes, the captured exonic regions were sequenced on the Illumina HiSeq2000 platform at IGA Technology Service in Udine . Paired-end reads of 100 bp were generated for each fragment .For each tissue, a strand-oriented RNA library was prepared to preserve information about which DNA strand was the original template during the synthesis of transcripts, thus offering strand orientation for detection of antisense transcription and providing information about regulatory relationships.The cytoplasmatic rRNA removal was performed for each total RNA sample using the Ribo-Zero rRNA removal Kit . The rRNA-depleted RNA was used to prepare the stranded-oriented RNA-seq library using the TruSeq Stranded Total RNA Sample Prep Kit , according to the manufacturer’s instructions. Briefly, each RNA was chemically fragmented prior to the random priming reverse transcription reaction for first strand cDNA generation. The fragmentation step resulted in a RNA-seq library including inserts ranging in size from approximately 100–400 bp. During the second strand synthesis, dUTP was incorporated in place of dTTP, thus preventing amplification of this strand during the subsequent PCR step and retaining strand information. cDNA libraries were sequenced on the Illumina HiSeq2500 platform at IGA Technology Services in Udine . Paired-end reads of 100 nt were generated for each fragment .Brain DNA, from same individuals for which RNA and exome sequencing was performed, was used to create three DNA libraries by the TruSeq DNA Sample Prep kit (Illumina), including inserts ranging in size from 200 to 400 bp approximately. Then, each library was sequenced on the Illumina HiSeq2000 platform at Personal Genomics Service in Verona . Paired-end reads of 100 bp were generated for each fragment .Indexed cDNA libraries from the RNA fraction at low molecular were prepared using the TruSeq small RNA sample Preparation kit  according to the manufacturer’s protocol and recommendations. Single end sequencing (1 × 50 b), after fluorimetric quantification, was performed on the Illumina Miseq platform.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and, subsequently, trimmed at 3′ end to remove adaptor sequence contamination by TrimGalore . Cleaned reads were then aligned to the GRCh37 human reference genome using GSNAPhttp://broadinstitute.github.io/picard/). Main mapping statistics were calculated using Picard and custom python scripts  were trimmed using TrimGalore, excluding reads with final length less than 50 bases.Cleaned reads were subsequently aligned onto the complete GRCh37 human genome by means of GSNAPhttp://support.illumina.com/sequencing/sequencing_software/igenome.html) and annotations for rRNA genes were downloaded from UCSC genome browser selecting the RepeatMask table.Transcriptome quantification and differential expression was performed using CufflinksRNA editing candidates were detected using a pipeline based on our REDItools suite20Resulting tables were subsequently updated adding genomic and exomic information by means of the REDItoolAddGenome.py script. Individual positions in updated table were finally annotated using the AnnotateTable.py script and the following databases: RepeatMask, dbSNP (v. 138) and RetroposedGenes from UCSC. For each table, we separated positions residing in Alu elements, repetitive non-Alu regions and non-repetitive regions using custom bash scripts. During the split, we retained only positions supported by at least 10 genomic reads and completely homozygous. DNA-RNA changes in retroposed genes were eliminated as well as sites in which the alternative nucleotide was supported by less than 2 RNA reads. RNA editing candidates in repetitive non-Alu regions and non-repetitive regions underwent more stringent filters. Indeed, we excluded positions in the first and last 6 bases of reads, with quality score less than 30, with DNA-RNA frequency change lower than 0.1, in homopolymeric regions longer than 5 bases, in which the alternative nucleotide was supported by less than 3 RNA reads. In addition, we removed duplicated reads and applied again the Blat filter. Resulting tables were used for downstream analyses by means of custom scripts.et al.RNA editing sites in hyper-edited reads were detected using the pipeline described in Porath All detected positions were finally annotated by ANNOVARhttp://beamish.ucc.ie/. Version 2 RADAR annotations were downloaded from http://rnaedit.com/.The comparison between our detected editing sites and available RNA editing databases, such as DARNED and RADAR, was performed by a custom script. DARNED annotations for human were downloaded from https://www.datatables.net/).A specialized database, containing the complete collection of human editing events with an interface designed to facilitate browsing and downloading of selected positions was designed in SQL using dedicated python scripts based on the sqlite3 module. Interactive tables showing results are dynamically generated using the JQuery plug-in DataTables .


Aspergillus niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes.Global climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from A. niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. niger  and three T. reesei  transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization.A hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.This study revealed the existence of a variety of xylose transporters in the cell factories The online version of this article (doi:10.1186/s13068-016-0564-4) contains supplementary material, which is available to authorized users. Aspergillus niger and Trichoderma reesei have become two of the main workhorses of today’s industrial biotechnology. Their high enzyme secretory capacity has already been industrially exploited during the last decades and both species have the capacity to efficiently degrade and utilize second-generation lignocellulosic feedstocks  was used for cloning experiments and plasmid propagation. It was grown at 37 °C on an LB medium , with 100 μg mL−1 ampicillin when required for transformants selection.A.niger N400 (CBS 120.49), NW199  -xylose or d-[U-14C]-glucose solution (Campro Scientific) was added. After exactly 20 s, the reaction was stopped by the addition of 1 mL of appropriate ice-cold quenching buffer , followed by vacuum filtration , and two subsequent washing steps with 5 mL of ice-cold quenching buffer. After 5 min of drying in the vacuum manifold, the filters were transferred to scintillation vials with 4 mL scintillation liquid , and activity was counted (Packard Tricarb 1600TR). All reactions were performed in triplicates. All values were corrected using triplicate negative control measurements without incubation, where the quenching solution was added prior to the addition of labeled substrate. Uptake rates at two typical substrate concentration ranges were measured; 1–100 μM and 0.1–40 mM. Substrate solutions with an activity of approximately 5–5000 Bq μL−1 were used. To determine kinetic parameters Km and Vmax, the data were fitted to the Michaelis–Menten model using the least squares method.Aliquots were incubated for 5 min at 30 °C in a heat block with vigorous shaking before uptake assay was started. To start the reaction, 10 μL of a 5 times concentrated A. niger [T. reesei [hist” transcript (gene ID 207921) [A. niger, and genes sar1 and act1 for T. reesei [RNA isolation from A. niger  and T. r. reesei  mycelium. reesei . Primer . reesei . Normali 207921)  for A. n. reesei .


The mangrove forest had a lower sound intensity of 70 dB re 1μPa.Hz-1 while the pass was characterized by a higher sound level with an average intensity of 91 dB re 1μPa.Hz-1. Habitats showed significantly different intensities for most frequencies, and a decreasing intensity gradient was observed from the reef to the shore. While habitats close to the shore showed no significant diel variation in sound intensities, sound levels increased at the pass during the night and barrier reef during the day. These two habitats also appeared to be louder in the North than in the West. These findings suggest that daily variations in sound intensity and across-reef sound gradients could be a valuable source of information for settling larvae. They also provide further evidence that closely related habitats, separated by less than 1 km, can differ significantly in their spectral composition and that these signatures might be typical and conserved along the coast of Moorea.As environmental sounds are used by larval fish and crustaceans to locate and orientate towards habitat during settlement, variations in the acoustic signature produced by habitats could provide valuable information about habitat quality, helping larvae to differentiate between potential settlement sites. However, very little is known about how acoustic signatures differ between proximate habitats. This study described within- and between-site differences in the sound spectra of five contiguous habitats at Moorea Island, French Polynesia: the inner reef crest, the barrier reef, the fringing reef, a pass and a coastal mangrove forest. Habitats with coral  were characterized by a similar sound spectrum with average intensities ranging from 70 to 78 dB re 1μPa.Hz Ambient sea noise is largely composed of sounds generated by abiotic sources (geophony), such as wind and waves, and biotic sounds (biophony) produced by various marine organisms –3. HowevData regarding the nature of marine soundscapes now exists for many locations globally, including sites within the Pacific –15, AtlaAlthough the key features of the acoustic signatures that distinguish some marine ecosystems have been identified, localized acoustic variability between adjacent habitats, i.e. those separated by less than 1 km, has rarely been characterized. On coral reefs for instance, how the soundscape of an inner reef crest differs from that of a barrier reef, and how temporally variable these soundscapes are is largely unknown. However, recent evidence suggests that spectral differences between spatially associated reef habitats can be largely due to variation in the sonic activity of marine organisms, i.e. soniferous fishes and snapping shrimps . AnotherThe aim of this present study was to describe the temporal and spectral acoustic features of five different, but spatially associated, marine habitats surrounding Moorea Island, French Polynesia. Because many coral reef-associated species have highly specialized habitat requirements we hypothesize that biotic variation between habitat types will create unique acoustic signatures that could provide key information for larval marine organisms during settlement.st to April 30, 2010 and 2011 at Moorea Island . Moorea has a large lagoon composed of five main habitat types: 1) the inner reef crest (CR), characterized by breaking waves, strong water flux, and a substratum comprised mainly of coral rubble with less than 10% live coral; 2) the barrier reef (BR), characterized by a water depth of 1–5 m and a substratum comprised of up to 30% live coral from a diverse range of species; 3) the fringing reef (FR) characterized by a water depth of 1–2 m and a substratum comprised of between 10–20% live coral from a limited number of species; 4) the pass (PA), a coral free area located in front of an opening in the reef crest, and canalizing the water flow to the ocean; and 5) the coastal mangrove forest (MG), comprised of a mud-sand substratum covered with mangrove trees located along the shoreline with a depth less than 1 m [The study was conducted from February 1than 1 m .Five GPS points were taken for RC, BR, FR and MG habitats, whilst only three GPS points were taken for PA habitat due to safety restrictions. In order to assess spatial and temporal variations, the study was repeated in two similar sites; one located on the North coast of Moorea and one located on the West coast . These sSounds were recorded from kayak using a HTI-96-MIN wide-band omni-directional hydrophone  connected to a calibrated Tascam DR-07 portable digital recorder . Sounds were digitized at 44.1 kHz (16-bit resolution). Replicate recordings at each GPS point were collected on the same day to control for potential between-day effects. At each GPS point, 1 to 3 recordings were taken so that the number of replicates varied between 5 and 15 for each habitat . RecordiRecordings were first cleaned by visually inspecting sonograms using Avisoft-SASlab Pro software  to detect and remove any unwanted anthropogenic sound sources  from the analysis. Power spectra  were then created from the analysis of 5s samples for each sound file and averaged to present the spectrum of each environment.Characteristics of each habitat were described on the basis of frequency modulations of power spectra. Mean values and standard deviations of intensities were calculated for all sampled frequencies, , for each sound file. Habitat sounds recorded on the North coast in 2011 were compared to each other (two-way ANOVA followed by Tukey's multiple comparison test). For each habitat, diel sound variations were determined by comparing mean intensities between day periods (day-dusk-night) for the North coast in 2010 and 2011, and for the West coast in 2010 (Two-way ANOVA followed by Tukey's multiple comparison test). Spatial variations within a given habitat type between each day period were determined between the North and West coasts in 2010 (Two-way ANOVA followed by Sidak's multiple comparison test). Finally, temporal variations within the same habitats and day periods were compared between both years for the North coast (Two-way ANOVA followed by Sidak's multiple comparison test). All analyses were performed using the GraphPad Prism 6 software .This study was conducted in accordance with the guidelines of the French Polynesia committee for publication ethics and no specific permissions were required for the recordings of the different habitats. Moreover, no organism was captured during the study. Thus, the recording experiments were approved by the CRIOBE animal ethics committee.-1. The power spectrum was characterized by a local maximum of 89 ± 4 dB re 1μPa.Hz-1 found at lower frequencies of ca. 200 Hz, before a slight intensity decrease down to a local minimum of 86 ± 3 dB re 1μPa.Hz-1 at frequencies from 700 to 1000 Hz. Intensities did not differ significantly between 1000Hz and 5000Hz (-1 (CR), 76 ± 8 dB re 1μPa.Hz-1 (BR) and 70 ± 11 dB re 1μPa.Hz-1 (FR) respectively. These habitats showed local peaks at ca. 170 Hz for intensities of 89 ± 7 dB re 1μPa.Hz-1, 82 ± 4 dB re 1μPa.Hz-1 and 84 ± 12 dB re 1μPa.Hz-1 for CR, BR and FR respectively. These peaks were then followed by a continuous decrease of intensities until local minima of 72 ± 6 dB re 1μPa.Hz-1 at ca. 900 Hz for the CR; 65 ± 4 dB re 1μPa.Hz-1 at ca. 950 Hz for the BR and 64 ± 11 dB re 1μPa.Hz-1 at 1120 Hz for the FR. At higher frequencies, intensities increased up to a plateau of ca. 80 dB re 1μPa.Hz-1 for the CR and the BR. Intensities of the FR at higher frequencies were lower, at ca. 75dB re 1μPa.Hz-1  (No significant daily differences were observed in the mangrove forest recordings (n  > 0.05) .North = 30; n West = 25; F2,511 = 62.00; P < 0.05). However, on the North Coast in 2011, no significant variation in intensity was observed for any frequency of the power spectrum between day periods  . These differences occurred for frequencies above 1600 Hz at dusk, and above 2100 Hz at night. There was no significant difference between sounds recorded at dusk and night . This difference was also identified in 2011 but only up to ca. 1700 Hz  , and for frequencies below 500 Hz at the North Coast in 2011. This difference was not found at the North Coast in 2010 where only frequencies between 1030 Hz and 2600 Hz differed between day and night. At the West Coast in 2010, significantly lower intensities were found at dusk for frequencies between 1000 Hz and 3000 Hz although no significant differences were identified between dusk and night at the North Coast in either year  with no difference observed between dusk and night. While no data is available for the North Coast in 2010, in 2011 intensity was significantly lower at night than during the day only for frequencies below 500 Hz   . At the nd night . At the  < 0.05) . Night rher year . Recordi < 0.05) .4,511 = 2; P > 0.05), or between the fringing reef and the mangrove forest , although during the day sound intensity tended to be lower at the fringing reef at frequencies between 250 Hz and 1800 Hz. During the day and night, sound levels were significantly higher at sites far from shore, the inner crest and barrier reef, than at those sites closest to shore, the fringing reef and mangrove forest . This tendency also occurred at dusk, although differences were not significant . Sounds of the barrier reef did not differ significantly between dusk and night although sound level was significantly higher at the North Coast during the day at frequencies below 500 Hz  (Sound acoustic signature of the mangrove forest did not differ between North and West Coasts at any diel period in 2010 (n  < 0.05) .North = 25; n West = 15; F2,511 = 48.0; P < 0.05) but there were no differences between sites at dusk and night   .Numerous studies have now shown that the ambient sounds produced by reefs can attract larval fishes and/or crustacean, suggesting that reef noise could play an important role during settlement , 35–38. In Moorea, reef habitats could be differentiated on the basis of their mean sound level alone. The pass had the highest sound level, most likely due to the rapid flow of water through the habitat that creates turbulence and friction against the substrate. In contrast, the relatively slow moving waters surrounding the coastal mangrove forest displayed the lowest sound level. Between these two habitats there was a declining gradient in sound intensity ranging from the inner reef crest (characterized by breaking waves and strong water flow), to the barrier reef , and the fringing reef . Therefore, it appears that high water flow and biodiversity-promoting coral cover may elevate mean sound levels with these factors resulting in louder and more frequent acoustic events. The pass, the inner reef crest and the barrier reef (i.e. the noisiest habitats) were further characterized by diel variations in sound intensity, with sound in the pass and the inner reef crest elevated at night, while the sound of the barrier reef was elevated during the day. The elevated nocturnal sound levels observed in habitats closest to the open ocean may help facilitate the detection of reefs by pelagic larvae, many of which approach and settle onto the reef at night. Only weak variations (i.e. at frequencies below 1000 Hz) occurred between day and night on the fringing reef while sound intensity in the mangrove forest did not vary at all. Thus, a spatial gradient of diel sound variation appears to exist, ranging from strong variations on the reef crest to little or no variation close to shore. The larvae of species from a variety of reef habitats appear to use reef noises to locate appropriate settlement sites. With this in mind, these results suggest larvae could potentially use the acoustic gradient that exists between the outer reef and shoreline to orientate themselves within the lagoon and promote movement towards or away from specific areas. Besides characterizing different habitats, acoustic variation may also be used by larvae to judge relative habitat quality as there is some evidence that sound intensity is greater on healthy reefs compared to those that are degraded .-1, 20–5000 Hz) likely due to the stronger water flow which can increase sound levels across a broad range of frequencies [ca. 10 dB higher sound level found at the North Coast, where the pass is only ~180 m wide, creating stronger water flow than is found on the West Coast where the pass is wider at ~220 m [The soundscape of the pass differed most substantially from other habitats, with the highest mean sound intensity  showed a similar spectral pattern with a local maximum peak at ca. 180 Hz decreasing to a local minimum between 690 Hz and 1035 Hz. This general pattern is similar to spectral curves recorded on reefs on the Great Barrier Reef, Australia [Habitats characterized by the presence of coral , it did increase the amount of coral debris and terrigenous inputs that may have resulted in some unrecognised change to lagoonal sound spectra, be it due to changes in biological activity  or via sOverall, these results highlight that, on coral reefs, variations in acoustic signatures can occur at small spatial scales due to habitat-specific biotic and abiotic factors. As many larvae have specialised habitat requirements, and use sounds to locate these habitats as they return to the reef, acoustic variations may help explain differential patterns of settlement. Although general similarities existed between habitats characterized by live coral, the presence of small but consistent spectral differences between these coral rich habitats suggest that larvae may be able to use these cues to orientate within heterogeneous environments.


There is evidence of a prominent role of Src in tumor progression-related events such as the epithelial–mesenchymal transition (EMT) and the development of metastasis. However, the precise molecular interactions of Src with metastasis suppressors remain unclear. Herein, we review known metastasis suppressors and summarize recent advances in understanding the mechanisms of how these proteins inhibit metastasis through modulation of Src. Particular emphasis is bestowed on the potent metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1) and its interactions with the Src signaling cascade. Recent studies demonstrated a novel mechanism through which NDRG1 plays a significant role in regulating cancer cell migration by inhibiting Src activity. Moreover, we discuss the rationale for targeting metastasis suppressor genes as a sound therapeutic modality, and we review several examples from the literature where such strategies show promise. Collectively, this review summarizes the essential interactions of metastasis suppressors with Src and their effects on progression of cancer metastasis. Moreover, interesting unresolved issues regarding these proteins as well as their potential as therapeutic targets are also discussed.A major problem for cancer patients is the metastasis of cancer cells from the primary tumor. This involves: (1) migration through the basement membrane; (2) dissemination via the circulatory system; and (3) invasion into a secondary site. Metastasis suppressors, by definition, inhibit metastasis at any step of the metastatic cascade. Notably, Src is a non-receptor, cytoplasmic, tyrosine kinase, which becomes aberrantly activated in many cancer-types following stimulation of plasma membrane receptors ( Metastasis is a complex cascade process that involves a number of sequential events by cancer cells in order to “escape” from the primary tumor, penetrate tissue barriers, migrate to distant sites through the circulation and invade new organs (secondary site) to form new tumors . HoweverSrc (v-Src) gene encoded by the Rous sarcoma virus was the first defined oncogene and encodes the first recognized tyrosine kinase . Th. Th36]. c-Src is normally present in its inactive form, in which Tyr527 is phosphorylated and stabilized by two key intra-molecular interactions, including: (1) binding of phosphorylated Tyr527 to its own SH2 domain; and (2) binding of the SH2-kinase linker to the SH3 domain Figure  39]. Th. Th39]. The regulation of c-Src activity by intra-molecular interactions suggests that it can also be regulated by interaction with molecules that compete with the functional domains. Indeed, c-Src binds to various tyrosine-phosphorylated proteins by recognizing specific phosphopeptide sequences via the SH2 domain . For exavia the ubiquitin-proteasome pathway  a aNDRG1 gNDRG1 is a multifunctional protein involved in tumorigenesis and tumor development, and its function differs in different tumor-types , 24, 141in vitro and in vivo [In vitro studies demonstrated that over-expression of NDRG1 significantly decreased the proliferation rate of MCF7 breast and EJ bladder cancer cell lines [in vivo, cell growth was not affected in vitro, potentially due to in vivo modulatory factors such as those associated with the stroma and angiogenesis [NDRG1 has been demonstrated to inhibit primary tumor growth  in vivo , 146. Inll lines . Moreovell lines . Furtherll lines . Subsequll lines . Interesogenesis .via attenuating the expression and phosphorylation of the inhibitor of κB kinase (IκBα) and subsequently NF-κB signaling [The establishment of metastatic lesions is dependent upon successful initiation of angiogenesis, a process that is essential for providing the oxygen and nutrients required for cell growth. Interestingly, NDRG1 was found to inhibit the process of angiogenesis by negatively regulating critical pro-angiogenic factors, such as IL-8, MMP-9 and VEGF1, in pancreatic cancer . Moreoveignaling .in vitro [in vivo [In addition to its effects on primary tumor growth, the role of NDRG1 as a metastasis suppressor has been demonstrated in vitro , 28 and [in vivo , 148. ND[in vivo  and to i[in vivo . NDRG1 e[in vivo . Further[in vivo . In cont[in vivo .via proteins including MMPs, which degrade extracellular matrix, and adhesion molecules, such as β-catenin and E-cadherin that form the adherens junction at the cell membrane [Recently, NDRG1 has been shown to suppress metastasis by a mechanism involving the modulation of the structural protein actin . In cancmembrane , 34, 154membrane , 34. Furmembrane . Togethevia decreasing EGFR expression, leading to loss of activated EGFR, and thus, preventing the EGFR-c-Src interaction  class, including di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Figure or cells , 183. Weor cells . Notablyor cells , 184, whAs the field grows, and additional novel strategies for therapeutic intervention are developed, the number and kind of targets are likely to increase. Moreover, future candidate metastasis suppressor genes may also prove tractable as pharmacological targets.This review has briefly summarized the known molecular interactions between the potent oncogene Src and a variety of metastasis suppressors. However, as discussed, the role of Src in cancer metastasis can be multifaceted, with this latter oncogene being able to negatively regulate a number of metastasis suppressors, while itself being a target for these proteins. This further indicates the complex relationship between Src and metastasis suppressors, which participate in a delicate balance that ultimately determines a cell's ability to invade and metastasize. Importantly, understanding the complex interaction between c-Src and key metastasis suppressors such as NDRG1 has resulted in the development of new anti-metastatic therapies such as the novel agents, Dp44mT and DpC.


RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant–virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces  Viral infection of plants triggers the expression of an RNaseIII enzyme that represses the production of siRNAs by cleaving their long dsRNA precursors. Read the accompanying Synopsis. Most eukaryotes produce essential regulatory molecules called small RNAs. These molecules are produced primarily by a class of RNaseIII enzymes called DICER, which excises small RNA duplexes from long double-stranded (ds)RNA precursor molecules. Plants also encode several RNaseIII enzymes called RNASE THREE-LIKE (RTL), but the function of these proteins is largely unknown. Here, we show that RTL1 represses small RNA production by cleaving dsRNA before DICER can process them. RTL1 appears to specifically act on the templates of a class of small RNAs called siRNAs, but not on miRNA precursors, suggesting that it cleaves long-perfect (or near-perfect) dsRNA, but not short-imperfect dsRNA. We also found that RTL1 expression is induced after virus infection, suggesting that RTL1 could act as an inducible antiviral defense by destroying dsRNA intermediates of viral replication. Our findings suggest that viruses have evolved to inhibit RTL1 activity, ultimately resulting in successful viral infection. Saccharomyces cerevisiae Rnt1 and S. pombe Pac1 proteins. Both of these yeast proteins are longer than bacterial RNaseIII proteins and contain an additional 100 amino acids at their N-terminus. Class III proteins have a DRB and two RNaseIII domains and include Drosha, which is involved in the first cut of miRNA precursors in animals but is incapable of producing small RNAs by itself. Class IV proteins correspond to animal and plant Dicer and contain an RNA helicase domain, a PAZ domain, either one or two RNaseIII domains, and one or two DRB domains. Animal and plant Dicer proteins are the only RNaseIII proteins that have been shown to produce small RNAs in the size range of 18–24 nt, with the exception of class II RNaseIII from a few budding yeasts [In eukaryotes, the biogenesis of small RNAs is either Dicer-dependent or Dicer-independent. Dicer-independent small RNAs resulting from the action of RNA-dependent RNA polymerases, exoribonucleases, Argonaute (AGO) proteins, or a combination of these factors have been found in fungi, invertebrates, and mammals but not in plants or protists –4. In cog yeasts .Arabidopsis encodes four Dicer-like (DCL) proteins [MIR genes. By contrast, DCL2, DCL3, and DCL4 produce 22, 24, and 21 nt small interfering RNAs (siRNAs), respectively, which derive from long, dsRNA precursors originating from either long inverted repeats or from the action of RNA-dependent RNA polymerases (RDR) on single-stranded RNA precursor. Multiple classes of siRNAs exist in plants. The largest class corresponds to POLYMERASE IV-dependent siRNA (p4-siRNAs), which are RDR2- and DCL3-dependent 24 nt siRNAs deriving from transposons and repeats. Trans acting siRNAs (ta-siRNAs) are RDR6- and DCL4-dependent 21-nt siRNAs, which derive from non-protein coding TAS genes. Endogenous inverted repeat-derived siRNAs (endoIR-siRNAs) do not depend on an RDR for their production because they derive from very long stem-loops. They come in different flavors, including DCL4-dependent 21 nt, DCL2-dependent 22 nt, and DCL3-dependent 24 nt siRNAs. At last, young miRNAs define a category of small RNA that is intermediate between DCL2-, DCL3-, and DCL4-dependent endoIR-siRNAs and DCL1-dependent old (conserved) miRNAs. Indeed, young miRNAs derive from foldback stem-loops of intermediate sizes, which are mainly processed by DCL4. Most 21 and 22 nt small RNAs are loaded onto AGO1 to guide the cleavage of complementary mRNA [The plant model proteins , which pary mRNA .DCL2, DCL3, and DCL4 also produce siRNAs in response to the entry of exogenous genetic material. For example, transient transgene expression leads to a consistent production of 21, 22, and 24 nt siRNAs, even when the transgene has not been designed to produce an RNA that can fold into a dsRNA structure . FollowiArabidopsis genome has the capacity to encode five proteins referred to as RNASE THREE-LIKE (RTL). RTL1, RTL2, and RTL3 harbor RNaseIII and DRB domains [Arabidopsis stock centers, and RTL1 function is unknown. RTL2, which contains one RNaseIII domain and two DRB domains, is ubiquitously expressed at low level [45S pre-rRNA and enhances the production of exogenous siRNAs when overexpressed [Aside from DCL1–4, the  domains , whereas domains ,21. RTL1 domains . Mutantsow level . RTL2 prxpressed ; howeverxpressed . RTL3 haxpressed . RTL4 anxpressed . RTL5 shArabidopsis wild-type plants grown under laboratory conditions, RTL2, RTL4, and RTL5 are expressed throughout the plant. In contrast, RTL1 is weakly expressed in roots, while RTL3 is below detectable levels in all tested tissues ([http://jsp.weigelworld.org/expviz/expviz.jsp). Moreover, RTL1 and RTL3 have no known biological or biochemical function. Given that several key components of antiviral RNAi, including the Arabidopsis AGOs AGO1, AGO2, and AGO5 and rice AGO2 and AGO18, are induced during virus infection [Arabidopsis (Col) plants with different single-stranded RNA viruses: turnip crinkle virus , turnip vein clearing virus , cucumber mosaic virus , and turnip yellow mosaic virus , and we measured the accumulation of RTL1 and RTL3 mRNA. The accumulation of RTL2 mRNA was also measured to examine a possible contribution of this protein during virus infection because it also harbors RNaseIII and DRB domains and is weakly expressed under regular conditions [RTL1 mRNA accumulation was observed in systemically infected leaves, whereas the level of RTL2 and RTL3 mRNA remained largely unchanged . Then, RTL1 localization was analyzed by immunostaining of root cells  instead of virus-infected plants. These plants exhibited a range of developmental defects, including smaller stature, yellowing and curled rosette leaves, late flowering, and sterility  were also sequenced for comparison. Flowers were chosen because it is the tissue producing the highest variety of small RNAs. The distribution of the 17 to 30 nt reads matching the nuclear Arabidopsis genome but excluding tRNA and rRNA sequences was examined  were identified. Assembling overlapping sliding windows defined 10,814 nonoverlapping regions showing a decreased accumulation of small RNAs in 35S:RTL1 plants and only 16 regions showing an increased accumulation of small RNAs in 35S:RTL1 plants  was introduced transiently into Nicotiana benthamiana leaves together with either 35S:RTL1 or a 35S:GFP control. As expected, 35S:GU-UG + 35S:GFP infiltrated leaves accumulated high levels of 21 and 24 nt GU siRNAs, whereas 35S:GU-UG + 35S:RTL1-infiltrated leaves did not accumulate detectable levels of GU siRNAs -, TCV-, TVCV-, CMV-, and TYMV-inoculated Col plants. TAS2 ta-siRNA accumulation was strongly reduced in TYMV-inoculated plants  were able to suppress the RTL1 overexpression phenotype conferred by the 35S:RTL1 construct, despite an abundant accumulation of the two amiRNAs . Consistent with the symptoms, viral RNA accumulated at high levels in inoculated Col and 35S:RTL1-Flag plants  revealed that RTL1 inhibits the accumulation of all types of small RNAs normally produced by DCL2, DCL3, or DCL4 from ~6,000 loci. Because RTL1 overexpression does not affect the accumulation of DCL1-dependent conserved miRNAs, which are processed from imperfectly-paired short hairpins, it is likely that RTL1 specifically affects perfectly paired long dsRNA, such as the precursors of ta-siRNAs and p4-siRNAs, which derive from RDR activity, or near-perfectly paired long dsRNA, such as the precursors of young miRNAs and of endoIR-siRNAs. Consistently, in vivo and in vitro assays show that RTL1 cleaves dsRNA. Because siRNAs are almost totally absent in 35S:RTL1 plants where the four DCLs are present, RTL1 likely has a very high affinity for long dsRNA and thus cleaves dsRNA before they enter processing by the DCLs. RTL1 constitutive expression does not result in the accumulation of a novel size class of small RNA, indicating that RTL1 does not act as a DCL enzyme and does not produce small RNA per se. Rather, it likely cleaves long dsRNA precursors into multiple fragments, which subsequently are degraded into nucleotides by 5’-to-3’ and/or 3’-to-5’ exoribonucleases. Exceptionally, RTL1-mediated cleavage of dsRNA precursors can give rise to molecules that are better processed by another DCL than the original dsRNA, likely because these cleavage products are stable and/or adopt a conformation that makes them a better substrate for one DCL. For example, the hairpin located at the 3’UTR of At3g18145 appears to be a bad substrate for DCL1, resulting in a very low production of the 21 nt miR3440b. In contrast, an abundant production of a 24 nt siRNA species is observed in 35S:RTL1 plants . For comparison of RTL1 expression between roots and leaves of Col, and between leaves of Col and Arabidopsis was transformed using the floral-dip method.For virus infection, 10-d-old in vitro plants were inoculated with TVC, TVCV, CMV, and TYMV 1 d after being transferred to soil. Virus inoculations were performed as previously described ,34. PlanN. benthamiana agroinfiltrations, 10 ml cultures of Agrobacterium tumefaciens C58C1 (pMP90) carrying the plasmids of interest were grown overnight at 28°C and then centrifuged at 6,000 rpm. The bacterial pellets were suspended at 10 mM MgCl2, 10 mM MES pH 5.2, 150 μM acetosyringone solution to a final OD600 of 1.0. The resuspended bacterial solution was incubated at 20°C for 3 h and then used to infiltrate leaves. Leaves were harvested 3 d after infiltration.For 35S:RTL1 and 35S:RTL1-tagged constructs, RTL1 (At4g15417) genomic sequence starting at the ATG and ending at the stop codon was PCR-amplified and cloned into the pDONR207 (Gateway Technology-Invitrogen) using attB1RTL1F and attB2RTL1R Gateway-adapted oligonucleotides . The G89 and D96 amino acids were replaced by an A using the RTL1m2-F and RTL1m2-R oligonucleotides  according to the manufacturer.For production of a His-RTL1 recombinant fusion protein, a full-length RTL1 cDNA was amplified by a reverse transcription polymerase reaction (RT-PCR) as described before . The RTL primers  with theRTL1 RT-PCR analysis, cDNA was synthesized using 2 μg of total RNA digested with DNaseI (Fermentas). 500 ng of final DNA-free RNA was used for a reverse transcription with oligo-dT (Fermentas). The PCR was performed using Taq DNA polymerase (Thermo Scientific-Fermentas) and 1 μl of RT reaction using RTL1-6 and RTL1-7 oligonucleotides . Gene expression was normalized to GAPDH expression. Primer sequences are listed in For quantitative RT-PCR, total RNA was extracted using RNeasy plant mini kit (Qiagen). 1 μg of total RNA was digested with DNaseI (Fermentas) and converted to cDNA using the Revertaid H minus reverse transcriptase (Thermo Scientific) and oligo dT. Plant protein extraction and western blots using Flag antibodies were performed as previously described . For wesThe recombinant His-RTL1 proteins were produced following Novagen's instructions. Briefly, lysis was performed in Binding buffer supplemented with DNase (20 ug/ul), MgCl2 , and Protease inhibitor cocktail (Roche). After cell disruption and centrifugation, the soluble fraction was purified using the Ni2+ resin. Following elution, purified His-RTL1 proteins were finally dialyzed against Sample Buffer .A. thaliana plantlets using TriZol reagent . After treatment with Turbo DNase (Ambion), 500 ng of RNAs were premixed in reaction Buffer , then were incubated with 100 ng of recombinant RTL1 or Sample Buffer, in Reaction Buffer for 10 min at 37°C, the reaction was stopped by heating samples at 70°C for 5 min. 50 ng of treated RNAs were used to realize reverse transcription (RT) (Promega) with IR71rt, IR2039rt: 3'UTRrev and U3rt primers, followed by 42 cycles of PCR with IR71fwd2 and IR71rev2, IR2039fwd and IR2039rev, 3'UTRfwd and 3'UTRrev, and U3fwd and U3rt primers, respectively, using the Gotaq PCR Kit (Promega).For the cleavage assay, total RNA was extracted from 14-d-old For denaturation, the RNA premix was boiled at 70°C for 5 min and immediately placed on ice at least 5 min before adding proteins. Recombinant RTL1 or Sample Buffer were added and incubated for 1 h on ice. Finally, the reaction was stopped by heating samples to 70°C for 5 min. RT reactions were performed with IR71rt and U3rt primers, followed by 42 cycles of PCR with IR71fw2 and IR71rev2 and U3fw and U3rt primers, respectively.http://code.google.com/p/mosaik-aligner/) and Bowtie [ad hoc scripts, and indicate normalized read counts. Data were normalized with respect to Col-0 under the assumption that the 27 conserved miRNA  [www.ncbi.nlm.nih.gov/geo (accession no. GSE49866).The 3' adapters of the reads were removed with S-MART  tools, td Bowtie  to the T08, 472)  where gl08, 472) . EndoIR-08, 472) , which d08, 472) . Young m08, 472)  and discArabidopsis RTL1 were retrieved using TblastN. DCL sequences were discarded based on the presence of RNA helicase and PAZ domains upstream of the RNaseIII and DRB domains. In each species, the RTL1 ortholog was identified based on the presence of a single RNaseIII domain and a single DRB domain. Proteins were aligned using MultiAlign.Plant protein sequences homologous to For the subcellular localization of RTL1-GFP, onion epidermal layers were transfected using the PDS-1000/He biolistic. GFP microscopic images were taken using a Zeiss Axioskop 2 microscope and recorded using a Leica DC 300 FX digital camera (Leica).For subcellular localization of native RTL1, immunofluorescence was performed using 8 d-old roots as described previously . TreatedS1 Data(XLSX)Click here for additional data file.S1 Fig35S:RTL1 (RTL1) and 35S:RTL1-Flag (RTL1-Flag) plants were hybridized each with a different probe and then rehybridized with U6 as a loading control. A simplified figure showing one representative U6 control is shown in Ten identical gel blots of total RNA from flowers of wild-type (Col), (TIF)Click here for additional data file.S2 FigSmall RNA abundance was normalized to the total amount of conserved miRNAs. The distribution of ta-siRNAs along the chromosome is shown on the left, and the size distribution is shown on the right. The sizes are indicated by different colors: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).(TIF)Click here for additional data file.S3 FigSmall RNA abundance was normalized to the total amount of conserved miRNAs. The distribution of endoIR-siRNAs along the chromosome is shown on the left, and the size distribution is shown on the right. The sizes are indicated by different colors: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).(TIF)Click here for additional data file.S4 FigSmall RNA abundance was normalized to the total amount of conserved miRNAs. The distribution of PolIV/PolV-siRNAs along the chromosome is shown on the left, and the size distribution is shown on the right. The sizes are indicated by different colors: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).(TIF)Click here for additional data file.S5 FigSmall RNA abundance was normalized to the total amount of conserved miRNAs. The distribution of young miRNAs along the chromosome is shown on the left, and the size distribution is shown on the right. The sizes are indicated by different colors: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).(TIF)Click here for additional data file.S6 FigSmall RNA abundance was normalized to the total amount of conserved miRNAs. The distribution of small RNAs along the chromosome is shown on the left, and the size distribution is shown on the right. The sizes are indicated by different colors: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).(TIF)Click here for additional data file.S7 FigA) The Arabidopsis Information Resource (TAIR) annotation of the genomic region spanning the At3g18145 gene. B) Predicted hairpin structure of the 3’ UTR of the At3g18145 RNA. C) Distribution of small RNAs along the At3g18145 locus in wild-type Col, 35S:RTL1, and dcl234. Reads are normalized to the total number of conserved miRNA reads. D) Size distribution of small RNAs from the At3g18145 locus in Col, 35S:RTL1, and dcl234 triple mutant. The number of reads of each size of small RNAs is indicated by a color code: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red). Reads are normalized to the total of conserved miRNAs. E) RNA gel blot detection of 24 nt siRNA from the 3’UTR of At3g18145 in wild-type (Col), Col transformed with the 35S:RTL1 construct (Col/RTL1), and dcl2dcl3dcl4 mutants transformed with the 35S:RTL1 construct (dcl234/RTL1). Transformants exhibiting the strongest RTL1 developmental phenotype were analyzed. LMW RNAs were hybridized with a probe complementary to the 24 nt siRNA from the 3’UTR of At3g18145 and with U6 as loading control.(TIF)Click here for additional data file.S8 FigSmall RNA abundance was normalized to the total amount of conserved miRNAs. The distribution of small RNAs along the chromosome is shown on the left, and the size distribution is shown on the right. The sizes are indicated by different colors: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).(TIF)Click here for additional data file.S9 FigA) Representative phenotypes of wild-type plants (Col) and transgenic plants expressing the indicated tagged-RTL1 constructs. B) Total RNA was extracted from two independent transformants expressing each of the indicated tagged-RTL1 constructs and hybridized with a TAS2 probe and with U6 as a loading control. C) The same constructs were introduced into line L1. Total RNA was extracted from two independent transformants expressing each of the indicated tagged-RTL1 constructs and hybridized with a GUS probe and with U6 as a loading control. Note that the images of (TIF)Click here for additional data file.S10 FigA) RNA gel blot detection of the artificial miRNA amiR-RTL1b in N. benthamiana leaves infiltrated with constructs under the control of the 35S (35S::amiRb) or UBQ10 (UBQ10::amiRb) promoter. Both constructs produced similar amounts of amiR-RTL1b. B) RNA gel blot detection of the artificial miRNAs amiR-RTL1a and amiR-RTL1b in a series of transgenic Arabidopsis carrying the UBQ10::amiRa or UBQ10::amiRb constructs.(TIF)Click here for additional data file.S11 Fig35S:RTL1-Myc and 35S:RTL1mR3-Myc plants. Ten-d-old plants were inoculated. Pictures were taken three weeks following inoculation.Pictures of mock- and TYMV-inoculated wild-type (Col), (TIF)Click here for additional data file.S12 FigArabidopsis thaliana (NP_680697.4), Brachypodium distachyon (XP_003560753.1), Cicer arietinum (XP_004502137.1), Cucumis sativus (XP_004159229.1), Fragaria vesca (XP_004287969.1), Oryza sativa (NP_001057601.1), Populus trichocarpa (XP_002301611.1), Solanum lycopersicum (XP_004243532.1), Sorghum bicolor (XP_002444209.1), Vitis vinifera (XP_002270948.2), and Zea mays (ACG34313.1).Alignment of RTL1 amino acids sequences from (TIF)Click here for additional data file.S1 TableAll reads: total number of reads per library. 17–30: number of reads ranging between 17 and 30 nt. Mapped: number of 17–30 nt reads matching the 5 Arabidopsis chromosomes. No rRNA-tRNA: number of 17–30 nt reads matching the 5 Arabidopsis chromosomes after eliminating rRNA and tRNA. Total miRNA: number of reads matching the hairpins of all registered miRNA. Conserved miRNA: number of reads corresponding to the mature form of the 27 conserved miRNAs(XLSX)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 TableMaximal distance admitted to define a locus = 1 kb.(XLS)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC. In eukaryotic cells, nascent mRNAs are transcribed as precursor mRNAs (pre-mRNAs) in the nucleus by RNA polymerase II (RNAPII). pre-mRNAs undergo processing steps to become mature mRNAs and form messenger ribonucleoproteins (mRNPs), complexes of mRNA and proteins that are transported to the cytoplasm through the nuclear pore complex (NPC) and translated into proteins.C-terminal domain of RNAPII, which promotes mRNA processing such as 5' capping, splicing, and 3' cleavage and polyadenylation [During transcription, mRNA processing factors are co-transcriptionally associated with the nylation . Trans-anylation ,3,4.The fully processed mRNP includes an export receptor that allows mRNP to pass through the NPC. Two major export receptors are implicated in two distinct mRNA export pathways across the NPC: a Nxf1-Nxt1/Tap-p15 (Mex67-Mtr2 in yeast) heterodimer and Crm1/Xpo1. Nxf1 harbors an RNA binding domain, but free Nxf1 forms a closed conformation that cannot bind RNA efficiently in human cells. Binding of TRanscription-EXport complex 1 (TREX-1) to Nxf1 causes Nxf1 to adopt an open conformation that can efficiently bind mRNA [The NPC is one of the largest protein complexes in eukaryotic cells, penetrating the inner and outer nuclear membrane. A fully assembled NPC has an estimated molecular mass of ~125 MDa in vertebrates . Its thrThe RNA molecules that are exported from the nucleus into the cytoplasm include not only mRNAs, but also ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and some types of viral mRNA. rRNA is a component of a ribosomal subunit and functions in translation. tRNAs, also essential molecules for translation, carry specific amino acids to the ribosome. snRNAs and proteins form small nuclear ribonucleic particles (snRNPs), which are the core component of the spliceosome. pre-mRNA splicing at the spliceosome requires at least five types of snRNP containing U1, U2, U4, U5, and U6 snRNA. Additionally, U3 snRNA is required for rRNA processing , and U7 In this review we also briefly introduce the mRNA export system of certain viruses whose genome is transferred into the nucleus as viral RNP. Although the export system of viral mRNA from the nucleus differs according to the type of virus, as examples we will describe the systems of Mason–Pfizer monkey virus (MPMV) and human immunodeficiency virus 1 (HIV-1) in this article.The concept of this review is to introduce the major export pathway of each type of RNA through the NPC . We direSchizosaccharomyces pombe is not essential for mRNA export, and overexpression of spMex67p inhibits mRNA export [The major export receptors of mRNA in mammalian cells are Nxf1 and Crm1. Nxf1 is thought to be the most important mRNA export receptor. Nxf1 forms a heterodimer with Nxt1 that can bind mRNA via TREX-1. TREX-1 is conserved from yeast to humans. Yeast TREX-1 binds mRNA in a transcription-dependent manner, whereas human TREX-1 is recruited to mRNA in a splicing-dependent manner ,13,14. MA export . In the A export .Drosophila [in vitro [The function of Nxt1 is still largely unknown. Nxt1 stimulates binding of the Nxf1-RNA complex to nucleoporin p62, and a Nxf1 mutant defective in binding to Nxt1 cannot promote mRNA export in humans . A recenosophila . Interesosophila  and promin vitro ,25. ThesSaccharomyces cerevisiae (AlyRef in humans) interacts with the 3' end processing factor Pcf11 [TREX-1 consists of AlyRef (Aly/Thoc4/Ref/Bef), Uap56, Cip29, pDIP3, ZC11A, and the THO subcomplex, which consists of Thoc1 (THO1/Hpr1/p84), Thoc2 (THO2), Thoc3 (THO3/Tex1), Thoc5 (THO5/fSAP79), Thoc6 (THO6/fSAP35), and Thoc7 (THO7/fSAP24). Recruitment of TREX-1 and export of mRNP requires the 5' capping of pre-mRNA because CBP80, the component of the cap binding complex (CBC) that binds on the 5' capping site, interacts with AlyRef and the THO subcomplex ,27. Cip2Recently, a novel TREX-1 component was identified and named Chtop . Chtop, D. melanogaster [D. melanogaster [M. musculus, R. norvegicus, and other organisms [A. thaliana, the putative orthologs of Thoc1 and Thoc3 are required for small interfering RNA (siRNA) biosynthesis [S. pombe; Mlo3, the essential factor for mRNA export in S. pombe, is required for siRNA production [Several components of TREX-1 participate in the biogenesis of small RNA. Uap56 functions in the export of Piwi-interacting RNA (piRNA) precursor in nogaster . piRNA fnogaster . Piwi prnogaster  and sincrganisms . In A. tynthesis ,36. siRNynthesis . A relatoduction ,39. The When an mRNP passes through the NPC with nuclear export receptor and enters the cytoplasmic face, a conformational change in the exported mRNP called “remodeling” occurs. This process is essential for mRNA dissociation in the cytoplasm.S. cerevisiae. The main player in the remodeling machinery is the DEAD-box helicase Dbp5 (DDX19 in vertebrates) [6) [6 and Nup159-triggered ADP release [The following insights were obtained from studies of ebrates) ,41,42. Debrates) ,44. The  release ,46. Afte release ,47.It remains unclear whether these regulatory mechanisms are fully conserved in vertebrates, although the human homologs of Dbp5 and Gle1 can be detected in the cytoplasmic filaments of the NPC and are thought to function in mRNP remodeling. In fact, depletion of DDX19 or hGLE1 results in nuclear accumulation of bulk poly(A) RNA ,48. DDX1i.e., silenced genes) associates with the inner nuclear membrane. The binding of genes to the NPC requires interaction with the mRNA export machinery and transcribed genes [In yeast, the chromatin in the nucleus does not diffuse randomly but is attached to the nuclear membrane. Genome-wide approaches have revealed that some components of the NPC associate with active genes ,52. In ced genes . The reled genes , whereased genes . Other red genes ,57. Sileed genes , implyinC-terminal domain.The recruitment of actively transcribed genes to the NPC depends on the association of Spt/Ada/Gcn5 acetyltransferase (SAGA) complex and TREX-2, which functions as an anchor. SAGA is one of the chromatin-modifying transcriptional co-activator complexes and consists of 21 proteins. SAGA thus forces active gene loci to physically contact the NPC via TREX-2. SAGA and TREX-2 are anchored into the nucleoplasmic side of the NPC via the NPC components Mlp1 and Nup1, respectively ,59. The D. melanogaster [A. thaliana [SAGA was first found in yeast, although homologous proteins have been identified in other eukaryotes. SAGA also functions as an anchor of transcription sites to NPC in nogaster , in whicnogaster . A homolthaliana . Human Tthaliana . Recent thaliana ,70, and thaliana . TREX-2 thaliana . Therefothaliana ,73,74.S. cerevisiae [Nxf1 functions in bulk mRNA transport, whereas rRNAs, U snRNAs, and a certain subset of mRNAs are exported in a Crm1-dependent manner ,76. The revisiae ,78, wherrevisiae . The rolrevisiae ,81. Crm1C-terminal domain that is required for binding of Nxf1 to the NPC. Instead, Nxf3 possesses a Crm1-dependent export signal and can tether RNA, and therefore functions in the Crm1-dependent RNA export pathway [To date, three adaptor proteins have been shown to function in the Crm1-dependent mRNA export pathway: RNA-binding protein human antigen R (HuR), leucine-rich pentatricopeptide repeat protein (LRPPRC), and nuclear export factor 3 (Nxf3) . HuR can pathway . In mice pathway . There a pathway .7G-cap and CBC [Like mRNA, spliceosomal U snRNAs are transcribed by RNAPII and have the same m and CBC . However and CBC . The hig and CBC .Many factors engage in mRNP transport via Crm1, including GTP binding Ran (Ran-GTP), RanBP1, RanBP2 (Nup358), RanBP3, and RanGAP. Crm1 interacts with the nuclear export signal (NES) of its cargo in a RanGTP-dependent manner and RanBP3 supports this interaction . Crm1 cacis-acting RNA transport element [Interestingly, RBM15 promotes the export of mRNAs containing the  element , and thi element . TherefoThe ribosome that serves as the machinery of protein synthesis (translation) is a large complex that consists of two subunits formed by ribosomal RNAs (rRNAs) and ribosomal proteins (RPs). The small subunit (the 40S subunit) contains 18S rRNA, and the large subunit (the 60S subunit) contains 5S, 5.8S, and 28S (25S in yeast) rRNAs . All rRNS. cerevisiae, the pre-60S subunit is exported by Crm1 [In  by Crm1 ,101, Mex by Crm1 , or Arx1 by Crm1 . The Mex by Crm1 . The pre by Crm1  or Mex67 by Crm1 . Ltv1 an by Crm1 ,108,109. by Crm1 ,110,111.In mammals, the pre-60S subunit is exported by Crm1  or ExporThe export of pre-ribosomes is closely related to their proper assembly. Therefore, it is often difficult to distinguish between the factors that are required to complete processing of pre-ribosomes in the nucleus and those that facilitate the nuclear export of pre-ribosome.Transfer RNA plays an essential role in translation by transporting amino acids to the ribosomal complex for peptide chain elongation. tRNA genes are scattered throughout the human and yeast genomes, and are transcribed by RNAPIII. At M phase of the cell cycle, the transcriptional level of tRNA peaks because of the association of tDNA with Nup2 and Nup60 via Cohesin . InteresS. cerevisiae [A export receptor is also required for tRNA to cross the NPC meshwork . The mairevisiae ,127,128.S. cerevisiae [A. thaliana Xpot homolog PAUSED is nonessential, and insects lack a Xpot homolog [S. pombe [The yeast los1∆ strain shows nuclear accumulation of end-processed intron-containing tRNAs. A defect in tRNA export results in such a phenotype because the splicing machinery for tRNA is located on the outer surface of the mitochondria ,129,130.revisiae . However homolog ,135,136.S. pombe ,139,140.Drosophila [S. cerevisiae msn5∆ strain shows nuclear accumulation of intronless tRNA, but not intron-containing tRNA [The importin-beta family member Msn5/exportin-5 (Xpo5) is also postulated to be involved in tRNA export in yeast and osophila ,131,141.osophila ,144,145.After transcription and export, tRNA is modified and contributes to translation in the cytoplasm. However, tRNA can also undergo retrograde import into the nucleus. This process is conserved from yeast to humans ,123,146.MicroRNAs are small RNA molecules of approximately 22 nt that regulate gene expression via RNA silencing. miRNA is processed in both the nucleus and the cytoplasm; therefore, the miRNA traversing the NPC is a precursor miRNA (pre-miRNA) rather than a fully processed miRNA.Most of the miRNA is transcribed as primary miRNA (pri-miRNA) by RNAPII in the nucleus, and pri-miRNA has a 5' cap, poly(A) tail, and local hairpin structure ,150. Mat7G-cap are exported in this pathway  [The pathway described above is a canonical pathway; in addition, three non-canonical pathways have been described, a Drosha- and DGCR8-independent pathway; a Dicer-independent pathway; and a TUTase-dependent pathway . For the snoRNA) ,163,164. snoRNA) ,166,167; snoRNA) , and the snoRNA) . These RViruses have evolved special mechanisms of gene expression to replicate. Most steps of viral gene expression depend on the machineries of its host cell and mRNA export is no exception. When a viral genome is replicated, some species retain their genome in the cytoplasm of the host cell, whereas others transport their genome into the nucleus. In the last section of this review, we discuss viral mRNA export, focusing on two retroviruses, MPMV and HIV. We will describe viral replication with a focus on the nucleocytoplasmic transport of RNA in accordance with the concept of this review. A retrovirus usually has a single promoter in its whole genome, which means it has just one primary transcript ; howeverThis is achieved by export of the viral mRNA in either intron-containing or spliced forms. Usually, intron-containing mRNA cannot translocate into the cytoplasm because of the mRNA quality control system. The retrovirus has special systems that enable the export of intron-containing mRNA, thus overcoming the quality control system.MPMV has two different mRNA export pathways: a splicing-dependent pathway and a splicing-independent pathway. The former is a simple pathway in which MPMV mRNA is exported to the cytoplasm using the bulk mRNA export pathway of its host cell. The MPMV primary transcript is recognized by the spliceosome, which presumably results in the sequential recruitment of TREX-1 and then the transporter Nxf1. The spliced MPMV mRNA that is exported in this way encodes the Env protein. In contrast, the splicing-independent pathway translocates MPMV mRNA to the cytoplasm in its intron-containing form. This pathway depends on the constitutive transport element (CTE), an RNA element located in a 3' untranslated region of the MPMV primary transcript ,171. CTENxf1 mRNA and Nxf1 proteins [Nxf1 contains the CTE in its alternatively spliced intron. This CTE-containing mRNA is translated into truncated Nxf1, the function of which is unknown.The interaction of Nxf1 with the CTE has also reported using endogenous proteins . The nasHIV-1 has evolved a more complicated and sophisticated mechanism of mRNA export than MPMV. HIV-1 produces 15 proteins, including regulatory and accessory proteins in addition to the structural proteins, enzymatic proteins, and envelope proteins . HIV-1 hin vitro [In the early phase, HIV-1 mRNAs are exported into the cytoplasm in a splicing-dependent manner. This type of mRNA is completely spliced and encodes two regulatory proteins, Tat and Rev, and one accessary protein, Nef . The Revin vitro ,186. Strin vitro . Accordiin vitro , matchinin vitro .By taking advantage of the mRNA export machinery of its host cell, HIV-1 exports as many as seven isoforms of mRNA derived from one primary transcript, thus producing multiple proteins.D. melanogaster larval neuromuscular junction [This review summarizes a broad range of RNA export mechanisms through the NPC. In general, RNA export receptors are selected according to the type of RNA, but there are many overlapping relationships between the type of RNA and the export receptor used. Studies using yeast are much more advanced than those using human or other mammalian cells, especially with respect to the interaction between RNA-protein complexes and the NPC components. Therefore, further studies of the RNA export mechanisms in humans are required in the future. There are two interesting studies reporting export of RNA-containing particles in an NPC-independent manner. In both cases, RNA export is achieved by nuclear envelope budding. One involves the egress of herpesvirus nucleocapsid  and the junction . We presStudies into the mechanism of mRNA export contribute to progress in various fields of research, such as viral infectious diseases, cancer, and efficient protein production systems using mammalian cells. Moreover, a lot of attention has been paid to the relationship between the defects in RNA metabolism and neurodegenerative diseases . Insight


While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5′-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5′-aldehyde. We conclude that hydroxyl radical abstracts a 5′-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5′ deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2′-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple. To understand the myriad roles of RNA in biology, it will be necessary to define the three-dimensional structures of a large (and growing) collection of RNA molecules . A partiInterpretation of the SHAPE experiment has benefited from extensive mechanistic investigations  that havThe hydroxyl radical produces a strand break in a nucleic acid molecule by abstracting a hydrogen atom from the sugar-phosphate backbone . There aHere we use two complementary experimental approaches to measure the reactivity of the hydroxyl radical with each of the ribose carbon–hydrogen atoms of the sarcin/ricin loop (SRL), a model RNA molecule . We firs2H]-ribose, D-[2″-2H]-ribose, D-[3′-2H]-ribose, D-[4′-2H]-ribose and D--ribose) were purchased from Omicronbio, Inc.  and were used without further purification. Deuterium incorporation in each was greater than 98%, as indicated by the manufacturer. We synthesized specifically deuterated ribonucleoside triphosphates    by adaptG, C, U) . Brieflyin vitro transcription using T7 RNA polymerase . The RNA molecules synthesized were 5′-GGGUGCUCAGUACGAGAGGAACCGCACCC-3′, a 29-mer RNA containing the sequence of the rat sarcin/ricin loop, and 5′-GGCCUGAGUAAGCGGCGAGCGAAAGGGCC-3′, a 29-mer RNA containing the sequence of Helix 13 from Thermus thermophilus 23S ribosomal RNA.RNA molecules were prepared by To synthesize an RNA molecule, a DNA strand coding for the SRL (5′-GGGTGCGGTTCCTCTCGTACTGAGCACCCTATAGTGAGTCGTATTA-3′), or ribosome Helix 13 (5′-GGCCCTTTCGCTCGCCGCTTACTCAGGCCTATAGTGAGTCGTATTA-3′), was annealed with a DNA strand containing the T7 promoter (5′-TAATACGACTCACTATAGG-3′) to generate a partial duplex for use in a run-off transcription reaction . PartialSRL RNA with deoxyguanosine incorporated at position 10, which we refer to as deoxyG10-SRL, was purchased from IDT  and used without further purification.32P]ATP (3000 Ci/mmol)  using T4 polynucleotide kinase (New England Biolabs). Enzymes were removed by phenol/chloroform extraction. Unincorporated [γ-32P]ATP was separated from radiolabeled RNA using a NucAway spin column.RNA was dephosphorylated by treatment with Antarctic Phosphatase  and 5′-end labeled with [γ-32P]  (Perkin Elmer) were mixed to a final volume of 30 μl in an Eppendorf tube. The ligation reaction was carried out overnight at 4°C. The enzyme was removed by phenol/chloroform extraction. Labeled RNA was separated from unincorporated pCp, [5′-32P] by passage through a NucAway spin column. Note that an additional nucleotide is added to the 3′ end of the RNA molecule in this labeling reaction, so the length of 3′-radiolabeled SRL RNA is 30 nt.RNA (10–20 pmol), 3-μl 10X T4 RNA ligase I buffer, 3-μl dimethyl sulfoxide (DMSO), 0.5-μl adenosine triphosphate (ATP) (75 mM), 2-μl T4 RNA ligase I (New England Biolabs) and 5-μl cytidine-3′,5′-bis(phosphate), Cleavage products were ethanol precipitated, heated to 90°C for 2 min, loaded onto a 20% denaturing polyacrylamide gel and electrophoresed for 3.5 h at 60 W constant power.β-elimination of a 5′-aldehyde terminus in a DNA molecule to produce a 5′-phosphate terminus, a free base and furfural , cleavage products were not heated at 90°C prior to loading on a denaturing polyacrylamide gel, because we noted that heating led to a decrease in the amount of 5′-aldehyde-terminated fragments with a concomitant increase in the amount of 5′-phosphate-terminated fragments. This behavior is analogous to the furfural .Following electrophoresis, the gel was dried and then exposed to a phosphor screen for 18 h. Gel bands were imaged using a Typhoon Trio+ Imager (GE).4 in buffer (50-mM potassium phosphate (pH 7.6), 100-mM NaCl and 500-mM KCl). After 2 min the reaction was stopped by addition of 3.5 v/v 100% ethanol and 1-μg carrier tRNA, and the products were precipitated.After gel purification, 3′-radiolabeled RNA was subjected to hydroxyl radical cleavage as described above. To the quenched reaction mixture was added 1 μl of freshly prepared 1-M NaBHkH/kD, the ratio of the reaction rates for a normal versus a deuterated sample, which is defined as the deuterium KIE  program . We evalrium KIE . Band inhttp://www.bioinf.manchester.ac.uk/naccess/) with a probe radius of 1.4 Å, corresponding to the radius of a water molecule. The reported values represent the average surface area computed from a set of 11 crystal structures of the SRL (see Supplementary Table S1). We used the PyMOL Molecular Graphics System  to produce images of the SRL and Helix 13.The solvent accessible surface areas of individual ribose hydrogen atoms in the SRL RNA molecule were calculated using the program naccess   specifically deuterated at one of the ribose carbons . The other three nucleotides were not deuterated. Each deuterated SRL was radiolabeled and subjected to hydroxyl radical cleavage. We compared the cleavage pattern of a deuterated SRL to the cleavage pattern of a control SRL RNA prepared with all natural (non-deuterated) nucleotides.To determine which ribose hydrogen atoms are abstracted by the hydroxyl radical, we performed a series of deuterium KIE experiments ,20. We b2) ,17. UsinkH/kD)  . For nonWe carried out similar hydroxyl radical cleavage experiments on SRL molecules containing the other 19 deuterated nucleotides . We then calculated KIEs for residues U7-G24 of the SRL. 5′ KIE values  ranged (except for residue U11) from 1.19 to 1.65. The 5′ KIE value for U11, 1.96, was substantially greater. 4′ KIE values  ranged from 1.0 to 1.21. We found that substitution of a deuterium atom at the 3′, 2′ or 1′ ribose position resulted in an apparent KIE near 1.0 for production of the 3′-phosphate-terminated product that is assigned to cleavage at that residue . We conclude from these experiments that hydrogen atom abstraction from the 5′ and 4′ carbons of ribose constitutes the predominant mode of reactivity of the hydroxyl radical with RNA.Recall that the deuterium KIE is much larger in magnitude for the 5′-dideuterated U11 residue of the SRL compared to any other KIE that we measured . Since U11 takes part in the GUA base triple motif, we asked whether an unusually large 5′ KIE is characteristic of base triples in other RNA molecules. We chose to study Helix 13 from 23S rRNA. Similar to the SRL, Helix 13 contains a GUA base triple, an S-turn and a bulged G motif . Although the GUA base triple in Helix 13 is capped by a larger loop than is the SRL, the overall folded structure is very similar. We prepared the 5′-dideuterated-U analog of Helix 13, radiolabeled the RNA molecule at the 5′ end, subjected the deuterated RNA to hydroxyl radical cleavage and measured the deuterium KIE . We found that the U residue of the GUA base triple in Helix 13 exhibits a 5′ KIE that is unusually large in magnitude (1.90) and comparable to the magnitude to the 5′ KIE of residue U11 in the SRL. We performed a similar experiment in which 5′-dideuterated-G was incorporated in Helix 13 , and found no KIE greater than 1.5, again similar to our results for the SRL . We conclude that the structural environment that exists in a GUA base triple leads to unusual reactivity of the hydroxyl radical toward the 5′-hydrogens of the U residue, revealed as a signature 5′ deuterium KIE for 5′-radiolabeled RNA. This large isotope effect likely results from nearly exclusive abstraction of a 5′-hydrogen atom from the U of the GUA triple .We obtained further evidence that the hydroxyl radical abstracts 5′-hydrogen atoms from RNA by analyzing the strand termini produced in the cleavage reaction. In DNA, abstraction of a 5′-hydrogen atom produces a 5′-aldehyde terminus at the site of cleavage ,11,13. ATo investigate whether similar cleavage chemistry occurs for RNA we prepared a sample of 3′-radiolabeled SRL, subjected it to hydroxyl radical cleavage and separated the cleavage products on a denaturing gel. The cleavage pattern of this sample consists of one band for each nucleotide of the SRL . Treatment of the cleavage products with sodium borohydride yielded two distinct bands per nucleotide . One of these bands, which comigrated with the original product, had mobility consistent with that of a strand terminated by 5′-phosphate. The second band, the product of borohydride treatment, comigrated with a 5′-hydroxyl-terminated fragment that was produced by alkaline hydrolysis .By analogy to DNA cleavage chemistry  we assigTo assign each gel band to the SRL nucleotide that was attacked by the hydroxyl radical, we took into account the chemistry that results in the strand break Figure . For 5′-We confirmed our band assignments by conducting deuterium KIE experiments on 3′-radiolabeled SRL . Deuteration of the 5′-carbon of G led to a substantial decrease in band intensity for only the 5′-hydroxyl-terminated fragments that we assigned as resulting from attack at the G residues of the SRL. Similarly, we found that 4′-deuteration of G resulted in a decrease in band intensity for phosphate-terminated strands that we assigned as the products of radical attack at the guanine residues of the SRL.It is clear from the prominence of the 5′-hydroxyl-terminated strands in the cleavage pattern of 3′-radiolabeled SRL RNA Figure  that mosWe note that two bands are especially prominent in the cleavage pattern of 3′-radiolabeled SRL Figure : the 5′-Because G10 and U11 are associated with the GUA base triple and the bulged-G structural motifs of the SRL, we investigated these observations in more detail.We now focus our attention on the GpU dinucleotide step within the GUA base triple of the SRL. It has been noted that the 5′-GpU dinucleotide platform is highly over-represented in natural RNAs , perhapsTo do this, we substituted a deoxyguanosine residue at position G10 in the SRL, to preclude formation of the 2′-OH/phosphate hydrogen bond in the GpU platform. We then performed hydroxyl radical cleavage experiments on 3′- and 5′-radiolabeled deoxyG10-SRL molecules. The cleavage pattern of 3′-radiolabeled deoxyG10-SRL Figure  was idenFor 5′-radiolabeled deoxyG10-SRL Figure  we also To resolve this apparent contradiction, we considered the possibility that the band running at the position of the A12 product might in fact be composed of more than one cleavage fragment. Recall that the cleavage pattern of 5′-radiolabeled RNA Figure  consistsWe calculated the solvent accessibilities of the ribose hydrogens of the SRL and found that the 2″- and 3′-hydrogen atoms of G10 are unusually exposed to solvent Figure . At no oGreenberg and coworkers have shown that abstraction of a 2″-hydrogen atom from the ninth nucleotide of a 19-mer RNA yields a fragment that migrates two nucleotides slower than a 3′-phosphate-terminated 8-mer . They asWhile as far as we know there has been no previous determination of the products of 3′-hydrogen atom abstraction from RNA, it has been proposed for DNA that the product is a strand terminated by 3′-phosphoglycaldehyde and missing the attacked nucleotide ,33. If sFrom these experiments we conclude that the hydrogen bond between the 2′-OH of G10 and the 3′-adjacent phosphate enforces the distinctive hydroxyl radical reactivity of the G and U residues of the GUA triple. The hydroxyl radical abstracts almost exclusively a 5′-hydrogen from the U, leading to an unusually large KIE. The 2′-OH/phosphate hydrogen bond  stabilizSince this hydrogen bond has been implicated in the structural stability of the GU platform of the GUA triple , we propOur experiments show that abstraction of a 5′-hydrogen atom is the major pathway for strand cleavage of RNA by the hydroxyl radical. How do these experimental results fit with structural features of RNA? The usual interpretation of the hydroxyl radical cleavage pattern is that the extent of cleavage at a given nucleotide corresponds to the solvent accessibility of its sugar hydrogen atoms ,9,34–36.Despite the overall correspondence of cleavage and solvent accessible surface area, one SRL nucleotide, tetraloop residue A15, is cleaved much less than its calculated solvent accessibility would predict. In SRL X-ray structures ,25 one sResidue A9, on the other hand, is apparently cleaved substantially more than its solvent accessibility would predict Figure . While wWe show here that the hydroxyl radical cleavage experiment can be used to quantitatively monitor the solvent accessibilities of single ribose hydrogen atoms in an RNA molecule. Single-atom resolution is achieved by taking advantage of the ability of gel electrophoresis to resolve RNA fragments of the same length that differ in the chemical nature of the strand termini  that are produced by the cleavage reaction . FurtherAt nearly all nucleotides in the RNA molecule we studied, the most abundant cleavage product comes from abstraction of a hydrogen atom attached to a ribose 5′-carbon. The main evidence for this conclusion is that the cleavage product produced in highest yield is an RNA strand terminated by a 5′-aldehyde moiety Figure . AdditioSince work over several decades on free radical cleavage of DNA ,13 has eWhile most nucleotides in the relatively small RNA molecule that we studied are cleaved to about the same extent, three stand out in the cleavage pattern. It is perhaps not surprising that these nucleotides, G10, U11 and A20, form a GUA base triple, the signature structural motif of the SRL . In the An unusual feature of the GUA triple is its structural rigidity, which is thought to be conferred by a G-ribose-OH/U-phosphate hydrogen bond Figure . The disHigh-throughput methods for RNA structure mapping are now in active development, to cope with the rapidly expanding universe of cellular RNAs ,43. New Supplementary Data are available at NAR Online.