AbstractVermetidae were recorded during the study and their substrate preferences identified.Coral reefs are often termed underwater wonderlands due to the presence of an incredible biodiversity including numerous invertebrates and vertebrates. Among the dense population of benthic and bottom-dwelling inhabitants of the reef, many significant species remain hidden or neglected by researchers. One such example is the vermetids, a unique group of marine gastropods. The present study attempts for the first time to assess the density and identify preferred reef substrates in the Gulf of Kachchh, state of Gujarat, on the western coast of India. A total of three species of the family  PageBreakone of the sessile gastropods, the vermetids. Vermetids do not have a normal coiling shell and become untwisted; in post-larval stages, the irregularly coiled shells are firmly attached to rock or any other hard substrates. These meandering tubes resemble the polychaete worm tubes  usually close to coral reefs diffusion boundaries . Presentniferans . The laro-Paciﬁc .In terms of biodiversity, coral reefs are an exceptionally diverse ecosystem showcasing a large number of species interactions . Such inThylacodesvariabilis Hadfield & Kay, 1972 (as Serpulorbis), from various locations inside and outside the Gulf of Kachchh i.e., Adatra, Okha, Porbandar, Bedi, Mithapur and Danda , Faviafavus , Poriteslutea , Poritescompressa , Turbinariapeltata  and Gonioporaplanulata  among others.Paga reef has abundant reef biodiversity including both fauna and flora except for the sand patch exposed at the western end of the reef. The coral assemblages show very diverse forms on this reef, including a variety of sponges, sea anemones, zoantharians, tube worms, crabs, gastropods, bivalves and echinoderms. Altogether, they contribute more than 200 species of marine invertebrates in this locality . It has i.e., Ceraesignum, Thylacodes and Petaloconchus that showed densities of 7.7, 5.3 and 0.4 individuals/100 m2 respectively.Vermetids were evident as soft-bodied organisms with a bright orange, pale red or dark magenta colouration and covered in an upright calcareous tube, which was sometimes slightly elevated. The vermetid shells, originally dull white to earthy grey in colour, were observed to be well-encrusted by epibionts such as calcareous algae and live coral. The length of the vermetid tube emerging out of the live coral substratum was 2–3 mm and ranged from 3–8 mm in other substrates, like rock covered by silt or sand. In some places, the irregularly coiled tubes of the vermetids were also found in completely exposed conditions. The present study summarises the occurrence of three genera of vermetids PageBreakviz., live coral, silt over rock, rubble, algae over rubble. One-way ANOVA was performed to evaluate the variation in vermetid densities among substrate types.Paga reef is located between 22°28.8' to 22°30.0'N latitude and 69°11.6' to 69°15.0'E longitude covering an area of 1472.4 ha which remains submerged during high tide and gets exposed only during low tides. Therefore, the field work was carried out during a small window of low spring tides falling in daylight. The reef area was surveyed using ten belt transects measuring 100 m × 1 m in the intertidal zone . The traVermetidae  was recorded from the study sites namely, Ceraesignum Golding, Bieler, Rawlings & Collins, 2014, Thylacodes Guettard, 1770 and Petaloconchus Lea, 1843  showed the highest density followed by Thylacodes spp.  and Petaloconchus spp. . The generic composition in the survey is 57% of Ceraesignum spp., 40% Thylacodes spp. and 3% Petaloconchus spp. Furthermore, the association of each genus with the substrate type was different, illustrated in the histogram , rubble, Favia Milne Edwards, 1857, Platygyra Ehrenberg, 1834, Porites Link, 1807, Goniastrea Milne Edwards & Haime, 1848 and algae over rubble (AoR). Maximum numbers of individuals were recorded on SoR and a minimum on AoR. However, there is no significant difference for substrate preference among the three genera . The species-specific preference of each vermetid species for a substrate is described below. All vermetid species were solitary: no gregarious species were found during the survey.Overall, the occurrence of vermetids was recorded on seven different substrates, Ceraesignum was 7.7 individuals/100 m2 found on six different substrates. It remained embedded in the massive and submassive coral PageBreakcolonies or rock, except a short part of the tube opening. The individuals of this genus showed the highest association with SoR (73%) and were recorded the least number of times on Favia Milne Edwards, 1857 (1%) and AoR , Goniastrea (4%), Favia (1%) and Porites (9%). It is noteworthy that three of the four coral genera belong to family Faviidae; among all the live corals, maximum individuals were found on the live colonies of Platygyra.The density of the large vermetid R Figure . If the Thylacodes was 5.3 individuals/100 m2, which is less compared to Ceraesignum spp. The individuals of this genus were found associated with six different substrates: PageBreakPageBreakSoR, rubble, Porites, Platygyra, Favia and Goniastrea , Platygyra (2%), Goniastrea (2%) and Favia (2%). The highest density of Thylacodes was recorded on Porites spp. It implies that more than 50% of the animals were found embedded in the live coral colonies of Porites. The animals were recorded without an operculum as bright orange “spots” in the dark green Porites. Thylacodes spp. shells, originally dull white to earthy grey in colour, were observed well-encrusted by epibionts such as calcareous algae and live coral. The length of the vermetid tube emerged out of the live coral substratum was 2–3 mm and ranges approximately 3–8 mm in records of presence in other substrates like rock covered by silt or sand.The density of Petaloconchus was represented by only four individuals and the density was 0.4 individuals/100 m2. The majority (75%) of the individuals were recorded on rubble and the remaining 25% on SoR  between the means of both the genera.The present study revealed that vermetid density on Paga reef varies with the genera; however, the results of ANOVA shows that there is no significant difference in the Ceraesignum spp. has resulted as 7.7 individuals/100 m2, which is much less compared to other studies worldwide. Ceraesignum  as high as 60,000/m2 on a water-levelled bench at Diamond Head, Oahu. Ceraesignummaxima on Luminao Barrier Reef, Guam. These studies revealed that the vermetid density between the coral heads ranged from 0–520 individuals/m2 and on the Poriteslutea coral heads it ranged from 15–520 individuals/m2, which are far higher than the records of the present study. This may be attributed to the large range? of substrate availability on Luminao Barrier Reef and Oahu. Additionally, The density of Poriteslobata. As well as providing the geological survey of a vermetid reef of the Salento Peninsula, Porites colonies and bio-clastic accumulations. Poriteslutea. This association preference is concordant with the present study consisting of an association of vermetids with the massive coral Poriteslutea and additionally, the new associations of vermetids with Faviaspeciosa, Goniastreapectinata and Platygyra sp., which has revealed novel research. Moreover, the authors have also observed a stronger association of vermetids with Poriteslutea and Poritescompressa on other reefs of the Gulf of Kachchh (unpublished work). Petaloconchuskeenae with Poritesi.e., 590 individuals/m2 of the Porites colony, whereas Petaloconchus spp. was recorded only on abiotic substrates in the present study.Dendropomamaximum, on the corals revealed that the vermetid gastropod reduces skeletal growth of corals by up to 81% and coral survival by up to 52%, presumably by an unknown mechanism involving the mucus nets  showed the highest association with Porites on Paga Reef. Porites is a dominant and frequently encountered coral genus and plays a significant role in community composition on this reef. It is frequently distributed at a variety of reef zones., starting from tidal pools at back reef areas and the reef flat to up to the reef edge with massive as well as submassive growth forms.The impact of the largest species of vermetid gastropod, cus nets . These scus nets . Hence, The present work brings forth the density, diversity and habitat preferences of three vermetid genera at Paga Reef, in the Gulf of Kachchh, India. The study reveals the occurrence of vermetids on variety of substances. The organisms are distributed in all the reef zones across the intertidal belt of the reef. In spite of the adaptability of vermetids to a large range of substrates and reef zones, their density remains limited on Paga Reef compare to other reef areas worldwide. The study recommends an urgent need to identify the organisms at more detailed taxonomic levels with their respective habitat preferences.


Serum, feces, and various tissues were collected from the mice after RNA consumption and analyzed for several miRNAs. Exogenous plant miRNAs were present in the sera, feces, and tissues of animals and these exogenous plant miRNAs were primarily acquired orally. MiR-172, the most highly enriched exogenous plant miRNA in B. oleracea, was found in the stomach, intestine, serum, and feces of mice that were fed plant RNA extracts including miR-172. The amount of miR-172 that survived passage through the GI tract varied among individuals, with a maximum of 4.5% recovered at the stomach of one individual, and had a range of 0.05–4.5% in different organs. Furthermore, miR-172 was detected in the blood, spleen, liver, and kidney of mice.MicroRNAs (miRNAs), a class of small RNAs, are important molecules that influence several developmental processes and regulate RNA interference (RNAi), and are abundant in animals, plants, and plant tissues that are traditionally consumed in the diet. The survival of plant small RNAs from the diet in animals, however, remains unclear, and the persistence of miRNAs from dietary plants in the animal gastrointestinal (GI) tract is still under debate. In this study, ICR mice were fed plant total RNAs in quantities of 10–50  MicroRNAs (miRNAs) are a class of small RNAs (19–25 nucleotides (nt) long) which have posttranscriptional activity in animals and plants  tract, and organs.In this report, we investigated the survival of exogenous RNA in the digestive system, blood, and several organs of mice after being fed RNAs extracted from Although the functional effect of those plant-specific miRNAs in mammals is under debate, first their ability to survive the mammalian GT tract and enter blood and organs needs to be verified. A plant specific miRNA, miR-172, was identified in the feces by polymerase chain reaction (PCR) and validated by TaqMan® assay. These results show that exogenous plant miRNA can survive in the murine GI tract, enter peripheral blood, and continue to other organs.B. oleracea (see below) in DEPC-treated water in quantities of 10 to 50 μg in a total volume of 500 μL. The solution was administered from a pipette tip directly into the oral cavity. In some of the feeding experiments, the same amount of RNAs solution was added to the food offered as diet to the animals. As a control, prior to feeding, a fecal sample from each experimental animal was examined to confirm the absence of miRNA sequences with homology to miR-172 (data not shown).All animal experiments were performed in accordance with permission from the ethics committee of Southeast University, China. All animal experiments were performed using ICR strain mice on a 12-h light/dark cycle in a pathogen-free animal feeding facility at Southeast University. Male and female ICR mice between the ages of 2–4 months were gavage fed total RNAs extracted from B. oleracea, Brassica rapa, rice, corn, Arabidopsis thaliana, and Solanum lycopersicum using Trizol®  according to the manufacturer's protocol. The concentration of total RNA was quantified by the absorbance at 260 and 280 nm. The total RNA was electrophoresed in 1.6%/Tris-Boric acid-EDTA buffer agarose gel to visualize small RNAs. A stem-loop reverse transcription polymerase chain reaction (RT-PCR) assay  was incubated at 37°C with freshly drawn serum or fecal suspension in DEPC-treated water from control mice. At various times after starting incubation , RNA samples were prepared, purified (see below), and analyzed for the presence of small RNA sequences by electrophoresis.To determine the degradation of plant small RNA in vitro, total RNA  after feeding the exogenous RNA solution to mice, fecal samples were collected. Mice were then anesthetized with ether, the abdominal fur was thoroughly washed with ethanol, and one set of surgical instruments was used to open the peritoneal and thoracic cavities by cutting the diaphragm. Finally, the still palpitating heart was punctured with a disposable 0.9-mm gauge needle, and blood (about 0.5–1 mL) was drawn with a syringe. For facilitation, in some of the experiments, blood was taken from the fundus of eyes in the mice. Thereafter, the contents of the stomach, intestines, liver, spleen, and kidneys were removed for analyzing. Control analyses on abdominal fur washing fluids performed by PCR methods revealed no evidence for detectable contamination.The contents of the stomach, intestines, and feces were dispersed in 0.5 ml DEPC-treated PBS, respectively. The suspensions were incubated at 37°C for 15 min, and subsequently nucleic acids were extracted according to the methods described above. These extracts were subsequently treated with 10 U of DNase per mL at 37°C for 30 min. Prior to use, the DNase was heated to 85°C for 10 min to destroy contaminating DNase.Total RNA was extracted using Trizol® from the liver, spleen, kidney, and blood according to the standard protocol. In some of the experiments, the blood cells were pelleted by low-speed centrifugation, and RNA was extracted separately from the cell pellet and serum by the SDS-proteinase K-phenol/chloroform procedure. Next, DNase treatment was performed to eliminate residual DNA contamination. Finally, after a second acid phenol:chloroform extraction, the RNA was resuspended in DEPC-treated water.μL total volume) was incubated with 1 μL of a cocktail containing 10 mmol/L of each of the antisense primers listed in the table To measure the maximal length of miRNA fragments resistant to GI tract degradation, gene-specific primers and reverse transcriptase were used to convert the pre-miRNA and mature miRNA from stomach and fecal samples to cDNA , 200 nmol/L of each primer, and water to a total volume of 20 μL. The reactions were amplified for 15 sec at 95°C and 1 min at 60°C for 40 cycles. The thermal denaturation protocol was run at the end of the PCR cycles to determine the number of products that were present in the reaction. Reactions were typically run in duplicate. The cycle number at which the reaction crossed an arbitrarily placed threshold (CT) was determined for each gene and the relative amount of each miRNA.Real-time quantitative PCR was performed using standard protocols on an Applied Biosystems 7500 Sequence Detection System . Briefly, 1.25 μg of RNA were treated with DNase and followed a similar protocol to that described above in the SYBR Green assay.To verify the SYBR Green results and to determine whether plant miR-172 can survive the mouse GI tract, a TaqMan® assay was conducted. TaqMan® probe is the “gold standard” in miRNA detection at present, which can distinguish a one nucleotide difference in sequence and provides a reliable means to assess the survival of miR-172 in the GI tract. The RNA samples from blood, fecal suspensions, and contents of the stomach, intestines, and organs of animals previously fed with 50, 30, or 10 To determine the percentage of originally administered plant RNA that was present after digestion, the TaqMan probe assay was used. Standard curves were constructed for mature miR-172 using a standard sample and quantifying 10-fold dilutions by real-time PCR. After collecting and quantifying the miR-172 present in the various tissues and samples, percentages of the originally administered RNA were determined.μg total RNA per gram of mature plant sample, with 371.6 μg being the average total RNA yield per gram of mature seeds or plant tissue from seven independent RNA extractions (range 214.2–905.3 μg per gram of sample) (B. rapa) than in mature seeds   . After eB. oleracea, B. rapa, A. thaliana, and S. lycopersicum as detected by semiquantitative RT-PCR analysis with 30 cycles  showed high levels of expression in Despite the lack of a low-molecular weight RNA marker, we can infer that the bright bands on the agarose gel are small RNA fragments in the range 20∼100 bp that survived degradation after different times of incubation  up to 36 h in the contents of the intestine Fig. B, up to In addition, from the experiments carried out, there was no evidence that differences in the sex of the animals or their age within the 2- to 4-month range affected the outcome of any of the experiments reported .RNA is an unstable molecule that degrades easily under normal environmental conditions . Despite the presence of mature miR-172 in fecal samples, pre-miR-172a and pre-miR-172b were not detected in mice feces 2 h after feeding. It is apparent that pre-miR-172 decomposed in 2 h, while miR-172 could survive between 2 and 72 h after feeding RNA. At later times after feeding, pre-miR-172 could no longer be detected in the mice. There was no evidence for long-term survival of pre-miRNA in the intestines of the mice.Next, miRNA survival in the GI was measured in mice fed exogenous plant RNA, as well as investigating the presence of miR-172 sequences in liver, spleen, kidney, whole blood, and serum of the animals fed with plant RNA. Between 2 h and 72 h after feeding 50 μg or 10 μg of RNA were fed to mice , the relatively large amount of exogenous miRNA detected in test samples reassures us that exogenous plant miRNAs did survive in the GI tract. Moreover, the transient survival of small RNA in the GI tract, feces, blood, and organs have been proved by TaqMan probe methods, and is a method generally accepted.μg. These values must be related to the body weight of mice (between 15 and 25 g), and thus constitute an approximate fraction of 2/106 of their body weight. For humans of average weights between 50 and 70 kg, one would thus have to postulate an intake of about 50–140 mg of RNA with the daily food uptake to parallel the situation simulated in these mice experiments. Based on the estimate of ∼500 μg of total RNA yielded per gram mature seeds, this would require about 280 g of food consumed, a reasonable human exposure.The amount of foreign RNA administered to mice was between 10 and 50 The stability of exogenous RNA in organisms and whether it can enter cells and elicit certain functional control in the recipient cells remains unclear. Zhang et al.  reportedμg of RNA to mice, and the result suggests that there is a good correlation between miR-824 and miR-172 (Fig. In this context, many questions remain to be investigated, none of which will be technically easy to control. Our data are consistent with the notion that a steady flow of exogenous genetic material persists in the GI tract, and a very small percent can enter the circulation and distribute in different organs (Zhang et al. 172 Fig. . The preWhile our results do not support general and consistent uptake of dietary plant miRNAs, additional studies are needed to establish whether or not plant or animal xeno miRs are transferred across the GI in sufficient quantity to regulate endogenous gene expression, and these experiments have been initiated (Witwer et al. This study shows that foreign plant miRNA ingested by mammals can survive through the GI system, and enter the bloodstream and various organs of mice. Experiments involving foreign miRNA entering into established mammalian, and probably other eukaryotic, genomes have to take into account the possibility that these foreign small RNA may have important consequences, with unexpected alterations of mammalian genome and expression patterns. It is important to note that we have not found phenotypic changes in all the mice fed with the foreign RNA.


Arabidopsis thaliana. An integrative framework that combined de novo assembly and genome-guided assembly was applied to reconstruct transcriptomes for the two datasets. Up to 44.8% of the de novo assembled transcripts in total RNA-seq sample were of low abundance, whereas only 0.09% in polyribosomal RNA-seq de novo assembly were of low abundance. The final round of assembly using PASA (Program to Assemble Spliced Alignments) resulted in more transcript assemblies in the total RNA-seq than those in polyribosomal sample. Comparison of alternative splicing (AS) patterns between total and polyribosomal RNA-seq showed a significant difference  in intron retention events: 46.4% of AS events in the total sample were intron retention, whereas only 23.5% showed evidence of intron retention in the polyribosomal sample. It is likely that a large proportion of retained introns in total RNA-seq result from incompletely spliced pre-mature mRNA. Overall, this study demonstrated that polyribosomal RNA-seq technology decreased the complexity and diversity of the coding transcriptome by eliminating pre-mature mRNAs, especially those of low abundance.Recent RNA-seq studies reveal that the transcriptomes in animals and plants are more complex than previously thought, leading to the inclusion of many more splice isoforms in annotated genomes. However, it is possible that a significant proportion of the transcripts are spurious isoforms that do not contribute to functional proteins. One of the current hypotheses is that commonly used mRNA extraction methods isolate both pre-mature (nuclear) mRNA and mature (cytoplasmic) mRNA, and these incompletely spliced pre-mature mRNAs may contribute to a large proportion of these spurious transcripts. To investigate this, we compared a traditional RNA-seq dataset  and a ribosome-bound RNA-seq dataset  from  As a result of the development of deep sequencing technology, our understanding of transcriptome complexity has been greatly improved during the past decade. An example of this improvement is alternative splicing analysis in plants. The first wave of genome-wide transcriptome analysis consisted of direct sequencing of cDNAs and ESTs on a large scale and suggested that 11 to 30% of the multi-exonic genes in plants were alternatively spliced . AfterwaIn many cases, RNA-seq libraries are constructed from total cellular RNA, which is a mixture of nuclear and cytoplasmic RNA and contains, in addition to protein-coding RNA, a variety of non-coding RNAs. In the nucleus, protein-coding RNAs are capped, polyadenylated and spliced before exiting to the cytoplasm as mature mRNAs. While some coding RNAs may be retained in the nucleus awaiting specific environmental or developmental cues, much of the nuclear RNA population consists of partially processed transcripts in which intron splicing is incomplete. In a total RNA-seq analysis, these incompletely spliced isoforms can contribute additional but potentially misleading variants of the annotated gene structures.Nicotiana benthamiana cells to correlate with the likelihood that they would be subject to NMD [Increasing evidence shows that a proportion of transcript isoforms, whether due to incomplete processing or spurious splicing of background transcripts may not contribute to the functional protein pool, likely due to nonsense-mediated mRNA decay (NMD) ,6. NMD it to NMD , hence tt to NMD .PERK10 (PROLINE-RICH EXTENSIN-LIKE RECEPTOR KINASE 10) encodes a proline-rich protein and the translational efficiency of its mRNA is increased during hypoxia based on ribosome-profiling evidence; however, sequencing of total mRNA extracts failed to detect this change [Another obvious yet less-discussed reason for spurious/low abundance isoforms is that in addition to various species of non-coding RNAs and non-specific background transcription, total cellular RNA isolated by current methods (which often include polyA+ selection) contains a mixture of pre-mature and mature mRNAs. Sequencing of such mixtures can lead to the reconstruction of incompletely spliced isoforms and the annotation of non-existent gene models. It can also lead to a lack of correlation between mRNA and protein levels . To circs change . Applicas change . For inss change .Medicago truncatula successfully demonstrated that differential translation of mRNAs significantly contributed to the regulation of gene expression during nodule formation [Despite its merit, one of the limitations of ribosome profiling technology is that it is particularly challenging to assemble complete transcripts from the short fragments isolated for sequencing, and it is therefore impossible to comprehensively identify alternative isoforms and monitor the translational dynamics of splicing in cells . RNA-bloormation . Recent ormation . Althougde novo and genome-guided assembly methods to reconstruct the transcriptomes for total RNA-seq and polyribosomal RNA-seq samples. Comparison of alternative splicing patterns between the two assembled data sets revealed a significant difference in intron retention. Our results reveal that polyribosomal RNA-seq decreased the complexity and diversity of transcriptome. We demonstrate here that polyribosomal RNA-seq technology is an efficient method to eliminate some pre-mature mRNAs likely resulting from incomplete splicing.In our study, we applied a combination of We compared the splicing patterns using RNA-seq data derived from 3–4 weeks old Col-0 leaves. The polyribosomal data consisted of a total of nearly 96 million reads  . Since tTo investigate whether polyribosomal RNA-seq is a nonbiased method for a broad survey of the transcriptome, we aligned total RNA-seq and polyribosomal RNA-seq reads to the Arabidopsis genome (TAIR10) using Tophat v2.0.8  and compde novo as well as a genome-guided assembly, and then combined the outputs using the PASA pipeline [de novo assembly is still popular even when the reference genome is available, as it can recover transcripts that are derived from segments of the genome that are missing from the genome assembly. It can also detect transcripts from an unknown exogenous source. The PASA pipeline is then used to take advantage of the high sensitivity of reference-based assembly while leveraging the ability of de novo assembly to detect novel transcripts [To obtain an accurate and comprehensive transcriptome database, we performed both a pipeline . The advpipeline . First, nscripts .de novo assembly resulted in 42,962 and 35,545 transcripts for total RNA-seq and polyribosomal RNA-seq samples respectively . This result revealed that de novo assembly for total RNA-seq generated more low abundance transcripts, whereas polyribsomal RNA-seq seemed to be consistent before and after filtering. Finally, we used PASA to integrate the de novo assembly and genome-guided assembly, generating comprehensive transcript databases  (The Trinity ectively . To avoiRNA-seq) .To evaluate whether the sets of comprehensive assembled transcripts correspond to known isoforms in TAIR10, we calculated how many genes with assembled transcripts had at least one isoform that resembled an annotated transcript in TAIR10. We required the assembled transcript to contiguously cover at least 80% of the total length of the annotated isoform and have at least 95% identity with TAIR10 cDNAs. We found that 50% of genes within total RNA-seq assembled transcript set and 45.6% of genes within the polyribosomal RNA-seq assembled transcript set had at least one transcript that is annotated in TAIR10 .de novo and genome-guided assembled transcripts. The number of intron-containing genes showing AS in the unfiltered transcript set  is only slightly higher than that observed after filtration. A more likely explanation is that the previous study analyzed mixed RNA isolated from flowers under different stages combined with 10-d-old seedlings, whereas we only identified AS in leaves. Therefore, it is expected that more alternative splicing events can be found in their study. Moreover, the previous study used Tophat to identify AS events, whereas we applied PASA. The PASA algorithm is based on alignment assembly, which is very stringent in its detection of alternative isoforms [Previous research on Arabidopsis using RNA-seq showed that up to ~61% of genes with introns had evidence of alternative splicing . To estiisoforms .It is reported that more than one-third of alternatively spliced isoforms harbored pre-mature termination codons (PTCs) in human and Arabidopsis ,6,22. ThTraditional mRNA extraction has the potential to mix RNA molecules from the cell nucleus and cytoplasm, generating a mixture of pre-mature mRNA and mature mRNA. It is likely that pre-mature mRNA contribute to an overestimated prediction of alternative splicing events. The recently developed polyribosomal RNA extraction technology has provided a new strategy to obtain polyribosome-bound mRNA, most of which are assumed to be mature mRNA . Thus, iTo investigate the different patterns of alternative splicing, we employed PASA  to identOverall, 10,135 and 5,281 alternative splicing events were identified, affecting 2,090 and 1,396 genes in total RNA and polyribosomal RNA samples, respectively. This marked decrease from the total to the polyribosomal sample suggests that the polyribosomal transcriptome is less complex and diverse. Prior research on Arabidopsis alternative splicing either based on cDNAs/ESTs or total RNA-seq reported that intron retention was the predominant type of alternative splicing in Arabidopsis . Our res–6). These results suggest that the longer introns tend to be spliced during pre-mRNA processing.It is reported that intron size is one of the factors that impacts splicing efficiency . In the In conclusion, this analysis revealed that many isoforms with intron retention AS events in total RNA-seq are not found in the polyribosomal fraction. Many of these are likely to be incompletely processed mRNAs. However, other scenarios should not be overlooked. Some primary mRNAs may be retained in the nucleus awaiting processing and transit to the cytosol upon a developmental or environmental cue. Other mRNAs may exit the nucleus but be recruited to ribosomes only in response to appropriate stimuli.Arabidopsis thaliana encodes 54 functional GSTs, classified into seven clades [ATGSTF11 (AT3G03190) encodes a glutathione S-transferase belonging to the phi class and has been proposed to be involved in the biosynthesis of glucosinolates [Glutathione-S-Transferases (GSTs) are a superfamily that play a prominent role in glutathione (GSH) metabolism of many living organisms . The genn clades . Gene ATinolates . Only onAFC2 (AT4G24740), which had been analyzed previously [AFC2 belongs to Clk/STY (LAMMER-type) protein kinases, which play important roles in the regulation of alternative splicing through phosphorylation and interaction with serine/arginine-rich (SR) proteins [AFC2 [AT4G24740). Two types of AS events were observed in the total RNA sample, including intron retention and exon skipping . Total and polyribosomal RNA-seq data were obtained from NCBI . For random selection of reads, 95.5 million reads were obtained from the 1st read in the file to the 95.5 millionth read.The reference genome, annotation and sequenced full-length cDNAs of Tophat v2.0.8  was implde novo and genome-guided assembly, we used the default parameters. For building comprehensive transcriptome in PASA, we fixed the maximum intron length to be 5,000 nt, which is similar to a previous study [To obtain accurate assembly of transcripts, we applied a well-developed framework, Trinity v20140413  and PASAde novo assembly using the Trinity de novo algorithm, which is a compilation of three distinct sub-programs. After that, we utilized a new feature, implemented in the Trinity package, to perform genome-guided assembly. This algorithm directly applies GSNAP to generate the read alignments, and then partitions the genome-aligned reads into subsets that will each be targeted for independent Trinity de novo assembly.This pipeline first performs de novo and genome-guided assembly results and built comprehensive transcriptome datasets for the total and polyribosomal RNA-seq.After assembly, transcript abundance estimation was performed using RSEM , supportS1 Table(DOCX)Click here for additional data file.


A non-classical nuclear localization signal (ncNLS) of influenza A virus nucleoprotein (NP) is critical for nuclear import of viral genomic RNAs that transcribe and replicate in the nucleus of infected cells. Here we report a 2.3 Å resolution crystal structure of mouse importin-α1 in complex with NP ncNLS. The structure reveals that NP ncNLS binds specifically and exclusively to the minor NLS-binding site of importin-α. Structural and functional analyses identify key binding pockets on importin-α as potential targets for antiviral drug development. Unlike many other NLSs, NP ncNLS binds to the NLS-binding domain of importin-α weakly with micromolar affinity. These results suggest that a modest inhibitor with low affinity to importin-α could have anti-influenza activity with minimal cytotoxicity. Influenza A virus is a serious pathogen with the capacity to cause seasonal epidemics and high mortality pandemics23The genome of the influenza A virus is segmented into eight negative-sense RNA molecules, each of which is associated with the vial RNA polymerase subunits  and many molecules of nucleoprotein (NP) to form eight viral ribonucleoprotein (vRNP) complexes. The nuclear import of vRNPs is mediated by importin-α:β heterodimer789101113161618202122The NP protein encodes an NLS at its amino terminus that has been shown to function as the major signal necessary for the nuclear import of vRNPs242524252527293127The fact that the major NLS of vRNPs is of a non-classical type suggests that targeting the host importin-α could be a viable strategy to develop novel antiviral compounds to inhibit nuclear import of vRNPs and free NP without interfering with nuclear import of many  of the host proteins. Here we report the crystal structure of importin-α bound to NP ncNLS. Structural and functional analyses identify key binding pockets on importin-α and suggest that a modest inhibitor that binds to these sites with micromolar affinity could be an effective anti-influenza drug with minimal cytotoxicity.To elucidate the structural basis for how NP ncNLS binds to importin-α to ultimately develop novel therapeutic strategies, we determined a 2.3 Å resolution crystal structure of the NLS-binding ARM-repeat domain of mouse importin-α1 in complex with NP ncNLS by molecular replacement . ElectroMutagenesis of importin-α verified that NP ncNLS binds to the minor-NLS binding site but not to the major NLS-binding site of importin-α in solution . In GST Alanine-scanning mutagenesis of NP ncNLS showed that not all ncNLS residues are equally important for importin-α binding  and nuclThe structural basis for NP ncNLS-importin-α interactions also provides a rationale to understand why nuclear trafficking of vRNPs can be regulated by phosphorylation/dephosphorylation of NP. In the late stage of influenza virus infection, the progeny vRNPs are exported from the nucleus to cytoplasm, and posttranslational modifications of NP have been proposed to be one of the mechanisms to prevent the exported vRNPs from going back to the nucleus. It is known that some of the NP ncNLS residues  are phosphorylated at late stage of infection, leading to reduction of the NLS activity34KD = 1.7 μM; KD = 5.1 nM; KD value measured for SV40 NLS is comparable to the affinities observed previously23363738394023363738394041An immediate suggestion from our structure is that one way to develop antiviral drugs would be to design small-molecule inhibitors that bind specifically to the minor NLS-binding site of importin-α. The binding pockets for the key residues  of NP are particularly attractive as the target binding sites for the inhibitors to block the NP binding effectively. The inhibitors directed against the ncNLS-binding site need to have sufficiently high affinity for importin-α to compete with NP. Interestingly, by using a solid phase binding assay, we found that NP ncNLS binds to the NLS-binding domain (ΔIBB importin-α) with micromolar affinity  to the minor NLS-binding site of importin-αet al., the “compound 17” has the highest affinity (KD = 0.9 mM)Recently, Holvey We conclude that, although it is unclear why influenza NP protein has evolved to hijack only the minor NLS-binding site, potentially, the structural and biochemical properties of the interactions between host importin-α and influenza NP ncNLS could be exploited to develop novel antiviral drugs.S-transferase (GST)-tagged ncNLS  of the NP of influenza virus strain A/Puerto Rico/8/34 (H1N1) was constructed by ligating complementary oligo-DNAs encoding the ncNLS into BamHI/XhoI sites of pGEX-TEVMus musculus (mouse) importin-α1  in complex with NP ncNLS, un-tagged mouse importin-α1 (residues 72–498)Escherichia coli host strain BL21-CodonPlus(DE3)RIL (Stratagene) at 20 °C from pET15b (Novagen), whereas GST-NP ncNLS was expressed separately in the same strain at 25 °C from pGEX-TEV. After harvesting, the two sets of cells were mixed, suspended in buffer A  and lysed by sonication on ice. All subsequent steps were performed at 4 °C. Tween20 was added to 0.1%, and the clarified lysates were incubated with glutathione-Sepharose 4B  for 4 h. After washing the beads extensively with buffer B , the GST-tag was removed with His-TEV protease (20 μg/ml) overnight. The importin-α1(72–498)-NP ncNLS complex released from the resin was finally purified over Superdex200  in buffer C . The complex was concentrated to 28 mg/ml using a 3 kDa molecular weight cutoff Amicon Ultra centrifugal filter (Millipore).An expression plasmid for glutathione H32 symmetry  with one complex in the asymmetric unit.Crystals of mouse importin-α1(72–498)-NP ncNLS complex were readily obtained by hanging drop vapor diffusion method using a screening kit suitable for crystallization of protein-protein complexesRfree from 48.5% to 38.5%, after which there was clear difference density corresponding to the NP ncNLS in the minor NLS-binding site. Iterative cycles of model building using COOTRfree of 24.6% (Rcryst 18.8%) that contained importin-α1 residues 76–105, 111–496, NP residues 3–14, and 80 water molecules. A TLSMD analysishttps://www.pymol.org). Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4ZDU.Diffraction data were processed using MOSFLM and CCP4 programsE. coli strain BL21-CodonPlus(DE3)RIL (Stratagene). GST-NLS fusion proteins were purified over Glutathione-sepharose 4B  and gel filtration over Superdex200 . His/S-ΔIBB importin-α1 was purified over Ni-NTA (Novagen) and gel filtration over Superdex200 . Mutants were created using the Quickchange system (Stratagene), and all constructs were verified by DNA sequencing.GST-NP ncNLS, GST-SV40 NLSGST pull-down assays were performed in buffer D . GST-NLS fusion proteins (50 μg) were immobilized on 10 μl of packed Glutathione-sepharose 4B  beads and incubated with His/S-ΔIBB importin-α1 (17 μg) in 50 μl of binding buffer for 1 h at 4 °C. Beads were then spun down and washed twice with 1 ml of binding buffer. Bound proteins were subsequently eluted with SDS-sample buffer, and analyzed by SDS-PAGE and Coomassie staining.2SO4. The signal was determined at 450 nm with an ImmunoMini NJ-2300 plate reader . Binding data were analyzed with GraphPad Prism (GraphPad Software) using nonlinear regression assuming one-site binding.Solid phase binding assays were carried out on Immulon 2HB microtiter plates (Dynex) essentially as described previously23404156EcoRI/SalI sites of pEGFP-PK2. For live cell imaging, NIH3T3 cells were transfected with expression plasmids for GFP-PK-NP ncNLS using Lipofectamine 2000 Transfection Reagent (Invitrogen). After two days, the GFP-PK-NP ncNLS proteins expressed in the transfected cells were observed under an Olympus IX81-DSU spinning-disk confocal fluorescence microscope with an excitation filter (460–480 nm) and an emission filter (495–540 nm).An expression plasmid for GFP-Pyruvate kinase (PK)-NP ncNLS fusion protein was constructed by cloning a PCR-amplified DNA fragment encoding a linker (GSDYDIPTTENLYFQGS) and the NP ncNLS (ASQGTKRSYEQMET) into How to cite this article: Nakada, R. et al. Structure of importin-α bound to a non-classical nuclear localization signal of the influenza A virus nucleoprotein. Sci. Rep.5, 15055; doi: 10.1038/srep15055 (2015).


Knowledge of the RNA three-dimensional structure, either in isolation or as part of RNP complexes, is fundamental to understand the mechanism of numerous cellular processes. Because of its flexibility, RNA represents a challenge for crystallization, while the large size of cellular complexes brings solution-state NMR to its limits. Here, we demonstrate an alternative approach on the basis of solid-state NMR spectroscopy. We develop a suite of experiments and RNA labeling schemes and demonstrate for the first time that ssNMR can yield a RNA structure at high-resolution. This methodology allows structural analysis of segmentally labelled RNA stretches in high-molecular weight cellular machines—independent of their ability to crystallize— and opens the way to mechanistic studies of currently difficult-to-access RNA-protein assemblies. The determination of RNA structures within high-molecular weight protein-RNA complexes in non-crystalline state is technically challenging. Here, the authors describe a solid-state NMR protocol for the determination of RNA structures at high resolution. We demonstrate that RNA structure is accessible at high resolution by ssNMR using a few, easy to prepare, nucleotide-type selectively labeled samples. This methodology opens the way to the structure of RNA stretches in large RNA-protein assemblies, independent of their ability to crystallize, and thus to mechanistic studies of yet inaccessible cellular machines.In gene expression regulation, stress response and pathogens infection, a multitude of non-coding RNAs and ribonucleoprotein complexes accomplish their function cycling through transient intermolecular contacts and related conformational changes. Taking influence on these processes requires a mechanistic understanding of the intermolecular interactions, which, in turn, necessitates structural information. Both naked RNAs and RNPs represent a challenge for structural biology. The conformational plasticity of the RNA restricts application of X-ray crystallography, while the high-molecular weight of the RNA (or RNP) of interest pushes solution-state NMR to its limits. Lately, solid-state NMR (ssNMR) spectroscopy, which is applicable to macromolecules of any size in non-crystalline form, has emerged as a powerful alternative to study the structure of amyloid fibrils13Pyrococcus Furiosus (Pf) as part of the complex with the protein L7Ae 15N,13C correlation . For the long-range carbon–carbon transfer, we tested different mixing sequences and finally settled on the PDSD (Proton-Driven-Spin-Diffusion) scheme due to its superior sensitivitySeventeen out of the 26 nucleotides were assigned site-specifically using correlations between the C1' or C6/C8 atoms of nucleotide e strand . To imprrelation , Methods13C,15N-TEDOR-13C,13C-PDSD  were assigned by substitution of the tetra-loop sequence GAAA with UUCG. This alleviated the overlap of G14 and A15 with the resonances of the GAAA loop, allowing their assignment, as well as the identification of G10 and of two adenosines of the A11–A13 stretch. The poor intensity of the GAAA tetra-loop resonances is indicative of conformational heterogeneity; likewise, the terminal G1, U25 and C26 spin-systems are not visible in any of the spectra and were not considered in the structure calculation.13C-PDSD  was appl13C-PDSD  and yiel13C-PDSD . As an e13C,31P correlation, which, with a TEDOR mixing time of 3.2 ms, should provide sequential C2'i/Pi+1 and C3'i/Pi+1 contacts. The mixing time was optimized for sensitivity of transfer over two to three bonds, ranging up to 4–5 Å distance .Finally, we could sequentially assign >90% of all carbon resonances of the 15N,15N through-space correlation 17The determination of RNA secondary structure requires the identification of base pairs. To this end, we used a 15N nuclei exploiting the spatial proximity of their attached protons lab-RNA yielded strong N6A22/C1'G4 and N6A5/C1'G21 signals. The last two correlations were also detected in a 13C-band-selective, 15N-TEDOR spectrum , is exclusively compatible with two N7-amino, N3-imino base pairs, which are typical of k-turn motifs.Secondary structure prediction suggests one U·U and two A·G base pairs . Initial19 protons . This st protons  on three protons ; laspectrum . This paThe U3·U23 base pair might be detected from the proximity of the two H3 atoms in a NHHN correlation. In our case, the chemical shift difference of only 1 p.p.m. between the U3-N3 and U23-N3 hindered the resolution of the weak cross-peak from the intense diagonal. Therefore, we resorted to the analysis of chemical shifts (CS), as indicators of secondary structure. The CS of U23-C2 (151 p.p.m.) and both U3- and U23-C4  deviate from the values of non-stacked disordered nucleotides (154.0 and 168.5 p.p.m.) as well as from the values of A-form helices  and carbon–nitrogen (6) distances, respectively; CHHC and NHHC experiments yielded distances between protons j distance ranges. A total of 91 inter-nucleotide cross-peaks were obtained from the 13C,15N-TEDOR-13C,13C-PDSD experiments, which were all incorporated in structure calculations, in addition to 46 intra-nucleotide restraints over ≥3 bonds -band-selective,15N-TEDOR, we obtained six inter-nucleotide cross-peaks from both the k-turn and helical regions  and to anti in the other cases. Only A19 displayed a χ angle in the syn conformation, in agreement with other k-turn RNA structuresIn addition to distance restraints, we obtained dihedral angles from analysis of ribose chemical shifts , one orthologous complex from Solpholobus solfataricusSs, PDB code 3PLA) and another L7Ae-Box C/D RNA from Pf with a different RNA sequenceφ angle of 23° that defines the k-turn geometry of the ssNMR structure is in very good agreement with the φ angles of the reference structures .The 26mer Box C/D RNA used in this study does not crystallize in complex with L7Ae; however, the crystallographic structure of two orthologous complexes from δ torsion describes the ribose pucker and is defined by the chemical shift analysis of ɛ and ζ angles of the same nucleotides of 1RLG deviate from the values of both our structures bundle and the other three crystallographic structures. The β, ɛ and ζ torsion angles are not directly determined by any NMR parameter, but rather restrained loosely by data base values (see Methods), 31P–13C and 13C–13C distances. Nevertheless, the distribution of these angles in the ssNMR bundle is quite narrow and in good agreement with the reference structures. The α and γ torsion angles are the least well defined by the NMR distance restrains in the stretch 18–21 of the Box C sequence. Interestingly, high variability is observed for these torsion angles among the four reference structures as well, indicating that the k-turn geometry is tolerant to different values , where the dynamic nature of the molecular complexes represents an obstacle to crystallization.Escherichia coli (LB medium) and purified over a Ni-Nta column. Nucleotide-type 13C, 15N selective-labeled Box C/D RNA was prepared by in vitro transcription with T7 polymerase produced in house. Labeling patterns of the RNA were obtained using NTP mixtures where only one or two nucleotide types were either 15N or 13C, or double 13C,15N labeled.The L7Ae–Box C/D RNA complex was assembled from protein and RNA in 1:1 ratio and purified by size exclusion chromatography. L7Ae was expressed in 13C,15N nucleotide-type selective-labelled RNAs: A,Clab-RNA, A,Glab-RNA, A,Ulab-RNA, C,Ulab-RNA, G,Clab-RNA and G,Ulab-RNA lab-RNA and lab-RNA lab-RNA . Next to−1 in buffer containing 25 mM HEPES and 120 mM sodium chloride at pH 7.5, and subsequently mixed with equal amount of precipitation solution , as reported previously1332The L7Ae–Box C/D RNA complex was concentrated to 20 mg ml1H/13C/15N probehead. 13C,31P TEDOR experiments were acquired at 600 MHz with a WB Bruker Avance III spectrometer equipped with a tunable 1H/X/Y probehead at Bruker Biospin in Rheinstetten. The temperature of all experiments was 260 K. 13C,15N-TEDOR-13C,13C-PDSD, 13C,31P-TEDOR, 13C,15N-TEDOR and 15N,15N-RFDR experiments were performed at 16 kHz MAS, while proton diffusion-based CHHC and NHHC experiments were performed at 13 kHz MAS.Solid-state NMR experiments were performed on a 700 MHz SB Bruker Avance III spectrometer equipped with 3.2 mm MAS 13C,15N-TEDOR-13C,13C-PDSD experiment . The 13C–15N dipolar coupling was reintroduced in a short TEDOR mixing time (1.5–2 ms), during which magnetization was transferred to nearby 15N nuclei, and then, after t1, back to the 13C. In t1, we recorded the frequency of nitrogens close to carbons, as for example that of N1/N9 directly bound to C1' and C8/C6. The following, long 13C,13C-PDSD step  transferred the 15N-chemical shift labelled 13C magnetization to nearby carbons. Finally, 13C magnetization was detected during t2. The ambiguity on the carbon from which the magnetization originates in the PDSD step, either C1' or C8/C6, was lifted in three-dimensional experiments, where the 13C frequency was recorded before the PDSD mixing. Alternatively, we evaluated the efficiency of the 15N1/N9-13C transfer, which in several instances was found to be better towards the C1' than towards C8/C6. As a third alternative, a 13C-band-selective-TEDOR transfer, with selectivity either on C1' or on C6/C8, can be used to resolve the ambiguity. Cross-peaks were evaluated and translated into distance restraints. Distance ranges (d) were applied for inter-nucleotide restraints as 3.5<d<9 Å, according to several previous studies3435d<6 Å, and intra-nucleotide ribose–ribose restraints, 3<d<4 Å, were determined from the nucleotides' geometry.In the periment  13C magn13C,31P-TEDOR experiment  can be applied after TEDOR to transfer the 13C magnetization to further carbon spins, such as C1'. This experiment was useful to identify the ribose spin systems through the better-resolved C1' chemical shift. 13C,31P-TEDOR spectra were recorded for Alab-RNA and G,Ulab-RNA; due to the limited signal-to-noise, only one TEDOR mixing time was recorded (3.2 ms). The spectra yielded 17 non-trivial restraints, which were classified as 3<d<5 Å, as appropriate for a mixing time of 3.2 ms.In the periment , after i13C-band-selective,15N-TEDOR experiment13C magnetization, the 13C–31N dipolar coupling was reintroduced in a TEDOR mixing time of 6–15 ms with band-selective 13C inversion pulses; the long mixing allows transferring magnetization between carbons and nitrogens as far as 5–6 Å. The 15N and 13C frequencies were recorded during t1 and t2, respectively. With a 13C,15N-TEDOR that was selective for C1' and C4', we obtained four G-C1',C4'/A-N6,N9 cross-peaks from the lab-RNA and two G-C1'/U-N1,N3 cross-peaks from the lab-RNA. Also in this case, we did not acquire multiple TEDOR mixing times, due to limited signal-to-noise. Distance ranges 3<d<5 Å and 3<d<7 Å were attributed to the strong and weak peaks, respectively, at a mixing time of 12 ms.In the 15N,15N-RFDR experiment  or 15N (NHHC). Next, the magnetization was transferred back to protons, from where, after a short proton mixing of 100–200 μs, it was transferred to nearby carbons with a 100-μs cross polarization step; finally the frequency of 13C was recorded in t2. Inter-proton distances 2<d<4 and 2<d<5 Å were attributed to the strong and weak signals, respectively, following previous studies19In the CHHC and NHHC proton spin diffusion-based experiments  for canonical base pairs and non-canonical base pair U3·U23. Flexible planarity was introduced for the base pairs A5·G21 and G4·A22 by defining the plane that involves one atom of the acceptor and four atoms of the donor base to allow for propeller twist and tilt, as described in ref. Structures were calculated using the Aria 1.2/CNS 1.1 set-up281142α, β, ɛ and ζ were restrained to the range typical for A-form helix  for nucleotides 2, 6–9, 14–17, 24, which are involved in canonical base pairs; the α, β, ɛ and ζ angles of the remaining nucleotides were loosely restrained to the allowed ranges . Dihedral angles α and ζ of nucleotides G4, G6, A18, U20 and G21 were additionally restrained to 0°±120° based on 31P chemical shiftsγ was restrained to the gauche+ conformation for nucleotides involved in base pairs.The ribose conformation was restrained through the analysis of ribose chemical shifts−1, and dihedrals—200 kcal mol−1, in the final cooling step.)Three hundred structures were calculated in one iteration without the automated assignment or the distance calibration options of Aria 1.2 using an assigned distance list. Before minimization, we randomized all backbone dihedral angles. The minimization protocol used the force-field DNA-RNA-allatom-hj-opls.top and the following parameters in the four steps of simulated annealing (SA), together with the PROLSQ nonbonded parametersThe final structures were analysed using MolMol13C,15N-TEDOR-13C,13C-PDSD of the lab-UUCG-RNA allowed identifying two adenosines of the GAAA tetra-loop of the wild-type RNA, which disappear in the mutant spectrum. In addition, we could confirm the assignment of A15, which does not overlap with any other spin-system in the UUCG-RNA. The 2D 13C,15N-TEDOR-13C,13C-PDSD spectrum of G,Clab-UUCG-RNA allowed the assignment of G10, whose spin-system is not present in the spectrum of the mutant RNA. In addition, the resonances of G14 shift slightly in the mutant with respect to the wild-type RNA, due to the different structure of the UUCG tetra-loop. This fact confirmed the assignment of the G14 spin-system.We measured two samples of the mutant Box C/D RNA containing the stable UUCG tetra-loop (UUCG-RNA) instead of the GAAA tetra-loop to aid and confirm the assignment. The 2D A.M. produced samples, developed and performed both NMR experiments and structure calculations, interpreted the results and wrote the manuscript together with T.C. B.S. contributed to the development of pulse programs and structural calculation protocols. G.A. helped in the acquisition of NMR data. T.C. designed the project, contributed to the development of pulse programs and structural calculation protocols, interpreted the results and wrote the manuscript together with A.M. All the authors discussed the results reported in the manuscript.Accession codes: The atomic coordinates of 10 lowest energy structures have been deposited in the Protein Data Bank under accession number 2N0R. The NMR chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank, entry 25534.How to cite this article: Marchanka, A. et al. RNA structure determination by solid-state NMR spectroscopy. Nat. Commun. 6:7024 doi: 10.1038/ncomms8024 (2015).Supplementary Figures 1-10, Supplementary Tables 1-2 and Supplementary References


Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude . Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein–RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of  Regulation by small non-coding RNAs (sRNAs) is crucial for orchestrating global changes in bacterial gene expression . The besPseudomonas aeruginosa has a conserved RNA recognition surface, despite a distinctly different polypeptide fold when compared with the domain-swapped dimeric structure of the CsrA/RsmE homologues  are specifically recognized by the protein backbone and thus, the shape of the protein, while the RNA stem is semi-specifically recognized on its major groove side by protein side-chains. Although this structure demonstrates the molecular basis for the CsrA/RsmE protein recognition by a specific GGA motif, which is close to the high affinity SELEX-derived consensus sequence  structure of a stem–loop encompassing the RBS of the ith RsmE . The strsequence , it is uin vitro transcription from double-stranded DNA templates using T7 polymerase and was subsequently purified by denaturing high-performance liquid chromatography (HPLC) followed by butanol extraction as reported earlier  in 95% H2O/5% D2O (v/v) at 283 K. Non-exchangeable RNA proton resonances were assigned using 2D 1H-1H NOESY (τm = 150 ms), 1H-1H TOCSY, 1H-13C HSQC, 3D 13C-edited NOESY (τm = 150 ms) and 3D HC(C)H TOCSY spectra in D2O with samples, in which the RsmE protein was only 15N-labelled and the RNA nucleotide-specifically 13C,15N-labelled   signals were assigned based on 2D 1H-1H nuclear Overhauser enhancement spectroscopy (NOESY) (τm = 150 ms), 3D 13C-edited NOESY (τm = 150 ms) and 3D  NOESY spectra (τm = 150 ms)  binding experiments were conducted on a VP-ITC instrument from MicroCal. The calorimeter was calibrated according to the manufacturer’s instructions. The RNA and the protein samples were dialysed against the same buffer batch . Concentrations were determined after dialysis using optical density absorbance at 260 and 280 nM for RNA or protein, respectively. The RNA  was titrated into the RsmE protein . ITC binding experiments were performed at 298 K and typically consisted of 30–40 injections of 4–10 µl with an injection speed of 2 s/µl and a 5 min interval between additions. The stirring rate was 307 rpm. All measurements were repeated at least twice. Using Origin 7.0, the raw data was integrated, corrected for non-specific heats and analysed according to a one-site or two-site model. The RNA sequences of all the constructs are summarized in In the slow exchange regime on the NMR timescale, the resonances corresponding to the free and bound macromolecules are simultaneously present as two separated peaks if their chemical environments change upon complex formation. Their relative integrals directly relate to the fraction of free and bound macromolecule (protein or RNA) present in the sample. Knowing the total concentration of macromolecules (protein and RNA), the dissociation constant can directly be determined using the equation:GA motif forming an intermolecular hydrogen bond) is observable for the unlabelled RNA in complex, the dissociation constants with their associated errors for the hcnA-GGA#1 and hcnA-GGA#2 RNAs binding to RsmE (As only a single well-separated resonance (imino of the second G in the G to RsmE  were dethcnA gene was subcloned into the vector pIVEX1.3-CAT (Roche) directly upstream of the chloramphenicol acetyl-transferase (CAT) open reading frame, yielding the vector pCFX100, which was amplified using a plasmid maxi prep kit (Macherey-Nagel). S30 cell extract was obtained from Escherichia coli BL21 (DE3) Star cells, which were genetically modified by introducing a C-terminal (His)6-tag in the csrA gene following the procedure by Datsenko and Wanner (E. coli BL21 (DE3) Star 6csrA::(His). In addition to the previously described extract preparation protocol . A total of 10 µl of the diluted solution was then mixed with 990 µl CAT reaction solution  and the increase in absorbance at 412 nm was followed for 20 min on a Cary 300 Bio spectrophotometer. The expression levels of the reporter enzyme were derived from the slope of absorbance at 412 nm against time and were then normalized to the largest value that was obtained after complete saturation of RsmE in the reaction mixture with RsmX RNA.Analytical scale cell-free expressions (50 µl) of the pCFX100 plasmid were set up in presence of various amounts of wild-type or mutant RsmX sRNA and 100 nM dimeric RsmE protein from P. fluorescens CHA0 is a sRNA of 127 nt composed of four stem–loops (SLs) and a ρ-independent terminator (P. fluorescens CHA0. All four stem–loops of RsmZ contain a conserved A(N)GGAX motif in the loop on top of a stem closed by two base pairs, a C-G followed by a U-A, except SL3 that has a G-C instead of a U-A as penultimate stem-closing base pair (39-41) is single-stranded and is also missing the nucleotide N of the consensus motif.The sRNA RsmZ from rminator . It contrminator . Here, wase pair . HoweverSupplementary Figure S1). Unexpectedly, the complex with the single-stranded 9 nt-GGA39-41 RNA is also in slow exchange indicating a slow rate of complex dissociation (<10/s). The binding affinities differ by one to two orders of magnitude. Whereas SL2 has an affinity of 16 ± 3 and 185 ± 3 nM for the binding of the first and second RNA molecule to the dimeric RsmE protein, respectively, the other RNA targets have affinities ranging from 1.5 to 3.5 µM .First, we characterized the binding of these RNAs to RsmE with NMR chemical shift titrations and ITC-binding experiments. All the protein and RNA resonances in the 4 SL complexes are in slow exchange relative to the NMR chemical shift timescale . In the single-stranded 9 nt-GGA39-41 complex structure, the nucleotides flanking the common A(N)GGAX binding motif, are not well defined and are not recognized by the RsmE protein . They are flexible as can be judged from the narrower line width of their aromatic and sugar resonances. An overview of all the structures is shown in To understand the molecular basis for these differences in affinity, we determined the structures of the corresponding protein–RNA complexes. The high quality of the spectra allowed us to collect enough intra- and intermolecular NOEs to solve the structures of all five complexes with an RMSD of 0.75–1.2 Å. The structural statistics are shown in A(N)GGAX motif is recognized in an identical manner in all five structures, also found in the structure of the 20 nt-RBS RNA bound to RsmE . As the recognition of the A(N)GGAX motif is largely achieved by many hydrogen bonds between the RNA bases and the protein backbone, the recognition is sequence specific and these bases cannot be substituted by others and still be accommodated by the given protein fold. In contrast to the core A(N)GGAX motif, the nucleotides N and X within and the nucleotides adjacent to it are variable and allow for a modulation of the binding affinity.The conserved  to RsmE  . A static structure allows a rationalization of the enthalpic contribution to the free energy of binding but does not provide any insight into the entropic contribution to binding, which might explain why the affinity of SL2 is not significantly higher than that of the 20 nt-RBS for RsmE. Remarkably, ITC-binding experiments show a significantly higher binding enthalpy for SL2 compared with the 20 nt-RBS RNA and SL4 . However, the favourable binding enthalpy is compensated by more unfavourable binding entropy for SL2 compared with the other two RNAs.Despite the larger number of hydrophobic contacts and the stacking of adenine A26 on Arg5015N-heteronuclear NOE experiments for all three complexes, which report on the protein backbone dynamics in the ps–ns timescale , the C-terminal nucleotides Leu55 to Ala57 become more rigid than when N is a cytosine (20 nt-RBS) or N is absent (SL4) ((N)GGAX motif results in a significant gain in binding affinity for P. fluorescens CHA0 RsmE. However, the energetic origin of this gain depends on the identity of the base of the looped out nucleotide.We then addressed the question of why SL2 binding is entropically less favoured when compared to binding of SL4 and the 20 nt-RBS RNA to RsmE. For this, we recorded imescale . The 15Nnt (SL4) . These rnt (SL4) . In summA side-chain with the carbonyl O6 of the guanine of the CG stem closing base pair is absent in SL1. However, this loss in hydrogen bond contacts between protein and the RNA major groove of SL1 is compensated by new hydrogen bonds of the looped-out G5 base with the side-chains of Arg6A and Glu46B (A(N)GGAX motif and with C4 in the stem (A(N)GGAX adenine (A8 in the 20 nt-RBS RNA) at an optimal place for forming strong hydrogen bond interactions with the backbone of the protein GGAX or the insertion of two nucleotides between the ANGGAX hexa-loop and the stem influence the binding affinity for RsmE by one to three orders of magnitude, several RNA constructs have comparable affinities despite significant structural differences .The 5′-nucleotide of the penultimate stem closing base pair is specifically recognized by the glutamine Gln29identity a–c. In S protons d, wherea protons e. Althou protons . In contosine C4 f. This nB contacts the phosphate backbone of the RNA loop but is not involved in stacking interactions with the RNA GGAX motif (39-41 RNA) or two additional nucleotides are inserted between the stem and the A(N)GGAX loop (SL1). Intriguingly, in the single-stranded 9 nt-GGA39-41 RNA, the Arg44B side-chain is not only stacking on the adenine A38 of the A(N)GGAX motif, but is also forming a hydrogen bond to the A38 2′-hydroxyl group . Strikingly, however, by titrating two equivalents of the mutant RNAs into one equivalent of RsmE protein homo-dimer, we could simultaneously observe the free and the bound protein amide resonances and notably also the free and bound RNA imino resonances (on in the order of 106–108/Ms (We determined structures of GGA motifs located either in loops of hairpins or single-stranded regions with affinities for RsmE covering more than two orders of magnitude (∼10–3500 nM). Yet, other GGA motifs are partially or entirely buried within secondary structures and would therefore not be expected to bind the CsrA/RsmE protein. Two such GGA motifs are present in SL1 of the 5′-UTR of the orescens . As NMR sonances . This sh6–108/Ms ,40]. Thu6–108/Ms . A smallIn summary, we demonstrate that not only the sequence surrounding a certain GGA motif contributes to the modulation of the binding affinity for RsmE but that the secondary structural context influences the range of binding affinities of the Csr/RsmE protein–RNA recognition system to extend over five orders of magnitude.ANGGAX placed directly on top of a stem . We performed NMR chemical shift titrations to observe the binding of 15N-labelled RsmE to an unlabelled minimal fragment of the hcnA mRNA 5′-UTR containing the three GGA#3–#5 motifs. Unsurprisingly, the first RsmE protein dimer binds simultaneously to GGA#3 and GGA#5, whereas a second RsmE dimer binds to GGA#4, which is the weakest binding motif. These findings suggest that one RsmE dimer binding to both GGA#3 and GGA#5, which overlaps with the RBS, is responsible for translation repression of the hcnA mRNA. These observations are in agreement with previous in vivo results demonstrating that mutations in GGA#3 and #5 significantly reduced translation repression by RsmE, while mutations in GGA#4 only slightly affected repression GGAX binding motif but the context of the sequence modulates the binding affinity for RsmE by more than two orders of magnitude. When the GGA motif lies partially or entirely within a base paired region, the affinity can further decrease by two to three orders of magnitude, resulting in a total modulation of the binding affinity of the Csr/Rsm system to more than five orders of magnitude. The degree of binding affinity attenuation is given by the extent by which the RNA secondary structure has to be disrupted in order to bind RsmE (compare in A(N)GGAX binding motif, which is recognized by many hydrogen bonds to the backbone of the RsmE protein. As this recognition involves binding of the nucleobases by the backbone and not the side-chains of the protein, this recognition is specific and these bases cannot be replaced by other ones and still be accommodated by the given protein fold. In contrast to the core A(N)GGAX motif, the nucleotides N and X and the nucleotides adjacent to the core motif are variable and allow for a modulation of the binding affinity.We have elucidated and compared the solution complex structures of RsmE bound to six different target RNAs, which are the four SLs and the single-stranded region between SL2 and SL3 of the sRNA RsmZ (this study) and a 20 nt RNA SL encompassing the RBS of the orescens . All RNA76-78 and GGA39-41) or more severely, occluding GGA motifs within secondary structures lead to lower binding affinity. Interestingly, AGGGA and AAGGA pentaloop sequences have very recently been shown to have high affinity for RsmE, thus suggesting that omitting the nucleotide X in A(N)GGAX when the nucleotide N is a purine also constitutes a high affinity binding target for CsrA/RsmE GGAX in the single-stranded 9 nt-GGA39-41 RNA has a C2′-endo sugar pucker conformation, while an equivalent adenine stacking on a stem has a sugar pucker C3′-endo conformation. RNA adaptation leading to recognition of different RNA sequences with equal affinity has been demonstrated recently. The solution structures of the oligonucleotides 5′-UCCAGU-3′ and 5′-UGGAGU-3′ in complex with the RRM domain of SRSF2 revealed that the cytosines having an ‘anti’-base conformation are recognized very similarly as the guanines having a syn conformation GGAX is missing does not change the binding affinity considerably. The single-stranded 9 nt-GGAe a stem a and thee a stem . Interesormation . One arg65  and the RsmN protein from P. aeruginosa . Simply, the looped-out adenine (corresponding to nucleotide N of the A(N)GGAX motif) has a different structure. This is due to the α-helix, which is only present in RsmE but not in RsmN.Notably, despite their distinct protein folds, the orthologues RsmE protein from ruginosa  bind SL2In contrast to the CsrA/RsmE system, in which the protein binding surface is almost identical in all the homologous proteins and the variety of RNA sequences are recognized by the same protein by adaptation of protein side-chain and RNA conformations, the coat proteins of single-stranded bacteriophages recognize their cognate RNA targets by very distinct binding modes. The co-evolution of coat protein and corresponding RNA structure ensures that each coat protein specifically binds only its cognate RNA target and discriminates against other RNA targets ,51.flhDC mRNA by binding to its 5′-UTR  to adjust its affinity according to its specific function in the cell. Different affinities of mRNAs for CsrA/RsmE allow for a fine-tuning of translation repression if CsrA/RsmE binds to the RBS or close to it. Strong binding of CsrA/RsmE to the RBS increases competition with the 30S ribosomal subunit for binding to the RBS, hence stronger translation repression b 11,17),1711,17)s 5′-UTR ,53 and mficantly . We specE. coli (hcnA mRNA are not assumed to be relevant for binding RsmE in vivo (dissociation constants of 300 µM and 2.7 mM). It has been shown that chromosomally transcribed mRNAs are not homogenously expressed in the cellular space of E. coli  of an mRNA could be modulated by several factors that could vary in a cell state-specific manner. It is credible that sRNAs base pair with the 5′-UTR of the mRNA as has been proposed for RsmY and the hcnA mRNA 5′-UTR in P. fluorescens (2+)  , the temens (2+)  or co-trens (2+)  could moIn conclusion, this work exemplifies the enormous diversity in the Csr/Rsm protein–RNA recognition and provides the basis for the next level of binding complexity, which is the assembly of several CsrA/RsmE homo-dimeric proteins on a sRNA or mRNA containing more than one GGA motif on a single molecule.39-41/RsmE complexes have been deposited in the Protein Data Bank under accession codes 2mfc, 2mfe, 2mff, 2mfg and 2mfh, respectively.Atomic coordinates, NMR chemical shifts and NMR restraints for the structures of the SL1/RsmE, SL2/RsmE, SL3/RsmE, SL4/RsmE and 9 nt-GGASupplementary Data are available at NAR Online.Swiss National Science Foundation ; the SNF-NCCR structural biology Iso-lab. Funding for open access charge: NAR Editorial Board members are entitled to one free paper per year in recognition of their work on behalf of the journal.The Conflict of interest statement. None declared.


Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5’-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different substrates. The enzyme arranges as a stable dimer maintained by mutual interactions between the active site cleft of one molecule and the flexible N-terminal tail of the symmetrically related RdRP. The latter, partially mimicking the RNA template backbone, is involved in regulating the polymerization activity. As expected from previous sequence-based bioinformatics predictions, the overall architecture of the TaV enzyme shows important resemblances with birnavirus polymerases. In addition, structural comparisons and biochemical analyses reveal unexpected similarities between the TaV RdRP and those of Flaviviruses. In particular, a long loop protruding from the thumb domain towards the central enzyme cavity appears to act as a platform for de novo initiation of RNA replication. Our findings strongly suggest an unexpected evolutionary relationship between the RdRPs encoded by these distant ssRNA virus groups.Thosea asigna virus (TaV), an insect virus belonging to the  Thosea asigna virus, has unveiled two essential elements controlling polymerization activity: (i) the protein N-terminus that invades the central cleft of the neighboring RdRP molecule, thus stabilizing a dimeric form of the enzyme with partially occluded template binding channels; and (ii) a long loop protruding towards the catalytic cavity which harbors the binding site of incoming nucleotides, thus providing a platform for de novo replication initiation. The close structural and functional resemblance between this enzyme and flaviviral RdRPs strongly suggests the existence of an unexpected evolutionary link between these two distant virus groups.RNA dependent RNA polymerases (RdRPs) are the catalytic components of the RNA replication and transcription machineries, and thus central players in the life cycle of RNA viruses. The in-depth understanding of both the structure and regulation of viral RdRPs displaying different replication-transcription strategies might provide essential clues for an effective control of virus propagation. The characterization of the first non-canonical RdRP of a positive-stranded RNA virus, the permutotetravirus  Birnaviridae and Permutotetraviridae families, harboring double-stranded (ds) and positive (+) single-stranded (ss) RNA genomes, respectively. In these enzymes, motif C is located upstream of motif A forming a non-canonical C-A-B arrangement with a unique connectivity of the major structural elements of the active site . Na. Napol wsolution . The crysolution . The RdRsolution . As experimposed .in vitro using both TaVpol and TaV rORF1 constructs in the presence of the TaV-derived ssRNA template described above. Auto-nucleotidilation of TaVpol has not been detected (pol(T157A) and TaVpol(S4A) mutants, where the predicted nucleotidylation residues UTP incorporation measured in counts per minute using a liquid scintillation counter .Polymerase activity assays were performed following a previously described protocol  with minvariants , were prpol or the full-length TaV rORF1 supplemented or not with 5 μl of a ssRNA template (0.2 mg/ml) were performed for 10 min under optimal conditions . Reaction products were subjected to 11% SDS-PAGE, and recorded by autoradiography following an 8 h exposure period.Reaction mixtures containing the TaVpol, apo-form and the Lutetium derivative were obtained by the sitting-drop vapor diffusion method as previously described  GTP, and incubated for 45 min at 35°C.Autoradiography of the polymerization products analyzed in a 7% acrylamide TBE gel. The first and second lines correspond to the activity of TaV(TIF)Click here for additional data file.S7 Figin vitro RdRP activities of full-length TaV rORF1 (top) and TaV rORF1(Δ27) (bottom). The reaction was stopped at different incubation times and the reaction products were analyzed in 7%TBE-PAGE and recorded after an exposure time of 8 h. Reactions were performed in presence of the ssRNA template of 311-nts harboring the TaV 3UTR’ sequence. The bottom image shows an 11% SDS-PAGE stained with Coomassie blue of the two proteins, used as protein loading controls.Autoradiograms of (TIF)Click here for additional data file.S8 FigA sample of the purified enzyme was negatively stained with 2% (w/v) uranyl acetate. Red and blue circles indicate dimer and monomer respectively. Scale bar, 3 nm.(TIF)Click here for additional data file.S9 Figpol is shown in cyan, the RNA-ATP-bound enzyme in magenta and the Lutetium derivative in gold. One monomer of the RNA-ATP-bound structure shows a -7° rotation around the Z axes with respect to the same position of the apo-form TaVpol. The same monomer of the Lutetium structure shows a 22° and -18° rotation around Z and X axes, respectively, in comparison to the apo-form enzyme. The structural comparisons of individual monomers also show a subtle opening of the central channel. (B) Different conformations adopted by the TaVpol N-terminal ends in the apo structure (blue and cyan) and in the RNA-ATP complex (red and pink). In the TaVpol-RNA-ATP complex, the polymerase N-terminus points outwards from the central cavity leaving enough space to accommodate a ssRNA template, modeled in stick representation.(A) The apo-form of TaV(TIF)Click here for additional data file.S10 Figpol template channel in the TaVpol-CCCAUUCGACUCCUG-ATP complex. The strong peaks of electron density (purple mesh) can be easily interpreted as the phosphate groups of a short oligonucleotide bound to the channel, in good agreement with template models derived from the superimposition of available RdRP-RNA complexes onto the TaV enzyme. The modeled template (shown as sticks in atom type color) was obtained by the superimposition of the HCV NS5B replication initiation complex (PDB ID 1WTA) onto the TaVpol active site. (B) A partial data set from other complex co-crystals, the TaVpol-GUAUACUACACCCAUUCGACUCCUG complex, have been obtained and analyzed . The X-ray data was collected using synchrotron radiation at the Swiss Light Source, PXI beam line. Unfortunately, these crystals were extremely sensitive to radiation and died before completing data collection . Attempts to merge data for different crystals failed due to the lack of isomorphism. The structure was solved by molecular replacement, using the coordinates of the unliganded TaVpol as search model. Analysis of the electron density maps revealed the presence of a partially ordered extra electron density to position a stretch of four nucleotides, most probably the 3’-end (5’-CCUG) of the template, occupying the template binding channel of the two polymerases molecules of the crystal asymmetric unit. Model refinement was performed with the program REFMAC5 [work of 22.3%, Rfree = 25% with good stereochemistry . The image shows a view of the template channel with the refined coordinates of the bound tetranucleotide modeled inside. Polymerase residues directly contacting the RNA are explicitly labeled. The template base at position 3’ appears tightly packed to motif B residues T443 and T444. A σA-weighted 2ІFoІ-ІFcІ map, contoured at 1.2σ, of the tetranucleotide template is shown as a green mesh. Inset corresponds to an autoradiograph of in vitro polymerase activity of TaVpol, analyzed in 7% acrylamide TBE gels, showing that mutations in residues T443 and T444 abolish RNA synthesis.(A) View of a σA-weighted |Fo|-|Fc| electron density map (3.5σ) around the TaV REFMAC5  applying REFMAC5 . The fin(TIF)Click here for additional data file.


Conventional procedures to assay RNA degradation by a protein with ribonuclease (RNase) activity require a step to isolate intact RNA molecules, which are used as a substrate. Here, we established a novel “In-cell RNA hydrolysis assay” in which RNAs within cells are used as a substrate for the RNA-hydrolyzing protein, thereby avoiding the need to prepare intact RNA molecules. In this method, the degree of RNA degradation is indicated by the fluorescence intensity of RNA molecules released from fixed and permeabilized cells following treatment with the potential RNase. A catalytic 3D8 antibody capable of degrading RNAs and pancreatic RNase A were used as model RNases. Our data demonstrate that the novel In-cell RNA hydrolysis assay is a reliable and sensitive method to analyze the activities of potential RNA-hydrolyzing proteins such as catalytic antibodies. Some anti-DNA antibodies that are found at high titers in humans and mice with autoimmune diseases, such as systemic lupus erythematosus and multiple sclerosis , 2, are Several methods have been developed to analyze the DNA-hydrolyzing activity of catalytic anti-DNA antibodies, in which plasmid DNA or synthetic nucleotides are used as a substrate –12. ThesHere, we developed an “In-cell RNA hydrolysis assay” in which RNA molecules within cells are used as a substrate, thereby avoiding the need to isolate intact RNA from cells or to synthesize RNA. Two proteins with RNase activity, 3D8 scFv antibody and ribonuclease (RNase) A, were used as model proteins to test this application. In this assay, degradation of cellular RNAs is simply indicated by the fluorescence intensity of RiboGreen-labeled RNA molecules released from fixed and permeabilized cells upon treatment with the RNase. This was verified by the finding that the amount of RNAs inside fixed and permeabilized cells was decreased after treatment with an RNase. The stringency of this method was confirmed by the finding that cellular DNA did not affect the fluorescence intensity of RiboGreen-labeled RNA. This assay provides a simple means of determining the RNase activity of potential RNases including catalytic antibodies.3D8 scFv and HW6 scFv proteins containing a protein-A tag were purified from the supernatants of bacterial cultures using IgG-Sepharose affinity chromatography [4 cells/well in a 48-well plate were washed three times with ice-cold PBS and fixed with 4 % paraformaldehyde prepared in PBS for 15 min at room temperature (RT). Cells were washed twice with PBS, and cell membranes were permeabilized with P buffer  for 30 min at RT. Thereafter, cells were treated with 100 μl of various concentrations of 3D8 scFv antibody, HW6 scFv antibody, or RNase A (Intron Biotech) prepared in PBS containing 1 mM MgCl2 (pH 7.2) for 2 h at 37 °C. Next, 50 μl of the conditioned medium was added to microplate wells containing 100 μl of Quant-iT™ RiboGreen® reagent  diluted 200-fold with TE buffer  and incubated for 2 min at RT in the dark. The reaction mixtures were excited at 485 nm and emission at 530 nm was measured using a fluorescence analyzer.To detect cellular RNA fragments released from cells using RiboGreen reagent, A549 human lung epithelial cells grown at a density of 5 × 10E. Coli (Invitrogen), or 1 μg/ml pUC19 plasmid DNA was incubated with RiboGreen reagent in the absence or presence of 2 U DNase I (NEB) prepared in PBS containing 1 mM MgCl2 (pH 7.2) or in DNase I reaction buffer .In some cases, conditioned medium of fixed and permeabilized cells, pure 16S and 23S rRNA from 5 cells/well in a 6-well plate were fixed and permeabilized as described above. Thereafter, cells were treated with 500 μl of 10 μM RNase A or 3D8 scFv antibody prepared in PBS containing 1 mM MgCl2 (pH 7.2) for 2 h at 37 °C. Then, 150 μl of conditioned medium from each well was applied to a protein precipitation kit  to remove protein contaminants, and A260 was measured using a UV spectrophotometer.To detect cellular RNA fragments released from cells by measuring ultraviolet (UV) light absorbance, A549 cells grown at a density of 5 × 10® RNA Alexa Fluor® 488 Imaging Kit (Molecular Probes). A549 cells grown on coverslips (5 × 104 cells/well in a 24-well plate) were incubated with 1 mM 5-ethynyl uridine (EU) for 20 h at 37 °C, allowing the incorporation of EU into newly synthesized RNAs. Cells were fixed and permeabilized as described above and then treated with 500 μl of 10 μM RNase A or 3D8 scFv antibody prepared in PBS containing 1 mM MgCl2 (pH 7.2) for 2 h at 37 °C. Cells were incubated with Alexa 488-azide solution, which ligates EU, for 30 min at RT in the dark, and washed three times with RNase-free PBS. Nuclei were stained with Hoechst 33342 (Vector Laboratories) for 30 min at RT. Images of intracellular green fluorescence were acquired by confocal microscopy (Carl Zeiss LSM 710).To analyze the level of RNA inside cells, confocal microscopy was performed using a Click-iT5 cells/well in a 6-well plate) were incubated with 1 mM EU for 20 h at 37 °C and then detached by trypsin treatment. Cells were washed, fixed, and permeabilized as described for confocal microscopy experiments. After treatment with 800 μl of 10 μM RNase A or 3D8 scFv antibody prepared in PBS containing 1 mM MgCl2 (pH 7.2) for 2 h at 37 °C, cells were incubated with Alexa 488-azide solution, which ligates EU, for 30 min at RT and washed three times with RNase-free PBS. Finally, cells were suspended in 4 % paraformaldehyde prepared in PBS, and intracellular green RNA fluorescence was analyzed using a FACSCanto II flow cytometer (Becton–Dickinson).To detect cells containing fluorescent RNA molecules, A549 cells  contained ~400 ng of RNA. This supports the observation that RNA released from untreated cells in a 48-well plate could not be detected by measurement of A260 but was detected by RiboGreen labeling (Fig. RNA degradation in fixed and permeabilized cells treated with various concentrations of proteins was quantitatively analyzed. A standard curve was generated using pure 16S and 23S rRNA to calculate the concentration of RNA released from fixed and permeabilized cells upon treatment with an RNase (Fig. Furthermore, the sensitivity of our method was assessed with RNase A. RNase A was diluted to various concentrations from a stock solution of 1 µM, and then 100 µl of each dilution was incubated with fixed and permeabilized cells, prior to detection of the RiboGreen signal. RNA hydrolysis was detectable with 3.91 nM RNase A (Fig. To verify the principle of the In-cell RNA hydrolysis assay, we examined whether the amount of RNA within cells decreased as RNA was released. Cells were incubated with EU, which is actively incorporated into nascent RNAs in the nucleus, fixed, permeabilized, and treated with RNase A or 3D8 scFv antibody. Following labeling of EU-incorporated RNAs with Alexa 488-azide, RNA remaining inside cells was analyzed by confocal microscopy (Fig. We established the novel In-cell RNA hydrolysis assay. This assay was designed to detect the release of degraded RNA generated by a RNA-hydrolyzing protein from fixed and permeabilized cells using RiboGreen, an ultrasensitive reagent to RNA in solution. We expected that the fluorescence intensity of RiboGreen would be directly proportional to the amount of RNA fragments released from permeabilized and fixed cells treated with an RNase; however, the fluorescence intensity of RiboGreen was decreased by treatment with an RNase due to the complete degradation of RNA molecules such that they were no longer recognized by RiboGreen Fig. b. TherefCells were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % saponin. Paraformaldehyde is a protein-crosslinking fixative that creates covalent bonds between proteins, primarily residues of the basic amino acid lysine, without affecting nucleic acids. This indicates that nucleic acids can be freely released from fixed cells, while the majority of proteins would be retained . SaponinThe level of RNA in nuclei was dramatically reduced in cells treated with RNase A or 3D8 scFv antibody Fig. b. This cOur study demonstrates that the In-cell RNA hydrolysis assay can be a useful alternative method to analyze RNase activity.


The tumour suppressor p53 is an important mediator of cell cycle arrest and apoptosis in response to DNA damage, acting mainly by transcriptional regulation of specific target genes. The exact details how p53 modulates this decision on a molecular basis is still incompletely understood. One mechanism of regulation is acetylation of p53 on lysine K120 by the histone-acetyltransferase Tip60, resulting in preferential transcription of proapoptotic target genes. PDCD5, a protein with reported pro-apoptotic function, has recently been identified as regulator of Tip60-dependent p53-acetylation. In an effort to clarify the role of PDCD5 upon DNA damage, we generated cell lines in which PDCD5 expression was conditionally ablated by shRNAs and investigated their response to genotoxic stress. Surprisingly, we failed to note a rate-limiting role of PDCD5 in the DNA damage response. PDCD5 was dispensable for DNA damage induced apoptosis and cell cycle arrest and we observed no significant changes in p53 target gene transcription. While we were able to confirm interaction of PDCD5 with p53, we failed to do so for Tip60. Altogether, our results suggest a role of PDCD5 in the regulation of p53 function but unrelated to cell cycle arrest or apoptosis, at least in the cell types investigated. Cells are constantly exposed to noxic insults and therefore have evolved complex mechanisms to respond to these challenges to secure survival and genomic integrity. One of these insults is genotoxic stress, which can lead to persisting mutations thereby contributing to the development of cancer789After DNA damage activates the DDR machinery, p53 is phosphorylated at several amino acids1113One of the modulators of the HAT activity of Tip60 described is programmed cell death 5 (PDCD5)17181920211624252627To explore the role of PDCD5 in cell death signalling in more detail we generated a PDCD5-specific antiserum that recognizes endogenous mouse and human PDCD5 protein as well as a series of human cancer cell lines where PDCD5 expression can be conditionally ablated by RNAi and studied their response to DNA damage.In an effort to study the role of PDCD5 in the DNA damage response, we first investigated the transcriptional regulation of PDCD5 in Hct116 colon carcinoma, A549 lung carcinoma and U2OS osteosarcoma cells. Cells were exposed to γ-irradiation or etoposide, two established triggers of p53 and the DDR machinery. However, neither treatment led to a consistent and significant increase in PDCD5 mRNA transcripts while the p53 target genes p21 and Bax where found increased . AltogetAs PDCD5 was reported to be detectable in the cytoplasm as well as in the nucleus and described to shuttle into the nucleus upon DNA damageAs PDCD5 was described to bind to both p53 and Tip60, we used co-immunoprecipitation assays to re-investigate these postulated interactions. In accordance with previous findings16In contrast, we were not able to reproducibly detect specific interaction between PDCD5 and Tip60 using several immunoprecipitation-protocols and different stimuli tested (data not shown). In fact, Tip60 was easily precipitated using pre-immune serum conjugated to sepharose-beads. Using different concentrations of salt during washing, we found a dose dependent decrease of this interaction while PDCD5/Ig complexes where highly stable, even at 400 mM of salt in the wash buffer . AlthougBax, Noxa and Puma as well as the CDK inhibitor p21 were upregulated to various extents in response to DNA damage and in a time dependent manner. However, no significant effect of PDCD5 knockdown was detected using cell lines harbouring one of two independent shRNAs targeting PDCD5  of the cell lysate was measured at 595 nm for the assessment of clonogenic potential relative to untreated cells. Again the data showed no difference between wild type and PDCD5 knockdown cells even in this long-term assay in both Hct116 , providiSince our approach so far was to investigate the loss of function of PDCD5 in cell death, we finally generated two acute lymphoblastic leukemia (ALL) cell lines (PreB697 and CEM) stably overexpressing 3xFlag-PDCD5. Overexpression in CEM cells was much more pronounced when compared to PreB697 cells but did not seem to affect cell growth. Despite increased levels of PDCD5 in these cell lines, there was no difference in viability as measured by MTT assay  or AnnexClearly p53 not only regulates cell death but also enforces cell cycle arrest. We therefore investigated the effect of PDCD5 knockdown on cell cycle distribution after DNA damage. Time-course analysis in response to irradiation showed a similar cell cycle distribution in Hct116 cells, as assessed by DNA staining, as well as cells in mitosis, quantified by intracellular staining of phospho-Histone H3 . All of The transcription factor p53 is considered to execute its tumour suppressing function via the modulation of two opposing but interwoven signalling pathways, i.e. cell cycle arrest and apoptosis13p21, but it is not clear if it is recruited to this promoter by p53 directlyet al.PDCD5 was proposed to be an additional intermediate fine-tuning this pathway by increasing the HAT activity of Tip60 after binding, thereby promoting K120 acetylation of p53 and promoting cell death in response to DNA damageDespite its ability to interact with p53 , we fail2O2, Nutlin-3, polybrene, propidium-iodide, doxocycline, puromycin , AnnexinV–APC, AnnexinV-488, 7-AAD (Biolegend), etoposide, camptothecin (Alexis). Antibodies: α-IκBα (#9242), α-PARP1 (#9532), α-GAPDH (#2118), α-Histone H3 (Ser10) (#9701), α-γH2A.X (#2577), pChk1 Ser345 (#2341), Chk1 (#2345) ; α-p53 (sc-6243), α-tubulin (sc-32293) (Santa Cruz Biotechnology); α-PDCD5 (Proteintech); α-LaminB1 (gift from Peter Gruber); α-Flag (M2) (Sigma), α-p21 (#554262) (BD Pharmingen); α-rabbit AlexaFluor 488 (Invitrogen), goat α-rabbit HRP, goat α-mouse HRP (Dako). SiRNA targeting human PDCD5 (5′-CUAAAGCAGUAGAGAAUUATT-3′) was synthesised by Microsynth and transfected into 293T cells using Metafectene (Biontex) according to the manufacturer′s instructions.Chemicals and cytokines used were staurosporine, cycloheximide, doxorubicin, DAPI, HE.coli with Glutathione-sepharose using standard protocols. Specificity was confirmed by siRNA experiments using 293T cells transiently transfected with Flag-tagged mouse PDCD5 . The antiserum, in contrast to the commercial antibody used, recognizes mouse as well as human PDCD5  and used for protein quantification with Bradford reagent (Pierce). Usually, 50 μg of the lysates were loaded and run on Tricine gelsNuclear Extracts were prepared as described previously2, 2.5 mM CaCl2 and protease inhibitors (Roche)) and immunoprecipitated with Flag-M2 beads (Sigma) or PDCD5 antiserum coupled overnight to Affi-Prep Protein A beads (Biorad). Mouse IgG (Santa Cruz Biotechnology) or pre-immuneserum served as controls, respectively. After incubating the lysate with antibody-coupled beads for 1 h, beads were washed three times in lysis buffer, unless otherwise indicated. Beads were boiled in SDS loading buffer and supernatant loaded on SDS-Page gels.293T cells were transfected with the indicated constructs , Flag-Tip60) for 24 h with Metafectene (Biontex) according to the manufacturer′s instructions and subsequently treated as indicated. Cells were lysed in IP-BufferΔΔCT method.RNA was isolated with Trizol (Invitrogen) and after DNAse digestion (Promega) reversely transcribed  using random hexamer primers. cDNA was amplified with DyNAmo Flash SYBR Green qPCR Kit (Finnzymes) on an Eppendorf Mastercycler ep realplex2 cycler . Primers used were hPDCD5 , hBax , hPuma , hNoxa , hp21  and actin . Fold induction compared to untreated controls was calculated by the Immunofluorescence staining was performed as previously described2) and analysed on a FACSCalibur instrument (Beckton Dickinson). Cells negative for 7-AAD and AnnexinV were considered as alive. For cell cycle analysis, cells were fixed in 70% EtOH/PBS, blocked in PBS with 1% BSA, permeabilised with 0.25% Triton X-100 in PBS and stained with rabbit α-Histone H3 (Ser10) antibody (1:100), followed by α-rabbit AlexaFluor 488 secondary antibody (1:100). After digestion with RNAseA and staining with 40 μg/ml propidiumiodide (30 min 37 °C), cells were analysed by flow cytometry on a FACScan instrument. For the MTT assay, 5.000 cells  or 10.000 cells  were plated in 96-wells in either medium containing the indicated drugs (Lymphoma lines) or were treated the following day as indicated. MTT assay (Roche) was performed 24 h after treatment according to the manufacturer′s instructions.Cell viability was determined by staining with 7-AAD and AnnexinV-APC or AnnexinV-488 (Biolegend) in AnnexinV-binding buffer .