Therefore, this interaction represents is an ideal target to develop antifibrotic drugs. A high throughput system to screen for chemical compounds that can dissociate LARP6 from 5'SL has been developed. It is based on fluorescence polarization and can be adapted to screen for inhibitors of other protein-RNA interactions. Screening of 50,000 chemical compounds yielded a lead compound that can inhibit type I collagen synthesis at nanomolar concentrations. The development, characteristics, and critical appraisal of this assay are presented.Fibroproliferative diseases are one of the leading causes of death worldwide. They are characterized by reactive fibrosis caused by uncontrolled synthesis of type I collagen. There is no cure for fibrosis and development of therapeutics that can inhibit collagen synthesis is urgently needed. Collagen α1(I) mRNA and α2(I) mRNA encode for type I collagen and they have a unique 5' stem-loop structure in their 5' untranslated regions (5'SL). Collagen 5'SL binds protein LARP6 with high affinity and specificity. The interaction between LARP6 and the 5'SL is critical for biosynthesis of type I collagen and development of fibrosis  RNA binding proteins regulate key cellular processes, such as transcription, splicing, polyadenylation, translation, mRNA stability, subcellular partitioning of mRNA, formation of stress granules, and processing bodies ,3,4,5,6.Type I collagen is the most abundant protein in human body. It is composed of three polypeptides, two α1(I) and one α2(I) polypeptides, which fold into rod-like heterotrimeric molecule. The heterotrimers of type I collagen are secreted into cellular medium, proteolytically cleaved, and polymerized into fibers . The fib5'SL consists of two double stranded stems, which flank a central single stranded bulge. 5'SL binds protein LARP6 with high affinity and specificity . LARP6 dIn fibrosis collagen synthesis increases several hundred fold . HoweverE. coli can give sufficient quantities for high throughput assays. However, two aspects must be kept in mind when using bacterially expressed mammalian RNA binding proteins. First, does the bacterially expressed protein make the same contacts with the RNA as the natural protein? It is likely that in a pure two-component system the bacterially expressed protein will bind its target, but does it make exactly the same contacts as the native protein. If not, then using such preparation for screening of drugs that will interfere with the binding of native protein is meaningless. One way to test the specificity is to make single point mutations in the RNA and analyze them for binding. If the recombinant protein binds the mutants with the same relative specificity as the native protein, it can be assumed that it is folded correctly, as well as that posttranslational modifications do not contribute to the specificity. This is reasonable approach for strictly sequence specific RNA binding proteins, like LARP6, but may present a problem for proteins that bind degenerate sequences or homopolymers. Second, what is the fraction of active protein present in the preparation? It is not uncommon that up to 99% of the protein may be biologically inactive [Fluorescence polarization (FP) is a method that measures the size of a fluorophore ,32. The inactive ,34,35. OFor the experiments in p + σn)/µp − µn[p and σn are standard deviations of FP of fully bound 5'SL RNA and free 5'SL RNA and µp and µn are the mean values, respectively. By using FP readings obtained from a 384-well plate /µp − µn, where σAfter incubation of 6 h at RT the FP readings of the same plate were repeated; they showed more variability between the wells after the prolonged incubation . At highThe assay was developed to screen a library of chemical compounds for a compound that can displace LARP6 from fl-5'SL RNA, as detected by a decrease in FP. To show that dissociation of LARP6 can be detected, competition experiments were performed in which unlabeled 5'SL RNA was added in increasing concentrations into the reaction containing fl-5'SL RNA and LARP6. The decrease in FP in concentration dependent manner indicated dissociation of LARP6 from fl-5'SL RNA and redistribution onto the competitor 5'SL RNA, while unrelated RNA showed no effect. We were able to compete out 80% of the bound LARP6 with 250-fold molar excess of the competitor. This verified that the assay can detect dissociation of LARP6 and that it is suitable for high throughput screening based on the inhibition of binding.Based on the titration curves , for theTo investigate how FP of a fluorescein labeled RNA change with the total fluorescence intensity we added increasing amounts of free fluorescein to the fixed amounts of fl-5'SL RNA and measured FP . FluoresThis example clearly indicates that a chemical compound with intrinsic fluorescence can negatively affect FP readings and present itself as a positive hit. This is a major drawback of the assay and, as high concentrations of compounds are used in the initial screens, the measurements based on decrease in FP must be carefully re-evaluated. We re-screened the positive hits obtained in our initial screen by the strategy similar as above. The positive hit compounds were added at concentrations from 40 µM to 1 µM to fl-5'SL RNA alone or to LARP6/fl-5'SL RNA complex and FP was measured. Compounds that were able to selectively decrease the FP of LARP6/fl-5'SL RNA complex were considered true hits. By this re-evaluation procedure we ended up with 19 compounds  that were active in dissociating LARP6 from fl-5'SL RNA. These compounds were directly tested in a cell-based assay for the ability to inhibit of type I collagen synthesis.1–8, 11–15, 17, 18) and that three were completely ineffective , as compared to controls (DMSO). Secretion of fibronectin was not affected, suggesting an effect specific for type I collagen. However, one compound (compound 9) showed dramatic inhibition at 10 µM concentration and was designated as a lead compound. It had similar structure to the three other compounds  that were less potent, but clearly active.Collagen secreted into the extracellular medium is responsible for fibrosis, so measuring the ability of cells to secrete type I collagen is a pertinent way to predict the antifibrotic potential of a drug ,40. TherThus, the screening was successful in identifying compounds that are inhibitory to collagen production in a cell-based assay. The lead compound could be classified to the same structural group with three other compounds, suggesting that the method has selected for compounds of similar structures. The molecular weight of the active compounds ranged from 250 to 400, the octanol/water partition coefficient (clogP) ranged from 0.58 to 4.72, the total polar surface area (t-PSA) ranged from 42 to 94.7 and the solubility, expressed as logSw, was between −2.11 and −5.54. The lead compound was further tested at lower concentrations in other types of fibroblasts and was effective in 500–250 nM range.in vitro, they spontaneously activate within seven days and increase their collagen synthesis 50–100 fold [in vitro. After two days in culture the cells were still quiescent and expressed type I collagen at barely detectable levels. The transformation into the activated state was initiated at day three to four in culture and the terminal activation into myofibroblast-like cells was achieved after seven to eight days. At this time point collagen expression was maximal and 50–100 fold higher than at day two. When the lead compound was added to quiescent HSCs in culture at day two after isolation and the cells were subjected to activation in the presence of the compound for additional five days, the collagen synthesis was inhibited by 50% at 100 nM and almost completely abolished at 250 nM (in vitro.Hepatic stellate cells (HSCs) are liver cells responsible for excessive collagen production in liver fibrosis ,42. They100 fold ,44,45. Wt 250 nM . Thus, tThe lead compound was identified by its ability to decrease FP of fl-5'SL RNA/LARP6 complex. However, is its activity in the cells also due to dissociation of LARP6 from the 5'SL of collagen mRNAs? To verify this we treated lung fibroblasts with various concentrations of the compound, prepared cell extracts and analyzed the binding of endogenous LARP6 to radiolabeled 5'SL RNA as probe using gel mobility shift assay. As shown in E. coli are mostly inactive in specifically binding the RNA, only small fraction of the molecules recognize the RNA in the sequence specific manner; (2) This may not an impediment for high throughput screening if active molecules are present in sufficient amounts to give a good FP signal; (3) Excessive purification may further decrease the fraction of active molecules, so it is a trade off that has to be considered on individual basis; (4) Intrinsic fluorescence of the chemical compounds that are screened may decrease FP readings, without affecting the RNA/protein complex; (5) The effect takes place even if the fluorescence spectrum of the compound is only partially overlapping with the spectrum of the fluorophore carried by the RNA; (6) This is the main cause of false positive hits. To recognize these artifacts fluorescence intensity readings must be performed together with the FP readings. The compounds with such properties must be additionally tested. (7) Identification of structurally related compounds in the screen is a good indicator that the screen has yielded functional molecules.A FP assay that was used to screen for chemical compounds that can dissociate LARP6 from a complex with collagen 5'SL RNA is described. This interaction is critical for high level of collagen synthesis and the inhibitors may be potential antifibrotic drugs. During development of the assay several observations were made that may have general implications for development of similar assays for other RNA binding proteins. (1) RNA binding proteins purified from Based on our experience, it is possible to use FP to identify small molecules that can dissociate RNA binding proteins from the RNA. Most of the compounds identified in our screen were effective in inhibiting collagen production, suggesting that such screens may be highly productive. In conclusion, this work has established FP as a legitimate tool for high throughput screening of compounds affecting RNA binding properties of proteins.


RNA post-transcriptional modification is an exciting field of research that has evidenced this editing process as a sophisticated epigenetic mechanism to fine tune the ribosome function and to control gene expression. Although tRNA modifications seem to be more relevant for the ribosome function and cell physiology as a whole, some rRNA modifications have also been seen to play pivotal roles, essentially those located in central ribosome regions. RNA methylation at nucleobases and ribose moieties of nucleotides appear to frequently modulate its chemistry and structure. RNA methyltransferases comprise a superfamily of highly specialized enzymes that accomplish a wide variety of modifications. These enzymes exhibit a poor degree of sequence similarity in spite of using a common reaction cofactor and modifying the same substrate type.Relationships and lineages of RNA methyltransferases have been extensively discussed, but no consensus has been reached. To shed light on this topic, we performed amino acid and codon-based sequence analyses to determine phylogenetic relationships and molecular evolution. We found that most Class I RNA MTases are evolutionarily related to protein and cofactor/vitamin biosynthesis methyltransferases. Additionally, we found that at least nine lineages explain the diversity of RNA MTases. We evidenced that RNA methyltransferases have high content of polar and positively charged amino acid, which coincides with the electrochemistry of their substrates.After studying almost 12,000 bacterial genomes and 2,000 patho-pangenomes, we revealed that molecular evolution of Class I methyltransferases matches the different rates of synonymous and non-synonymous substitutions along the coding region. Consequently, evolution on Class I methyltransferases selects against amino acid changes affecting the structure conformation. O-methylases which act essentially in tRNAs with very few exceptions , where Lt is equal to the length of the sequence aligned in the target, Lp is the total length of the query amino acid profile, and S is the alignment score. This Similarity Index was used as a measurement of the sequence relationships among the MTases reflecting the edges in the protein networks.High quality alignments were used to built respective amino acid profiles were constructed using the HMMER3 algorithm and default parameters . The prohm (MCL) . The cluhttp://genomes.urv.cat/UPGMA/index.php[http://www.r-project.org/) and an ANOVA test with Bonferroni correction was used. The second approach performed to disclose the evolutionary model for each RNA MTase family analyzed. Likelihoods for 120 empirical models (containing 15 different matrices) implemented in ProtTest v3.3 were calculated [http://www.tcoffee.org) [Relationships among the RNA MTAses were analyzed by two approaches. First, occurrence probabilities for all the amino acids in each MTase family. The HMM profiles built with the HMMER3 software. All probabilities were set as variables in a similarity matrix, and a dendrogram was constructed using the UPGMA algorithm with Pearson’s coefficient and 100 bootstrap replicates on the following web server: index.php. The mullculated -99. The fee.org) .http://www.r-project.org/) and the ggplot2 package [Presence of different RNA MTases and related proteins was massively detected in almost 12,000 fully-sequenced bacterial genomes publicly available in the Pathosystems Resource Integration Center (PATRIC). Approximately 50 million encoded proteins were tested to match the RNA MTases using probabilistic inference methods, as previously stated. The alignment hits and respective Similarity Index were clustered according to RNA MTase similarity. Then the violin density plots were drawn in R v3.0 ( package . AccordiAcinetobacter baumannii (186 genomes), Staphylococcus aureus (438 genomes), Pseudomonas aeruginosa (47 genomes), Mycobacterium tuberculosis (75 genomes), Enterococcus faecalis (271 genomes), Enterococcus faecium (229 genomes), Helicobacter pylori (243 genomes) and Salmonella enterica (393 genomes). They were respectively aligned using iterative and accurate methods [http://www.r-project.org/) using an ANOVA test with Bonferroni correction.The intra-species molecular evolution of the RNA MTases in human pathogens was investigated by analyzing the genetic variability in these genes in almost 2,000 genomes. Consequently, the coding sequences for all the RNA MTases studied, when presented, were extracted from pangenomes from eight common human pathogens:  methods ,104. The methods  and by c methods . As a reThe authors declare that they have no competing interests.ABP designed this study. JMR and SCB carried out the sequence and phylogenetic analyses. MC and JDP assisted sequence and phylogenetic analyses and computing performance. ABP and JMR worked in manuscript preparation. All authors read and approved the final manuscript.rlmN and rsmG. A comparative analysis done with the genes and pathogens for the distribution of the synonymous and non-synonymous substitutions in genes rlmN and rsmG is shown. Those genes are well known to be associated to antibiotic resistance. Critical sites for the protein function are highlighted in gray. The correlation between the high accumulation of the non-synonymous substitutions and hotspots for the functional inactivation of RsmG are more clearly inferred.The Cumulative Substitution Rate plots for genes Click here for filersmG. Two plots showing the distribution of the synonymous and non-synonymous substitutions at amino acid sequence level in rsmG genes from H. pylori and P. aeruginosa. Red lines show cumulative synonymous substitutions and green lines show non-synonymous substitutions. Hotspots for protein inactivation (gray shaded amino acid positions) were compiled from [Close view for Cumulative Substitution Rate in led from ,85.Click here for file


Plasmodium and many other parasitic apicomplexan species, is a remnant chloroplast that is no longer able to carry out photosynthesis. Very little is known about primary transcripts and RNA processing in the Plasmodium apicoplast, although processing in chloroplasts of some related organisms  shows a number of unusual features, including RNA editing and the addition of 3′ poly(U) tails. Here, we show that many apicoplast transcripts are polycistronic and that there is extensive RNA processing, often involving the excision of tRNA molecules. We have identified major RNA processing sites, and have shown that these are associated with a conserved sequence motif. We provide the first evidence for the presence of RNA editing in the Plasmodium apicoplast, which has evolved independently from editing in dinoflagellates. We also present evidence for long, polycistronic antisense transcripts, and show that in some cases these are processed at the same sites as sense transcripts. Together, this research has significantly enhanced our understanding of the evolution of chloroplast RNA processing in the Apicomplexa and dinoflagellate algae.The apicoplast, an organelle found in  Plasmodium and other members of the Apicomplexa group of parasitic eukaryotes. The apicoplast is a secondary plastid, resulting from an endosymbiosis event between the ancestor of the Apicomplexa and a member of the red algal lineage . None of the eight transcripts clones corresponded to a polycistronic transcript, although two included the full intergenic region at both the 5′ and 3′ ends of the SSU rRNA gene, extending as far as, but not including, the adjacent tRNA sequences (transcript b). The remaining six clones were derived from transcripts containing just the SSU rRNA. However, all were missing 1 bp at the 5′ end of the gene and either 6 bp (three clones) or 7 bp (three clones) at the 3′ end. These results suggest that the majority of the SSU rRNA sequences are monocistronic. In addition, the annotated 5′ and 3′ ends of the coding sequence may not be correct.Total RNA was circularized using an RNA ligase, and cDNA synthesized. Following RT-PCR and cloning of products, eight clones were found to contain sequence from the LSU rRNA and rps4 genes was tested. cDNA generated from all of the reverse primers gave products in the test PCR, indicating the existence of a polycistronic RNA molecule spanning the whole region from LSU rRNA to rpl23 and thus containing rRNA, tRNA and protein sequences.  using the circular RT-PCR technique . These revealed that the majority of transcripts (18/24) commenced 98 bp upstream of sufB, exactly consistent with the 5′ end of the trnT gene, although a substantial minority of transcripts (5/24) commenced 25 nt upstream of sufB, exactly consistent with the 3′ end of the adjacent trnT gene . The remaining transcript commenced 104 nt upstream. This would suggest that a long, initial primary transcript is created, which is cleaved at the 5′ and 3′ ends of trnT, releasing both the tRNA and a sufB mRNA with a 5′ UTR of 25 nt.echnique . Twenty sufB transcripts were heterogeneous. All transcripts extended past sufB into the adjacent orf51 gene, while the longest included both orf51 and orf101 together with a small portion of rpoB. Together, these results indicate that there is a transcript spanning from at least trnT into rpoB which is cleaved at a limited number of 5′ sites associated with the tRNA. The 3′ ends within protein coding sequences are much more heterogeneous, either as a result of exo-or endo-nucleolytic cleavage, or transcription termination.The downstream ends of the rps2-orf105-clpC-tufARegion. We also studied the orf105 region , while those downstream are located on the opposite DNA strand. The gene itself overlaps at the 5′ end with the trnS gene (24 bp) and at the 3′ end with rps2 . We performed circular RT-PCR and amplified products corresponding to transcripts of the orf105 gene . At the 5′ end of orf105, the majority of sequences recovered (21/28) represented transcripts with a 5′UTR of 61-64 nt, corresponding closely to the start of the trnS gene (at -62 nt). This site is shown with an arrow in transcript a. A minority (6/28) of cloned transcripts had a 5′ UTR between -7 (i.e. within the orf105 gene) and +9 nt. The other transcript extended into orf79. At the 3′ end, 21/28 clones corresponded to transcripts ending at +105/106nt, with a further clone corresponding to a transcript ending at +101nt (as shown by an arrow in transcript a). No transcripts extended as far as rpoC2. The results are consistent with a primary RNA molecule being cleaved upstream of trnS and also close to the 5′ end of orf105 to give either orf105 or tRNA-Ser, but not both (as the genes overlap). The strong 3′ terminus within rps2 could represent either a transcription termination site, or a cleavage site.5 region . (Note tas ycf93 ). This grps2  gave rise to 39 clones. Of these, only 11 contained the full rps2 coding sequence. The rest of the clones were missing the first 100-250 nt of rps2 and did not have a specific 5′ end . However, the vast majority (32/39) of clones had a 3′ end at +175-177 nt , corresponding to a transcript ending within orf105 (which is on the opposite strand). No clones extended past this point, suggesting that it is a strong cleavage or transcription termination site.Circular RT-PCR and amplification of cDNA corresponding to clpC and orf79 genes . The majority (19/26) of clpC transcripts were polycistronic. The longest products, which also contain orf79, (8/26) ended immediately prior to trnS. This is consistent with the same cleavage site identified with orf105 circularization  revealed that 22/25 sequences recovered corresponded to transcripts ending at +315/317 nt, immediately at the start of the trnQ gene. The remaining three ended at 0, +7 and +214 nt from the 3′ end of tufA. No transcript extended into trnQ. There were 19 different 5′ end sites ranging from 0 to +739 nt.RNA circularization analysis for Extensive polycistronic antisense transcription. Antisense transcripts of the apicoplast genome have been reported for the apicomplexan Toxoplasma, but it is not known if they occur in Plasmodium as well. We therefore tested for the presence of antisense transcripts in the Plasmodium apicoplast. Note that in this section 5′ and 3′ are used in accordance with the associated gene (i.e. as if the transcript were transcribed from the sense strand).rpoB, rpoC, clpC, tufA, orf105, SSU rRNA, rps2, and rpl2), and this was used in PCR. For each gene, a band of the same size was seen for both sense and antisense transcripts when analysed by agarose gel electrophoresis. This indicates that both sense and antisense transcripts existed for each gene. rps2.cDNA was synthesized using either a forward or a reverse primer (thus specific for sense or antisense transcripts) for eight genes , and then tested if it was possible to amplify sequences downstream of LSU rRNA from this cDNA by PCR. We successfully amplified a region from to rps19 .sufB. Analysis of transcripts containing the sense sequence of sufB had revealed that the gene is transcribed as a polycistronic molecule, extending at least as far as the adjacent trnT gene on the upstream side, and downstream through orf51 and orf101 into rpoB (transcript a). In order to determine if there were also antisense transcripts spanning this region, cDNA was synthesized using a forward primer at the extreme 3′ end of the adjacent LSU rRNA gene. PCR was then carried out on the cDNA using the same forward primer but with reverse primers at intervals through the five genes . Products of the expected size were obtained with all the reverse primers (data not shown), indicating that antisense transcripts extend from the 5′ end of LSU to at least 358 bp into rpoB (transcript b). The antisense transcript thus covers at least four genes in full: trnT, sufB, orf51 and orf101, as with the sense transcript previously identified.We also tested the presence and extent of antisense transcripts for Processing of antisense transcripts. Circular RT-PCRs to analyse sense strand transcripts had revealed that many transcripts are formed from the cleavage of polycistronic transcripts at processing sites associated with tRNA molecules. We therefore wished to determine if antisense processing occurs in the same way as sense transcript processing. Circular RT-PCR was therefore carried out to map antisense transcripts of tufA, clpC, sufB, orf129, rpl16 and rps2. The same primer sets were used as before, except that cDNA was first synthesized with the forward primer (i.e. the opposite of that used in mapping sense transcripts). The majority of antisense circularization reactions did not give rise to corresponding recognizable product in the subsequent PCR. This was presumably because the levels of antisense RNA levels were too low, or because transcripts were too long to be identified using the RNA circularization technique. Products were obtained for tufA, rps2 and rpl2, although success rates (in terms of clones obtained which contained recognizable apicoplast sequences) were much lower than for sense circularization experiments.tufAantisense. The circularization assay for antisense transcripts of tufA gave rise to six clones (transcript f). At the 5′ end, one clone extended 123 nt upstream of the gene, four clones extended 78- 83 nt upstream, and one clone finished +8 nt (i.e. within the gene). None of these sites corresponded to the 19 different tufA sense cleavage sites previously observed. In contrast, at the 3′ end, five of the six antisense transcripts ended 315-317 nt after the end of the gene, as did 23/25 of the sense transcripts. This site maps immediately before the start of trnQ, suggesting that this is a major cleavage site for both sense and antisense transcripts.rps2antisense. The rps2 and orf105 genes are adjacent genes, but on opposing strands. They overlap by 14 bp at the 3′ end. Twenty eight clones generated from rps2 antisense transcripts were identified and fully sequenced (transcript g). None of the sequences covered the whole of the gene, and 19/28 clones corresponded to an antisense transcript with a 3′ end 118-121 nt before the 3′ end of the rps2 gene (i.e. within the gene). This exactly corresponds to the 3′ end of the orf105 sense transcript at +105-106nt . Therefore, it seems likely that the majority of rps2 antisense transcripts arise as a by-product from cleavage of a long, primary transcript containing the sense orf105 mRNA.SSU rRNA/ orf79), two sites are within the rps2 and orf105 genes, at the point where the orientation of genes on the apicoplast genome switches from one strand to the other. The remaining seven processing sites are immediately adjacent to tRNA sequences. The cleavage sites associated with the 3′ ends of trnS, trnT, trnF, trnW, trnG all coincided with the presence of an adjacent UUAU motif (UUAA for trnG), trnG also coincided with an associated UUAA motif, while the cleavage site associated with rps2 was associated with a UUAG motif, while a UUAU motif was identified in orf105. No such motif was found near the trnQ cleavage site, which is conserved in both sense and antisense transcripts. These observations indicate that RNA cleavage is usually associated with a specific UUAA/U/G motif.Analysis of the results from all circular RT-PCR experiments had indicated the presence of twelve major RNA processing sites . The clones were of different lengths, and therefore represent independent transcripts, making it unlikely that this event was caused by reverse transcriptase or PCR error. The clones containing the editing event were obtained from multiple independent circular RT-PCR experiments using RNA from different extractions .Alignments of sequence data revealed the presence of single point substitutions in individual transcripts. Although these could be caused by very low levels of RNA editing, we could not exclude that these were errors in reverse transcriptase, PCR or sequencing, and so did not analyse them further. However, an alignment of circularization data from Plasmodium libraries corresponding to four time points . We next examined RNA-seq data from four rpoC2 gene which encodes a subunit of the RNA polymerase . This gene appears to contain a reading frame shift at position 1570-1575, where five A nucleotides encode either one or two lysine residues. No transcripts covering this region were identified in any of the four RNA-seq libraries. We therefore carried out RT-PCR across this region of rpoC2 and cloned the PCR products into E. coli. All three sequenced clones contained the genomic version of rpoC2 and none contained an edited version, suggesting that this gene is not edited despite the presence of a frame-shift mutation.We next examined the Plasmodium remnant chloroplast are polycistronic, confirming previous research  suggest that at least some transcription can proceed through them. Major cleavage sites were associated with an UUAU motif. The mechanism of RNA cleavage remains to be elucidated.There were two consistently observed instances of transcript ends within coding sequences. These were the 3′ ends of transcripts containing Plasmodium  Note that the different location of the 5′ end of the rps2 antisense transcript from the 3′ end of the sense transcript confirms that the antisense transcript was not an artifactual amplification of the sense transcript. Many other antisense transcript processing sites corresponded closely to sense processing sites. For example, both tufA antisense and sense transcripts had a cleavage site corresponding with the start of the trnW gene. This may indicate that cleavage in these cases is primarily dependent on secondary structure rather than sequence (which will be different between the sense and antisense transcripts).Antisense transcripts could be generated either by direct antisense transcription or by read-through from a gene located on the opposite strand. The exact coincidence of the start of the antisense transcript of Plasmodium are stage-dependent, leading to speculation that these molecules could be involved in stage-specific regulation of gene expression  and not in cDNA derived from linear RNA, which includes RNA which has not yet been processed. If correct, this interpretation would also suggest that RNA which has not yet been processed constitutes a relatively large fraction of the RNA pool.The occurrence of an RNA editing site in Plasmodium apicoplast has not previously been reported. In plant chloroplasts, RNA editing is restricted to C to U , and is generally uncommon. Some dinoflagellate algal species show extensive RNA editing, affecting around 5% of all nucleotides in Karenia mikimoti, although other species have very low (or absent) rates of editing, and the mechanism by which editing occurs is unknown , as shown in Supplementary figure S2. It will be important to determine if this editing site occurs in other Plasmodium and/or Apicomplexan species.To our knowledge, RNA editing in the  unknown . The edirpoC2, despite the gene apparently requiring a frame-shift for translation. It is unclear how the frameshift is removed, though it is possible that this occurs during translation. Ribosomal frame-shifting in chloroplasts is not common; to our knowledge, the only report to date involves an artificially introduced E. coli gene in tobacco. Translation of this gene was successful in tobacco, suggesting that all the signals necessary for frame-shift were present in the gene, and that prokaryotic-style 70S ribosomes can carry out translation including a frameshift , and only a single site (or a few at most) is involved. It will be interesting to see if this is unique to Plasmodium or occurs in other parasitic Apicomplexa, such as Toxoplasma. The construction of a series of stage-specific RNA-seq libraries  could help answer these questions.The occurrence of editing in the Plasmodium is complex. Some features, such as polycistronic transcription by a single RNA polymerase, appear to be conserved across Plasmodium, photosynthetic Apicomplexa, dinoflagellates and the red algae. Other features, such as RNA editing and ribosomal frameshifting, may be unique to specific lineages, and editing has apparently been acquired more than once. It is unclear whether RNA processing at tRNAs (Punctuation Processing) is an ancestral or derived characteristic, as very few tRNA genes have been identified in dinoflagellate chloroplasts (Plasmodium chloroplast ultimately derives). Punctuation processing is usually carried out by RNAseP, first identified in human mitochondria .RNA extraction: Total RNA was extracted from P. falciparum according to g for five minutes and the supernatant removed. For every 300 μl of infected red blood cells, 5 ml Trizol (Invitrogen) was added. This mixture was incubated at 37 °C for five minutes with occasional shaking. One-fifth Trizol volume of chloroform (Sigma-Aldrich) was added with vigorous shaking, and left to stand at room temperature for three minutes before centrifugation at 1400 g at 4 °C for 30 minutes. Three-fifths Trizol volume isopropanol (Sigma-Aldrich) was added to the aqueous layer. The mixture was divided into 1.5 ml aliquots and each was spun at 16000 g at 4 °C for 30 minutes and the supernatant discarded. The glassy white pellet was resuspended in ice-cold 75% ethanol, centrifuged at 16000 g at 4 °C for 30 minutes, and the supernatant discarded. The pellet was resuspended in non-DEPC treated RNase free water (Invitrogen) and DNase treated . RNA was purified using the Qiagen RNeasy mini spin column, eluted in RNase free water and stored at -80 °C until required.RNA circularization: RNA circularization was carried out essentially according to cDNA synthesis: All primers are listed in the Supplementary Material, supporting Table S1. For each experiment, 1000-5000 ng RNA (either linear or circular) was used, to which was added 1 μl of 2 μM gene-specific reverse primer, 1 μL 10 mM dNTPs (Bioline) and non-DEPC treated water to 12.5 μl. The reaction was incubated at 65 °C for 5 minutes, and snap cooled on ice. 4 μl Superscript reverse transcriptase buffer (Invitrogen), 2 μl 0.1 M DTT, 0.5 μl recombinant RNasin (Promega) were added and the mixture incubated at 37 °C for 2 minutes prior to the addition of 1 μl Superscript II or III reverse transcriptase. The reaction was incubated at 37 or 42 °C for 50 minutes and the enzyme inactivated at 70 °C for 15 minutes. A no-RT control reaction was carried out for every cDNA synthesis reaction, where 1 μl dH2O was added in place of the Superscript reverse transcriptase.RNA circularization experiments were performed in either duplicate or triplicate, from separate RNA preparations, and the data aggregated. All linear RT-PCR reactions were performed in triplicate, from separate RNA preparations.PCR, cloning and sequencing: PCR was carried out on DNA, cDNA (or no-RT control) using GoTaq DNA polymerase (Promega). All primers were designed with an annealing temperature of 50 °C – 53 °C and all reactions were carried out with an extension temperature of 60 °C. Products were analysed by agarose gel electrophoresis, and sequenced where required. Where required, PCR products were cloned into pGEM-T-easy (Promega) and used to transform chemically competent E. coli TG1. Plasmids were sequenced using Sanger sequencing at the Depatment of Biochemistry, University of Cambridge sequencing facility.RNA-seq analysis: Libraries containing transcriptome data for 10 hr, 20hr, 30hr and 40 hr post-infection were downloaded from the EBI. These correspond to study accession PRJEB3309, libraries ERR174301 (10 hr), ERR185969 (20 hr), ERR185970 (30 hr), ERR185971 (40 hr) sequences on an HiSeq2000 (Illumina) (llumina) . Sequencllumina) .


Hence, before using these tissues, it is important to carry out pre-analytical studies to ensure that they are representative of the in vivo state. In particular, tissues of the gastrointestinal tract have been thought to have low RNA stability. Therefore, this study aimed to determine if extended warm or cold ischaemia times and snap-freezing or banking in RNA stabilization solution affects RNA integrity or gene expression in human ileum mucosa. In short, ileum mucosa was collected for up to 1.5 h and 6 h of simulated warm or cold ischaemia respectively. Subsequently, RNA integrity and gene expressions were determined. It was found that RNA integrity remained high over the course of warm and cold ischaemia examined and there were in general no significant differences between snap-freezing and banking in RNA stabilization solution. Following the same trend, there were in general no significant changes in gene expressions measured . In conclusion, RNA in the ileum mucosa is maintained at a high integrity and has stable gene expression over the examined time course of warm or cold ischaemia when banked in RNA stabilization solution or snap-frozen in liquid nitrogen. As the average warm and cold ischaemia times imposed by surgery and the process of tissue banking are shorter than the time period examined in this study, human ileum mucosa samples collected after surgeries could be used for gene expression studies.The use of banked human tissue, obtained with informed consent after elective surgical procedures, represents a powerful model for understanding underlying mechanisms of diseases or therapeutic interventions and for establishing prognostic markers. However, donated tissues typically have varying times of warm ischaemia  Unlike the use of animal models, human tissue is typically obtained from resected tissues from elective surgeries after obtaining informed consent from the donor. As such, there will be varying warm and cold ischaemia times associated with each tissue dependent on the blood arrest time during the operation or the transport time from the operation suite to the pathologist and finally to the tissue bank [Comparative quantification of gene expression, through real-time quantitative polymerase chain reaction (RT-qPCR) or gene expression microarrays, is essential for understanding the molecular bases of diseases. For such studies, human tissue banks such as the one at the Surgical Clinic in the Hospital of the University of Munich, provide powerful models by banking leftover tissue in excess of what is required for diagnostic tests by the pathologists. For these tissues to provide relevant experimental results, the banked tissue must be of a high quality representative of the sue bank .Studies have been undertaken by various investigators to examine the effect of temperature, time and banking method (snap-freezing/ banking in RNA stabilization solution) on RNA integrity and gene expression in a variety of tissues  Tables. N = 52). Therefore, this study aimed to examine the effects of extended warm or cold ischaemia time and banking method (snap-freezing in liquid nitrogen or banking in RNA stabilization solution) on RNA integrity and gene expression.For ileum collected in this hospital, the average warm ischaemia time (37°C) was 12 ± 13 min and the average cold ischaemia time (on ice) was 22 ± 15 min with values expressed as means ± standard deviation (http://www.klinikum.uni-muenchen.de/Chirurgische-Klinik-und-Poliklinik-Grosshadern/de/0800-gewebebank/index.html) located in the Hospital of the University of Munich, which operates in accordance with the European Union-compliant ethical and legal framework of the Human Tissue and Cell Research (HTCR) Foundation (http://www.htcr.org) [The tissues and data used in this study were provided by the Biobank (tcr.org) . The proFrom each donor with colon carcinoma, a piece of terminal ileum was collected in the operation room during right hemicolectomy by the staff from the Biobank. During the surgical procedure, the terminal ileum part of the preparation was dissected out after the detachment of the branches of the middle colic artery and the right colic artery and before the complete resection of the whole preparation. The terminal ileum was dissected as follows. While the vascular structure remains intact, a 5 cm length of terminal ileum was isolated between two staplers. The vascular pedicle still supplying this part of the ileum was then clamped using an Overholt and the isolated ileum was then immediately separated and put on a back table for the preparation of ileum mucosa samples by the staff from the Biobank. The surgeon then completes the surgical intervention and removal of the remaining hemicolectomy preparation. During this procedure, no additional ileum tissue had to be resected as only the piece of terminal ileum, which is part of the hemicolectomy preparation, was separated in situ by slightly adapting surgical procedures with no effect on the therapeutic intervention.When the staff from the Biobank obtained the terminal ileum in the operation room, warm and cold ischaemia times remained less than 1 minute. As such, samples collected immediately were labeled as “time 0”. The whole ileum mucosa collection procedure is shown in FFPE ileum samples were sectioned at 6 μm thickness. These sections were stained according to the instructions for use that came with the Mayer’s haematoxylin solution  with some modifications. The modifications were that the slides were rinsed in 1% HCl in 70% ethanol for 2 seconds after haematoxylin staining and a dip in distilled water was done after each rinse in running tap water before the next step.The extraction of RNA from the ileum mucosa was performed using the RNeasy Mini Kit  according to the manufacturer’s protocol. In brief, 30 mg of ileum mucosa was homogenised in the provided lysis buffer using the TissueLyser LT bead mill  according to the manufacturer’s protocol. The homogenate was then transferred into a RNeasy spin column, which binds RNA using a silica-membrane. In order to remove any potential DNA contamination, an on-column DNA digestion step was done using the RNase-free DNase Set . The bound RNA was finally eluted from the spin column in 30 μl of RNase free water.Assessment of nucleic acid purity was done using the NanoDrop 2000 spectrophotometer . To quantify RNA, the Quant-iT Ribogreen RNA Assay Kit  was used according to the manufacturer’s protocol. RNA was quantitated with a fluorescent nucleic acid stain that can be detected using the Filtermax F3 microplate reader  operating with excitation / emission wavelengths of 480 nm / 520 nm respectively.To determine the RNA integrity, 200 ng of RNA from each sample was loaded on the RNA 6000 Nano Chip  according to the manufacturer’s protocol. The RNA integrity was assessed with the Agilent 2100 Bioanalyzer system, which generates a RNA Integrity Number (RIN) .RNA (1 μg) was transcribed into complementary DNA (cDNA) in a final volume of 20 μl using the SuperScript VILO cDNA Synthesis Kit  according to the manufacturer’s protocol. The synthesis of cDNA was primed by using random oligodeoxyribonucleotides. The newly synthesised cDNA was diluted 10 times for subsequent use in RT-qPCR. Both diluted and undiluted cDNA were stored at -20°C until further use.MYC, Hif1α, CDX2, HMOX1 and IL1β), a screening was done to determine appropriate reference genes from a panel  using geNorm [-ΔΔCT method with the inclusion of a normalisation factor obtained by geNorm [The RT-qPCRs were performed using a StepOnePlus Real-Time PCR System  with TaqMan Gene Expression Assays. All primers, probes and TaqMan Fast Advanced Master Mix were purchased from Life Technologies . The primers and probes used in this study are described in g geNorm . The rely geNorm .MYC, Hif1α and CDX2 gene expressions obtained from 6 donors . For 6 donors , statist260/280 ratios with values between 2 and 2.1 show that there is no protein contamination of the isolated RNA samples . HoweverH & E staining showed that the epithelium of the mucosa remains intact and the tissue retains characteristic tissue and cellular structure over the time course of warm or cold ischaemia .The RNA integrity of each sample was assessed using the RIN assigned by the Bioanalyzer. Electropherograms representing the highest (9.8) and lowest (7.9) average RIN obtained in this study are shown in HPRT1, GUSB, PSMB6, TBP and RPL13, were tested. GeNorm output indicated that RPL13 was the least stable reference gene . In short, the 4 inducible genes examined included an immediate early gene (MYC), the hypoxia-inducible factor 1α (HIF1α), an inflammatory response gene (IL1β) and oxidative stress inducible gene (HMOX1). Real-time-qPCR analysis showed that none of these 5 genes showed significant differences in relative gene expression when compared across various time points for both warm and cold ischaemia  to 10 (fully intact RNA) . Variouset al.  stated t [et al.  found th [et al.  could be [et al. , ≥7 [16, [et al.  or >5  , rectal et al.  found thet al.  or in saet al. .MYC after 90 minutes of cold ischaemia when banked using RNA stabilization solution. This result could be due to cold ischaemia triggering a transient overexpression of MYC at 90 minutes. However, this scenario is not very likely as a corresponding increase in MYC expression should have occurred in the snap-frozen samples. Instead, this increase could be due to an inadvertent sampling of cancerous tissue at this time-point, although the tissue obtained is from the resection margin, as it is known that colorectal carcinomas frequently have overexpression of MYC [For gene expression, there was only a significant increase in the relative expression of n of MYC , 26. Othn of MYC , 27–31. n of MYC , 32, then of MYC .In conclusion, ileum mucosa has a high RNA integrity and stable gene expression over a time course of warm or cold ischaemia whether banked in RNA stabilization solution or snap-frozen in liquid nitrogen. Since the average warm and cold ischaemia times imposed by surgery and the process of tissue banking is shorter than what was examined in this study, human ileum mucosa samples could be used for gene expression studies. Nonetheless, it is important to check that one’s genes of interest are not in the small proportion of genes affected by such conditions before proceeding. As to whether snap-freezing or banking in RNA stabilization solution should be done, both methods offer different advantages and disadvantages. RNA stabilization solution offers ease of use compared to liquid nitrogen and is also more operator-friendly compared to snap-frozen samples, which should not be thawed at all during processing steps. On the flip side, snap-frozen samples offer more versatility as samples banked using RNA stabilization solution have been found to provide poor microscopic images due to a loss in tissue morphology , 33 and S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


RNA editing is a molecular event that alters specific nucleotides in RNA post-transcriptionally. RNA editing has the potential to impact a variety of cellular processes and is implicated in diseases such as cancer. Yet, the precise mechanisms by which RNA editing controls cellular processes are poorly understood. Here, we characterize sequences altered by RNA editing in patient samples from lymphoma, neuroblastoma and head and neck cancers. We show that A-to-I RNA editing sites are highly conserved across samples of the same tissue type and that most editing sites identified in tumors are also detectable in normal tissues. Next, we identify the significant changes in editing levels of known sites between tumor and paired “normal” tissues across 14 cancer types (627 pairs) from The Cancer Genome Atlas project and show that the complexity of RNA editing regulation cannot be captured by the activity of ADAR family genes alone. Our pan-cancer analysis confirms previous results on individual tumor types and suggests that changes of RNA editing levels in coding and 3′UTR regions could be a general mechanism to promote tumor growth. We also propose a model explaining how altered RNA editing levels affect microRNA-mediated post-transcriptional regulation of oncogenes and tumor-suppressors. RNA editing is a post-transcriptional process that alters nucleotide sequences, and thereby potential function, of virtually all RNA types in eukaryotic cells. Several independently evolved editing mechanisms have been classified, including co- and post-transcriptional base insertions, deletions, substitutions or modifications. RNA editing sites are normally detected by identifying mRNA sequences that do not match the corresponding encoding DNA sequences. Current approaches to RNA editing site detection rely on the comparison of paired RNA-Seq and DNA-Seq data and on the identification of variant sites supported by the former but not the latter. In human, there are two predominant nucleotide substitution types (A to I and C to U) catalyzed by specific enzymes families. The adenosine deaminases acting on RNA (ADARs) catalyze Adenosine (A) deamination in double-stranded RNA to generate inosine (I). As inosine has similar base-paring properties as Guanosine (G), it is read as guanosine by translating ribosomes and reverse transcriptases used to generate next-generation sequencing librariesAZIN1 mRNA has been shown to stabilize the corresponding protein and to promote cancer proliferationRNA editing can alter protein coding sequences, splicing, transcript stability and expression levels45Despite this growing evidence, few transcriptome-wide studies have explored RNA editing events in cancer. In this work, we first evaluated whether a significant number of novel  RNA editing sites appeared in cancer samples. We leveraged publicly available parallel sequencing datasets (with both DNA-Seq and RNA-Seq data) to characterize RNA editing in cancer samples, and selected neuroblastoma (NB), Diffuse large cell lymphoma (DLBCL) and Head and Neck Squamous cell Carcinoma (HNSC) to cover tumors with various mutation rates and hematological and solid tumors9Given the lack of evidence for the presence of cancer-specific (somatic) RNA editing sites, we next focused on those sites that are significantly altered between tumors and paired normal samples. For this analysis, we focused on known RNA editing sites annotated in the Rigorously Annotated Database of A-to-I RNA Editing (RADAR), which is the most up to date RNA editing sites database1214RNA-Seq based Alignment Artifact Removal (RAAR) pipeline (https://github.com/bioliyezhang/RAAR) to eliminate these potential sequencing artifacts , and multiple alignment-related artifacts 16Improved Accuracy. We optimized our algorithms to select the best alignment out of Blat realignment output. We compared our method with the GATK realignment methodGATK realigned output (56.1%)  No reliance on genome annotation. Our pipeline works without genome annotation, while most existing tools depend on exon-intron annotations16Easy to use. We adopted the most commonly used VCF and BAM formats as the input/output of the pipeline; we also aimed to simplify installation by making the tool dependent only on commonly available tools such as samtools, bedtools and Blat.The main advantages of our pipeline are: 1)  (56.1%) . We alsoTo characterize RNA editing sites in 13 DLBCL samplessite if there is at least one sample with a minimum of 5 RNA editing supporting reads covering the site, and if at least 5% of all reads manifests the substitution  in all samples. For each of these sites, we counted the number of samples showing presence (non-zero level) of editing. The resulting distributions are displayed in We wanted to ascertain whether RNA editing could function as an additional mechanism contributing to malignant transformation by generating novel  A-I RNA editing sites that are unique to cancer samples. As the majority of A-I RNA editing sites could be seen in all samples, this seemed unlikely.Since RNA-Seq data from paired normal samples for DLBCL and NB were unavailable, we could not address this question directly in these tissues. However, based on the assumption that cancer specific RNA editing should not be detected in normal cells, we checked for the presence of the newly discovered RNA editing sites in the Illumina Human Body Map (HBM) data 2.0Thanks to the availability of paired normal samples in the HNSC dataset, we next examined whether the novel RNA editing sites identified in the tumors could also be observed in the paired normal tissues. As shown in Given the lack of evidence for the presence of somatic A-I RNA editing sites, we focused on the significant editing level changes between normal and tumor samples of known RNA editing sites included in the RADAR databaseOur analysis identified a total of 166 differentially edited genes  showed increased editing with nominal P-value ≤0.05   .RNA editing sites in the protein coding regions only account for a small proportion of total RNA editing sites in the genome. The majority of RNA editing sites resides at intronic region, and several of these intronic sites can affect RNA splicing to regulate the functions of genes2829MDM4 and CASP8 . We first focused our attention on cancer census genes listed in the COSMIC databasend CASP8 . Similarnd CASP8 . Taken tGiven the abundance of RNA editing sites in 3′ UTR and the important role microRNAs play in tumor suppressionand whose expression is known to be repressed by microRNAs. We further restricted our analysis to those genes that showed consistent editing level increase/decrease across the entire 3′ UTR. The resulting top ranking gene was MDM2, a key oncogene in the p53 pathway with elevated expression in multiple tumor typesMDM2 expression levels. To evaluate whether RNA editing may regulate MDM2, we first identified the microRNAs predicted to bind at RNA editing sites with significant editing level changes in at least one tumor type. We found three microRNAs matching these criteria  sites showed a significantly higher positive correlation in the former  (http://bioinfo.mc.vanderbilt.edu/TSGene/), we found 5 additional RNA editing sites whose levels are positively correlated with corresponding mRNA levels in at least one cancer type  . When wecer type . While mI and IV . Taken tIn summary, our RNA editing profiling of three tumor types found no evidence of cancer specific or novel RNA editing events. On the other hand, our pan-cancer analysis of TCGA datasets corresponding to 14 cancer types identified RNA editing sites recurrently altered in coding and 3′ UTR that may facilitate tumor progression, and predicted the potential regulation mechanisms for many of such alterations. These results can serve as a starting point to further understand the mechanisms of RNA editing regulation and the functional roles of RNA editing sites that significantly change in tumors vs. normals.https://cghub.ucsc.edu/) using the GeneTorrent application The dbGaP accession numbers of the accessed datasets are: phs000235.v2.p1 for the lymphoma datasetTrim-galore . The RNA-Seq reads were aligned to hg19 reference genome using Tophat v2.0.4samtools v0.1.18 rmdup functionmpileup file was generated by samtools for each sample and used to call variants using Varscan 2.2.11The adaptor trimming was performed on the FASTQ files from the illumina human body map by using When retrieving the lowly edited and low coverage RNA editing events based on a relaxed threshold for the previously identified highly confident sites, we discarded any event with a total coverage lower than 10 reads.To validate the novel RNA editing sites we identified in DLBCL, we calculated the read depth and editing levels for each of these sites in the HBM dataset (n = 16), and for each sample we excluded those sites with read depth below 10 reads. When the read depth was ≥10, the number of RNA editing supporting reads was ≥1 and the allele frequency was ≥1%, we considered the sample as supporting the RNA editing event. When an RNA editing event was detected in more than two samples, we considered the corresponding site a real RNA editing site based on the HBM dataset. When the RNA editing event was detected in only one sample, we considered this site likely to be a real RNA editing site. When no sample met the minimum read coverage threshold, we concluded we did not have enough information to make a call on this site. When at least one sample had sufficient coverage but no sample supported the RNA editing event, we considered this site not to be an RNA editing sites based on HBM. To validate the potential cancer-specific RNA editing sites in HNSC samples in their paired normal tissue samples, we adopted the same criteria.http://mircode.org/info.phpmiR-EdiTar to visualize the binding between a microRNA and its target 3′ UTR sequencesWe downloaded the predicted microRNA binding data of highly conserved microRNA families from For all the tumor-normal pairs in 14 TCGA datasets, we calculated the RNA editing level  for each coding and 3′ UTR RNA editing sites in RADAR database. Then we used the paired student’s t test to evaluate whether tumor samples showed a significantly increased or decreased editing level compared with normal samples for each site within individual tumor type. The FDR (B-H) cutoff of 0.05 was adopted to control for multiple hypothesis testing.MDM2 are frequently observed in tumors and can confound our correlation analysis, we first removed the tumor samples where a copy number alteration is detected by GISTIC2. We then measured the association between changed log2MDM2 expression level and changed MDM2 RNA editing levels by Pearson correlation for each site in each tumor type. We performed the Student’s t-test to compare of the correlation distributions within the three hsa-miR-200b sites and in the remaining (non hsa-miR-200b) sites.As copy number gains in Human breast cancer lines MDA-MB-231 and BT474 cells were purchased from ATCC, and grown at 37 °C in DMEM and RPMI 1640 media. Media were supplemented with 10% FBS and 1X pennicillin/streptomycin. Cells were transfected with 50 nM control microRNA or miR200 by Lipofectamine RNAiMAX reagents .Lysate was collected 72 h after transaction, and analyzed by immunoblotting using anti-MEM2  and anti-GAPDH  as previously describedHow to cite this article: Zhang, L. et al. Altered RNA editing in 3′ UTR perturbs microRNA-mediated regulation of oncogenes and tumor-suppressors. Sci. Rep.6, 23226; doi: 10.1038/srep23226 (2016).


Insilico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking.Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export.  The nucleocytoplasmic export of macromolecules (RNA and protein) and RNA-protein (RNP) complexes is one of the critical steps necessary to ensure normal cellular function. The study of retroviral RNA export has derived a great deal of information on this highly regulated process that represents a key step in the replication cycle that could serve as a viable therapeutic target ,2. For icis-acting Rev-responsive element (RRE) that are bound by the viral protein Rev to program nucleocytoplasmic RNA trafficking [While the nuclear RNA export factor 1 (NXF1) is required for the constitutive export of the 2-kb, completely-spliced viral RNA species , the 9-kfficking ,11. RNA fficking . The Srcfficking ,14. Nuclfficking . While tfficking ,17. Thusfficking .UPF1 is an RNA-binding protein that possesses RNA helicase and ATPase activities, Zn-coordinating finger motifs as well as acidic and basic amino acid clusters ,20. It sdenovo vRNA synthesis, immediately following transcription [In addition to transcripts carrying a PTC, substrates for NMD include, among others, mRNAs harbouring long 3'-untranslated regions (UTR), introns in the 3'-untranslated region (3'-UTR) and frameshift sequences ,39,42. Hcription , observacription ,45. All cription . It was cription ,46.UPF2 is a phosphoprotein that interacts with UPF1 and UPF3b to trigger NMD. In at least one study, the interaction between UPF2 and UPF1 was shown to be mediated by a conformational change of UPF1 from an RNA-binding to an RNA unwinding mode dampening UPF1’s ability to bind RNA . The othinsitu imaging analyses and insilico modeling of protein-protein interactions, we revealed that the association between UPF1 and UPF2 plays a critical role in the regulation of vRNA nucleocytoplasmic export. These results concretely explain why HIV-1 excludes UPF2 from its nuclear export and cytoplasmic HIV-1 RNP but also identify an unsuspected regulatory circuit involving multiple host UPF proteins in determining the fate of the HIV-1 vRNA.Since our earlier work strongly supported a nuclear role for UPF1 , in thisWT) and pCI-FLAG were described earlier [WT) and UPF1 mutants were described earlier [WT, FLAG-UPF21173 and FLAG-UPF21–1096 were previously described [1173 has point mutations which abolish its interaction with UPF1; FLAG-UPF21–1096 has a deletion at the C-terminus of the protein and does not bind UPF1 [Cell culture of HeLa cells and transfections of proviral DNAs, pNL4-3 and pMRev(−) as well as UPF1 siRNA and rescue experiments were performed as described before . Total c earlier . Bryan C earlier  and Alan earlier . Because earlier . FLAG-UPescribed ,49,53,54ind UPF1 ,53,54,55ind UPF1 . MonocytAntisera to UPF1, UPF2 and UPF3b were generously supplied by Jens Lykke-Andersen . Antisera to DDX3 was generously supplied by Luc DesGroseillers . Mouse anti-p24 and mouse anti-GAPDH antisera were purchased from Intracell and Techni-Science, respectively. Mouse anti-Digoxin was purchased from Sigma-Aldrich and sheep anti-Digoxin was purchased from Roche. Mouse and Rabbit anti-FLAG antibodies were purchased from Sigma-Aldrich and mouse anti-GFP was purchased from Roche. Rabbit anti-CRM1 was purchased from Santa Cruz. Rabbit and mouse anti-HA was purchased from Santa Cruz and Roche, respectively. The Nup62 antibody was purchased from Sigma-Aldrich and rat anti-RFP  was from Chromotek and the anti-SMG6 was from Abcam. For western analysis, horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were purchased from Rockland Immunochemicals . Signal intensities were scanned by densitometry using either ImageJ software  or using the Gel Doc System and quantitated by the Quantity One Software version 4.4.1  . Standarinsitu hybridization and immunofluorescence (FISH/IF) co-analyses were performed as described earlier [invitro transcription with digoxigenin-labeled UTP . Microscopy was performed on a Zeiss LSM5 Pascal laser-scanning confocal microscope (LSCM) or on a WaveFX spinning disk confocal microscope system . Microscopy techniques, filter sets and laser wavelengths were completely described earlier [vs. cytoplasmic phenotypes were defined only when >80% of the vRNA was localized in the nucleus or in the cytoplasm. Representative phenotypes are shown in the figures in the manuscript. The observed phenotypes were obtained in 78% to 85% cells (n > 50 cells per condition) in each experiment. Data were analysed by one-way analysis of variance followed by a Dunnett post-test using GraphPad Prism 5.0 software. This test compares the mean of each condition with that determined for the respective control. The histograms shown in the manuscript represent the fold-change in vRNA localization  in either nuclear or cytoplasmic compartments with respect to control conditions. Quantitative evaluation of the localization was achieved by visual inspection of the central confocal plane of laser scanning confocal images; cells were excluded from the calculation when either the signal intensity for HIV-1 or UPF antigens was poorly detectable or when the distribution was not distinct such that it spanned the nucleus, nuclear periphery and cytoplasmic compartments. In most experiments, distinguishing the nucleus/cytoplasm limits was readily observable and in some experiments the use of the chromatin stain, DAPI or immunofluorescence of Nup62 was used to confirm the cytoplasm/nucleus boundary. Only cells in which HIV-1 and the co-expressed proteins were considered in the calculations, where applicable. A p value of <0.05 was considered statistically significant. The RNA-specific dye SYTO14 was used to localize bulk mRNAs in the study as described earlier [mCherry mRNA localization, a representative unrelated mRNA (to HIV-1 RNA). Overexpression of UPF constructs had little effect on bulk mRNA localization  to generate a RT-PCR product of about 483 bp. Signal intensities of PCR signals (cycle 25–30) were quantitated exactly as described above [gapdh mRNA signal intensities.HeLa cells were transfected as described above and were described earlier . For celn etal. . mCherryed above ,62 usingg for 2 h, 4 °C. 12–500 µL fractions were collected and the abundance of FLAG-UPF1, pr55Gag, CRM1 and UPF3b were characterized by Western blotting. The vRNA was extracted using TriZol according to the manufacturer  and quantitated by RNA slot blotting analyses, exactly as described [500 µg protein from lysates derived from cells expressing HIV-1 pNL4-3 and pCi-FLAG-UPF1 was immunoprecipitated using FLAG-M2 agarose beads (Sigma-Aldrich). Immunoprecipitates were eluted using FLAG-3X peptide as per the manufacturer’s protocol. The eluted FLAG-UPF1 complexes were then carefully layered on a 5%–50% glycerol gradient and centrifuged at 80,000× escribed .etal., 2014) with default parameters and without specifying amino acids for interaction. Then, the models with the best score were refined using the Rosseta server [Protein-protein docking was performed using the structure of UPF1 (PDB: 2WJV) and DDX3 (PDB: 2I4I), previously described ,67. To oa server . For alaa server . DrawingWT) or the trans-dominant negative (TDN) NMD-null RNA helicase mutant, UPF1R844C, increased HIV-1 Gag and vRNA levels, indicating that UPF1’s function in vRNA metabolism is independent of its role in NMD [WT and two shuttling mutants, one lacking a nuclear export signal (∆NES) and one lacking a nuclear localization signal  co-analyses coupled with laser scanning confocal microscopy (LSCM) [WT resulted in the translocation of the vRNA to the cytoplasm as evidenced by the prominent cytoplasmic signal of the vRNA , suggese in NMD . To detey (LSCM) ,60. As ethe vRNA D. In shar export E,F. The r export  and was r export . This ecWT promoted a significant translocation of vRNA to the cytoplasm in Rev− conditions  and immunoprecipitated FLAG-UPF1WT. Rev, DDX3 and CRM1 all co-immunoprecipitated with UPF1WT in the presence and absence of Rev  with or without proviral DNA pNL4-3. FLAG complexes were then immunoprecipitated and the eluted complexes were assessed for FLAG (UPF1), UPF2, UPF3b, Gag and GAPDH by Western blotting analysis  with proviral DNA pNL4-3 and assessed vRNA localization by FISH/IF co-analyses (1–1096 exhibiting a complete loss of UPF1 binding failed to block vRNA export (1173), which exhibits an incomplete association to UPF1, resulted in a clear 50% phenotype between nuclear and cytoplasmic localization of the vRNA , and examined vRNA localization by FISH/IF co-analyses D. In conanalyses E,F. Wildanalyses E, similaA export F. A poinexport 11, which e1–1096 does not bind UPF1. Our results show that all of the UPF2 constructs immunoprecipitated with UPF3b but bound UPF1 differentially  will then acquire additional nuclear trafficking proteins including DDX3, DDX1, eIF5A, amongst others, that are important for vRNA nuclear export, and will position itself for nuclear export through the nuclear pore (The findings presented in this manuscript do not represent the first UPF2-independent function of UPF1 ,41. Our UPF2 does not play a functional role in several UPF1-containing mRNPs such as the UPF2-independent NMD RNP , the UPFHIV-1 commandeers UPF host proteins for critical steps of the viral replication cycle. In this manuscript we demonstrate that HIV-1 ensures nuclear export of the genomic RNA by the recruitment of UPF1 but by the exclusion of UPF2.


These ions can possibly become strongly correlated in the close vicinity of RNA surface. Most of the currently available software packages, which have widespread success in predicting ion effects in biomolecular systems, however, do not explicitly account for the ion correlation effect. Therefore, it is important to develop a software package/web server for the prediction of ion electrostatics in RNA folding by including ion correlation effects.Metal ions play a critical role in the stabilization of RNA structures. Therefore, accurate prediction of the ion effects in RNA folding can have a far-reaching impact on our understanding of RNA structure and function. Multivalent ions, especially Mghttp://rna.physics.missouri.edu/tbi_index.html provides predictions for the total electrostatic free energy, the different free energy components, and the mean number and the most probable distributions of the bound ions. A novel feature of the TBI server is its ability to account for ion correlation and ion distribution fluctuation effects.The TBI web server By accounting for the ion correlation and fluctuation effects, the TBI server is a unique online tool for computing ion-mediated electrostatic properties for given RNA structures. The results can provide important data for in-depth analysis for ion effects in RNA folding including the ion-dependence of folding stability, ion uptake in the folding process, and the interplay between the different energetic components. Because RNA backbone is highly negatively charged, the folding of RNA requires counterions to neutralize the backbone charge and to reduce Coulomb repulsion. As a result, RNA folding is sensitive to the ionic condition, such as ion type, size, valence and concentration –12. The 2+ ions, which are essential for the stabilization of RNA tertiary structure, we developed the Tightly Bound Ion (TBI) model  ion binding  from 0 M to 5 mM, the Mg2+ ion uptake increases from 0 to 0.035 per nucleotide. 2+ ion uptake curve as a function of [Mg2+].Since the ion binding distribution can be sensitive to the RNA structure, we need to generate explicitly the ensemble of discrete hairpin conformations –35. This2+ ions, are crucial for RNA tertiary structure folding. For the multivalent ions, ion correlation and fluctuation may play a important role in RNA folding. This demands a web server that can treat ion correlation and fluctuation effects in RNA folding. TBI is such a server. For a user provided RNA structure and ionic condition, the server computes ion binding properties, electrostatic free energies and various components. The TBI server provides results can be used to predict the ion-dependence of folding stability and ion uptake/release in the folding process. In the future development, we plan (a) to expand the server to treat other types of ions beyond the Mg2+ and Na+ ions, (b) to use a more detailed charge distribution  to model RNA charges, and (c) to include ion dehydration effects for ion binding.Accurate prediction of ion-mediated forces in the stabilization of RNA structure is critical to understanding RNA structure and function. Multivalent ions, especially Mg


Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Furthermore, we showed that miRNA of NGS-sufficient quality can be recovered from blood samples after >10 years of storage at −80 °C. This allows retrospective analyses of miRNAs from archived samples.The online version of this article (doi:10.1186/s13028-016-0224-5) contains supplementary material, which is available to authorized users. Non-coding microRNAs (miRNAs), amongst other small RNAs, play an important role in shaping a cell’s transcriptome profile by regulation of mRNA transcription and/or translation  and may In equine medicine, the knowledge about miRNAs in physiologic and pathologic conditions is still very limited restricted to certain diseases only, and thus warrants further investigation in this species –6.In contrast to mRNA, miRNA molecules are surprisingly stable—even in tissue samples of compromised quality —which suFor miRNA studies using equine EDTA blood, the use of PAXgene blood RNA tubes in combination with the PAXgene blood RNA extraction kit was recommended . HoweverFor miRNA extraction from serum samples we assessed how RNA yield is affected by the performance of three column-based and one column-free method, two different lysis reagents, and the effects of storage time at room temperature (RT), serum input volume, performing a second phenol extraction, and a second column elution. We performed our analysis on serum samples from three horses. As we assessed a large number of different parameters, we did not analyse all possible 576 combinations, but rather chose an incomplete matrix of 60 combinations that allowed us to address each of the six investigated parameters .RNA isolation was performed according to the protocols of the four different manufacturers: ZR Whole-Blood RNA MiniPrep, Zymo Reasearch; Direct-zol RNA MiniPrep, Zymo Research; miRNeasy Serum/Plasma Kit, Qiagen), and standard TRIzol method . The kitP = 0.12)  and after the second extraction RNA was no longer detectable and therefore none of those steps were considered as advantageous. Increased serum volume improved the RNA yield in samples processed with QIAzol, therefore, 1 ml of serum was chosen as the minimum and 2 ml as the optimum volume balancing RNA yield and the time required for the procedure.To increase the efficiency of the RNA extraction with miRNeasy serum/plasma kit the following procedures were tested: 1) second extraction: by adding 600 μl RNase-free water to the remaining organic phase, and further steps as indicated by the manufacturer; (2) second elution: re-using the first eluate; and (3) increased serum volume: 5× or 10× multiplication of 200 μl serum processed individually for the phase separation and then loaded sequentially on a single column  for next generation sequencing on a MiSeq  whereas the miRNA in small RNA ratio  was increased if the PAXgene blood miRNA kit was used , the RNA quality was significantly lower in long-term stored samples (Wilcoxon rank sum test P = 6.10e−4). Phase separation in long-term stored samples did not have a significant effect on RNA concentration (Wilcoxon rank sum test P = 0.31), but had a significant effect on RNA quality (Wilcoxon rank sum test P = 1.02e−4)  storage time and 2) sample handling before freezing on the RNA quality and quantity using the PAXgene blood miRNA kit and EDTA blood samples derived from 25 horses  measured by Fragment Analyzer in samples used for pilot experiments during method optimization and with already optimized method.


The SOLE folds into a stem–loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton.  Drosophila melanogaster, oskar mRNA localization in the oocyte determines where the abdomen and primordial germ cells will form. oskar mRNA transport to the posterior pole requires a polarized microtubule cytoskeleton and its associated motor kinesin , a protein complex that is deposited on the mRNA concomitant with splicing 20–24 nucleotides (nt) upstream of exon–exon junctions , have beior pole . Consistlization . Later wlization , is esselization .trans-acting factors  for localization, suggesting that this structural element participates in the recognition of  factors . In cont factors . Howeveroskar mRNA transcript, not requiring splicing for its formation, the mRNA is mislocalized . Conversely, the R2/R1 ratios of residues 14–20 and 24 are lower than average, which demonstrates fast (picoseconds) internal dynamics. All in all, the relaxation parameters are indicative of a complex internal dynamics in the MSL region.To gain more understanding of the dynamic properties of the MSL region, we measured relaxation parameters. C8 atoms E are almC8 atoms A, indicaAnalysis of the chemical shifts of both base and ribose carbons and protons  reveals The presence of 2 G-N1 and 4 U-N3 imino protons, as well as hydrogen bond mediated G-HN1/C-N3 or U-HN3/A-N1 correlations confirmed the presence of a stable stem for nucleotides (nt) 1–7 and 26–32 .i + 1) − H2′(i) NOEs, accompanied by weak H6/H8(i + 1) − H1′(i) NOEs were detected in these regions, as well as cross-strand NOEs between A20-H2 and C13-H1′, A11-H2 and U22-H1′, A24-H2 and C9-H1′. The A20-H2−C13-H1′ and A11-H2−U22-H1′ NOEs indicate base-pairing between G10−U22, A11−G21, G12−A20 and C13−G19. Therefore, in the structure calculation base-pairing was imposed between U8–A25, C9–G23, G10−U22, A11−G21, G12−A20 and C13−G19. The G10–U22 base pair was assumed to be cWW (cis Watson–Crick/Watson–Crick) (trans Hoogsteen/Sugar-edge) and cWW  , as this and cWW . The tHS and cWW . The spe3JH1′,H2′ coupling constants, measured in an HCCH-E.COSY spectrum, displayed values >4 Hz for A14 and G23, which is indicative of conformational equilibrium between the C3′-endo and C2′-endo ribose conformations.Next we applied chemical shift analysis of the ribose carbons to determine the ribose pucker and the exocyclic angle γ  All nuclThe structure was calculated using 769 NOEs, 206 dihedral angles, and 76 residual dipolar couplings (RDC) restraints (30 for the ribose and 46 for the base C–H vectors). The 10 lowest energy structures converged to a precision of 0.7 Å all heavy atom root mean square deviation (RMSD) , shows helical geometry with a widened major groove B. InclinIn summary, the stretches 3–13 and 19–30 form a continuous helix with a progressively widening major groove that offers a large interaction surface to proteins or peptides.13C relaxation rates. Here, we used the program ROTDIF , measured at a magnetic field of 14.1 T (150.9-MHz carbon frequency), and model both the diffusion tensor and the parameters of internal dynamics. We performed the analysis with two sets of data, comprising either all residues or only the PS stem. Both analyses resulted in an axially symmetric tensor , with average correlation time τC reducing from 6.58 ± 0.11 ns, when fitting the PS stem residues only, to 6.34 ± 0.13 ns with all residues included. This result reflects the dynamic nature of the MSL element. The τc value is in excellent agreement with the predicted one, after correcting for the higher viscosity of the D2O buffer used in our relaxation study . Additional chemical exchange contributions Rex are required to fit the relaxation rates of at least one of the carbons in the region 9–13 and 19–24, in agreement with the line broadening observed for these residues in the 2D 13C–1H correlations  formation of the SOLE, a 28-nt RNA sequence, which consists of 18 nt of exon 1 and 10 nt of exon 2, and (2) deposition of the EJC complex 20–24 nt upstream of the exon–exon junction. Both events are required for the correct localization of the oskar mRNA transcript , which acts in K10 mRNA transport by the dynein motor, displays A′-form conformation. In the K10 RNA two spatially registered widened major grooves seem to be required for transport.RNA recognition by proteins occurs mainly in the minor groove or at nonhelical elements. The deep and narrow shape of the A-form RNA major groove does not allow interactions with either peptides or proteins. However, in the past years several structures were solved that show RNA helices in a different geometry, with a widened major groove . The so oskar transcripts  CDE (constitutive decay element) stem–loop RNA facing its major groove and interacting with the apical part of the stem, the loop and the backbone on one side of the helix . The CDE524G−539A, 525C−538G, 526G−537U, 527C−536G, 528A−535A), which would stack on the PS stem without interruption. The elongation of the stem could serve the purpose of stabilizing the unwound helical structure of the PS region; alternatively, it could provide an additional interaction surface to cofactors.In the SOLE element, the RNA helix extends from the PS to the MSL region, where the major groove is widened further by the presence of two purine–purine base pairs . This geK10 mRNA necessitates two spatially registered widened major grooves: In the K10 TLS, A′-form helices are induced in the upper and lower parts of a long stem by a continuous stack of >3 purines in one strand. In contrast, the sequence of the SOLE RNA lacks a continuous purine stack in the PS stem and does not suggest the formation of A′-form helix. The unwinding of the helix from A- to A′-form starts only at the center of the PS stem and is consolidated by the purine stacking of the noncanonical base pairs in the MSL region. In addition, the short dimension of the SOLE element does not support the formation of two spatially registered widened major grooves.As for the K10 TLS, SOLE-dependent mRNA localization does not require a specific nucleotide sequence, but rather depends on RNA secondary structure. However, the structural details and the pattern of intermolecular recognition seem to diverge for the K10 TLS and the SOLE RNAs. Localization of cis WW A–G base pair in the HIV RRE (Rev response element) RNA stem functions as recognition site for the Rev peptide helix, which deeply penetrates the widened RNA major groove . The identity of the base pair 524C−538G, as well as the bulged out 539A, is consistently preserved; the next 525G−537U base pair can be formed in all but three species , while the noncanonical 526A−536G base pair is conserved either as such or as the isosteric A–A base pair . The 527G−535A base pair is present in all but two species (D. pseudoobscura and D. persimilis) as G–A or A–A, while the last base pair of the stem is less consistent. Most important, the disordered AUCAA loop is conserved in all species, suggesting that these exposed nucleotides might build a recognition element for proteins or nucleic acids. Nevertheless, its mutation to UUUUU does not affect localization . In Culex quinquefasciatus a 10-bp helix can form between position −16 of exon 1 and +10 of exon 2; the helix comprises four noncanonical G–U, A–G, and A–A base pairs and is capped by a pentanucleotide loop. In A. aegypti, a continuous stack of 10 bp, comprising six noncanonical G–U, G–A, A–A and possibly C–U pairs, is capped by a trinucleotide loop. The SOLE elements of the three organisms share common structural features: (1) The helix is 10–11 steps long and starts between position −18 and −15 of exon 1; (2) the apical part of the helix contains two purine–purine base pairs, which consistently widen the major groove; (3) the helix is poor in G–C content, suggesting a dynamic equilibrium between different structures. Interestingly, the loop is consistently rich in A's and U's, but its length is not conserved. Conversely, the SOLE element of Nasonia vitripennis is predicted to fold in a stem–loop structure with a few different features: The stem is only 8-bp long and contains one purine–purine base pair in the basal rather than in the apical part; however, similar to the Drosophila SOLE stem, an A′-form helical structure with a widened major groove is predicted for the stem of N. vitripennis, due to the continuous stack of three or more purines on one strand. With 10 nt, the apical loop is the longest among all species.The structure of the SOLE RNA solved here allows us to revisit the structure predictions of putative SOLE elements in other organisms  or unlabeled (Sigma-Aldrich) rNTPs and plasmid double-stranded DNA templates. The RNA was purified by denaturing 12% polyacrylamide gel electrophoresis. Unlabeled trans-acting hammerhead RNA was used to obtain RNA with homogenous 3′ end . All other experiments were performed in 99.96% D2O (Sigma-Aldrich). For residual dipolar coupling (RDC) experiments, 10–15 mg/mL Pf1 phages (ASLA Biotech) were added, resulting in a splitting of the deuterium solvent line of ∼13 Hz.NMR samples at 0.3–0.4 mM concentration were prepared by dissolving the RNA in 0.35 mL buffer . NMR experiments involving exchangeable protons were performed in a H13C–1H correlations of the base and ribose regions revealed that at low temperatures a conformational exchange process causes the disappearance of several resonances between nucleotides 10 and 25. At 308 K all resonances become visible: This temperature was chosen for all following experiments.NMR spectra were recorded on Bruker Avance 600 and 800 MHz spectrometers. 2D 1H, 13C, 15N) HbCNb  HsCNb  edited NOESY   HbCNb , 3D  HsCNb , 3D HCCHN) HsCNb , and 3D 00 msec) . The rib protons . Seven sR1, R1ρ and heteronuclear NOE , 100, 200, 400, 700(×2), and 1000 msec and 0, 8, 16(×2), 24, 32, 40, 48, 56, and 64 msec, respectively. To ensure proper alignment of the magnetization along the spinlock field, separate R1ρ experiments were recorded for C1′, C2, C5, and C6/C8 resonances, leading to a maximal resonance offset of 980 Hz from the spinlock frequency; only residues within ±76 Hz were evaluated for the lower spinlock field strengths . Relaxation rates were fit to a mono-exponential decay and errors estimated using a Monte Carlo approach within nmrview , H6–C6 (Py), H5–C5 (Py), H2–C2 (Ade), and C1′–H1′ (ribose). Seventy-six RDCs could be measured (45 in the bases and 31 in the riboses).Residual dipolar couplings (RDCs) were calculated as the difference between C3′-endo range by estimating the magnitude of the 3JH1′–H2′ scalar couplings in the HCCH-E.COSY 3D experiment. Fourteen γ dihedral angles were restrained to the gauche+ range in the PS stem, as confirmed by analysis of the can2 coordinate . During the calculations, hydrogen bonds were maintained by distances restraints, while planarity was enforced through weak planarity restraints .Hydrogen bonds of WC base pairs were detected in HNN correlations and in NOESY experiments for stretch 1–7 and 26–32 . The six base pairs of stretch 8–25 could not be verified experimentally, as the imino protons of this “unstable” part of the stem exchange too rapidly with water at 308 K. However, both the NOEs and the RDCs clearly indicated the formation of a stem with regular base stacking. Therefore, in the final calculations, hydrogen bonds were imposed for U8–A25, C9–G23, G10–U22, A11–G21, G12–A20, and C13–G19. The A–G base pairs were set to be One hundred structures were calculated without using the automated assignment or the distance calibration options of Aria 1.2. The simulated annealing (SA) protocol starts with a high-temperature torsion angle simulated annealing phase with 100,000 steps at 20,000 K (time step of 27 fs). This is followed by a torsion angle dynamic cooling phase from 20,000 K to 2000 K in 100,000 steps and by two Cartesian dynamic cooling phases with a time step of 3 fs .r) and axial (Da) components of the alignment tensor were obtained by evaluating the RDCs pattern distribution; an intensive grid search was performed around these values for both Da and r, where the dipolar coupling energy term was evaluated as a function of the alignment tensor; the energy profiles revealed a minimum for Da = 19.1 and r = 0.23 and these values were used in the refinement. The final ensemble of 10 structures  was refined in a shell of water molecules .The RDC refined structures showed no NOE (>0.5 Å) or dihedral angle (>3°) violations. The final structures were analyzed using MolMol ; figuresR1, R2, and heteronuclear NOEs were analyzed using ROTDIF  and J(ωC). Instead of the R2/R1 ratio, ROTDIF uses the related ratio of spectral density components ρ = 4J(0)/3J(ωC), which is initially estimated from the relaxation rates and subsequently refined while modeling the diffusion tensor and internal dynamics. The robust least-squares method was used for estimating the experimental diffusion tensor. In small RNA molecules the 13C relaxation rates of proton bound carbons are dominated by chemical shift anisotropy (CSA) and the dipolar contribution of the directly attached proton. For an overall tumbling correlation time τc = 6 ns, the dipolar contributions of neighboring carbons are smaller than 1% (11∼δ33), but the unique axis δ22 is rather perpendicular than collinear to the dipolar interaction tensor (The relaxation rates g ROTDIF . For τc  than 1%  and can n tensor . Nonethen tensor . To avoiSupplemental material is available for this article.