The understanding of RNA structure is a key feature toward the comprehension of RNA functions and mechanisms of action. In particular, non-coding RNAs are thought to exert their functions by specific secondary structures, but an efficient annotation on a large scale of these structures is still missing.By using a novel high-throughput method, named chemical inference of RNA structures, CIRS-seq, that uses dimethyl sulfate, and N-cyclohexyl- N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate to modify RNA residues in single-stranded conformation within native deproteinized RNA secondary structures, we investigate the structural features of mouse embryonic stem cell transcripts. Our analysis reveals an unexpected higher structuring of the 5′ and 3′ untranslated regions compared to the coding regions, a reduced structuring at the Kozak sequence and stop codon, and a three-nucleotide periodicity across the coding region of messenger RNAs. We also observe that ncRNAs exhibit a higher degree of structuring with respect to protein coding transcripts. Moreover, we find that the Lin28a binding protein binds selectively to RNA motifs with a strong preference toward a single stranded conformation.This work defines for the first time the complete RNA structurome of mouse embryonic stem cells, revealing an extremely distinct RNA structural landscape. These results demonstrate that CIRS-seq constitutes an important tool for the identification of native deproteinized RNA structures.The online version of this article (doi:10.1186/s13059-014-0491-2) contains supplementary material, which is available to authorized users. Drosophila melanogaster that both MLE and MSL2 proteins of the MSL complex act by binding to conserved structural domains of the roX1/2 ncRNAs, which then mediate targeting to the X chromosome to regulate dosage compensation in fruitfly [Hoxa6/7 genes, and its recruitment to chromatin is mediated by interaction with a stem-loop structure located in the 3′ region of the Mistral lncRNA [The development of high-throughput methods for the analysis of the epigenome and transcriptome have led to the discovery of thousands of previously unannotated transcripts ,2, many fruitfly ,12. Furtl lncRNA .in vitro folding may be representative of native RNA structures in vivo. While for certain small RNAs the in vitro folding landscape recapitulates well the in vivo one [in vitro to the prevalence of kinetically trapped intermediates and misfolded structures [in vitro folding of the RNAse P ribozyme is a slow process that takes several minutes and requires escape from a kinetic trap [in vivo and in vitro probing data on human telomerase RNA revealed that while the 3′-terminal small nucleolar RNA (snoRNA)-like domain folds into comparable structures in the two conditions, the 5′ template domain exhibits very different foldings [The growing number of annotated transcripts has outpaced the efficient analysis of their structure; at present, structural information exists for only a very tiny minority of annotated RNAs. To address this need, over the past few years various enzymatic- and chemical-based approaches have been proposed for the discovery of secondary structures for thousands of RNAs at a time -18; howevivo one -21, longructures -24. For tic trap ,25. Compfoldings .in vitro and in vivo. The first is based on the assumption that, in the cell, most nascent transcripts are likely to fold during transcription [in vitro, two recent reports analyzed the structures of Saccharomyces cerevisiae and Arabidopsis thaliana RNAs by treating the cells with dimethyl sulfate (DMS) [Two main scenarios can explain the differences observed in RNA folding cription ,27,28. Icription . The seccription -33. To ote (DMS) ,35.We present here a new method, named chemical inference of RNA structures followed by massive parallel sequencing (CIRS-seq), that allows genome-wide investigation of native deproteinized RNA secondary structures by exploiting the capacity of DMS and N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) to specifically react with RNA unpaired bases. Our approach, applied to mouse ESCs, allowed us to obtain single-base resolution structural information for thousands of transcripts in their native deproteinized conformation, revealing the structural complexity of the mammalian transcriptome.The CIRS-seq method Figure  is based6 deep-sequencing reads for each treatment, across two biological replicates.To perform transcriptome-wide probing of RNAs in their native deproteinized conformation, we lysed mouse ESCs in an isotonic buffer, and treated the lysate with Proteinase K to unmask regions of RNAs bound by proteins, without affecting the RNA structure  and CMCT  treatments, respectively  [Despite the respective strong preference of DMS and CMCT for A/C and G/U residues, we also observed non-canonical reactivities in both treatments. Our data are in agreement with previous reports showing DMS reactivity with G/U residues ,45-47 anhomolog) , a 15.5 homolog) ,53, and homolog) ; these rhomolog) ,54. WhenCollectively, this analysis proves the high accuracy of CIRS-seq, and provides a nucleotide-resolution panorama of the mouse ESC RNA structurome.de novo secondary structures. Constraints derived from chemical probing data may significantly improve the accuracy of RNA secondary structure prediction tools [Next, we verified the ability of CIRS-seq to infer on tools ,56. We con tools ,58, or con tools  to devisThanks to the high resolution enabled by CIRS-seq, we then investigated the structural features of mouse mRNAs, and looked for structural differences across transcript regions. We selected approximately 9,500 mRNAs, in which the DMS/CMCT treatment induced, on average, at least one RT stop per nucleotide . These results differ from what has been recently observed in A. thaliana, where the coding region is more structured than the UTRs [P-values 4.0e-24 and 6.5e-8, respectively, Wilcoxon rank sum test; Figure Meta-analysis of average reactivity across UTRs and coding regions revealed a strong reduction of reactivity scores in the 50 nucleotides of the 5′ UTR immediately preceding the Kozak sequence (average 0.165) compared with the first and last 100 nucleotides of the coding region , while the first nucleotide was the less structured and significantly more reactive to DMS/CMCT treatment than the second and third . Taken together these results suggest a deep involvement of RNA secondary structures in driving and regulating translation efficiency.We next analyzed the first and last 99 nucleotides of mRNA coding regions to determine if the previously reported periodic signal of three nucleotides ,34 was cP-value 1.1e-87, Wilcoxon rank sum test), snRNAs , tRNAs , and long intergenic non-coding RNA  compared with mRNAs (average 0.366) . Concordant with this observation, analysis of putative Lin28a binding sites revealed that the target A/G-rich motifs tends to assume a single-stranded conformation within the loop regions of hairpin-like structures  supplemented with 15% heat-inactivated fetal bovine serum, 0.1 mM NEAA, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 25 U/ml penicillin, 25 μg/ml streptomycin and 1,500 U/ml LIF, as previously described .Real-time quantitative PCR was performed using the SuperScript III Platinum One-Step Quantitative RT- PCR System  as previously described . The priFor RNA-seq library preparation, approximately 1 μg of TRIzol (Invitrogen) isolated total RNA from ESCs was subjected to ribosomal RNA depletion using the Ribo-Zero Gold Kit . rRNA-depleted RNA was used as the input for the RNA-seq library preparation using the TruSeq RNA Sample Prep Kit (Illumina) following the manufacturer’s instructions.Cell lysis and chemical probing, library preparation, and sequencing are detailed in the supplementary Materials and methods in Additional file RNA sample quality was assessed with the Agilent Bioanalyzer 2100. All of the samples had an RNA integrity number ranging from 9.9 to 10.CIRS-seq data analysis, normalization and background subtraction, and transcript analysis are detailed in the supplementary Materials and methods in Additional file CIRS-seq and RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE54106. Additional datasets and the source code for the analysis tool are available at .Additional file 1:PDF file containing supplementary Materials and methods, Figures S1 to S5), Table S1, and supplementary references.


E. coli NusA-NTD, located opposite to its RNA polymerase-binding domain (the β-flap domain), directly interacts with N in the antitermination complex. We hypothesized that this N-NusA interaction induces allosteric effects on the NusA-RNAP interaction leading to transformation of NusA into a facilitator of the antitermination process. Here we showed that mutations in β-flap domain specifically defective for N antitermination exhibited altered NusA-nascent RNA interaction and have widened RNA exit channel indicating an intricate role of flap domain in the antitermination. The presence of N reoriented the RNAP binding surface of NusA-NTD, which changed its interaction pattern with the flap domain. These changes caused significant spatial rearrangement of the β-flap as well as the β′ dock domains to form a more constricted RNA exit channel in the N-modified elongation complex (EC), which might play key role in converting NusA into a facilitator of the N antitermination. We propose that in addition to affecting the RNA exit channel and the active center of the EC, β-flap domain rearrangement is also a mechanistic component in the N antitermination process.Interaction of the lambdoid phage N protein with the bacterial transcription elongation factor NusA is the key component in the process of transcription antitermination. A convex surface of  E. coli NusA is a ∼55 kD protein, having multiple functional domains; N-terminal RNAP-binding domain (NTD), three RNA binding domains  and two C-terminal acidic repeats (AR1 and AR2)   site of the mRNA, where N and NusA make specific interactions  resistant to terminators. This phenomenon is called transcription antitermination ,14; Supp; Supp14;if  and nusA–V8A mutants induces synthetic defects in N-mediated in vivo antitermination, pHYD3011 plasmid expressing either WT or V8A NusA was transformed into the strains, RS1452 (having rpoB-G1045D and lac-PH-19B R′-lacZYAnutR-T reporter cassette) and RS1018 (having WT rpoB and lac-PH-19B R′-lacZYAnutR-T reporter). Following that chromosomal WT nusA of these E. coli strains was deleted by P1 transduction R(nusA::Cam). β-galactosidase assays were performed following the standard procedures using microtiter plates , pRS106  and pRS22 . The lacO sequence is present in the downstream oligos RS177, RS333 and RS994. The ops pause sequence from E. coli  by overlapping PCR amplification using the primers RS83, RS267 and RS276. The his pause sequence was incorporated after the nutR site  also by overlapping PCR method using the primers, RS83, RS263, RS264, RS265 and RS275. In both these cases, pRS22 was used for amplification, and the oligos RS275 and RS276 contained the lacO sequence so that both these templates have a lac operator sequence at their downstream edge. The template used in RNase H cleavage assays, Template VII, was amplified from pRS22 using RS83/RS404 oligo pairs. Template VIII, used in the Fe-BABE cleavage assays, was made from pRS385 using oligo pairs RS83/RS2. RS404 has the lac operator sequence. This operator sequence is cloned downstream of the H-19B nutR site in pRS385 (road-blocked complex) were formed at the operator site in the presence of Lac repressor. In some cases, pRS25 , 10 mM magnesium glutamate, 50 mM potassium glutamate, 1 mM DTT and 100 μg/ml BSA) at 32°C. The reactions were initiated with 175 μM ApU, 5 μM GTP, 5 μM ATP, 2.5 μM CTP and [γ-32P]CTP (∼3000Ci/mmol) to make a 23-mer EC (EC23). Then it was chased with 250 μM each of all the NTPs in the presence of 300 nM of NusA and 200 nM NusG. The reactions were stopped by phenol extraction followed by ethanol precipitation. Samples were loaded onto a 6% sequencing gel and analyzed using FLA 9000 phosphorimager (Fuji).For the 23 was formed at first in the same way as described before, except that the reactions were performed in T-Cl buffer  and in the presence of 100 nM Lac repressor. EC23 was then chased with 250 μM of NTPs for 2 min to form the RB. The RB was then washed to remove excess NTPs and used in different assays. The transcription reactions were carried out at 37°C.To form the RBs for different assays, we have used different DNA templates  immobilized to the streptavidin-coated magnetic beads via a biotin group at its 5′ end. These templates also contain a 22-bp lac operator sequence at its downstream end. On these templates, EC23 was chased in the presence of 10 nM of 32P-labeled WT NusA for 2 min to form the RBs on the immobilized Templates I, II and III. Excess NTPs were then removed by washing the beads, which was followed by the addition of 100 nM of unlabeled (cold) WT NusA with or without N as competitors. After 5 min, 15 μl of samples were removed and separated into supernatant (S) and pellet (P) fractions by keeping the tubes on magnetic stands. Half of the supernatant (S) and rest of the sample  / (b + x), where, x = [Competitor], y = fraction of dissociated NusA, b = concentration of the competitor at the half-maxima and a = maxima.To compare binding affinity of WT NusA for the stalled ECs made of WT and G1045D RNAP, fractions of dissociated radio-labeled WT NusA were plotted against increasing concentrations of cold NusA as competitor following the same experimental procedures as above. The curves were fitted to a hyperbolic equation of the form: his pause and 0, 30, 45, 60, 90, 120, 180, 240″ for the ops pause) and mixed with formamide loading dye (Ambion). RNA products were separated on 8% sequencing gel. Fraction of paused complex was calculated as: (Intensity of the paused RNA) / . Plots obtained from plotting the fraction of paused products against the time were fitted to an exponential decay curve of the form: y = yo + A.exp−λt, where A = amplitude, λ = rate constant, y = fraction of paused complex and yo value corresponds to the plateau of a curve representing unchased complexes. The pause half-lives (t1/2) were measured from t1/2 = ln2/λ.For pause assays, RBs made of WT or G1045D RNAP were at first formed on the Template V or VI at the respective pause sites in the same way as described above. Concentrations of DNA and RNAP were 10 nM and 50 nM, respectively. The RBs were then washed thoroughly to remove the excess NTPs, following which they were chased out of the stalled position in the presence of 100 μM each of UTP, CTP, ATP, 10 μM GTP and 1 mM IPTG. When required, 300 nM of NusA was added to the chase. Aliquots were removed during the specified time points , this reagent generates hydroxyl radicals. Three single Cys derivatives of NusA; S29C, S53C (in the NusA NTD domain) and S251C (in the SKK domain of NusA) were chosen to be conjugated to Fe-BABE.Fe-BABE (p-Bromoacetamidobenzyl-EDTA) is a cleaving agent that can be specifically conjugated to a cysteine residue of a protein. Under reducing conditions  protein was first dialyzed against metal removing buffer  and then against the conjugation buffer . 0.4 mM of Fe-BABE (Pierce) was incubated with 20 μM NusA in the conjugation buffer for 1 hr at 37°C. Unconjugated Fe-BABE was removed by passing the reaction mixture through a desalting column followed by equilibrating in the storage buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol and 0.1 mM EDTA). Efficiency of Fe-BABE labeling was estimated from its sensitivity to DTNB .32P]ATP (3000 Ci/mmole). RB was formed in a similar way on the Template VII (T7A1-H-19B R1-lacO-TR′nutR/t) for RNase H cleavage assays, except that RNAP (either β or β′) was radiolabeled instead of RNA. 600 nM Fe-BABE-NusA either in the presence or absence of 600 nM N were incubated with the RB at 37°C for 10 min in the cutting buffer  prior to initiation of the cleavage reaction. Cleavage was initiated by adding ascorbate and hydrogen peroxide  and the reaction was stopped by addition of one volume of 6X SDS sample buffer after incubation at 37°C for 5 min. Samples were heated to 95°C for 4 min prior to loading on 8% SDS-PAGE. Gels were exposed overnight to a phosphorimaging screen and the bands were analyzed in a Phosphorimager (Fuji).HMK (Heart Muscle Kinase)-tagged RNAP (either rpoB or rpoC) were radiolabeled using protein kinase A and [γ-23 instead of radio-labeled RNAP.For Fe-BABE-NusA mediated cleavage of nascent RNA in the RB, all the procedures were same as described above, except that RNA was radiolabeled during the formation of ECIn our previous study , we havenut site containing RNA, SKK domain makes specific contacts with the spacer region of this site . To test whether N increases the affinity of NusA for the EC, we measured the dissociation of radiolabeled NusA (P32-NusA) protein bound to the ECs stalled either close to the λ nutR site . With t32-NusA were measured from the supernatant fractions (S) . (iv) This N-effect was absent when the P32–NusA was part of the EC stalled on Template III (devoid of nutsite). These data indicated that the N-induced enhanced stability of NusA for the EC was dependent on the presence of nut site in the vicinity.We used ∼10-fold molar excess of cold (non-radiolabeled) NusA as competitor, to each of the stalled ECs. The amounts of dissociated P) Figure –E. We obnut site, we measured the dissociation of P32-NTD domain of NusA from the EC stalled on Templates I and III, both in the presence and absence of N. NusA-NTD interacts with RNAP but not with the nascent RNA  that were defective for N-mediated antitermination . In the P Figure , G1045D in vivo antitermination assays. We performed in vitro antitermination assays with all the β flap mutants using a DNA template (Template IV) where H-19B nut site is cloned upstream of a triple terminator cassette, R′-T1-T2T  of H-19B N were measured from the signal of the run-off (RO) products from the autoradiograms . Isolation of three flap domain mutants defective for N antitermination further reinforce the proposition that this domain of β-subunit of the RNAP is involved in this process.As NusA binds to the β-flap domain, we hypothesized that defect of N-mediated antitermination of the β flap mutant might be NusA mediated. To have a more detailed analysis of the involvement of β-flap domain in N antitermination as well as the role of NusA in the process, we engineered two more mutations in the β-flap domain, P1044A and V1046E Figure . We alsos Figure . G1045D,s Figure  and C. HR′T terminator. But when both V8A NusA and G1045D RNAP were expressed together in a strain (RS1452), the combination was severely defective for antitermination, thus exhibiting a strong synthetic defect . We found that G1045D mutation reduced the half-life of pausing significantly  .It has been established that the interaction of NusA and the β flap-tip helix (β 900–909 amino acids), located at the outer ridge of the RNA exit channel Figure , is the ) Figure –C. The phis pause hairpin-flap domain as well as the NusA-flap domain interactions. As G1045D mutation is located away (∼40Å) from the flap tip-NusA interaction site, it might have affected this interaction allosterically.Reduction in pause half-life both in the absence and presence of NusA indicated that the G1045D mutation caused defect in the 32-NusA from the ECs made either of WT or of G1045D RNAP stalled at the immobilized Template III DNA in the same way as described in Figure This pausing defect of G1045D RNAP might have arisen from its weaker interaction with the NusA-NTD. To test this, we measured the dissociation of Phis hairpin, which reduced the pause half- life. As this mutation does not affect intrinsic termination properties  the flexible flap-tip might have undergone spatial re-orientation in the G1045D mutant; and (ii) the nature of flap domain-NusA interaction is also changed in this mutant. These altered conformations of the flap domain affected the interactions with the operties , these cThe structural model of the EC ; Figure  revealednut site using lac repressor as a road block , -11 (RS550), -12 (RS551) and -14 (RS1106) Figure  positionWe observed the following. (i) In all the cases, RNA:DNA hybrid formed by -10 and -11 oligos were least accessible to RNase H cleavage, whereas that formed by -14 oligo was the most sensitive to the cleavage. This is consistent with the configuration of the exit channel, where -14 oligo is likely to form the hybrid at the outer edge of the channel Figures  and 4A. Therefore, G1045D mutation induces conformational changes in the RNA exit channel Figure . As thisrpoB-G1045D change.In this context, it should be noted that even though the reduced pausing exhibited by the β-G1046D RNAP is similar to that by N protein, the mechanism does not involve hindrance of pause hairpin formation in the exit channel (which is the case for N). Most likely the pause-reduction has occurred as a consequence of the altered NusA-flap tip interactions due to the We repeated the same RNase H cleavage assays  N enhances the NusA mediated constriction of the exit channel. (ii) Unlike Q , N did nThe concave surface of the NusA-NTD contacts with both the RNAP-flap and the nascent RNA . So we d32. The Fe-BABE-conjugated NusAs were functionally active in the in vitro N mediated antitermination assays .We designed NusA derivatives with single Cys residues at the positions 29, 53 and 251 Figure , and conWhen the stalled EC was formed with radiolabeled RNA and Fe-BABE conjugated NusA derivatives, we observed a broad cleaved band between -15 and -20 positions  of the nascent RNA, only when the Fe-BABE conjugated S29C NusA was present Figure , lane 6.These results indicated that in the presence of N, either the concave surface having the 29C of NusA moved away from the exiting RNA or N itself protected this part of the RNA. We ruled out the second possibility because the RNA:DNA hybrid formed by the -14 oligo Figure  was highNext we monitored the cleavage patterns of the radiolabeled β and β′ subunits of the RNAP present in the stalled EC in similar ways as described above Figure  and E anThese cleavage sites are located close to the β flap-tip helix (890–910 amino acids) and in the β′ dock domain (381–414 amino acids), which form the two sides of the RNA exit channel Figure . CleavagN-NusA interaction is the most important component in the N antitermination process . Till daWe have shown earlier that RNAP mutations in and around the RNA exit channel perturb N binding as well as its action 6 also inhis pause RNA hairpin formation in the channel.N-induced antipausing  and antiX. oryzae phage Xp10, binds RNAP near the β′-zinc-finger domain that forms the wall of the RNA exit channel located at the opposite side of the flap domain (Recent studies have revealed that targeting in and around the RNAP β-flap domain and modulating the RNA exit channel conformations are the common modes of action of different factors affecting the elongation and termination properties of the EC. The antiterminator, Q, binds to the β-flap domain of the RNAP and modulates the NusA-EC interaction in such a way that it forms an extended shield on the RNA exit channel ,35,36. Rp domain . The unip domain ,39.RNA exit channel is the nucleation site for the terminator RNA hairpins and is presumably the approach path of the terminator, Rho, to invade the interior of the ECs ,41. TherSupplementary Data are available at NAR Online.


The standard method for the storage and preservation of RNA has been at ultra-low temperatures. However, reliance on liquid nitrogen and freezers for storage of RNA has multiple downsides. Recently new techniques have been developed for storing RNA at room temperature utilizing desiccation and are reported to be an effective alternative for preserving RNA integrity. In this study we compared frozen RNA samples stored for up to one year to those which had been desiccated using RNAstable  and stored at room temperature. RNA samples were placed in aliquots and stored after desiccation or frozen (at −80°C), and were analyzed for RNA Integrity Number (RIN), and by qPCR, and RNA sequencing. Our study shows that RNAstable is able to preserve desiccated RNA samples at room temperature for up to one year, and that RNA preserved by desiccation is comparable to cryopreserved RNA for downstream analyses including real-time-PCR and RNA sequencing. In order to perform genomic research, nucleic acids must first be isolated, purified, and stored before downstream analyses can be performed. DNA is a very stable molecule and preserves well, conversely RNA is highly labile and degrades quickly if stored in improper conditions. Aqueous RNA can be degraded by spontaneous phosphodiester bond cleavage as a result of acid or base catalyzed transesterification from the intramolecular nucleophilic attack of the 2′ hydroxyl group on the phosphorous atom For years researchers have been investigating more effective means of storing and shipping RNA http://www.biomatrica.com/rnastable.php). Our study aims to test the effectiveness of RNAstable of preserving high quality RNA for long term storage, and most importantly, focus on the preservation of messenger RNA and the suitability of desiccated RNA for RNA sequencing.Research studies have examined the efficacy of the RNAstable system for storing desiccated RNA at room temperature, but these mainly focused on short term storage. RNAstable has been shown to be effective for preserving and maintaining desiccated viral RNA levels for up to 92 days as assayed by real time PCR TBP and compared the results of next generation whole transcriptome RNA sequencing (RNA-Seq).To study the effectiveness of RNAstable's ability to preserve RNA at room temperature, we performed RNA integrity analysis, quantitative RT-PCR, and next generation RNA sequencing with subsequent bioinformatics analysis. RNA Integrity Number (RIN) has been traditionally used as a measure of RNA quality, but a large part of the RIN calculation stems from ribosomal RNA integrity, therefore RIN represents an incomplete analysis of RNA degradation and quantification of message RNA in a specimen destined for gene expression analysis. RIN score has, for example, been found to be uncorrelated with the PCR efficiency of RNA samples undergoing qPCR RNA-Seq is a novel genomic sequencing technique that allows for thorough mapping and quantitating of the transcriptome. RNA-Seq allows one to quantitatively analyze a whole organism's transcriptome, and to analyze novel sequences, transcript isoforms, and all sizes and types of non-coding RNAs to a single base resolution We analyzed the integrity of the stored RNA samples by Agilent 2100 RIN number. Paired frozen and desiccated aliquots of total human RNA samples were analyzed every two months (except at 10 months) for one year of total storage. The integrity of RNA samples that were frozen at −80°C remained consistent, with average RIN scores ranging from 8.8 to 9.1. The integrity of RNA samples that were desiccated and then stored at room temperature were found to maintain average RIN values of between 8.7 and 9.1 at the five tested time points Fig. 1B.TBP at levels close to or exceeding the expression of TBP from the source-matched frozen RNA aliquot. Desiccated-to-frozen TBP expression ratios of one indicate equivalent expression of TBP in the frozen and desiccated aliquots, and ratios above one indicate that there are higher TBP message levels in the desiccated aliquots. The average of the relative TBP expression ratios of the paired desiccated to frozen samples was found to be above one after two and six months of storage. After one year of storage, five paired desiccated and frozen RNA samples were analyzed and only one RNA source had a TBP desiccated-to-frozen expression ratio above one but the average of these five ratios was still within half a standard error of the mean to a value of one  are shown in Table S1. All RNA samples, whether frozen or desiccated, were found to have a higher Ct value after 12 months of storage compared to after two months of storage. From the two month checkpoint to the year checkpoint the Ct value of the desiccated RNA samples increased on average by 2.85 cycles whereas the corresponding frozen samples showed an increase of 1.85 cycles (Table S1). The trend of increasing Ct number did not hold for each sample, as fluctuating values were seen across the RNA specimens over the yearlong study. All Ct values from frozen and desiccated samples stayed within the range of the upper 20 s to mid-30 s in cycle numbers representing positive TBP expression.Over the yearlong study the CACTB, GAPD and GUSB message in addition to TBP. Relative desiccated-to-frozen RNA expression level ratio averages were very consistent across the four genes after a year of storage , and high and low ratio outliers were also consistent across the four genes tested.qPCR validation experiments were then conducted using three additional primer/probe sets from the Roche Applied Sciences Universal ProbeLibrary Set. cDNA from RNA which was stored desiccated or frozen for 12 months was analyzed for relative expression of 2 = 0.93) . Furthermore, the gene expressions obtained from desiccated  and frozen (HD6F and MB2026F) methods are nearly identical (R2 = 0.997 and 0.999) , after 76 days of storage desiccated at room temperature  or after being frozen at −80°C . Nearly 97% of all sequence tags from fresh, frozen and desiccated RNA aliquots of both samples could be mapped to HG19 Table 1.TBP from desiccated RNA samples were lower on average than the Ct values from the same RNA which had been stored frozen at −80°C, indicating that desiccation can even be superior to −80°C frozen storage in terms of preserving RNA for downstream analysis.Total extracted RNA which had been desiccated and stored for up to one year at room temperature was found to maintain very high overall integrity. RIN analysis showed that total RNA can remain stable for one year of desiccated storage and that desiccation is comparable to −80°C frozen storage; the industry standard storage method for preserving total RNA integrity. Desiccating total RNA also preserves mRNA for downstream reverse transcription and qPCR analysis. RNA which was stored for up to one year was found to contain a sufficient amount of high quality mRNA transcripts of the genes tested to allow for qPCR detection after rehydration and reverse transcription. In fact, after six months of storage, when analyzed by qPCR, threshold cycles for t values after exponential DNA amplification. Running samples in triplicate and averaging the results helps to reduce possible errors but qPCR should not be relied on completely. Individual samples varied in calculated TBP Ct value across the time points (Table S1) in ways that would not be predicted from mere difference in storage length. Ct numbers would not be expected to go down after an RNA sample is stored for more time, but for some of the samples tested, Ct numbers dropped from month two to month six or from month six to month 12. The fact that the measured desiccated-to-frozen gene expression level ratios as measured by qPCR were lower at the 12 month time point could well be accounted for by the qPCR Ct variability. Regardless of any possible errors or variability, Ct values for TBP expression remained in the low to mid 30 s throughout the yearlong study for both desiccated and frozen samples representing preservation of mRNA.After 12 months of storage the desiccated-to-frozen RNA gene expression ratios dropped relative to the two and six month checkpoints, though the differences between these ratios are not statistically significant. qPCR carries with it an inherent variability, which may explain the differences seen. Small pipetting errors can result in highly varied CAfter being stored for 76 days at either room temperature in a desiccated state or at −80°C, RNA aliquots of two whole RNA extracts stored at these conditions were compared by RNA sequencing on gene expression of 14,114 genes. Transcriptome-wide expression profiles from RNA samples which were stored desiccated or frozen were very similar, indicating that the RNA stored by the desiccated method maintained the same gene expression profiles as the RNA stored in the traditional manner.In summary, desiccation appears to be a very promising technique for long term RNA storage. Storage in a desiccated state preserves RNA integrity and allows for robust downstream standard and next-generation transcriptomic analysis, and as such should be seriously considered in any modern biotechnical institute that deals with RNA. As desiccated room temperature storage has been shown to be effective at preserving high quality RNA, desiccation of RNA may also one day supplant ULT freezers for RNA storage, and would thus reduce laboratory reliance upon carbon-based power grids. Therefore, use of RNA desiccation techniques may save research groups energy costs, while simultaneously reducing their carbon footprint and significantly allaying the present fears associated with the real possibility of freezer failures.Blood was collected by the University of Colorado Skin Cancer Biorepository. Peripheral blood was collected in PAXgene RNA blood tubes (Qiagen/Becton Dickson) from 12 patients in the Cutaneous Oncology Clinic at the University of Colorado Hospital (UCH). Informed written consent was obtained from all subjects and this study was approved by the Colorado Multiple Institutional Review Board (COMIRB) No: 05-0309 at the University of Colorado Denver.2O. RNA yield was quantified using a NanoDrop 1000 spectrophotometer. Each isolated RNA sample was divided into 12 aliquots of 6 µl each, half of which were pipetted into 1.5 ml Eppendorf tubes and immediately stored at −80°C. The remaining six samples isolated from each blood sample were desiccated in Biomatrica RNAstable 1.5 ml tubes by room temperature vacuum centrifugation using an Eppendorf Speed Vacuum Concentrator . The desiccated RNA aliquots were then stored in a special humidity controlled dry storage cabinet. Paired aliquots of each sample (frozen and desiccated) were analyzed every two months (except at 10 months), RNAstable tubes were rehydrated in 6 µl of nuclease-free H2O, and the RNA quality was analyzed using an Agilent 2100 bioanalyzer to obtain RINs and by quantitative real time PCR at two, six, and 12 months .Total RNA was extracted from PAXgene RNA blood tubes using the PAXgene Blood RNA Kit  according to the manufacturer's protocol using only RNase-free equipment and solutions. The RNA was eluted in 80 µl of nuclease free HcDNA was synthesized from 100 ng of total RNA from both frozen and rehydrated samples using the Roche Transcriptor First Strand cDNA Synthesis kit according to the manufacturer's instructions.TBP), Beta-actin (ACTB), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPD), and beta-glucuronidase (GUSB) were assessed by qPCR. Primer and probe mixes were obtained from Roche Applied Science as part of the Universal ProbeLibrary Set, Human Reference Gene Assays. For the real-time-PCR assays, 20 µl reactions were run, each in triplicate. The PCR conditions were set for the UPL Roche designed protocol of two minutes at 50°C then 10 minutes at 95°C then 44 cycles of denaturation at 95°C, primer annealing at 60°C and polymerization at 72°C for ten, thirty, and one seconds respectively. qPCR reactions were performed on a BIORAD CFX 96 Real-Time System c1000 Thermal Cycler. A standard threshold of 32 RFU was set for comparison of RNA samples run on different plates, and RNA levels were compared pairwise as delta Ct ratios of desiccated to frozen samples.Message levels of the housekeeping genes TATA-Binding Protein .Frozen RNA was thawed and desiccated RNA was rehydrated after two, six, and 12 months and was reverse transcribed into cDNA. RT-qPCR was performed and the expression of t values of a given desiccated RNA sample and F being the average of the triplicate threshold cycle Ct values from the frozen RNA aliquot of the same sample.For D being the average of the triplicate threshold cycle CThe RNA Integrity or RIN value was calculated using the Agilent 2100 Bioanalyzer with RNA Nano chips at the University of Colorado Denver Genomics and Microarray Core.2O. RNA yield was quantified by a NanoDrop 1000 spectrophotometer. Each RNA sample was pipetted into three, 12 µl aliquots. One aliquot from each sample was sent immediately for RNA-Seq, another was placed at −80°C and the last aliquot was desiccated and stored as described above. After 76 days the desiccated samples were rehydrated in 12 µl of nuclease free H2O and the frozen aliquots were thawed on ice. These aqueous RNA samples were sent on ice for whole transcriptome sequencing (RNA-Seq) .Total RNA was extracted from blood samples HD6 and MB2026 stored in the University of Colorado Skin Cancer Biorepository. Both RNA samples were eluted twice with 20 µl nuclease free HTotal RNA was extracted and converted into a sequencing library according to the manufacturer's protocol . These libraries were sequenced on the Illumina HiSeq 2000 sequencer with 1×50 . On average, 56 million (range: 39–84 million) reads were obtained from each sample. RNA-Seq analysis was performed as previously described Figure S1The desiccated to frozen (D:F) ratios of three genes relative to the TBP D:F ratio from five RNA samples. Box and whisker plots are shown, indicating the spread of the data from the five samples. A value of one indicates that the preservation of RNA in the desiccated sample relative to its frozen aliquot is the same for TBP and the listed gene.(DOCX)Click here for additional data file.Figure S2Gene expression comparisons between fresh RNA and RNA stored by desiccated and frozen methods for 76 days. The top panels illustrate the comparison of RNA from HD6D  and HD6F  to HD6 . The bottom panels illustrate the comparison of RNA from MB2026D  and MB2026F  to MB2026 . X- and Y-axis represent the gene expression values in FPKM values.(DOCX)Click here for additional data file.Figure S3Flow chart of desiccated-frozen RNA evaluation scheme. In the diagrams of microcentrifuge tubes, grey indicates aqueous RNA, yellow indicates desiccated RNA, and white indicates frozen RNA.(DOCX)Click here for additional data file.Figure S4RNA-Seq sample preparation flowchart. In the diagrams of microcentrifuge tubes, grey indicates aqueous RNA, yellow indicates desiccated RNA, and white indicates frozen RNA.(DOCX)Click here for additional data file.Table S1t) values for five frozen and desiccated samples using Roche TBP primer/probe mixes after two, six, and 12 months of storage.qPCR threshold cycle (C(DOCX)Click here for additional data file.


Gemin5 is a RNA-binding protein (RBP) that was first identified as a peripheral component of the survival of motor neurons (SMN) complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs) through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs). Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E). Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES) elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis. Ribonucleic acid-binding proteins (RBPs) are important players of gene expression control in all organisms, from Bacteria and Archaea to Eukarya. RBPs associate with nascent transcripts and are subsequently involved in all aspects of the RNA life cycle. Indeed, these factors co-ordinately regulate all steps of gene expression, from transcription, to RNA processing, export, localisation, translation and stability ,2.RBPs are defined through their ability to interact with their RNA target, which may be sequence specific or structure-dependent and occasionally sequence promiscuous. Often, RBPs interact with RNA, forming large ribonucleoprotein particles (RNPs). The composition of the RNPs undergoes dynamic remodelling during the cellular response to intra- and extra-cellular environment changes, leading to a reprogramming of their functional properties ,4.The number of novel RBPs is growing due to the implementation of highly sensitive identification methods. A large number of the proteins recently described as RBPs were previously shown to participate in cell metabolism pathways and other cellular events, unrelated to RNA life span. This is an indication of the multifunctionality of RBPs. Moreover, the diversity of activities performed by RBPs can also be indicative of their property to link processes occurring in the nuclear and the cytoplasmic compartment of the cell, as exemplified by RNA splicing factors, RNA transport and translation. In agreement with this, many RBPs, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), are shuttling factors ,6,7.RBPs often are multidomain proteins containing motifs endowed with the capacity to interact with different targets, either proteins or nucleic acids (RNA and DNA). The interaction with the RNA partner takes place through the RNA-binding domain (RBD) of these proteins. The most common classes of canonical RBDs are the RNA recognition motif (RRM), the double-stranded RNA binding domain (dsRBD), the K homology domain (KH), the Pumilio homology domain (PUM-HD) and the zinc fingers . BrieflyS. cerevisiae ,21S. cerGemin5 is an abundant protein, predominantly distributed in the cell cytoplasm, as shown by immunofluorescence microscopy and subcellular fractionation studies . Co-locaGemin5 was described as a peripheral RBP of the SMN complex . The proBeyond its role in the snRNPs assembly, Gemin5 has been shown to participate in the alternative splicing process and in tumour cell motility. The MDA-MB-435 tumour cell line modified to maintain the metastatic property (C-100) or to supress it (H1-177) was used to analyse the global mRNA splicing profile ,28. This2O2 and tumour necrosis factor-α (TNFα) driven apoptosis  and at least one stem-loop (SL) located at the 3' end of the Sm site  . FurtherThe vast majority of Gemin5 is outside of the SMN complex , suggestQuantitative proteomic analysis of the human cap-binding complex led to the identification of Gemin5 as a novel eIF4E-binding partner . Gemin5 As mentioned above, Gemin5 has cap-binding capacity. The cap-recognition motif resides in the 13WD repeat domain according to cap-affinity chromatography of C-terminal and N-terminal truncated proteins . Since eThe ability of Gemin5 to interact with both eIF4E and the cap structure suggests that Gemin5 may interfere with the recruitment of the eIF4F complex to mRNAs. This hypothesis may explain the downregulatory effect of the protein in cap-dependent translation initiation. Further studies will be necessary to elucidate the mechanism underlying the role of Gemin5 in cap-dependent translation control.Two independent approaches, riboproteomic analysis and immunoprecipitation of photocrosslinked factors, showed that Gemin5 interacts with the IRES elements of two RNA viruses, FMDV and HCV . The intXenopus homologue of human TAR-RNA binding protein (TRBP) (xlrbpa) [The FMDV IRES consists of five structural RNA domains  . Domain (xlrbpa) , TRBP [6(xlrbpa)  or micro(xlrbpa) ,63.Dissection of the Gemin5 protein into various fragments has shown that the C-terminal region of Gemin5 has RNA binding capacity ,45. It iCurrently, there is no experimental evidence of the structure of the RBS2 region (residues 1383 to 1508). Prediction models for the C-terminal portion of the protein have shown that RBS2 comprises four helices (H1–4). Interestingly, the helix H2 is enriched in leucine, and the helix H3 is a glutamine-rich sequence  Figure . ProteinPicornaviruses induce a shutdown of cap-dependent translation in infected cells . Cleavagpr°) of FMDV recognises two sequences, RKAR and TKRL, within Gemin5 (pr° substrates [pr° and the peptide G5/1078-1438-FLAG containing the RAGHR sequence in transfected cells yielded a p25 proteolytic product. The lack of this cleavage product in double and triple mutants of this motif generated by substitution of basic residues to glutamic acid (RAGHR to RAGEE and EAGEE) demonstrated that Lpr° is the protease recognising this motif.Given that one of the factors proteolysed in picornavirus-infected cells was the RNA helicase of the SMN complex, Gemin3 ,75, the n Gemin5 A. These bstrates . Interesbstrates . The putThe p85 product results from the cleavage at the RKAR sequence  and inclin vitro and in living cells, confirming that RBS2 is the region responsible for the downregulatory effect of the protein [Overexpression of the p85 product produces a moderate upregulatory effect of IRES-dependent translation . A simil protein .pr° renders the p85 fragment. This polypeptide, which does not contain the amino-terminal region of the protein, could undergo a local conformational change within the RBS1 domain, resulting in a different crosstalk between the RBS1 and RBS2 moieties. We propose that the local structural rearrangement of the RBSs present in C-ter could be responsible for a moderate upregulation effect of IRES-dependent translation initiation, as observed in p85.To explain the role of Gemin5 in IRES-dependent translation in infected cells, we propose the following hypothesis B. The fuThe structural rearrangement of the RBS1 may take place in two different manners. One, the lack of the N-terminal region, may originate a local refolding of the RBS1 domain due its intrinsically unstructured flexibility . Two, foIn this review, we have discussed recent advances in the understanding of the functions of Gemin5. This protein performs a key role, not only in the biogenesis of snRNPs, but also in alternative splicing, stress response and translation control. The distinct roles of Gemin5 are related to its capacity to recognise different RNA targets, the snRNAs (by means of the snRNP code), the cap residue (m7 GpppN) of mRNAs or a specific domain within the FMDV IRES element . According to the identification of the domain of the protein involved in the recognition of the different RNA motifs, the N-terminal region participates in snRNAs recognition, while the most C-terminal region determines not only the interaction with the FMDV IRES, but also harbours the capacity to downregulate translation. Interestingly, the region of the protein containing the repressor domain is not stable in infected cells, indicating that viruses have developed mechanisms to subvert host factors and benefit the viral replication cycle.Most RNA-binding proteins bear conserved motifs, known as the RBDs. However, neither the N-terminal region nor the C-terminal region of Gemin5 harbours a canonical RBD. The fifth WD repeat domain has been demonstrated to be involved in the recognition of the snRNP code, as well as in the recognition of the cap. In contrast, a bipartite non-canonical RNA binding domain within the C-terminal region is responsible for the interaction with the IRES element and, moreover, for the negative effect on internal initiation. This non-canonical motif, however, appears to have an intrinsically disorganised structure, which can confer flexibility to the protein and, thus, the possibility to select different conformations depending on the function exerted by the protein. To date, the network of RNAs and proteins interacting with this region of Gemin5 is unknown. Future studies aimed at disclosing these networks will undoubtedly reveal new unanticipated roles of Gemin5 in other cellular processes.


Trypanosoma brucei, and found that it localizes to the nucleus and is essential for viability. The subset of RNAs bound to RBP33 was determined by immunoprecipitation of ribonucleoprotein complexes followed by deep sequencing. Most RBP33-bound transcripts are predicted to be non-coding. Among these, over one-third are located close to the end of transcriptional units (TUs) or have an antisense orientation within a TU. Depletion of RBP33 resulted in an increase in the level of RNAs derived from regions that are normally silenced, such as strand-switch regions, retroposon and repeat sequences. Our work provides the first example of an RNA-binding protein involved in the regulation of gene silencing in trypanosomes.We have characterized the RNA-binding protein RBP33 in  Genome organization in these organisms is unusual for a eukaryote, as open reading frames are organized in long polycistronic transcription units (TUs) that are transcribed by RNA polymerase II from only a few initiation sites in the chromosome Trypanosoma brucei genome, including retroposons INGI/RIME and SLACs, and satellite-like repeats such as CIR147 T. bruceiThere are numerous repetitive sequences within the Saccharomyces pombeArabidospsis thalianaS. pombe and S. cerevisiaeIn other eukaryotes, non-coding RNAs (ncRNAs) and RNA-binding proteins (RBPs) are emerging as important factors regulating gene silencing and chromatin structure through RNAi-dependent and independent mechanisms. Examples include RNAi-mediated formation of heterochromatin at non-coding and repeated DNA in in vitroIn this work we describe the characterization of the RNA-binding protein RBP33, which was first identified in a search for proteins that were able to bind to a uridine-rich RNA element T. brucei RNA-binding protein RBP33 (Tb927.8.990) is 317 amino acids long, and contains an RNA-recognition motif, a putative nuclear localization signal and two intrinsically disordered regions  in a tetracycline-inducible fashion. Although RNAi was not very efficient at the mRNA level, a marked reduction in protein abundance was observed after 48 h of tetracycline induction  with respect to input RNA, and ∼170 transcripts enriched 8-fold or more (log2FC>3) (bona fide proteins according to the annotated reference genome (http://www.geneDB.org/Homepage/Tbruceibrucei927) and available ribosome profiling data To gain insight into RBP33 function, we sought to identify the set of RNAs associated to RBP33 using immunoprecipitation of RBP33 ribonucleoprotein complexes followed by RNA-seq deep sequencing. In the immunoprecipitated material, we could identify ∼430 transcripts that were enriched 4-fold or more (logog2FC>3) . Examplebona fide proteins, the pteridine transporter Tb927.10.9080 and the flagellum protein FTZC (Tb927.10.8590). Silencing of RBP33 did not have a major effect on the abundance of either transcript; FTZC protein levels were not altered either  prevents read-through at RNA polymerase II termination sites L. tarentolae cells seem to require base J for viability T. brucei can survive without J T. bruceiT. bruceiIn this work, we have undertaken the characterization of the RNA-binding protein RBP33. We found that the protein is localized in the nucleus, and is essential in both bloodstream and procyclic trypanosomes. Using RNA immunoprecipitation and deep sequencing, we were able to identify a cohort of transcripts associated with RBP33 and thus obtain valuable information about the function of this protein. Although RBP33 binds to mRNAs encoding known or conserved proteins, most of its RNA targets are likely to be non-coding and/or present at minimal levels in the cell. Indeed, 90% of the RBP33-associated RNAs annotated as coding for ‘hypothetical proteins’ or ‘hypothetical proteins, unlikely’ were not detected by ribosome profiling via chromatin conformation. The source of ncRNAs could be SSRs, genes whose products are annotated as ‘hypothetical proteins (unlikely)’ or even transcripts coding for known proteins. In this regard, we have shown that although RBP33 binds to translatable transcripts, its depletion has no apparent effect on the levels of target mRNAs or in the protein product. However, RBP33 could be involved in the processing of cryptic ncRNAs located within the untranslated regions of a defined set of mRNAs. Alternatively, RBP33 could be part of a surveillance mechanism that selectively degrades RNAs transcribed from silenced regions in the genome. Another plausible interpretation is that RBP33 depletion results in an increased maturation or alternative processing of transcripts derived from SSR and retroposons, which is supported by the observation that slower-migrating INGI/RIME and CIR147 repeats transcripts decrease in abundance upon RBP33 silencing in bloodstream forms  or 5 µg/ml (bloodstream trypanosomes) hygromycin. For RNAi induction, tetracycline was added to the culture medium at a concentration of 1 µg/ml.T. brucei RBP33 were raised in rabbits and affinity-purified using a full-length version of the protein  tails, 20 µg of total RNA obtained from RBP33-depleted samples were incubated with 300 pmoles of oligo(dT)20, heated at 65°C for 10 min, transferred to a water bath at 42°C, cooled to 30°C, treated with 5 U of RNAseH (Invitrogen) for 30 min at 37°C and subjected to Northern hybrization. Samples lacking oligo(dT)20 were treated identically and analyzed in parallel.Northern hybridizations and quantitative RT-PCR analysis were done as described 9 procyclic trypanosomes were washed in ice-cold Voorheis's-modified phosphate-buffered saline , resuspended in 1 ml of vPBS and UV-irradiated twice at 400 mJ/cm2 in a Stratalinker apparatus (Stratagene). Cells were resuspended in 2 ml of 10 mM Tris-HCl, pH 7.4, 2 mM DTT, 1 mM EDTA, 0.1% Igepal, 50 U/ml RNaseOUT (Invitrogen) and protease inhibitors  and lysed by passing the suspension through a 27-gauge syringe thrice on ice. The cell extract was centrifuged at 16,000×g for 10 min at 4°C, and NaCl was added to the supernatant at a final concentration of 150 mM. This final lysate was incubated with 2 µg of affinity-purified anti-RBP33 antibody for 3 h at 4°C. To capture RBP33 ribonucleoprotein particles, 150 µl of 50% protein A agarose slurry  were added to the lysate. After an incubation of 1.5 h at 4°C, beads were transferred to a disponsable column (Biorad), washed twice with 10 ml of washing buffer  and once with 2 ml of washing buffer containing 1 M urea, resuspended in 450 µl of 10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.5% SDS, 1 mM EDTA and incubated in the presence of 100 µg of proteinase K (Ambion) for 30 min at 50°C. RBP33-associated RNAs were obtained by phenol:chloroform extraction and ethanol precipitation, and were DNaseI-treated prior to sequencing.To identify the cohort of transcripts bound to RBP33, 2×10T. brucei TREU 927 genome v4 using Bowtie http://www-huber.embl.de/users/anders/HTSeq/doc/index.html). Replicates were compared with the input RNA sample using the edgeR package http://www.ebi.ac.uk/ena/data/view/PRJEB6768).cDNA libraries for high-throughput sequencing were obtained from two biological replicates using the Illumina mRNA-seq kit according to manufacturer's instructions. Libraries were sequenced on an Illumina platform at EASIH  and the reads of 36 nucleotides in length were aligned to the Figure S1Alignment of RBP33 from T. brucei, T. cruzi and Leishmania major.(PDF)Click here for additional data file.Figure S2Assesment of anti-RBP33 antiserum specificity and RBP33 abundance. (A) A single immunoreactive band is visible in total cell extracts of bloodstream and procyclic trypanosomes in western blot assays. (B) RBP33 specific staining is no longer detected when the protein is ablated by RNAi in immunofluorescence analysis. (C) Quantitation of RBP33 protein levels in procyclic trypanosomes. The abundance of RBP33 present in different amounts of trypanosome cells (left) was compared to known amounts of recombinant His6-CBP-RBP33 (PDF)Click here for additional data file.Figure S3Examples of Artemis plots of RNA-seq data. (A) Regions corresponding to genes FTZC (Tb927.10.8590), RBP33 (Tb927.8.990) and Tb927.7.6010 are shown. (B) Transcripts derived from the SSR located in between genes Tb927.10.2390 and Tb927.10.2400 increase in abundance upon RBP33 depletion. Probes used for Northern hybridizations are indicated. Probe A is identical to that used in (PDF)Click here for additional data file.Figure S4RBP33 depletion has no effect on the levels of PT-X and FTZC mRNAs or FTZC protein. (A) Total RNA was obtained from trypanosomes grown in the presence or absence of tetracycline for 48 h, transferred to Nylon membranes and hybridized with probes designed to detect the PT-X and FTZC mRNAs. 7SL RNA was used as a loading control. (B) Total cell extracts were obtained from parasites grown in the presence or absence of tetracycline for 48 h and subjected to western blot analysis using an antiserum against FTZC (PDF)Click here for additional data file.Figure S5RNaseH treatment. To assess whether the transcripts detected with probe B in (PDF)Click here for additional data file.Table S1List of transcripts associated with RBP33. Transcripts with a log2FC enrichment >3 (Sheet 1) or >2 (Sheet 2) with respect to the input sample are shown.(XLSX)Click here for additional data file.Table S2Oligonucleotides used in this work. List of the oligonucleotides designed for Northern and quantitative RT-PCR assays.(XLSX)Click here for additional data file.


We report onstructure-function studies of the RRM domain of RBPMS bound to a CAC-containingsingle-stranded RNA. These results provide insights into potential topologies ofcomplexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sitesas detected by photoactivatable-ribonucleoside-enhanced crosslinking andimmunoprecipitation. These studies establish that the RRM domain of RBPMS formsa symmetrical dimer in the free state, with each monomer bindingsequence-specifically to all three nucleotides of a CAC segment in the RNA boundstate. Structure-guided mutations within the dimerization and RNA-bindinginterfaces of RBPMS RRM on RNA complex formation resulted in both disruption ofdimerization and a decrease in RNA-binding affinity as observed by sizeexclusion chromatography and isothermal titration calorimetry. As anticipatedfrom biochemical binding studies, over-expression of dimerization or RNA-bindingmutants of Flag-HA-tagged RBPMS were no longer able to track with stressgranules in HEK293 cells, thereby documenting the deleterious effects of suchmutations  RNA recognition motifs (RRMs) constitute the most abundant RNA-bindingdomains in higher vertebrates that play diverse roles in post-transcriptional geneexpression processes ranging from mRNA and rRNA processing to RNA transport,localization and stability (reviewed in A systematic study of transcriptome-wide mRNA targets of the RRM domain ofRBPMS using photoactivatable-ribonucleoside-enhanced crosslinking andimmunoprecipitation (PAR-CLIP) has identified RNA targets composed of tandem CACtrinucleotide motifs separated by variable spacer segments  length.We have also investigated the impact of structure-guided dimerization andRNA-binding mutants of RBPMS RRM on RNA-binding affinity and oligomerization, aswell as localization of RBPMS to cytoplasmic stress granules under oxidative stressconditions.We have solved the X-ray crystallographic structure of the RRM domain ofRBPMS in its free form and in complex with CAC-containing UD.melanogaster (CPO protein) and C. elegans (MEC8protein)  is associated withdimer formation according to the Complexation Significance Score 1·0calculated with PDBePISA .The RRM domain of RBPMS is conserved from humans to protein) a, andbaprotein) . The strprotein) . The cry-helices b. The st-helices , and anoβ-strand . The lat12A3C4CU5-3′by molecular replacement using our solved structure of the RBPMS RRM L81M mutantin the free state as a search model  that is involvedin either protein-protein contacts in the RNA-free structure  or in prβ sheet surface in the RBPMS RRM-RNA complex, withkey intermolecular contributions by conserved aromatic amino acids (Phe27 andPhe65) projecting from the two central β strands of theRNA-binding surface of the RRM domain  to measure RNA-bindingaffinities of RBPMS RRM mutations of key amino acids involved in CAC RNArecognition in the complex. We selected the PAR-CLIP-identified, natural targetRNA segment Ghiometry g. Mutatihiometry g. Furthehiometry f, or revhiometry d, also rhiometry g. A doubhiometry d, reducehiometry g.The RBPMS RRM dimeric arrangement within the asymmetric unit in thecomplex with RNA c, as welWe therefore studied homodimerization of the RBPMS RRM domain, as wellas full-length RBPMS isoform A (ENSP00000318102) b and S2aα-helix 1 and the loops betweenβ1 and α1, and betweenα2 and β4  lining the electrostatic surface of thedimer interface c,d. We in vivo RBPMS mRNA binding, we generated stable HEK293 celllines inducibly expressing Flag-HA-tagged R38Q, K36E/R38E, F65A, K100E, as wellas wild-type full-length RBPMS isoform A (ENSP00000318102). We had previouslyshown that wild type Flag-HA-tagged RBPMS co-localized with poly(A) RNA incytoplasmic granules after oxidative stress treatment using 400μM arsenite, similarly to the known stress granulemarker and mRNA-binding protein G3BP1  with differences observed for the C-terminal loopsegment (residues 103–111), that is involved in either protein-proteincontacts in the RNA-free structure or in protein-RNA interactions in the complex. TherelThe RBPMS RRM domain shares 90% amino acid identity with itsparalog RBPMS2 RRM a. An NMRThe RBPMS RRM dimer interface is stabilized by electrostatic andhydrophobic interactions as outlined in the Results section and is virtuallyidentical to RBPMS2 RRM dimer interface in solution  and resuThe RBPMS and RBPMS2 RRM homodimerization interface  are distβ-sheet of the RRM acts as a platform for RNArecognition, with base-specific recognition limited to two adjacent nucleotides, we propose a modelwhere each CAC segment targets its binding site on the RBPMS RRM dimerexhibiting a large interface involving parallel alignments of the helices a, as see helices c. Thedi helices  , we proposea model where CAC segments target their binding sites on the separate RBPMS RRMdomains b, as seeEscherichia coli strain BL21-CodonPlus (DE3)-RIL(Stratagene) grown in Luria-Bertrani (LB) medium supplemented with 50 mgml−1 kanamycin. Single and double mutations of RBPMSL81M, F27A, F65A, K100E, R38Q, E97A/K100A, T103A/K104A, K36E/R38E and K36/E39Awere introduced into the plasmid in one or two rounds of mutagenesis using theQuikChange II XL kit (Agilent) according to the manufacturer’sinstructions. The SeMet substituted RBPMS L81M mutant was expressed by growingcells in a M9 minimal medium using a standard protocol to saturate thebiosynthetic pathway for methionine production (The PCR-amplified cDNA fragments encoding the RRM domain of human RBPMS(14–111) were cloned into a modified pRSF-Duet1 (Novagen) vectorencoding 6His-Sumo tag at N-terminus between the BamHI and XhoI restrictionsites. The plasmid containing the DNA insert of interest was transformed intooduction . The recC2221, with two protein molecules per asymmetricunit. The crystals of the protein-RNA complex belonged to space groupP21 with two protein–RNA complexes perasymmetric unit. Crystal and diffraction data characteristics are summarized inRNA oligonucleotides were commercially synthesized (Dharmacon Research),deprotected and desalted according to the manufacturer’s instructions.Crystals of the RBPMS RRM and the complex of RBPMS RRM with5′-UCACU-3′ RNA were grown by sitting-drop vapor diffusion.Crystallization conditions were determined with sparse matrix screens  using a Mosquito crystallization robot (TTP Labtech). Theprotein was crystallized by mixing equal volumes(0·2–0·4 ml) of 2 mM protein solution in 25 mM Tris-HClpH 7·5, 0·5 M NaCl and reservoir solution containing 0·2M lithium sulfate, 0·1 M Tris-HCl pH 8·5, 40% (v/v) PEG400. The complex was crystallized by mixing equal volumes(0·2–0·4 ml) of 0·8 mM complex (1:1 protein toRNA molar ratio) solution in 25 mM Tris-HCl pH 7·5, 0·5 M NaCland reservoir solution containing 0·1 M Bis-Tris-HCl pH 6·5,28% (w/v) PEG 2000 MME. Droplets were equilibrated against 0·1ml reservoirs at 20 °C. For data collection, crystals were flash-frozen(100 K) in liquid nitrogen. The data were collected on the 24-ID beamline at theAdvanced Photon Source (APS) and processed by using HKL2000 . The SADThe structure of the RBPMS RRM L81M mutant was determined by SAD phasingusing the anomalous diffraction data collected at Se peak wavelength. A total offour Se sites were located using SHELEXD , and theITC measurements were performed at 25 °C using an iTC200 calorimeter. Protein and RNA samples were dialyzed in 20 mM Tris-HClpH 7·5 containing 100 mM NaCl. The protein concentration range in thecell of volume 200 ml was 0·03–0·05 mM. The RNAconcentration range in the injection syringe of volume 60 ml was0·26–0·5 mM. The data were analyzed with the MicrocalORIGIN software using a single site-binding model.http://www.addgene.org; 59388, http://www.addgene.org.RBPMS mutations were introduced into the plasmidpFRT-TO-FLAG/HA-RBPMS_KDR. This plasmid was generated from pFRT-TO-FLAG/HA-RBPMS(available on μgml−1 blasticidin, and 100 μgml−1 hygromycin.Flp-In HEK293 cells expressing Flag-HA-tagged wild-type and mutant RBPMSwere prepared as previously described (μg ml−1 doxycycline for 24 h.Arsenite was added to the cells at a final concentration of 400μM and incubated for 30 min at 37 °C.Slides were fixed and processed as previously described (HEK293 cells inducibly expressing Flag-HA-tagged wild type and mutantRBPMS were grown on Lab-Tek II chamber slides and induced with 1The atomic coordinates and structure factors for the RBPMS RRM L81Mmutant and RBPMS RRM–RNA complex have been deposited in the ResearchCollaboratory for Structural Bioinformatics PDB with accession codes 5CYJ and5DET, respectively.Supplemental


Z. Adjusted logistic regression models showed a negative association between SRH  and measures of heart rate variability (HRV). Glycemic markers were positively associated with poor SRH. No adjusted association was found with inflammatory markers, BP or lipids. In both unadjusted and adjusted linear models Pearson’s correlation strength was significantly higher between SRH with HRV measures compared to SRH with other biomarkers. This is the first study investigating the association of ANS function and SRH. We showed that a global measure of SRH is associated with HRV, and that all measures of ANS function were significantly more strongly associated with SRH than any other biomarker. The current study supports the hypothesis that the extent of brain–body communication, as indexed by HRV, is associated with self-rated health.The present study aimed to investigate the possible mechanisms linking a single–item measure of global self-rated health (SRH) with morbidity by comparing the association strengths between SRH with markers of autonomic nervous system (ANS) function, inflammation, blood glucose and blood lipids. Cross–sectional comprehensive health–check data of 3947 working adults (age 42±11) was used to calculate logistic regressions, partial correlations and compare correlation strength using Olkins  Self-rated health (SRH), a simple question asking individuals to rate their health in general, has consistently been found to predict mortality, morbidity, and other health outcomes . This isThe single item SRH measure has demonstrated good reproducibility, reliability, and strong concurrent and discriminant scale performance with an established more detailed subjective health status measure . Its valOne of the explanations proposed for this independent association of SRH with mortality and morbidity is that SRH is more inclusive than the covariates typically used in studies and as a single item, provides an optimal integration of many types of information, including external (e.g. diagnoses) and internal information (e.g. bodily sensations) ,10. ThusIn line with these self-reports, SRH has been found to be associated with a wide range of biomarkers ,17, inclst April 2014), the relationship between SRH and HRV has not been investigated.A possible but as yet uninvestigated mechanism that could link the various sources of information that feed into SRH is autonomic nervous system (ANS) activity. One widely used measure of ANS activity is the beat-to-beat variations in the cardiac rhythm as indexed by heart rate variability (HRV). HRV may be more than just an index of healthy heart function and may in fact serve as an easily measured output of the brain’s integrative system for adaptive regulation . Like SRIt is hypothesized that SRH would be associated with ANS activity as indexed by HRV and that this association would be more pronounced in comparison to other frequently used but less inclusive biomarkers.Therefore, the following analysis aimed to investigate (1) the associations between a single–item measure of global SRH and HRV in a large sample of working adults; and (2) the relative strength of associations between global SRH and HRV versus other frequently used biomarkers including inflammatory markers. A total of 9730 workers employed in four distinct plants were invited between September 2009 and May 2011 to a voluntary comprehensive health–check comprising a questionnaire and a medical examination in Southern Germany. A total of 4881 employees  accepted the invitation to the health-check (response rate 50.2%). Complete information on all used measurements was necessary to rule out composition effects when comparing coefficients between measurements resulting in an analysis sample of N = 3947 (80.9%). All participants were Caucasian and assessed by an agent independent from the employer who conducted the health risk assessments and data collection . The Ethical Committee of the Mannheim Medical Faculty, Heidelberg University approved this secondary data analysis (2010-296E-MA). All participants gave written informed consent prior to examination. For more details on measurements and population please see ,30.SRH was assessed using a single item asking about overall health status based on the past four weeks: “In general, would you say your health is: Excellent (1), Very Good (2), Good (3), Fair (4), Poor (5)”.Raw beat–to–beat intervals (IBI) from 24h HR-recordings were analyzed by researchers at the Centre for Neuropsychological Research  according to the Task Force Guidelines . The 24-Demographic, medical and lifestyle variables were obtained from an online questionnaire. The questionnaire had to be completed prior to being able to schedule the medical examination. All participants were enrolled and examined between 10 a.m. and 5 p.m. on a typical workday (Monday to Friday) during work hours. Upon arrival a medical examination was performed and the HR-recorder was attached. The next morning, between 7 a.m. and 9 a.m., a fasting blood sample was collected from all individuals. Samples were transported to a commercial laboratory  within 2 hours of sample collection and analyzed within 24-hours.1c]), and blood lipids  were determined using routine laboratory analyzers , blood glucose  Blood pWork stress was assessed by a validated short version of the Effort–Reward Imbalance (ERI) questionnaire . AccordiSubjective sleep quality was assessed by a continuous score based on a translation of the 4–item Jenkins sleep quality scales with higher values indicating worse sleep quality . Socio-eThe components of the MetS were defined according to the joint interim statement to harmonize the MetS . We cons2 test for categorical variables. A four-stage approach was applied:The distribution of all continuous variables was determined and transformations were used as necessary to meet the assumptions of modeling. Differences between the full sample and the analysis sample were determined by t-test for continuous and χb. Compared to Pearson’s correlation, Kendall τb does neither assume a normal distribution nor equidistant scaling of the correlated variables. In addition, Kendall τb makes adjustments for ties. Values of τb range from −1 (100% negative association) to +1 (100% positive association). A value of zero indicates the absence of association. This could be important, since the various biomarkers are usually neither normally distributed nor equidistant to a five-point Likert scale (SRH). On the other hand, Pearson correlations are easier to compare.First, bivariate associations between SRH and continuous biomarkers were assessed using Bonferroni-corrected Pearson’s correlation as well as nonparametric Kendall’s rank correlation coefficient τb were used to reflect the association between SRH and a biomarker independent of covariates  in the model.Second, multiply–adjusted partial correlation coefficients and partial rank correlation coefficients Kendall τZ to systematically compare the correlation strength (gained from step one and two) between the associations of SRH and each biomarker additionally adjusting for multiple comparisons by Bonferroni-correction. The null hypothesis of Olkins Z is that the correlation strength between SRH and biomarker A is equal to the correlation strength of SRH and biomarker B.Third, we used one sample Olkin’s Fourth, biomarkers were categorized into either clinically relevant categories or into quantiles if no clinical cut offs exist  to show Imputing missing SRH values (2.01%) by missing value regression had no impact on any results and are reported here. A total of 3947 individual data sets with complete data regarding questionnaire, medical data, and HRV assessment remained. STATA 12.1MP  was used for data management and analysis.The study sample consisted primarily of male workers (78%). The majority of participants were skilled workers (64%) as well as project or division managers (29%) without (78%) and with (22%) leadership responsibilities. Healthy behavior seems to be prominent with 81% non-smoker, 62% drinking alcohol maximal twice a week, 70% doing sweat–rich activities at least once a week, and having less than three components of the MetS (77%). We found some differences between the full sample and the analysis sample regarding blood lipids and glucose levels but the magnitudes of the differences are below clinically relevant levels (e.g. FPG-difference = 2.08mg/dl) .b) indicate the highest significant associations between our indices of ANS function and SRH. Higher SRH values  were significantly and negatively associated with vagally–mediated HRV  as well as with mixed sympathetic and parasympathetic measures . Additionally, rank order Kendall τb correlations reveal a comparable order of association strength  or inflammatory markers (CRP r = 0.13 and WBC r = 0.07). It is important to note that correlations of SRH and ANS function measurements were significantly stronger (compared using Olkins Z) during nighttime recordings  compared to daytime recordings  of HRV (data not shown). Every correlation of SRH with ANS function measures was significantly larger when compared with every correlation between SRH and other biomarkers using Olkins Z (Olkins Z .b) with sleep quality, work stress, age, and physical activity. Also work stress is significantly and positively correlated with all measures of blood glucose, blood lipids (not HDL), diastolic BP, as well as negatively correlated with all measures of ANS function. All correlations had a small effect size  and both sympathetic and parasympathetic mediated HRV . Glycemic markers  were positively associated with poor SRH. No adjusted association was found with any inflammatory markers, BP, and blood lipid measures . This means, the higher the self-reported work-stress the higher the odds ratio for worse SRH. The OR of the HRV variables were slightly attenuated after entering work stress and sleep quality into the models but remained significant and of a relevant effect size. Full models had a pseudo–R2 between 17–19%.The forest plot  presentsmeasures . Work stThis is the first study to show that a global measure of SRH was associated with ANS function as indexed by HRV. The strength of associations of traditional biomarkers with ANS function measures were compared and it was shown that all measures of ANS function were significantly more strongly associated with SRH than any of the other assessed biomarkers in a healthy working population. These findings provide valuable information about the mechanisms by which SRH is related to mortality and morbidity.1c) had significant bivariate associations with SRH. The magnitudes of the associations in the present study are similar to those previously reported [Consistent with prior research, the present study found that biomarkers associated with inflammation (CRP and WBC) and the MetS  a period of rest where the parasympathetic nervous system and hence the vagus is expected to be dominant. Better sleep quality is associated with better SRH  and insuThe exact neural concomitants of these bodily functions have also been a topic of investigation. A network of neural structures has been identified that is important in the integration and regulation of many systems of the body and has been termed the medial prefrontal-brainstem axis . An impoA possible explanation for the stronger negative association of HRV and SRH can be drawn from the neurovisceral integration model of cardiac vagal control. This model integrates autonomic, attentional, and affective systems into a functional and structural network . One of Additionally, the bi-directional communication between the peripheral nervous system and the brain has been clearly enunciated in the neurovisceral integration model –68. The Four possible factors have been proposed to explain the validity of SRH as a predictor of health outcomes . ImportaThis study adds to the ample evidence of the validity of a single SRH item as a measure of one’s health. However, a single item may have also its limits, which should be further explored. For example, research has identified certain subpopulations such as women or Blacks where the SRH–mortality relationship is less pronounced. Mastery and a more active lifestyle in a variety of domains such as cognitive, physical and social domains has been proposed as a potential explanation in this regards . There aThis study has several strengths. This is a large occupational sample from four distinct geographical regions within Germany comprised of employees with differing socioeconomic backgrounds rather than being limited to one particular professional group. Furthermore, all participants were from a similar cultural background, so genetic and cultural heterogeneity that might affect SRH are likely minimized. Finally this study accounts for many potential covariates in the statistical analyses and HRV was recorded over 24h.In sum we report for the first time that poor SRH was inversely associated with vagally-mediated HRV in a large, young healthy working cohort. It has been shown that HRV was more strongly associated with SRH than any other assessed biomarker. The present study provides important insights into the mechanisms by which SRH is able to provide information not readily available from other sources about the health status of individuals. The common ability of both SRH and HRV to provide an integrative index of exteroceptive and interoceptive information relevant to health related perceptions and behaviors offers new insights into SRH and HRV. Future research is needed to investigate the temporal and causal association of ANS function and self-rated health.


Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.The effects of spermidine analogues  on cell growth were studied using  E. coli, wild type W3110 (A-type)  are present at millimolar concentrations in both prokaryotic and eukaryotic cells, and play important roles in cell proliferation and viability –4. To st(A-type) , the amo(A-type) , 7. Poly(A-type) , and alslyamines . The mec systems , 14, butE. coli, the major triamine is SPD  by triamines ], which lacks bulged-out region, were measured. CD spectra of wild type OppA RNA, OppA + U RNA, wild type RpoE RNA, and RpoE -20(ΔU) RNA at 42°C were shown in yntheses , 34. As riamines  was lessE. coli MA261 by four triamines is different at 32°C, 37°C and 42°C. This is probably due to the difference of the ability of triamines to cause the structural change of RNA and that of the uptake activities of triamines at different temperatures. In the latter case, it is noted that HSPD is effectively taken up at 42°C, because the inhibition of the transcription of potABCD operon by PotD-triamine complex was the weakest in PotD-HSPD  at different temperatures  by measuring effect of triamines on cell growth and protein synthesis . The N-N2+. The degree of SPD stimulation of OppA and RpoE synthesis through stabilization of the bulged-out region of double-stranded RNA at 37°C was 5.1- and 2.3-fold, respectively, when MA261 cells were cultured in the presence of putrescine and harvested at A540 = 0.5 [540 = 0.3. So, the degree of polyamine stimulation was slightly different from that reported in previous studies [We have previously shown that effects of polyamines are mainly caused by stabilization of a bulged-out region of double-stranded RNA, whose structure is not stabilized by Mg40 = 0.5 , 37. In  studies , 37.Thermus thermophiles can grow at 78°C in the presence of long and branched polyamines [Many bacteria probably adapt to the environmental temperature through synthesizing suitable polyamines to interact with, and stabilize RNA. For example, lyamines . It woulS1 FigCD spectra were recorded as described in Materials and Methods.(PPTX)Click here for additional data file.


Arabidopsis thaliana protoplasts, despite chromatin decondensation, RNA polymerase II transcriptional activity is reduced. The subsequent investigated stages displayed a clear decrease in the quantity of 25S ribosomal RNA (rRNA) first and then poly(A+) RNA, particularly in the cytoplasm. Therefore, the reduced transcription activity and the removal of these RNA transcripts from the cytoplasm is a crucial process in obtaining totipotency in plant cells. After the cytoplasm cleaning of transcripts derived from mesophyll cells, we observed the resynthesis of these RNAs. An increase in the amount of examined molecules to a level similar to that in differentiated mesophyll cells precedes the divisions of already undifferentiated cells. In this work, we show changes in RNA polymerase II transcription dynamics and the quantity of poly(A+) RNA and 25S rRNA during dedifferentiation and re-entry into the cell cycle.The mechanisms of plant cell dedifferentiation and the acquisition of totipotency are poorly understood. One of the methods to induce the dedifferentiation process in plant cells is simple and requires the removal of the cell wall. After cell wall removal in protoplasts, large-scale chromatin decondensation is observed (Tessadori et al. in J Cell Sci 120:1200–1208,  Arabidopsis thaliana interphase cell nucleus has heterochromatin organised in so-called chromocenters, which contain heavily methylated, mostly repetitive DNA sequences  remain unchanged  showed changes in cell nucleus architecture similar to A. thaliana, but in this system, two distinct phases of chromatin decondensation were observed. The first phase occurs in freshly isolated protoplasts, and the second one occurs when cultured cells re-enter the cell cycle and progress into the S-phase  and Mediator complex   to 52 (Homo sapiens) tandem repeats of the consensus sequence Y1S2P3T4S5P6S7  are strictly associated with the phosphorylation pattern of the RNA POL II C-terminal domain (CTD) of its largest subunit Rpb1  and poly(A+) RNA and 25S ribosomal RNA (rRNA) transcripts in A. thaliana Col-0 seeds were washed in 70 % ethanol for 2 min, sterilised in 6 % calcium hypochlorite solution for 13 min and washed 10 times for 3 min in sterile water. Then, the seeds were sown in 75 % Murashige and Skoog medium supplemented with 0.7 % (w/v) agar. The growth conditions were continuous light with 45 μmol m−2 s−1 irradiance and an ambient temperature of 24 °C. Protoplasts were isolated from 14-day-old plantlets. The isolation and culturing of protoplasts and all necessary solutions were prepared according to the protocol of Chupeau et al. .All plant material used for fluorescence in situ hybridisation (FISH) and immunofluorescence analysis was fixed in 4 % formaldehyde  diluted in PIPES  for 1 h (leaves were placed in a vacuum for 1 h to remove air from the intercellular spaces). After fixation, the material was washed five times in PBS for 3 min. In the case of leaf mesophyll cells, we modified the protocol of nuclei isolation  hybridisation buffer , 30 % (v/v) formamide  and 20 % (v/v) H2O. For the analysis of 25S rRNA localisation and amount, we used the oligo probe 5′Cy3-AGCTACTAGATGGTTCGATTAGTCTTTC3′; the hybridisation step was performed at 37 °C. Transcripts with a poly(A+) tail were visualised using a 30-nt Cy3-oligothymidyne probe, and FISH was performed at 26 °C. The post-hybridisation steps in the case of 25S rRNA were washes in 4×, 2× and 1× the concentrations of SSC for 5 min each; for poly(A+) RNA, the FISH materials were washed three times in 4× SSC for 5 min and once in 2× SSC. For FISH double-labelling  and poly(A+) RNA), both probes were applied simultaneously in the hybridisation medium, and hybridisation was conducted at 30 °C using antisense U2 snRNA 5′ rhodamine green-ATATTAAACTGATAAGAACAGATACTACACTTG. Control reactions were conducted without oligo probes. DNA was visualised by 4,6-diamidino-2-phenylindole  staining in all reactions.FISH was conducted for a minimum of 16 h (with a 1-h pre-hybridisation step in the same buffer) using hybridisation buffer with the following composition: 50 %  diluted 1:300 in 1 % BSA in PBS for at least 16 h at 4 °C. Next, the examined materials were washed three times in PBS and incubated with a goat anti-rat secondary antibody IgG labelled with Alexa Fluor 488  and diluted 1:500 for 1 h at 35 °C. The secondary antibody was washed three times for 5 min in PBS, and the samples were stained with DAPI . The control reactions were performed without primary antibody.U test with Bonferroni correction was used.Protoplasts and cultured cells were observed using a Nikon TS-100 light-inverted microscope equipped with a PRIOR Lumen 200 fluorescent lamp and a Nikon DS-U3 digital camera. The results of FISH and immunolocalisation were analysed with a Nikon PCM-2000 confocal microscope and fluorescence-inverted Nikon Eclipse TE 2000-E microscope. A 100×  Plan Fluor DIC H/N2 oil immersion lens was used. For each material  and each reaction, we obtained three-dimensional optical sections with a 0.5-μm step interval from 22 to 46 cells, depending on the stage. The obtained data were corrected for background autofluorescence determined based on negative control signal intensities. All measurements were conducted at the same magnification, area and time of laser scanning. For the image processing and analysis, NIS-Elements F 3.2 , EZ Viewer software package  and ImageJ  RNA detection in leaf mesophyll cells, a signal was visible in the nucleus and cytoplasm Fig. . In cellIn protoplasts, the signal pattern was very similar to mesophyll cells Fig. . DespiteAfter 24 h of CDP culture, a strong homogenous signal was observed in cell nuclei, and the fluorescence decreased in the cytoplasm Fig. . The cytAfter 72 h in CDP, the fluorescence signal decreased in the entire cell and occurred mainly in the cell nucleus; the cytoplasmic signal was almost not visible Fig. . The cytAfter 120 h of culture, in non-divided CDP Fig.  and diviIn both non-divided and divided CDPs, the poly(A+) RNA level in the cytoplasm increased compared to the 72-h CDP and recovered to an amount similar to mesophyll cells Fig. . HoweverRecently, Kołowerzo et al.  showed tAfter FISH treatment for 25S rRNA transcripts, in all tested cells among every stage, the fluorescence was present in nucleoli and at a lower level in the nucleoplasm and cytoplasm Fig. . The nucIn leaf mesophyll cells, fluorescence was present in nucleoli and the cytoplasm Fig. . The fluAfter removing the cell wall, we observed a significant decrease in fluorescence intensity in whole cells, nucleoli and the cytoplasm in protoplasts Fig. . A quantAt 24 h after cell wall removal, 25S rRNA transcripts were observed in a very small area of the CDP cytoplasm in the form of single aggregates Fig. . The cytAfter 72 h of culture, fluorescence was observed in CDP nucleoli, and in the cytoplasm, 25S rRNA transcripts began to be visible again at a high level Fig. . The CDPAfter 120 h of culture, the fluorescence signal was very strong in nucleoli of non-divided CDP and was present in a pattern similar to that observed in mesophyll cells Fig. . Among aA. thaliana mesophyll protoplast culture, fluctuations in the quantity of the active form of RNA POL II, poly(A+) RNA and rRNA were observed. The analysis of the level and distribution of these molecules resulted in establishing a time-space model of the expression of genes transcribed by RNA POL II (poly(A+) RNA) and RNA POL I (25S rRNA) during dedifferentiation and the repeated progression into the mitotic cycle of the cultured cells.In the process of isolation and the subsequent 120 h of A. thaliana  gene in A. thaliana protoplasts, indicated that chromatin decondensation does not cause an increase in the number of transcripts in the investigated sequences , showed that 1728 genes were upregulated in protoplasts. Our results showed that, in protoplasts, the amount of actively transcribing POL II is strongly reduced, and the observed single foci of fluorescence may be the location of transcription of these upregulated genes. The lack of a significant decrease in the quantity of transcripts, despite a decrease in the amount of POL II EF, at this stage, may be related to the impaired maturation and degradation of RNA  RNA and 25S rRNA particularly in the cytoplasm. Initially, a gradual decrease in 25S rRNA, which was already visible in the protoplasts, was observed, and the decrease in the number of transcripts of poly(A+) RNA appeared in later stages of culturing, i.e. after 24 h. In the case of poly(A+) RNA, the level in the nucleus did not correlate with the decrease of POL II transcription activity and in no stages was it lower than that in the nuclei from leaf mesophyll cells. Thus, this result is an indication of the presence of mechanisms allowing the accumulation/retention of poly(A+) RNA in the cell nucleus in the isolated and cultured protoplasts. In plant cells that are exposed to heat and ethanol stress conditions, the number of nuclear proteins conjugated with SUMO proteins increases, and poly(A+) RNA accumulates in the cell nucleus  RNA was observed in the cytoplasm, with the lowest value observed after 72 h of isolation. At this stage, the poly(A+) RNA transcripts are almost undetectable in the cytoplasm. An increase in the ribonuclease level observed in tobacco protoplasts after 24 h following isolation may be responsible for the process of removing poly(A+) RNA from the cytoplasm  RNA from the cytoplasm, as shown by us, is likely a crucial process in plant cells in obtaining totipotency. These processes lead to the removal of ribosomes and protein-coding transcripts derived from mesophyll cells. Hence, following the introduction of a new model of expression enabling a cell fate switch, the newly formed mRNA transcripts are translated on a newly formed translational apparatus.A. thaliana displayed an increase in gene transcripts whose products are involved in RNA polymerase activity, the mRNA of ribosomal proteins and nucleolin involved in pre-rRNA processing  RNA in the cytoplasm sharply decreased after 72 h and only increased after 120 h following isolation. A study of the transcriptome of the cultured protoplasts of


Picornaviridae family. The structural data currently available from these viruses provided high-resolution snapshots for a range of conformational states associated to RNA template-primer binding, rNTP recognition, catalysis and chain translocation. As these enzymes are major targets for the development of antiviral compounds, such structural information is essential for the design of new therapies.RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication within the infected cells. RdRP function is critical not only for the virus life cycle but also for its adaptive potential. The combination of low fidelity of replication and the absence of proofreading and excision activities within the RdRPs result in high mutation frequencies that allow these viruses a rapid adaptation to changing environments. In this review, we summarize the current knowledge about structural and functional aspects on RdRP catalytic complexes, focused mainly in the  RNA dependent RNA polymerases (RdRPs) are the catalytic components of the RNA replication and transcription machineries and the central players in the life cycle of RNA viruses. RdRPs belong to the superfamily of template-directed nucleic acid polymerases, including DNA-dependent DNA polymerases (DdDP), DNA-dependent RNA polymerases and Reverse Transcriptases (RT). All these enzymes share a cupped right hand structure, including fingers, palms and thumb domains, and catalyze phosphodiester bond formation through a conserved two-metal ion mechanism . A strucPicornaviridae family is one of the largest virus families known, including many important human and animal pathogens. Picornaviruses are non-enveloped RNA viruses possessing a single-stranded RNA genome (7–8 kb) of positive polarity, with a small peptide  linked to its 5′-end. Their genomes have a long highly structured 5′ nontranslated region (NTR), a single large open reading frame (ORF) and a short 3′ NTR, terminated with a poly(A) tail. The ORF is translated in the cytoplasm of the host cell into a polyprotein, which is proteolytically processed by viral proteases to release the structural proteins (VP1-4), needed to assemble virus capsids and the nonstructural proteins (2A-2B-2C-3A-3B-3Cpro-3Dpol and in some genera L) as well as some stable precursors necessary for virus replication in host cells , for the aphthovirus FMDV and for the cardiovirus EMCV, either isolated or bound to different substrates bis(5-nitro-benzonitrile), with broad-spectrum antiviral activity against both enteroviruses and cardioviruses [pol-GPC-N144 co-crystals revealed that the binding site of the compound was located at the junction of the palm and the fingers domains, partially overlapping with the binding site of the templating nucleotide  were readily selected in the presence of suboptimal concentration of GPC-N114 . The reay Tyr195 E. In conpol might serve as a starting point for the design of new antiviral compounds targeting the template-binding channel.In summary, the identification of this novel drug-binding pocket in the picornaviral 3Dpol. 3Dpol also catalyzes the covalent binding of two UMP molecules to a tyrosine on the small protein VPg. Uridylylated VPg then serves as a protein primer for the initiation of RNA synthesis.RNA-dependent RNA polymerases (RdRPs) play central roles in both transcription and viral genome replication. In picornaviruses, these functions are catalyzed by the virally encoded RdRP, termed 3DThe ever growing availability of structures of picornaviral catalytic complexes provided an increasingly accurate picture of the functional steps and regulation events underliying viral RNA genome replication. Data currently available provides high-resolution pictures for a range of conformational states associated to template and primer recognition, VPg uridylylation, rNTP recognition and binding, catalysis and chain translocation. Such structural information is providing new insights into the fidelity of RNA replication, and for the design of antiviral compounds.pol possibly at different stages of the virus replication initiation process. Although the number of 3Dpol-VPg structures available show individual snapshots of the process, to obtain a global picture of the assembly, regulation and dynamics of complete replication initiation complexes, requires further analyses of high order assemblies formed not only by the polymerase and VPg but also involving different viral and host proteins, protein precursors and RNA templates. Such structures should provide a more detailed view of the molecular events underlying the initiation of picornavirus genome replication.Protein primed mechanism of replication initiation mediated by VPg appears to be a process that involves more than one VPg binding site in 3DThe structures of a large number of picornavirus replication-elongation complexes captured subtle conformational changes associated with nucleotide selection and active site closure. Among these movements, the motif B-loop assists in the positioning of the template nucleotide in the active site. Binding of the correct nucleotide then induces the β-strands realignment in the palm subdomain and repositioning of the motif A aspartate for catalysis. Steric clashes between the motif B-loop and the template RNA would finally promote translocation.