Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qβ replicase while maintaining the encoded gene function.Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qβ RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qβ replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of β-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qβ replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an  In the field of in vitro synthetic biology or artificial cell synthesis, in vitro synthesis of several types of biological functions is being attempted to understand the design principles of biological systems or to develop new technologies ,4,5,6,7.Recently, we have coupled an RNA replication system with a reconstituted gene expression system to develop a translation-coupled RNA replication system, wherein an artificial RNA genome replicates by the Qβ RNA replicase encoded on itself . When weOne of the possible strategies is designing the RNA structure by using only synonymous mutations. In a previous study, we attempted this strategy and succeeded in developing an RNA sequence encoding the α-domain gene of β-galactosidase to be replicable at a certain level . HoweverserS as model genes, we performed a combinatorial selection cycle, consisting of two types of selection, replication selection, in which more replicable RNAs are selected, and functional selection, in which RNAs encoding the functional genes are selected. We repeated this cycle for 10 rounds, and obtained more replicable RNAs that encode the functional genes.In this study, we attempted to establish an evolutionary strategy to obtain a replicable RNA while maintaining the encoded gene function. Using RNAs encoding the α-domain gene or Escherichia coli strain expressing the ω-domain of β-galactosidase for α-complementation. The cells were then inoculated on an agar plate containing a colorimetric substrate for β-galactosidase and an antibiotic to select the plasmid-harboring cells. If an E. coli cell has a plasmid harboring the functional α-domain gene, it produces a blue colony . (7) Abelection  and was We randomly chose four blue colonies in the final round and extracted each plasmid clone, which was used to prepare each RNA clone (m1–m4). Sequencing analysis showed that various different mutations were introduced in these RNAs . To examE. coli cells expressing the ω-domain gene and streaked them on an agar plate containing the colorimetric substrate of β-galactosidase. The E. coli containing no plasmid exhibited white colored streaks, whereas those containing the original α-domain gene and m1 to m4 exhibited blue colored streaks  After 10-h incubation, the small colonies were transferred on to a new agar plate containing 5% sucrose for selection of cells that lost the original complementary plasmid. An E. coli cell harboring the plasmid encoding the functional target gene would produce a large colony on this plate. All large colonies were picked up and pooled. (7) Plasmids were extracted from the colony mixtures.We next attempted to apply the combinatorial selection method to other genes. Given that the present method employing blue-white selection is not applicable to other genes, we developed an alternative in vivo selection method, as described in serS and glnS. The plasmid encoding each gene was mixed with the same amount of the plasmid encoding the non-functional mutant counterpart and applied to in vivo functional selection  but disappeared after selection (lane A). Thus, we demonstrated that functional gene is selected using the in vivo selection method.To test the applicability of the second selection cycle for selection of a functional gene, we first performed model selection experiments for two genes, n  was approximately 1.7-fold higher than that of the original RNA before selection cycle  revealed that the affinity between its minus strand and Qβ replicase was improved attributed to at least two point mutations, A87C and G116U. In addition, we developed another type of combinatorial cycle applicable to a broader range of genes and obtained more replicable RNA encoding functional E. coli. This method would contribute to obtain replicable RNAs that encode various gene types. Such replicable RNAs would allow the development of more complex RNA self-replication systems that would mimic the natural living systems.The first combinatorial selection cycle  relies oThe mechanism of how the mutations introduced in the selection cycle increased RNA replication is presently unknown. Our previous studies show the importance of strong RNA structures throughout the template RNA, given that in their absence the newly synthesized RNA strand hybridizes with the template RNA strand to form double-stranded RNA—a dead-end product for RNA replication ,24,25. A−3 per base per replication -UTP. The solution was incubated for 5 min  and JW0666 , were constructed under the complementation condition as follows. First, we constructed the low copy plasmid vector, pFE604, a derivative of F plasmid as shown in n method . The prisucB gene into the complementary plasmid by ligating the fragment carrying the sucB gene with vector fragments using In-Fusion cloning kit . The sucB gene fragment was amplified from plasmid pCDSSara [GGAAGCTAAAGCGGAGCCTATGGAAAAACG and AACTGCCTTAGGATATCGGCATTTTCTTTTGC, while the vector fragments were amplified from each complementary plasmid with primers TATCCTAAGGCAGTTATTGGTGCCCTTAAAC and TCCGCTTTAGCTTCCTTAGCTCCTGAAAATC.We then introduced pCDSSara  by PCR wserS gene was introduced into an E. coli strain JW0876 by the standard heat-shock method and streaked on to a Luria-Bertani (LB)-agar plate containing 50 μg/ml ampicillin and covered with nitrocellulose filter. After 10-h incubation at 37°C, small colonies appeared on the filter. The filter together with the small colonies was transferred on to a new LB-agar plate containing 5% sucrose and incubated at 37°C for another 12–24 h. An E. coli cell harboring the plasmid carrying the functional target gene and without the original complementary plasmid would produce a large colony on this plate. All large colonies were collected.Steps (1–4) and (7) were the same as the first selection cycle described above except for the omission of PCR amplification and mutagenesis. The RNA was transcribed directly from plasmid. The ampicillin resistance plasmid encoding S1 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


A paper recently published in Science . LwaCas13a was heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference, and improved specificity , which was the most effective at inactivating RNA. They successfully engineered a deactivated variant of PspCas13b that still binds to specific nucleosides of RNA but lacks its “scissor-like” function. Then, the deactivated variant of PspCas13b was fused to ADAR2 deaminase domain, which is involved (with ADAR1) in the A → I conversion in RNA transcripts. Hence, in the novel system, the deactivated form of the Cas13b enzyme was able to recognize a target sequence of RNA, whereas the ADAR2 element was performing the base conversion without cleaving the transcript or relying on the native cellular apparatus.To create REPAIR, Cox et al.  systematCox et al.  modifiedInterestingly, Cox et al.  further REPAIR presents a promising RNA editing platform with broad applicability for biotechnology research and therapeutics. Cox et al.  demonstrThe REPAIR system is an excellent research tool. Introducing specific sequence modifications into RNA molecules could allow to answer questions about alternative splicing mechanisms, translation, and even editing. In addition, by editing RNA rather than DNA, it might be possible to confer temporary, reversible genetic edits, rather than the CRISPR's permanent genome edits. This would allow the potential for temporal control over editing outcomes, as well as avoid the ethical issues that have arisen around genome editing.However, due to RNA specific properties, the RNA base editors would have to be repeatedly administered to function as a therapeutic approach. In addition, despite the large number of recent findings and novelties to improve CRISPR via REPAIR (Cox et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer EP and handling Editor declared their shared affiliation.


The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study RNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control, and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. On the other hand, the study of the differential stability of mature RNAs has been hampered by the fact that different methods have often yielded inconsistent results. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability. Subsequently, the degradation can proceed through two major pathways . The mRNXRN1 have been frequently associated with susceptibility to bacterial diseases, such as Staphylococcus aureus infections, and have been also implicated in the altered defense against viruses -adenine and [32P]-phosphate /t1/2.Just like radioactive decay, the decay of mRNA molecules is typically described by an exponential function.p.When the initial level of the RNA is zero then the time to reach the steady-state (equilibrium) is determined also by the decay rate, and not—as often incorrectly assumed—by the synthesis rate, The above equation and the half-life in it can be fitted to the time series of (labelled) RNA levels upon induction of gene expression or upon addition of modified nucleotides. The time to reach the half-saturation corresponds exactly to the half-life ,2,3.Equation (1) is used to fit half-lives in transcriptional inhibition and gene control experiments, while both Equations (1) and (2) are used for in vivo labelling experiments.rpb1-1 allele alters the mRNA stability even at permissive temperature -phosphate [In one of the first comparative studies, the half-lives of 11 mRNAs were measured using the temperature sensitive polymerase  than for weakly expressed genes (R = 0.15 at the 10th percentile), which suggests that the weak signal intensity may introduce considerable measurement error.This study was followed by the first genome-wide comparison , which erpb1-1 allele of the polymerase) indicated a low reproducibility of the method, with the Spearman’s rank correlation coefficient between five different studies ranging between 0.28 and 0.77 [A more recent study assessed both internal consistency and inter-method reliability. Two variants of the metabolic labelling employing different pulsing protocols yielded uncorrelated half-lives . The comand 0.77 . Furtherrpb1-1 allele increases mRNA stability even at permissive temperature [To explain the discrepancy between the transcriptional inhibition and in vivo labelling, the study using a variant of the metabolic labelling, the cDTA, found that the perature . Similarperature . Thus, tperature ,53. Thisperature .FUR4 gene are reduced upon exposure to uracil [Since two studies using metabolic labelling did not correlate with each other and with the inhibition studies , it was o uracil . Thus, tIt is somewhat surprising that there is no correlation between two variants of the metabolic labelling . What maThe methods applied to measure the half-life can perturb the cell or RNA turnover, and each method can have a differential effect on the stability of individual mRNAs. Therefore, it is possible that a sequence motif identified with a particular method reflects the interaction of the perturbation with the cell rather than the original half-life. Thus, using two independent methods or cross-validated methods can ensure that a motif identified reflects the parameter to be measured. This is particularly important because RNA decay may be determined not only by simple sequence motifs but also by RNA secondary structure  and by tR = 0.64), having median half-lives of 7.6 and 4.6 h. A metabolic labelling study with 4sU labelling reported considerably shorter mRNA half-lives in dendritic cells, in the range between 10 and 70 min [A systematic comparison of mRNA stabilities in mammalian cells is hampered by the large variety of cell lines and differentiation states. A study using global transcriptional inhibition with actinomycin D reported a median mRNA half-life of 10 h . Two stud 70 min . Thus, cSaccharomyces cerevisiae.Next, we will review how the major components of the mRNA degradation machinery were discovered, from a historical-methodological perspective. All the major components, the Xrn1 and exosome, and the major surveillance pathway (nonsense mediated decay) were identified in the yeast XRN1 in the regulation of the half-life of the unstable MFA2 and stable PGK1 mRNAs [rpb1-1 was used to show that the glucose-dependent stability of an mRNA is Xrn1-dependent [The Xrn1 protein was identified biochemically as a 5′-to-3′ ribonuclease activity on uncapped mRNA. Capped mRNAs were shown to be quite resistant to degradation by the purified Xrn1p . The 5′ K1 mRNAs . The metependent . Xrn1 alependent .Little is known about the regulation of Xrn1; subcellular sequestration of Xrn1 may play an important role. The protein is diffusely distributed in the cells in exponentially growing cultures but becomes localized to the cytoplasmic P-bodies at the diauxic shift. Subsequently, at the post-diauxic stage, Xrn1 is localized to the plasma membrane, which prevents mRNA degradation, as shown by the reduced decay of the MFα2 mRNA using a TET-based gene control method .XRN1 among related yeast species [KEM1 alias, which refers to Kar-enhancing mutations: haploid cells with xrn1 deletion have a very low mating efficiency due to a defective karyogamy [xrn1 null diploid cells difficult.It has been shown that the Xrn1p undergoes rapid evolution, as evidenced by the large sequence divergence of  species . This maaryogamy . The defSKI genes; the Ski proteins bridge the exosome and the RNA. The in vivo activity of the exosome was studied by combining the inactivation of the decapping protein Dcp1 with the deletion of SKI8 [GAL and MFA2 RNAs as studied by the gene control method, using the GAL promoter. However, when both of these proteins were inactivated, the half-life of the mRNAs lengthened around 6–7 times [The main component of the second pathway of mRNA decay is the exosome, which is a multi-protein complex with a 3′-to-5′ ribonuclease activity . The exo of SKI8 . Individ–7 times .rpb1-1 mutant [PGK1 mRNAs was, the more strongly the mRNA got destabilized [So far, few stability sequence motifs have been identified with dominant effect on stability when transferred to other mRNAs. A simple sequence that consistently changes the half-life of the mRNAs is the premature stop codon ,73,74. J1 mutant . The cloabilized . There aabilized . Second,abilized ,46. NMD abilized .In vivo labelling and inhibition of RNA expression have been used for at least four decades to measure RNA half-lives. Surprisingly, only recent studies unveiled that the two methods yield half-lives with no or minimal correlation. A more recent study showed that there is a high correlation between specific variants of the in vivo metabolic labelling and gene control methods. The consistency between two independent methods indicates high inter-method reliability. Thus, some variants of the genetic control and metabolic labelling may reflect the half-lives the most faithfully. However, there is no guarantee that using a method class, let it be metabolic labelling or gene control, will deliver the half-lives faithfully. If the conditions are not optimal, such as the concentration of the labelled nucleotide or the expression level of the activator that controls the gene, the measured half-lives and RNA synthesis rates may be inconsistent.The most robust methods are expected to have played a major role in the identification of the molecular machinery of mRNA degradation. In most cases, in vitro biochemical studies were first undertaken. Subsequently, the in vivo activities of the identified components were confirmed by most of the major methods. Thus, all methods capture RNA decay, at least qualitatively. However, current evidence indicates that specific variants of the gene control and metabolic labelling methods can be used with high reliability to quantify the differential stability of mature mRNAs. We expect that the confirmation of mRNA stabilities by two different methodologies and further developments in the analysis of inter-method reliability will help to identify mRNA stability determinants.


Science , the exact species used in the presented study, can cause serious flaccidness in silkworm, posing a big burden in sericulture development in East Asia. The genome of BmCPV is composed of 10 segments of double-stranded (ds) RNA. The naturally mature virions encapsulate their own RNA-dependent RNA polymerases (RdRps) into the capsid and transcribe the minus-strand RNA to mRNA inside the intact capsid, which usually occurs in host cell’s cytoplasm. In comparisons with other members of Reoviridae having a T = 13 layer (here T is the triangulation number), CPV virion only has a single layer capsid, adopting T = 1 lattice, with ~70 nm in diameter (Fig. The CPV belongs to the To reveal the structure of CPV, both X-ray and single-particle cryo-EM were applied. Probably due to the intrinsic flexibility of some viral components, CPV had eluded the efforts of the X-ray method, while cryo-EM advanced the structural studies while steadily improving resolutions (Hill et al. Aiming to build the structure of the genome and RdRps of CPV, the authors developed a so-called symmetry-mismatch reconstruction in their study. At the heart of the method, it finds out the accurate positional parameters for particles containing the genome and enzymes as a normal single-particle analysis dose Frank . The stuWith their new strategy, Liu and Cheng solved the structures of both the NCPV and TCPV in the presented paper. The arrangements of genome and RdRps are alike in both virions, while the interactions between genomic RNA and RdRps differ in the two states. Their genome and RdRps adopts a pseudo-D3 symmetric organization, by which further reconstruction enhanced the resolutions to allow building a Cα model. Some extra densities are seen in the active sites in NCPV and TCPV and nucleotides can be assigned into them. From both maps, a fragment of dsRNA is observed to bind to each of the 12 RdRps bracelets, which shifts in NCPV compared to that in TCPV, which is interpreted as virion transcription at initiation stage. The channels for the exit of transcript and RNA template are partially blocked in NCPV RdRps, but completely open in TCPV RdRps. A loop in RdRps adopts a retracted conformation in NCPV and becomes extended in TCPV, thus it is interpreted as the switch loop. These details about the viral functional enzymes and genome are not seen in previous virus structure studies, which have been going on for nearly four decades (Harrison et al. Reoviridae viruses, Liu and Cheng proposed a dsRNA virus replication and transcription model, although structures of some potential states were still lacking in this study. According to their model, the replication initiates from the plus-strand RNA, which passes through the template entry channel of RdRps. The resulting dsRNA then exits at the template channel exit and binds to the bracelet domain of RdRps to prepare for transcription. Upon transcription, dsRNA disassociation from the binding site triggers the opening of the transcript and template exit channels, as well as the extension of the switch loop. The dsRNA is dissembled at the entrance of the template entry channel likely catalyzed by RdRps, and then the minus strand enters into the channel serving as template for synthesizing mRNA, while the plus strand stays outside the channel by binding its 5′ cap to the RdRps. When the finished transcript exits the transcript channel, the template associates with the original plus-strand RNA to form dsRNA for next round transcription. This four-channel coordinated model for dsRNA virus replication and transcription is a principle finding of Liu and Cheng’s structure analysis.On the basis of the internal structures of NCPV and TCPV plus previous knowledge on the Regarding the methods, as the authors mentioned, similar idea had been used elsewhere including solving the bacteriophages local symmetry mismatched details (Tao et al. The current work’s data were collected on a CCD camera instead of a superior direct detector, which contributes a lot to image quality improvement and image processing (Liu and Zhang Further application of direct detectors for data collection, combining with other image-processing algorithms, and improved specimen-preparation methods should bring other advances in the use of cryo-EM for structural virology. With the outstanding success of the current study, the gate is open for structural studies of internal contents of other viruses like phages and other infectious viruses (Dai et al.


Even with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved.in situ at -80°C.The goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage Tissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes .Fresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6–7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months.in situ at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts.These results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue  Challenges continue to exist in regards to acquiring high quality samples from animal tissues despite recent technical advances. From the time spent collecting the samples to the use of stabilizing or preserving agents, and ultimately to the storage conditions and duration prior to actual analysis, each step is critical and requires attention. Timing plays a key role in obtaining high quality samples. Ideally, samples are harvested immediately post-euthanasia by trained and experienced staff, and the time between death and sample stabilization is minimized to limit sample degradation. If the samples are not analyzed at the time of euthanasia, then additional factors that may affect sample quality are of concern, including freezing rate and duration of storage. Understanding these challenges faced in the laboratory aids the researcher to optimize protocols to maximize sample quality and accomplish science objectives; translating those practices for use in the space environment, however, raises new challenges.Several factors potentially affect sample quality, and ultimately, the downstream experimental results. The procedure to obtain high quality RNA varies between different laboratories . The mosRNA is subject to degradation by ubiquitous RNAses, and high quality RNA is needed for analysis of gene expression, next-generation sequencing, gene profiling and other “Omics” analyses in the study of molecular mechanisms. The addition of an RNA stabilization solution to tissues, either by immersion or perfusion, has become standard practice for RNA stabilization , 10, 11.In the studies reported here, we investigated various time points and storage conditions for retrieving tissues from mice, and then determined the impact that these conditions had on sample quality. By measuring RNA Integrity Number (RIN) and enzyme activities in tissues under various recovery scenarios, we have characterized and established an acceptable timeline for rodent tissue isolation, preservation, and storage to optimize science return from both ground based and on-orbit flight sample collection.Adult female C57Bl/6J mice from Jackson Laboratory  were used in all the experiments. All animals were euthanized by intraperitoneal injection of Euthasol  followed by cervical dislocation. All efforts were made to minimize suffering. The Institutional Animal Care and Use Committee at the NASA Ames Research Center approved the animal care and experimental procedures.Fresh tissue samples: To provide positive control tissues representing preservation by standard laboratory conditions, spleens and livers were harvested immediately post-euthanasia then were either promptly (within 5 min) frozen in liquid nitrogen (liver) or preserved in RNAlater (spleen). To simulate the anticipated dissection timing by astronauts on-orbit, spleens were removed at 6 or 22 minutes after euthanasia, and livers were removed at 9 or 25 minutes post-euthanasia , then plTime-delay between euthanasia and stabilization: To bracket the possible time frames that the astronaut crew may need to dissect then preserve the samples, spleens were harvested at 1, 20, 40, 60, 80 and 100 minutes post-euthanasia and preserved in RNAlater. Livers were harvested from the same mice at 2, 25, 45, 65, 85, and 105 minutes post-euthanasia and frozen in dry ice. Samples were stored at -80°C for 1 month prior to analysis.Whole Carcass (WC): Mice were euthanized by intraperitoneal injection of Euthasol followed by cervical dislocation. The carcasses were then wrapped in two layers of foil and placed in Ziploc bags. The carcasses were frozen using an aluminum block that was pre-chilled with liquid nitrogen, to simulate the freezing kit used on-orbit, and transferred to a -80°C freezer for storage (up to 10 months). Various tissues  were collected from 2.5, 4 and 6–7 month frozen WC after thawing at room temperature for 20–30 minutes, and then preserved in RNAlater. Positive control WC were immersed in liquid nitrogen immediately after euthanasia and were stored in a 80°C freezer for 4 and 6–7 months.Partially Dissected Carcass (PC): Spleens and livers were removed following euthanasia, and utilized for a different study. The dissected carcasses then were wrapped with two layers of aluminum foil, and sealed in a Ziploc bag before freezing on dry ice. The time from euthanasia to the carcass freezing in dry ice was about 2 hours. PC were stored at -80°C for 2.5 months prior to harvesting the tissues . PC were removed from the -80°C freezer, and placed at room temperature with the aluminum foils open for partial thawing to the extent that dissection commenced, which was up to 35 minutes after removal from -80°C. Tissues were preserved in RNAlater.RNA was extracted from homogenized tissues using TRIzol reagents . RNA concentration and purity were assessed using a NanoDrop 2000 UV-Vis Spectrophotometer . RNA quality was measured by calculating the RNA integrity number (RIN) using the Bioanalyzer 2100 , where a RIN of 10 is considered intact and 0 totally degraded RNA [™ Pierce™ Micro BCA™ Protein Assay Kit, Pierce Biotechnology, Rockford, IL). Catalase protein levels were measured using the Catalase Human ELISA Kit , and specific activity of the enzyme calculated . Glutathione reductase activity levels were determined using the Glutathione Reductase (GR) Assay Kit  and GAPDH activity levels were determined using the colorimetric GAPDH assay kit . The activity levels were also normalized by the total protein concentrations using the Micro BCA Protein Assay Kit. Positive controls were obtained according to standard laboratory protocols where dissections were followed by immersion of the samples in liquid nitrogen.The activities of three hepatic enzymes  were measured to assess sample quality after various collection conditions. Catalase activity was measured using the colorimetric assay for Catalase  according to the manufacturer’s instructions, and normalized to total protein levels . In non-parametric boxplots, medians within the interquartile (boxes) and full range (whiskers) are displayed. p<0.05 was considered significant throughout.To determine if harvest times post-euthanasia alters RNA quality, freshly-dissected spleens and livers were isolated at various time points, preserved as shown in Activity levels of three liver enzymes also were measured from the time-delayed dissections. Glutathione reductase  and GAPDTo assess the influence of storage duration on sample quality, spleens and livers were collected per standard laboratory procedures (up to 5 minutes post-euthanasia), and then stored at -80°C for 1, 3.5, 4.5, and 11 months prior to analysis. At all time points investigated, no significant degradation was found in the RNA quality of any of the tissue samples (RIN > 8) . These dTo assess RNA quality from whole frozen carcasses (WC), we analyzed the spleen, liver, heart, kidney, and lung dissected from WC that were stored at -80°C for 2.5, 4, and 6–7 months. Results from spleens showed that increased duration at -80°C from 2.5 to 6–7 months caused a 25% decline in RIN values. Long durations of storage (4-7months) had minimal effects on RNA quality of liver, heart, kidney, and lung although RIN values were 6% lower when comparing WC liver at 6–7 months  to freshUsing a separate group of WC we dissected numerous additional tissues and determined the RNA quality. High quality RNA was recovered from various tissues ; RIN values were greater than 7.5 in all tissues . Small iTo determine if the carcasses that were stored after select tissue dissection also can yield high quality samples of other tissue types after long-term storage, tissues were harvested from partially dissected carcasses stored at -80°C for 2.5 months. Similar to the results found as in WC; PC tissue samples resulted in high RNA quality recovered from brain, adrenal glands, eye, thymus and hindlimb muscles. Small intestine and bone marrow samples showed severe degradation similar to that seen in WC .Our results demonstrated that high quality RNA (RIN> 7) could be isolated from most of the tissues, even from tissues stored for a prolonged period of time  after 65 minutes post-euthanasia. These data demonstrate that tissues collected with appropriate timing that is typical for laboratory dissections  can yield samples of acceptable quality to achieve exceptional science return, as expected . These fTesting methods in ground based experiments by applying anticipated on-orbit scenarios, reduces the risk of recovering samples of inadequate quality from high cost spaceflight experiments. Spleens and livers collected at various time points post-euthanasia to simulate challenging conditions for dissections in microgravity yielded samples with high RIN values, thus anticipating return of samples with high quality from space even if preservation post-euthanasia is delayed up to 1 hour. When plant samples were placed into a -20°C freezer, severe RNA degradation resulted; however, when the plant samples were preserved in RNAlater and stored at either 4°C or -20°C, the integrity of RNA was maintained. Furthermore, wheat samples preserved in RNAlater from an experiment performed on the ISS, yielded RNA quality comparable to fresh samples, even after undergoing fluctuations of storage temperatures . More reWe showed that multiple tissues collected from frozen carcasses yielded high quality RNA, which could be applied to RNAseq, gene profiling and qPCR. High quality sample recovery for RNA and at least some enzymes is possible from most tissues, despite either delayed preservation post-euthanasia or prolonged storage. Based on our RIN results, it is our recommendation that the collection of tissue samples occur within 45 minutes for studies requiring high quality RNA and within 85 minutes for usual RNA studies. Should sample retrieval occur from frozen carcasses, our studies show that many, but not all tissues yield high RNA quality up to 7 months storage time at -80°C.S1 FileData sets for this study are fully available without restriction.(PDF)Click here for additional data file.


Earlier studies showed that offset errors can be clinically relevant over different systems, and are not consistently reduced by protocol optimization , 2. Corr-of-view . Complem-of-view , this stData from six 1.5 T MR systems were evaluated, with two systems from each of the vendors GE, Philips and Siemens. Aortic and main pulmonary artery 2D flow studies were acquired during routine clinical or research examinations. Each site used their local standard scan protocol, with limited spatial wrap around, retrospective ECG gating, and without other phase offset correction methods (i.e. Philips LPC filter off). Within the same day a phantom measurement was obtained with identical acquisition parameters.in-vivo and phantom measurements. Only studies with an agreement within 0.6 cm/s between these check ROIs were used in the following analysis.To verify the phantom acquisition, a ROI at stationary tissue in the thorax wall was placed and compared between in-vivo data were calculated using the algorithm of Walker et al. [Interpolated velocity offsets from the r et al. , 5, aftein-vivo and phantom was -0.2 ± 0.6 cm/s. 27 studies were further excluded due to too large deviation of the phantom scan at the thorax wall. Before correction the offset at the vessel (as assessed in the phantom) was -0.7 ± 1.4 cm/s, and resulted in a -6 ± 16% error in cardiac output. The optimal order of the correction plane was 1st order, except for one GE system (system 6) at which a 2nd order plane was required. After application of the interpolation correction the remaining offset velocity was 0.0 ± 0.5 cm/s and 0 ± 5% error in cardiac output.A total of 123 flow measurements in 80 subjects were included . At the thorax wall the agreement between This study shows that interpolation-based offset correction reduces the offset with comparable efficacy as phantom measurement phase offset correction without the time penalty imposed by phantom scans.


Escherichia coli and Salmonella enterica has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ‐dependent sRNAs are largely unknown. Here, we report in Salmonella Typhimurium the mode‐of‐action of RaiZ, a ProQ‐dependent sRNA that is made from the 3′ end of the mRNA encoding ribosome‐inactivating protein RaiA. We show that RaiZ is a base‐pairing sRNA that represses in trans the mRNA of histone‐like protein HU‐α. RaiZ forms an RNA duplex with the ribosome‐binding site of hupA mRNA, facilitated by ProQ, to prevent 30S ribosome loading and protein synthesis of HU‐α. Similarities and differences between ProQ‐ and Hfq‐mediated regulation will be discussed.Research into post‐transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria  ProQ has a double role in this regulation: (i) it is necessary for the intracellular stabilization of RaiZ, and (ii) it together with the RaiZ‐hupA duplex prevents 30S ribosome loading. These results lay the foundation for a mechanistic exploration of target regulation by the new large class of ProQ‐associated sRNAs.Here, we report the characterization of a ProQ‐dependent sRNA and the associated molecular function of the protein. We show that the RaiZ sRNA  of which it covers the last third of the CDS and the entire 3′ UTR. The RaiZ RNA sequence is conserved in several enterobacteria that are closely related to Salmonella  or in a growth medium that induces the Salmonella pathogenicity island‐1 (SPI‐1) and less in the exponential phase or under Salmonella pathogenicity island‐2 (SPI‐2)‐inducing conditions  and a 122‐nt processed sRNA (RaiZ‐S), with a cumulative abundance of up to 50–60 copies per cell  decrease in HU‐α production. These results were corroborated by the use of fluorescent reporter constructs . Importantly, combination of both mutant partners, which restores base pairing, rescued wild‐type levels of repression  hupA mRNA level dropped ~4‐fold in the proQ+ strain compared to the same strain carrying the control plasmid, it remained constant at ~75% of control in the ΔproQ background  in the presence of ProQ  or have been proposed to function as trans‐encoded base‐pairing sRNAs (SraL)  has been shown to behave as a sponge to control the level and activity of other sRNAs in E. coli  expression  and grown overnight at 37°C with vigorous shaking. The overnight cultures were then diluted 50‐fold with the same medium and grown in the same way for 12 h until OD600 ~0.3.Detailed information on bacterial strains and their construction is listed in in vitro RNA transcription template generation and northern blot hybridizations are listed in Oligonucleotides used for strain construction, cloning, Bacterial cultures were harvested and mixed with 0.2 volume RNA stop mix (5% acidic phenol in ethanol) and flash‐frozen with liquid nitrogen. Total RNA was extracted with the TRIzol reagent (Invitrogen), and 10 μg RNA was analysed by northern blotting. RNAs were detected by hybridization with the 5′‐labelled oligonucleotides, and autoradiographies were analysed by phosphorimager on a Typhoon FLA 7000 instrument with the help of the AIDA software .Salmonella RNase E thermosensitive strain JVS‐07000  to abrogate transcription. RNA samples were collected at indicated time points and quantified by northern blot analysis, as described above. The experiments were performed in biological triplicates.Bacterial cultures were grown to an OD600 of 0.5, when L‐arabinose was added to a final concentration of 0.2%. RNA samples were collected at 0 and 10 min after induction, as described above. Total RNA was treated with DNase I (Fermentas), and cDNA library generation and sequencing were performed by Vertis Biotechnologie AG, Germany, as described  and incubated at 95°C for 5 min. After denaturation, 0.02 OD samples were resolved by SDS–PAGE and Western blotting was performed as described  were synthesized with MEGAscript High Yield Transcription Kit . DNA templates with T7 promoter sequences were generated by PCR with oligonucleotides listed in 32P‐γ‐ATP  and 1 unit of polynucleotide kinase (Fermentas) for 1 h at 37°C in a 20 μl reaction. Unincorporated nucleotides were removed with Microspin G‐50 Columns , followed by purification of the labelled RNA on a denaturing polyacrylamide gel. Upon visualization of the labelled RNA with phosphorimager, the RNA band was excised from the gel and eluted with 0.1 M sodium acetate, 0.1% SDS, 10 mM EDTA at 4°C overnight, followed by phenol extraction and precipitation as before. Final concentrations were checked by NanoDrop 2000.Transcripts used for Salmonella Hfq and ProQ purification and EMSA were performed as described . For EMSA, 5 nM of labelled RNA was used. Both labelled and unlabelled RNA were denatured at 95°C for 1 min and slowly cooled down to room temperature, then mixed together and/or with purified Hfq or ProQ protein in a final volume of 20 μl of 25 mM Tris–HCl, pH7.5, 150 mM KCl, 1 mM MgCl2, and incubated at 25°C for 20 min. Reactions were resolved at 4°C on native 6–8% polyacrylamide (19:1) gels (in 0.5× TBE) at constant current of 50 mA for 3 h. Gels were dried and signals were analysed by phosphorimager as described above.in vitro‐synthesized 5′‐labelled RNA. Briefly, 0.2 pmol labelled RNA (in 5 μl) was denatured at 95°C for 1 min and chilled on ice for 5 min, followed by addition of 1 μl of 1 mg/ml yeast RNA (Ambion AM7118) and 1 μl of 10× structure buffer . Unlabelled partner RNA or purified ProQ protein were then added at various ratios and incubated at 37°C for 10 min. Then, samples were either treated with 0.1 U RNase T1 (Ambion) for 3 min, or with 5 mM lead (II) acetate (Fluka) for 1.5 min, or with 1.3 U RNase III (New England Biolabs) for 6 min, or with 0.02 U RNase V1 (Ambion) for 5 min, or left untreated. Reactions were stopped by addition of 12 μl denaturing loading buffer . RNase T1 sequencing ladders were prepared by using 0.4 pmol 5′‐labelled RNA denatured at 95°C for 2 min in the presence of 1× structure buffer and chilled on ice; RNA was digested with 0.1 U RNase T1 for 5 min at 37°C. Alkaline (OH) sequencing ladders were prepared by incubating 0.4 pmol 5′‐labelled RNA at 95°C for 5 min in the presence of alkaline hydrolysis buffer (Ambion). Reactions were stopped by addition of 12 μl denaturing loading buffer. Samples were denatured prior to loading  and separated on denaturing 6% sequencing gels containing 7 M urea in 1× TBE at constant power of 40 W. Gels were dried and signals were analysed by phosphorimager with the help of the AIDA software , as described above.Structure probing and mapping of ProQ‐binding regions were performed on et al, hupA 5′ UTR‐gfp fragment and 0.5 pmol of 5′‐labelled JVO‐01976 primer, complementary to the gfp fragment, were annealed together in annealing buffer  for 1 min at 95°C and chilled for 5 min on ice. Then, magnesium acetate and NTPs were added to final concentrations of 10 and 0.5 mM, respectively. For inhibition analyses, 1 or 2 pmol of RaiZ‐S was added. ProQ was added to a final concentration of 500 nM. All subsequent steps were carried out at 37°C. After 5 min of incubation, 2 pmol of 30S ribosomal subunits , pre‐activated for 20 min, was added. After incubation for 5 min, uncharged tRNAfMet was added to a final concentration of 1 μM, and incubation continued for 15 min. Reverse transcription was carried out with 100 U SuperScript II (Invitrogen) for 20 min. cDNA synthesis was terminated with 100 μl of stop buffer , followed by phenol–chloroform extraction. Aqueous phases were added 5 μl of 3 M potassium hydroxide and incubated at 90°C for 5 min. After ethanol precipitation, cDNA was dissolved in 10 μl of loading buffer II (ThermoFisher Scientific). Sequencing ladders were generated with DNA Cycle Sequencing Kit (Jena Bioscience) according to the manufacturer's protocol on the same DNA template as used for T7 transcription and the same labelled primer as in the toeprinting reactions. cDNAs and sequence ladders were separated on a 6% polyacrylamide/7 M urea gel. Autoradiographs of dried gels were obtained as described above.30S ribosome toeprinting assays were performed as described .Sample size considerations and applicability of the Motulsky . ConformAS and JV conceived the project, AS and CW designed experiments, CW, LLD and AS performed experiments, AS and JV wrote the paper, and all authors edited the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.


The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity for major satellite repeat RNA. To analyze an RNA-dependent interaction with chromatin, we purified native nucleosomes from mouse ES cells and detect that Suv39h1 and Suv39h2 exclusively associate with poly-nucleosomes. This association was attenuated upon RNaseH incubation and entirely lost upon RNaseA digestion of native chromatin. Major satellite repeat transcripts remain chromatin-associated and have a secondary structure that favors RNA:DNA hybrid formation. Together, these data reveal an RNA-mediated mechanism for the stable chromatin interaction of the Suv39h KMT and suggest a function for major satellite non-coding RNA in the organization of an RNA-nucleosome scaffold as the underlying structure of mouse heterochromatin.DOI:http://dx.doi.org/10.7554/eLife.25293.001 The classic distinction between euchromatin and heterochromatin is used to define decondensed, gene-rich and transcriptionally active regions of the genome vs. more compacted, gene-poor and transcriptionally silent domains . HoweverNuclear non-coding RNA have been proposed to facilitate recruitment of chromatin factors by serving as guide RNA  and/or tXist and long intergenic nuclear element (LINE) transcripts during X inactivation  digestion and fractionation in sucrose density gradients. The Suv39h KMT exclusively accumulate in the poly-nucleosomal fractions and this association was attenuated upon RNaseH incubation and entirely lost upon RNaseA digestion of the MNase-processed input chromatin. These data reveal an RNA component to be important for the recruitment of the Suv39h KMT and suggest that an RNA-nucleosome scaffold is the physiological template for the stable association of Suv39h enzymes to chromatin. In addition, RNA preparations that were purified from MNase-solubilized chromatin display sensitivity towards RNaseH, when they are examined with MSR-specific DNA probes. We propose a model, in which mouse heterochromatin is composed of a higher order RNA-nucleosome scaffold that contains MSR RNA:DNA hybrids and significant portions of single-stranded MSR-repeat RNA.Suv39h1 and Suv39h2. The mouse Suv39h2 gene contains an additional exon in the 5'UTR region  cells  and probed them with antibodies against the endogenous proteins. Immunoblotting with a Suv39h1 antibody indicated endogenous Suv39h1 (48 kDa) in wild type but not in n) cells . By cont Suv39h2  that als Suv39h2 . We concSuv39h dn mouse ES cells with plasmid constructs that drive expression under the control of the β-actin promoter  full-length Suv39h1 or full-length Suv39h2 into promoter . In addipromoter . Whole cpromoter . By contSuv39h dn mouse ES cells  , was notHeterochromatin is not transcriptionally inert and major satellite expression transiently increases at the G1/S phase of the cell cycle , with MSAt condensing chromosomes and during pro-metaphase, full-length Suv39h1-EGFP exhibits a broad and diffuse chromosomal pattern (data not shown), but then is largely absent from mitotic chromosomes, although pericentric H3K9me3 persists . In striSuv39h dn ES cells, MSR transcripts are similarly derepressed as compared to interphase cells  for increasing time points . Recovered proteins from the soluble (S) and insoluble (P=pellet) fractions were isolated for further analysis by Western blotting. For this, we included wild type ES cells as a source to compare the endogenous Suv39h1 and Suv39h2 enzymes. Progressive solubilization from chromatin by MNase digestion was quantified by measuring the relative presence of the endogenous or of the Suv39h-EGFP products in the soluble (S) versus the insoluble (P) fraction. A low S/P ratio indicates chromatin retention whereas a high S/P ratio would reveal chromatin release.In interphase cells, neither endogenous Suv39h1 or Suv39h2 nor full-length Suv39h1-EGFP or full-length Suv39h2-EGFP were significantly released into the supernatant, even after 10 min incubation with MNase . The SuvWe then analyzed the MNase mediated chromatin release profile in nocodazole-arrested cells. In pronounced difference to interphase, endogenous Suv39h1 and Suv39h1-EGFP showed rapid solubilization already during the first 4 min of MNase digestion . By contThe basic domain of Suv39h2 harbors 22 basic residues, of which 19 are arginines see . Amino aD = 0.8 μM) and of MSR-R RNA (KD = 0.4 μM) with full-length 6xHis-MBP-Suv39h2 (D = 0.4–0.6 μM) RNA interaction  construct  and RNA oligonucleotides (35 nt) and complementary DNA oligonucleotides from subunit 2 of the MSR. This allowed us to prepare single-stranded (forward and reverse) or double-stranded RNA or DNA or RNA:DNA hybrids as possible substrates for in vitro binding by GST-Suv39h2basicD(1-117). The data show  that theD ≤ 2 μM), intermediate binding to pRNA, Oct4P4 RNA, minor satellite repeat RNA and LINE L1MdA 5'UTR forward RNA (KD > 2 μM) but did not interact with LINE L1MdA 5'UTR reverse RNA, SINE B1 repeat RNA or with an unstructured poly(A) RNA control  from different repeat classes  and from pRNA of the rRNA cluster  or from  control . For com control . The HP1nd 38 μM .10.7554/Collectively, these data indicate that the extended basic domain of Suv39h2 provides an ssRNA recognition module that confers preferred selectivity to bind to pericentric (MSR) and telomeric (TERRA) RNA but not to other (e.g. some LINE and SINE B1) RNA  and doesBased on these in vitro RNA binding data, we next explored whether the association of Suv39h enzymes to native chromatin and nucleosomes may be dependent on an RNA component. For this, we first isolated chromatin by MNase digestion and separated the nucleosomal fragments on a sucrose gradient . Such an approach has been used to demonstrate nucleosomal interaction of DNMT3A  and of oFor this nucleosome association analysis, we compared the Suv39h-EGFP products with endogenous Dnmt3a and HP1α. In RNase-untreated samples, all three Suv39h-EGFP products are primarily enriched in poly-nucleosomal fractions and not in the nucleosome-free fractions , Dnmt3a In RNaseH-digested samples , although parts of the nucleosomal ladder persist this RNase treatment . TogetheSuv39h dn ES cells as compared to wild-type ES cells (We next performed quantitative PCR (RT-qPCR) with MSR- and LINE L1MdA 5'UTR- specific primers on nucleic acid material that was purified from MNase solubilized nucleosomal fractions. As above, nucleosomal fractions were separated on a sucrose gradient  and the ES cells . This enSubcellular isolation of RNA populations from cytoplasmic, nucleoplasmic and chromatin fractions further corroborated that around 80% of the total MSR RNA remain chromatin associated . AlthougHiseq RNA sequencing data with nuclear RNA preparations from mouse ES cells further show that major satellite repeats are expressed from both strands  and thatSuv39h dn ES cells. We observe pronounced reduction of hybridization signal for both MSR strands upon RNaseH treatment and complete loss after RNaseA  digestion  or reverse (pyrimidine-rich) MSR transcripts were used for Northern hybridization with RNA preparations that were either untreated or digested with RNaseH or RNaseA . In agreement with previous observations , we deteSuv39h dn ES cells, but RNaseH digestion only results in a minor reduction of the hybridization signal  of the input material to be enriched by the S9.6 antibody  as a novel RNA interaction motif that binds with high affinity to single-stranded transcripts from the MSR RNA in vitro . PrelimiTogether, the cumulative data indicate that the association of the Suv39h KMT to native chromatin is governed by an RNA-mediated process. We suggest that non-coding MSR RNA will organize a higher-order RNA-nucleosome scaffold as the physiological template for the Suv39h enzymes (see below). The RNA dependence of association with poly-nucleosomes is much less pronounced for HP1α and not observed for Dnmt3a see . In factThese multiple interactions may also explain that we could not observe dispersion of Suv39h1-EGFP or Suv39h2-EGFP from heterochromatic foci in interphase upon RNaseA incubation of permeabilized mouse ES cells, nor define conclusive in vivo interaction of Suv39h enzymes and MSR RNA (rather than MSR DNA) by using PAR-CLIP and RNA-IP. This difficulty may be further complicated by the inherent property of the MSR sequences to form RNA:DNA hybrids. In independent work, Mouse pericentric heterochromatin has long been described to have an RNA component important for the localization of HP1α and for the accumulation of H3K9me3 , 2011. WS.pombe  was affinity-purified over a HisTrap NHS  affinity matrix coupled with 2 mg of the Suv39h2-basicDomain double-branched peptide. Bound antibodies were eluted with 100 mM glycine, NaCl 0.5 M pH 2.7, and further diluted with PBS pH 7.4 supplemented with 0.05% v/v ProClin. The specificity of the affinity-purified antibody (2.0 mg/ml) was confirmed by immunoblotting of Suv39h2 proteins from different sub-cellular fractions of wild type and Suv39h double null mouse ES cells.Suv39h2-basicDomain rabbit polyclonal antibodies were generated by the immunization of rabbits with a double-branched peptide (#C14dbA) comprising amino acids 24 to 39 of the basic N-terminus of Suv39h2 [C-PSuv39h double null mouse ES cells from one confluent maxi dish were trypsinized and collected by centrifugation at 1200 rpm for 3 min in 15 ml falcon tubes. The cell pellet was resuspended in PBS and the cell number determined using a CASY-counter. 2 × 107 cells were resuspended in 200 μl of cold cytoplasmic lysis buffer  and incubated on ice for 5 min. The lysate was layered onto 500 μl of cold sucrose buffer , and centrifuged in microfuge tubes at 14,000 rpm for 10 min at 4°C. The supernatant recovered after this spin (~700 μl) represents the cytoplasmic fraction. The nuclear pellet was gently resuspended in 200 μl of cold glycerol buffer  was added to the samples, followed by pulsed vortexing and incubation on ice for 1 min. Samples were then centrifuged in microfuge tubes for 2 min at 14,000 rpm at 4°C. The supernatant from this spin represents the soluble nuclear fraction (nucleoplasm), while the remaining insoluble part represents the chromatin pellet. The following antibodies were used for immunoblotting: α-GFP (Invitrogen A11122), α-Gapdh (Sigma G8795), α-H3 (Abcam ab12079), α-H3K9me3 , α-H3S10p (Abcam ab5176).The experimental procedure was adapted from Suv39h double null (Suv39h dn) mouse ES cells using the Xfect reagent (Clontech). Polyclonal cell populations were kept under puromycin selection (1 μg/ml) at all times during subsequent passages. The isolation of wt and Suv39h dn ES cell cultures has been done in the Jenuwein laboratory  mutant were subcloned to generate Suv39h1-EGFP, Suv39h2-EGFP and Suv39h2-D(T3K81)-EGFP fusions and then transferred into the pCAGGS vector that drives expression under the control of the chicken β-actin promoter and confers puromycin resistance. Expression plasmids were transfected into boratory  and thes4 ES cells were attached to coated glass slides by using a cytospin (ThermoScientific) at 500 rpm (low acceleration) for 5 min. ES cells were fixed in 4% para-formaldehyde for 15 min at RT, washed three times with 1X PBS and permeabilized in a 0.05% triton-X solution for 5 min. Permeabilized cells were then washed twice with 1X PBS and incubated in blocking solution  for 30 min at RT. The slides were reacted with a α-H3K9me3 rabbit polyclonal antibody  O/N at 4°C at a 1:1000 dilution. Slides were then washed with 1X PBS and incubated for 1 hr at RT with a secondary goat α-rabbit antibody that was coupled to Cy5 (Life Technologies). Slides were mounted with VECTASHIELD mounting medium containing DAPI. Condensing and mitotic chromosomes presented in the cytospins were examined using a Confocal Spinning Disc microscope (Zeiss Observer Z1) to detect the EGFP signal of the Suv39h-EGFP products and the Cy5 signal for H3K9me3. Images were taken at 63x magnification, analyzed with Zen software 2011 SP3 (Black version) and are shown as ‘maximum intensity’ projections from Z stacks of representative ES cells (n = 40).Unsynchronized ES cell cultures expressing the various Suv39h-EGFP products were processed for indirect immunofluorescence to detect EGFP signals and H3K9me3 at heterochromatic foci as described . ImagingChIP was performed as described  and ChIPWhole cell lysates from unsynchronized and nocodazole-synchronized mouse ES cells were digested with 10 U of micrococcal nuclease (MNase) (Fermentas) for increasing time-points. The reaction was stopped and the samples were centrifuged at 15’000 g for 5 min. Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1  and α-Suv39h2 (LifeSpan BioSciences LS-C116360).600 was 0.8, incubated overnight at 16°C, harvested by centrifugation and lysed in a buffer containing 25 mM Tris-HCl pH 8.0, 350 mM NaCl, 10 mM imidazole, 1% glycerol, 1 mg/ml lysozyme, 0.1 mM PMSF and cOmplete, EDTA-free protease inhibitor cocktail tablets (Roche). Lysate was digested with 25 U/ml Universal nuclease (Thermo Fisher), sonicated  in a Branson sonicator and centrifuged at 39'000 g for 30 min at 4°C.The 6xHis-MBP-fused full length mouse Suv39h1, full-length mouse Suv39h2, the Suv39h2ΔBD(116-477) mutant and the extended basic domain of Suv39h2 {Suv39h2basicD(1-117)} were obtained as follows. A TEV cleavage site sequence (Integrated DNA Technologies) was cloned into the destination vector p6xHis-MBP using BplI and NotI restriction sites. The p6xHis-MBP-Suv39h1, p6xHis-MBP-Suv39h2, p6xHis-MBP-Suv39h2ΔBD(116-477) and p6xHis-MBP-Suv39h2-basicD(1-117) plasmids were then constructed by cloning PCR products that had been amplified from synthetic Suv39h1 and Suv39h2 coding sequences (Integrated DNA Technologies) into the modified p6xHis-MBP vector and verified by sequencing. 6xHis-MBP fusion proteins were expressed in Rosetta bacterial cells. Cells were grown in LB medium containing 100 μg/ml ampicillin and 0.4% glucose (only required for 6xHis-MBP-Suv39h2), induced with 1.0 mM isopropyl-β-D-1-thiogalactopyranoside when ODDifferent protocols were then used to optimize purification of the various 6xHis-MBP-Suv39h products. For 6xHis-MBP-Suv39h1 and 6xHis-MBP-Suv39h2ΔBD(116-477), supernatants were affinity purified using 1 ml HisTrap  and size purified using Superdex200  columns. The 6xHis-MBP-Suv39h2 product was affinity purified using a 1 ml MBPTrap column  followed by cation exchange purification with a MonoS column . The 6xHis-MBP-Suv39h2basicD(1-117) was affinity purified using a 1 ml HisTrap column  followed by cation exchange purification with a MonoS column .600 was 0.8, incubated overnight at 16°C, harvested by centrifugation and lysed in 11 ml (per 0.5 l starting culture) of a buffer containing 40 mM Tris-HCl pH 8.0, 9% glycerol, 2.5 mg/ml lysozyme and cOmplete, EDTA-free protease inhibitor cocktail tablets. Lysate was digested with 25 U/ml benzonase, mixed with 0.5 M KCl, 0.1% NP40, 0.2% Tritonx100, sonicated  in a Branson sonicator and centrifuged at 12'000 g for 30 min at 4°C. This lysate supernatant (20–30 ml) was affinity-purified by incubation with 0.6 ml Glutathione Sepharose 4B resin  at 4°C for 2 hr and 45 min with rotation. The Glutathione Sepharose 4B resin was washed 3 times with 15 ml of buffer containing 40 mM Tris-HCl pH 8.0, 0.5 M KCl, 9% glycerol, 0.1% Tritonx100, 0.1% NP40 and 3 more times with 15 ml of the same buffer supplemented with 0.05 mM ZnCl2. GST-fused proteins were eluted from the washed Glutathione Sepharose 4B resin by incubation with rotation for 30 min at 4°C in 0.6 ml buffer containing 20 mM Tris-HCl pH 8.0, 0.5 M KCl, 9% glycerol, 1 mM DTT and 10 mM reduced glutathione pH 8.0. This elution step was repeated five times and the eluates were combined.The GST-fused mouse Suv39h2basicD(1-117) was constructed by cloning a PCR product that had been amplified from the synthetic Suv39h2 coding sequence (Integrated DNA Technologies) into the pGEX-6P1 plasmid and verified by sequencing. GST-fused proteins were expressed in BL21 gold bacterial cells. Cells were grown in 2xYT medium containing 100 μg/ml ampicillin, induced with 0.4 mM isopropyl-β-D-1-thiogalactopyranoside when ODRecombinant proteins for full-length Suv39h1, full-length Suv39h2, the Suv39h2ΔBasicD(116-477) mutant and the extended basic domain of Suv39h2 {(Suv39h2basicD(1-117)} were expressed as 6x-His-MBP-fusions in Rosetta bacterial cells and purified. In addition, the extended basic domain of Suv39h2 and HP1α were also expressed as GST-fusions in BL21 gold bacterial cells and purified. Single-stranded RNA corresponding to either the forward or reverse transcript of one unit (234 nt) of the major satellite repeat (MSR) or of the forward or reverse transcript of one unit (208 nt) of LINE L1MdA 5'UTR (LINE) was generated by in-vitro transcription with T7 RNA polymerase (Thermo Fisher) of PCR products that had been amplified from the pSAT or pEX-L1MdA plasmids using primers containing the T7 promoter sequence. In-vitro transcribed RNA was purified with Qiagen RNasy Mini Elute Kit followed by 3’ end-labeling with a Cy5 fluorophore (Jena Bioscience) using T4 RNA ligase (NEB).2, 1 mM EDTA pH 8.0, 5% glycerol, 0.05% NP40, 2 mM DTT, 50 ng/μl yeast tRNA (Thermo Fisher) and 2.5 ng/μl BSA (NEB). Samples were incubated at 4°C with rotation for 1 hr and then resolved on a 4% polyacrylamide (60:1) gel  in 12.5 mM Tris-HCl and 100 mM glycine. The Cy5 signal was scanned on a Typhoon FLA 9500 fluorescence scanner and quantified using ImageJ software. Sequence information for the RNA and DNA oligonucleotides is shown in 35 nt 5’-Cy5 labeled and HPLC-purified RNA oligonucleotides for MSR, LINE L1MdA 5'UTR, SINE B1, minor satellites, pRNA, Oct4P4, TERRA and poly(A) were purchased from Sigma. For the MSR, also 5'-Cy5 labeled or unlabeled DNA oligonucleotides were purchased (Sigma). To generate dsDNA, dsRNA or RNA:DNA hybrids, equimolar amounts of forward and reverse ssRNA and ssDNA oligonucleotides were mixed in 1xSSC buffer  and incubated for 2 min at 90°C in a Thermomixer. The temperature was decreased to 60°C for 5 min, then reduced to 20°C for 30 min. For EMSA, 50 nM of nucleic acids were mixed with increasing concentrations (16 nM to 2 μM) of recombinant proteins in a buffer containing 20 mM Tris-HCl pH 8.0, 100 mM KCl, 3 mM MgClSHAPE-directed RNA secondary structure modeling was performed as described , with thIn silico structural predictions of RNA oligonucleotides were done with the RNAfold Webserver software (University of Vienna).Purified nuclei from mouse ES cells were incubated with 50 U of MNase (Fermentas) for 10 min at 25°C. The reaction was stopped with 10 mM EDTA and incubated on ice for 10 min. After centrifugation at 15'000 g for 5 min, the supernatant containing soluble chromatin was either left untreated or incubated with RNaseH or RNaseA. Treatment with RNaseH (Epicenter 10 U/μl) was with 50 U of enzyme to 1 ml of MNase solubilized chromatin for 2 hr at 37°C. Treatment with RNaseA  was with 100 μg of enzyme per 1 ml of MNase-solubilized chromatin for 2 hr at 37°C in a buffer containing either 350 mM or 100 mM NaCl. Samples were then loaded on a linear (5%–40%)%) sucrose density gradient, fractionated by ultra-centrifugation at 36’000 rpm for 14 hr at 4°C and individual fractions (600 μl) were collected. 50 μl aliquots from each fraction was used for DNA analysis. Proteins were concentrated with Amicon Ultra centrifugal filters and processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Dnmt3a (Abcam ab23565), α-HP1α (Millipore 05–689-clone15.19s2) and α-H3K9me3 (Abcam ab8898).2, 0.5 mM CaCl2 and 15 mM NaCl) until a final concentration of 500 ng DNA per μl is obtained. 1 ml of these nuclei was then incubated with 200 U of DNase1  for 30 min at 37°C. The reaction was stopped in 10 mM EDTA and incubated on ice for 10 min. After centrifugation at 15’000 g for 5 min, the supernatant containing soluble chromatin was left untreated or incubated with 100 μg of RNaseA (Fermentas) at low salt for 2 hr at 37°C. Soluble chromatin was then fractionated by ultra-centrifugation in a linear (5%–40%) sucrose density gradient as described above.A nuclear pellet was resuspended in DNase1 digestion buffer  and 5'-Cy3 (for RNA) labeled oligonucleotides (Sigma) as described above (see RNA binding assays). Single-stranded and double-stranded nucleic acids were incubated with 10 U of RNaseH (Epicenter) in a buffer containing Tris-HCl pH 7.4 and 100 mM NaCl for 30 min at 37°C. RNaseA treatment was carried out with 10 μg of RNaseA (Fermentas) in a buffer containing Tris-HCl pH 7.4 and 350 mM NaCl  or 100 mM NaCl  for 30 min at 37°C. Reactions were stopped in a final concentration of 12 nM EDTA, 0.6% SDS and 48% glycerol and incubated on ice for 10 min. Oligonucleotides were diluted to a concentration of 200 nM before separation on a 10% native PAGE gel. Gels were pre-run for 30 min at 150 V at 4°C in 0.5X TBE and the samples were loaded in an equal volume of native loading buffer (30% (v/v) glycerol, 80 mM HEPES-KOH pH 7.9, 100 mM KCl, 2 mM magnesium acetate) and electrophoresed in 0.5X TBE at 150 V using an XCell Sure Lock Midi-Cell Electrophoresis System. After electrophoresis, the gels were scanned in a phosphorimager . Sequence information for the 5'- Cy5-DNA and 5'-Cy3-RNA MSR oligonucleotides are listed in 32P  using T4-PNK and approximately 500,000 c.p.m. of each probe were used for Northern hybridization.Subcellular fractionation of mouse ES cells was performed as described . ChromatGapdh, Hprt, actin, tubulin) are indicated in For RT-qPCR detection of chromatin-associated RNA, RNA was extracted with TRIzol from the chromatin pellet. RNA was then incubated with TurboDNase and reverse transcribed  using random hexamers. Primers for the specific amplification of repeat elements  and housekeeping genes (RNA was purified from chromatin and nucleoplasmic fractions of wild-type mouse ES cells and converted into non-poly(A) selected, ribosomal RNA depleted  or poly(A) selected (TrueSeq mRNA) cDNA libraries following Illumina protocols. The cDNA libraries were sequenced on a HiSeq2500 (Illumina) platform using a 100 bp paired-end approach to give a coverage between 75–88 million reads per cDNA preparation. For each sample, three independent biological replicates were analyzed, resulting in a total number of 268 million reads (chromatin-associated RNA) and 237 million reads (nucleoplasmic RNA) for non-poly(A) selected, ribosomal RNA depleted cDNA libraries or 231 million reads (chromatin-associated) and 226 million reads (nucleoplasmic RNA) for poly(A) selected cDNA libraries. The reads were aligned to the mouse genome build mm10 using TopHat2  with defhttp://www.repeatmasker.org; Using the RNA-seq alignment files, coverage over repeat elements  present in the RepeatMasker mouse mm10 genome annotation was determined using RepEnrich , with th7 mouse ES cell nuclei and sonicated with a Bioruptor (Diagenode) for 5 min using 30 s. sonication cycles. TRIzol purification was then used to extract RNA and associated nucleic acids into the aqueous phase. 7.5 μg of these nucleic acids were either left untreated or incubated with 20 U of RNaseH (Epicenter) for 2 hr at 37°C. Reactions were stopped in lysis buffer and immunoprecipitated with 5 μl of the monoclonal S9.6 antibody (Kerafast) for 3 hr at 4°C and then processed for directed PCR with primers that are specific for MSR, LINE L1MdA and SINE B1 sequences (A chromatin pellet was prepared from 2 × 10equences .Hprt or 28S rRNA are listed in Cultures of wild type mouse ES cells were treated with 100 μg/ml of α-amanitin (Sigma) for 2 hr or 0.1 mM 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole  for 3 hr to inhibit RNA polymerase II transcription. Total RNA isolated from treated and untreated ESC was digested with the TURBO DNA-free kit (Ambion) and reverse transcribed using random hexamers and reverse transcriptase SuperScript II (Invitrogen). Negative controls were done without including reverse transcriptase during first-strand cDNA synthesis. Specific primers for the amplification of MSR, Hprt. Amplification products were separated on a 1% agarose gel.To test for the presence of a 5’ cap (m7GpppN), total RNA isolated from wild type ES cells was treated with 5 U of terminator-5’-phosphate dependent exonuclease (Epicenter) for 1 hr at 30°C. This enzyme selectively digests RNA with a free 5’-monophosphate (pN) but cannot digest RNA containing a 5’-triphosphate (pppN), 5’ cap (m7GpppN) or 5’ hydroxyl group. Following incubation with the terminator exonuclease, the RNA was reverse transcribed into cDNA using random hexamers and the cDNA was further amplified using specific primers for MSR and Hprt. Amplification products were separated on a 1% agarose gel.Total RNA isolated from mouse ES cells was incubated with non-coated beads or with Oligo(dT) coated beads (Thermo Fischer) to enrich for poly(A)+ RNA. Control RNA and poly(A)+ enriched RNA were digested with TurboDNase before being reverse transcribed into cDNA using random hexamers and then amplified using specific primers for MSR and DNA and RNA oligonucleotides (35 nt) spanning the second sub-repeat of the major satellite consensus sequence were used in the formation of single and double stranded molecules or of RNA:DNA hybrids, as described above (see RNA binding assays). Following separation on 10% native PAGE, gels were transferred to a positively charged nylon membrane (Hybond N+ Amersham) with a semy-dry system. Nucleic acids were then fixed to the membrane with 150 mJ UV light (254 nm) and visualized with SYBRGold (Invitrogen). The membranes were blocked overnight in 0.1% PBS-Tween at room temperature followed by immunoblotting with the S9.6 antibody (Kerafast ENH001) in BSA/PBS overnight at 4°C. After incubation with the secondary goatα-mouse HRP-coupled antibody (Jackson Immuno Research), signal was detected using ECL reagent . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by three reviewers, one of whom, Jessica Tyler, also served as Senior and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "MSR RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The compaction of repetitive DNA elements into heterochromatin is critical for genome stability in eukaryotes. Heterochromatin is molecularly defined by the abundance of histone H3 lysine 9 trimethylation (H3K9me), to which several H3K9me-binding proteins bind, recruiting silencing activities. The Suv39 family of lysine methyltransferases (KMT) catalyze H3K9 methylation in eukaryotes. How Suv39h proteins are recruited to heterochromatin has been the subject of several studies in plants, the fission yeast. and germ cells of flies, worms and mammals. These studies have shown that small and long nuclear noncoding RNAs cooperate to recruit the Suv39 family proteins to repetitive DNA elements, but whether and how nuclear RNAs also play a role in the recruitment of Suv39 proteins in mammalian somatic cells remains unknown. The paper by Camacho et al. addresses this question in mice, demonstrating that the mouse Suv39h2 N-terminal region possesses an RNA binding domain, which can bind to single-stranded long heterochromatic transcripts (such as major satellite repeat (MSR) and TERRA) with high affinity in vitro. They also show that MSR RNAs are tethered to chromatin and that these chromatin-associated RNAs are required for the stable association of Suv39 KMTs to polynucleosomes. These experiments are generally well-controlled and redundantly support each other, and the data are of excellent quality. Unfortunately, these data indicate no functional role for the N-terminal RNA binding domain of Suv39h2 in vivo. However, there is a concern that potential overexpression of the reintroduced Suv39h1 and Suv39h2 may have obliterated the ability to observe a functional requirement for both Suv39h1 and Suv39h2 or to see the functional requirement for the N-terminus of Suv39h2 in vivo.Essential revisions:1) How do the expression levels of the reintroduced GFP tagged Suv39h1 and h2 proteins compare to endogenous levels, given that they have more robust silencing and more H3 K9 methylation of major satellites in NZ arrested cells than the wild type ? It woul2) With regards to 1, the data in 3) Data in 4) How does Suv39h1 achieve repression of major satellites in NZ arrested cells without it binding to mitotic chromosomes? .5) It will be important for them to show that RNaseH /A treatment of cells leads to loss of Suv39h1 and 2 from heterochromatin by immunofluorescence. Summary:The compaction of repetitive DNA elements into heterochromatin is critical for genome stability in eukaryotes. Heterochromatin is molecularly defined by the abundance of histone H3 lysine 9 trimethylation (H3K9me), to which several H3K9me-binding proteins bind, recruiting silencing activities. The Suv39 family of lysine methyltransferases (KMT) catalyze H3K9 methylation in eukaryotes. How Suv39h proteins are recruited to heterochromatin has been the subject of several studies in plants, the fission yeast. and germ cells of flies, worms and mammals. These studies have shown that small and long nuclear noncoding RNAs cooperate to recruit the Suv39 family proteins to repetitive DNA elements, but whether and how nuclear RNAs also play a role in the recruitment of Suv39 proteins in mammalian somatic cells remains unknown. The paper by Camacho et al. addresses this question in mice, demonstrating that the mouse Suv39h2 N-terminal region possesses an RNA binding domain, which can bind to single-stranded long heterochromatic transcripts (such as major satellite repeat (MSR) and TERRA) with high affinity in vitro. They also show that MSR RNAs are tethered to chromatin and that these chromatin-associated RNAs are required for the stable association of Suv39 KMTs to polynucleosomes. These experiments are generally well-controlled and redundantly support each other, and the data are of excellent quality. Unfortunately, these data indicate no functional role for the N-terminal RNA binding domain of Suv39h2 in vivo. However, there is a concern that potential overexpression of the reintroduced Suv39h1 and Suv39h2 may have obliterated the ability to observe a functional requirement for both Suv39h1 and Suv39h2 or to see the functional requirement for the N-terminus of Suv39h2 in vivo.We disagree that there would be no functional role for the basic domain of Suv39h2 in vivo. The novel data on confocal imaging of mitotic chromosomes new  and the Essential revisions:1) How do the expression levels of the reintroduced GFP tagged Suv39h1 and h2 proteins compare to endogenous levels, given that they have more robust silencing and more H3 K9 methylation of major satellites in NZ arrested cells than the wild type ? It woulWe have added the Western analysis for the endogenous Suv39h1 and Suv39h2 protein levels new . The dat2) With regards to 1, the data in As indicated above, we have added a new paragraph in the Discussion  that clearly describes the physiological role of the basic domain of Suv39h2 in providing "an additional RNA affinity to the Suv39h2 enzyme, which then allows for more robust heterochromatin association of Suv39h2 that persists during mitosis".3) Data in This is a correct criticism and we have clarified this with a revised paragraph in the Discussion . We clearly state that several modules in the Suv39h proteins contribute to RNA interaction and also cite the independent work by the laboratories of Yoichi Shinkai and Aaron Straight that shows RNA binding by the chromo domain of mouse Suv39h1 and of human SUV39H1. In our work, we have focused on the definition of the basic domain of Suv39h2 as a novel RNA interaction motif that donates an additional RNA affinity to Suv39h2.4) How does Suv39h1 achieve repression of major satellites in NZ arrested cells without it binding to mitotic chromosomes? .Although there is transient dissociation of Suv39h1 from mitotic chromosomes, H3K9me3 persists during mitosis .5) It will be important for them to show that RNaseH /A treatment of cells leads to loss of Suv39h1 and 2 from heterochromatin by immunofluorescence.As already discussed in the original manuscript, we have done RNase treatment of permeabilized cells but have not observed dispersion of Suv39h enzymes from heterochromatic foci in interphase. We have revisited this and repeated RNaseA and RNaseH treatments under various salt and pH buffer conditions, but again have not seen dispersion from heterochromatic foci in interphase.


The development of these methods enables incremental studies on the function of RNA structure to be performed, revealing new insights of novel regulatory mechanisms of RNA structure in plants. Genome-wide scale RNA structure profiling allows us to investigate general RNA structural features over 10s of 1000s of mRNAs and to compare RNA structuromes between plant species. Here, we provide a comprehensive and up-to-date overview of: (i) RNA structure probing methods; (ii) the biological functions of RNA structure; (iii) genome-wide RNA structural features corresponding to their regulatory mechanisms; and (iv) RNA structurome evolution in plants.The dynamic structure of RNA plays a central role in post-transcriptional regulation of gene expression such as RNA maturation, degradation, and translation. With the rise of next-generation sequencing, the study of RNA structure has been transformed from  Table 1)  . Howeverin vivo chemical probing (p-toluene sulfonate (CMCT), which targets the unpaired N3 position of uracil and the unpaired N1 position of guanine  cleave either single-stranded (ss) RNA regions or double-stranded (ds) RNA regions to indicate RNA base-pairing status. The most commonly used enzymatic probing reagents include RNase V1 (for dsRNA), RNase S1 (for ssRNA), RNase A (for C/U in ssRNA), and RNase T1 (for G in ssRNA) . The RNa probing . Another guanine ; while 3 guanine . Among t guanine . The oth guanine . A partiTable 1) , COOLAIR, revealed the remarkable complexity of RNA structure up to 750 nt . This method achieved “attomole” sensitivity allowing RNA structure probing of low abundance RNAs in living cells  assays were traditionally used to measure the modified pattern of both enzymatic and chemical reactions . HoweverTable 1) . A receno 750 nt . A furthng cells . By subsng cells . For insperiment . The devTable 1). It was developed in yeast by measuring the catalytic activity of two enzymes, RNase V1 (for dsRNA) and S1 (for ssRNA)  . Both methods reveal in vivo RNA structures are more single-stranded than in vitro and in silico computational predicted RNA structures. Use of the Structure-seq method was recently extended to rice , was developed in mouse by using the SHAPE chemical reagent with the power of four-nucleotide probing (Table 1) (Table 1)  (r ssRNA) . This me sapiens . An enhaTable 1) . A furthbidopsis , 2015 inbidopsis  (Table 1 to rice . A folloTable 1) . In addiTable 1) .in vivo RNA structure map over tens of thousands of RNA with single nucleotide-resolution. These technological advances create an unprecedented scale for the in-depth study of the global impact of RNA structure in gene regulation. For example, regulatory RNAs are able to act as a master regulator in gene expression. In general, these regulatory RNAs directly turn on or off gene expression by altering RNA secondary structure. A recent study of RNA structure characterization on a range of regulatory RNAs in Arabidopsis is illustrated below .These powerful genome-wide methods can provide an accurate and quantitative THIC . With a low TPP concentration, the 3′ end processing of THIC mRNA results in a short 3′ UTR that permits high expression of the THIC gene. Conversely, with a high TPP concentration, TPP binds directly with the 3′ end of the RNA and induces a structural change that prevents splicing. This results in a long 3′ UTR inducing RNA degradation, subsequently reducing THIC gene expression , which resides in the 3′ UTR region of the thiamin biosynthetic gene THIC   contains a 5S rRNA mimic structural element in one of its exons . When ribosomal protein L5 binds to this 5S rRNA mimic, it triggers exon skipping in TFIIIA mRNA to control TFIIIA levels , inhibiting its translation when forming stable GQS structures . Interestingly, potassium concentrations in plant cells can dramatically increase under drought stress  also play an important role in gene expression regulation. RNA GQSs are typically more stable in the presence of potassium or sodium. Tens of thousands of putative GQSs were identified in  species . A recenructures  , in response to vernalisation. The distal COOLAIR isoform in Arabidopsis  is highly-structured with numerous secondary structural motifs, an intricate multi-way junction, and two unusual asymmetric 5′ internal loops . Interestingly, a single nucleotide polymorphism (SNP) in the natural variation accession, Var2-6, is able to change the structure to affect the RNA stability, resulting in a late-flowering phenotype in Var2-6 . A recent RNA structure characterization study by NMR shows the upper stem of a double-stranded region of pri-miR156 is important for Dicer processing at different temperatures, that substantiates the structure-determined Dicer processing feature  (Arabidopsis nuclei (Alternative splicing is an important process in RNA maturation. More than 40% of isoforms . The firtention) . PIP-seqs nuclei . Interess nuclei . Thus, tArabidopsis  that is found in over 60% of mRNAs in bidopsis . In vivoage site . These page site . Howeverve sites . This suin vitro RNA structurome analysis in Arabidopsis. Unlike yeast, highly-structured mRNAs are more likely to be degraded in Arabidopsis, probably via specific siRNA processing  prefers single-stranded conformations rather than double-stranded structures  sites. For example, NA⋅U pair . Cellulathylated . Recent ructures . A genomR region . Interesrofiling . A studytructure . This inAnother key player in post-transcriptional regulations is RBP. Unlike DNA binding protein, RBP associates not only with the primary sequence motifs, but also RNA structural patterns. A recent study combining genome-wide RBP profiling and RNA secondary structure profiling shows that RBP binding sites tend to be more single-stranded . Interesin vivo RNA structurome between plant species, to assess the evolutionary adaptation of RNA structure (in vivo RNA secondary structure conservation does not correlate with sequence conservation between rice and Arabidopsis. The conservation and divergence in both sequence and RNA secondary structure are highly relevant with specific biological processes (From an evolutionary perspective, the conservation and diversity of RNA structurome between species remains poorly understood. A recent study compared, for the first time, the conservation and divergence of tructure . This strocesses . This inin vitro conditions for studying RNA secondary structure. These new methods, with single-nucleotide resolution, genome-wide scale and high sensitivity, significantly accelerate the study of in vivo RNA structure and associated biological functions. Plants are more sensitive than animals to varying environmental conditions, such as changes in temperature, salinity, acidity, and heavy metal concentrations (Recent methodology advances have overcome previous limitations of both low-throughput and trations . These ftrations . By applXY, MY, HD, and YD wrote the manuscript. MY produced the figure. XY, HD, and YD made the corrections. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MORC7 (a gene implicated in epigenetic gene silencing), was markedly different. Long non-coding RNAs (lncRNAs) also underwent AS during the transition from milk seed to mature embryo and endosperm, with a complex gene structure, and were more extensively processed than predicted by current genome annotation. Exon retention of lncRNAs was enhanced in embryos. Searching all 5,515 lncRNAs in the NCBI genome annotation uncovered gene families based on highly conserved regions shared by groups of 3–35 lncRNAs. The homologies to other lncRNAs, as well as homologies to coding sequences, and the genomic context of lncRNAs provide inroads for functional analysis of multi-exonic lncRNAs that can be extensively processed during seed development.Alternative splicing (AS) is a key modulator of development in many eukaryotic organisms. In plants, alternative splice forms of non-coding RNAs (ncRNAs) are known to modulate flowering time in Arabidopsis and fertility in rice. Here we demonstrate that alternative splicing of coding and long non-coding RNAs occurs during rice seed development by comparing AS in immature seeds vs. embryo and endosperm of mature seeds. Based on computational predictions of AS events determined from a Bayesian analysis of junction counts of RNA-seq datasets, differential splicing of protein-coding, and non-coding RNAs was determined. In contrast to roots, leaves, flowers, buds, and reproductive meristems, developing seeds had 5.8–57 times more predicted AS. Primers designed to span introns and exons were used to detect AS events predicted by rMATs in cDNA derived from early (milk) seed, embryo, and endosperm. Comparing milk seed vs. mature embryo and endosperm, AS of  Alternative splicing (AS) is a fundamental mechanism for the functional diversification of the eukaryotic transcriptome. As more forms and functions of RNA are discovered, the importance of AS is being revisited as a means of not only creating multiple protein isoforms, but of producing and regulating short and long non-coding RNAs Ulitsky, . In planThe breadth of RNA-seq data available provide a powerful resource to analyze AS. Myriad algorithms are available to determine the abundance of different isoforms present in RNA-seq output, but not all are capable of or excel at distinguishing AS between different conditions or developmental stages , HID1 on photomorphogenesis (intron binding), and APOLO on auxin action (chromatin looping). COOLAIR and LDMAR, for example, also function by modulating the epigenetic states of their target promoters  approach was used to determine the nature of lncRNA gene families in the rice genome, and to explore homology of lncRNAs to protein coding sequences within and outside of the https://www.ncbi.nlm.nih.gov/sra) to test their suitability for alternative splicing analysis with rMATS. Detailed analysis was performed of datasets from the following studies: developmental time course GSE56463  and the above mentioned.gtf file. RNA-seq experiment FASTQ files (including replicates when available) were obtained with the SRAtoolkit fastq-dump command.For rMATS analysis, a.gtf file was constructed from the NCBI Reference IRGSP-1.0 Primary Assembly: GCF_001433935.1_IRGSP-1.0_genomic.gff. Chromosome names were converted from NCBI to IRGSP convention, and consequently the Cufflinks gffread tool was used to convert the.gff file to a.gtf file. STAR was used to generate genome files from the IRGSP FASTA genome sequences (p < 0.005 and FDR < 0.05) using PANTHER's statistical overrepresentation test with the default PANTHER GO-slim Biological Process annotation and Bonferroni correction settings  for 8 weeks followed by growth 12 h light (short day) to induce flowering. Milk seeds were harvested 8–9 days post-pollination, and mature seeds were harvested 7 days later. Embryos were manually separated from endosperm with a razor. Samples were collected in triplicate. RNA was isolated from whole milk seed and isolated embryo and endosperm using a modified TriZol method  mix. PCR was performed for 25–30 cycles . Primers flanking predicted splicing events were designed using PrimerSeq  first called a genome assembly from the NCBI , then searched for all features annotated as “ncRNA” and tabulated their locus ID, length, and number of exons. When run with rMATS output, it rejected all LOCs not found in one of the rMATS output files with p < 0.005 and FDR < 0.05. When run without rMATS output, it either filtered out all but the longest or all but the shortest isoforms. It then collected BLASTN results from the NCBI BLAST tool searching the refseq_RNA database with the longest lncRNA isoform. Consequently, alignments to ribosomal RNA were filtered out, and the results split into matches to O. sativa ncRNA, O. sativa protein-coding RNA, or alignments to RNA from organisms other than O. sativa. The NCBI nucl_gb.accession2taxid table was used to append the species name of the match to the non-species file. Lastly, for determination of lncRNA families, results were filtered by length (≥100 bp significant match length) and screened against the Type I transposon dataset downloaded from the RiTE Oryza transposable elements database to remove spurious ncRNA families associated with TE matches  are considered “fully supported” (https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Oryza_sativa_Japonica_Group/101/).Preliminary analysis of reproductive meristem RNA-seq data clusters , retained intron (RI), and differential 5′ or 3′ splice site (DSS) events. Surprisingly, relatively few events were detected in reproductive meristems 13 events) and a full series of plant tissues, 44 in root vs. leaf, 126 in leaf vs. bud, from 7 in bud vs. flower and 30 in flower vs. early seed   and via spliceosome (10.10-fold). These data hinted at interactions between AS of components of the splicing machinery and processes involved in methylation or chromatin modification  that may play a role in seed development. The rMATS analysis of the milkseed vs. mature seed RNA-seq experiment Figure  identifiMORC7  the coding potential was not affected by the presence or absence of the exon. However, in the case of LOC9270986 , the coding potential requires the absence of an intron, the presence of another, and the presence of an exon   proteins has been known for more than a decade   revealed a specific effect on proximally polyadenylated COOLAIR transcripts . Because we were searching against RNA and not genomic sequence, it is possible that the homologous regions are involved in splicing competition or competition for other factors necessary for RNA processing. As mentioned above, in some cases regions with homology to coding RNAs can be found in exons and introns that are selectively spliced from lncRNAs. During Arabidopsis root development, lncRNAs affect AS of transcripts by competing with RNA-binding proteins and modulating the output of splice variants (Bardou et al., Taken as a whole, these data demonstrate extensive AS during rice seed development that includes complex processing of lncRNAs. Using the same RNA-seq data, Wang et al.  employedEK and EL performed molecular biology experiments, designed primers, developed RNA protocols; AG wrote scripts and performed bioinformatic analyses; MK designed the study, reviewed the results and oversaw the manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease that is clinically characterized by progressive muscle weakness and impaired voluntary movement due to the loss of motor neurons in the brain, brain stem and spinal cord. To date, no effective treatment is available. Ample evidence suggests that impaired RNA homeostasis and abnormal energy status are two major pathogenesis pathways in ALS. In the present review article, we focus on recent studies that report molecular insights of both pathways, and discuss the possibility that energy dysfunction might negatively regulate RNA homeostasis via the impairment of cytoplasmic-nuclear shuttling in motor neurons and subsequently contribute to the development of ALS. Amyotrophic lateral sclerosis (ALS) is a rare motor neuron disease that occurs in adults with a prevalence of ~5 per 100,000. Patients with ALS develop muscle weakness and impaired voluntary movement, largely because of degeneration of the upper and lower motor neurons , probably as a result of higher energy consumption and lower food intake  impairs mitochondrial function by disrupting the respiratory transport chain machinery  has been found to improve motor function in mSOD1-expressing or TDP-43 mutant-expressing worms  encode RNA-binding proteins that have been found to affect multiple levels of RNA processing, including mRNA transcription, RNA transport, mRNA stabilization and miRNA biogenesis  G-quadruplexes, thus suggesting the importance of the hexanucleotide repeat expansion  involved in nucleocytoplasmic transport  in a Expression of the C9ORF72 repeat expansion and the resultant DPRs is associated with abnormal mitophagy and impaired mitochondrial function (Lopez-Gonzalez et al., As noted by several recent studies and reviews, the loss of nuclear functions of many RNA-binding proteins and nuclear transport components and the gain of function and/or aggregate formation of these proteins in the cytoplasm may contribute to ALS pathogenesis (Zhang et al., Importins are key molecules in the control of nucleocytoplasmic transport because they are needed for transporting macromolecules larger than 40 kDa (such as proteins; Görlich and Kutay, Multiple lines of evidence suggest the involvement of importins in ALS. First, we have previously demonstrated that AMPK activity is higher in motor neurons of the ALS spinal cord when compared with that of non-ALS subjects (Liu et al., This alteration in the function of importin-α1 induced by AMPK phosphorylation is likely to be a common pathogenic pathway that may contribute, at least partially, to the RNA toxicity observed in ALS. In accordance with the importance of importins in ALS, up-regulation of importin α has been found to be beneficial in a fly model of ALS that exogenously expresses the C9ORF72 repeat expansion (Zhang et al., Proper RNA transport is critical for cellular RNA homeostasis. In highly polarized cells, such as motor neurons, mRNA can be transported to neurites. Motor neurons are known to have local protein synthesis in their synapses after exposure to stimuli (Yoo et al., As described above, many ALS genes encode RNA-binding proteins (RBPs, Figure C9ORF72, P-YT wrote the first draft of section Importins and AMPK in Nucleocytoplasmic Transport and RNA Transport, YC organized the manuscript and wrote the final draft.Y-JL wrote the first draft from section Abnormal Energy Homeostasis in ALS to section The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.