The unexpected ability of an RNA polymerase ribozyme to copy RNA into DNA has ramifications for understanding how DNA genomes evolved. Related research article Samanta B, Joyce GF. 2017. A reverse transcriptase ribozyme. eLife6:e31153. doi: 10.7554/eLife.31153For as long as history has been recorded, humanity has tried to answer the ancient question of our origins. The ‘central dogma’ of molecular biology, first stated by Francis Crick in 1958, represented a major step forward in our efforts to answer this question . In thisThe flow of information from DNA to RNA to protein is thought to have evolved out of a simpler evolutionary period when genetic information was stored and transmitted solely by RNA molecules. This theory, known as the ‘RNA world hypothesis’, posits that an RNA enzyme or ‘ribozyme’ capable of copying RNA molecules existed early in evolution, and that protein synthesis by the ribosome  evolved out of this system . The theIn modern metabolism, protein-based enzymes called reverse transcriptases can copy RNA to produce molecules of complementary DNA. Other enzymes can promote the production of DNA nucleotides (the building blocks of DNA molecules) from RNA nucleotides via challenging chemical reactions. So how did the first DNA genomes come to be? There are two possibilities within the framework of the RNA world. In the first, protein enzymes evolved before DNA genomes. In the second, the RNA world contained RNA polymerase ribozymes that were able to produce single-stranded complementary DNA and then convert it into stable double-stranded DNA genomes.A number of laboratories around the world are trying to build ribozymes that can sustain RNA replication . RecentlWhether DNA genomes existed very early in evolution fundamentally rests on whether DNA nucleotides were available in the RNA world. There are plausible routes by which RNA and DNA nucleotides could have been synthesized before life emerged, meaning that they are likely to have been available at the dawn of an RNA world . LikewisDemonstrating that DNA polymerase ribozymes are able to rapidly use such DNA nucleotides would represent a major step forward for the early DNA genome model. While the field of artificial RNA polymerase ribozymes has made rapid strides, their ability to add multiple nucleotides rapidly is still very limited. Current ribozymes are significantly longer and more complex than the sequences that they are able to copy, but to make self-evolving systems, ribozymes need to be able to copy sequences that are longer and more complex than themselves. It will therefore be exciting to see if the techniques that have created such RNA polymerases are also able to evolve DNA polymerase ribozymes that have the potential to make self-replicating systems using DNA and not RNA as a source of genetic material. Such a system would bring us closer to understanding the transition from an RNA world to a type of life that respects the rules of the central dogma of modern biology.


It allows investigators to simultaneously visualize and sort through every guide targeting the protein-coding regions of any mouse or human gene.Guide Picker (Utilizing a multidimensional graphical display featuring two plots and four axes, Guide Picker can analyze all guides while filtering based on four different criteria at a time. Guide Picker further facilitates the CRISPR design process by using pre-computed scores for all guides, thereby offering rapid guide RNA generation and selection.The ease-of-use of Guide Picker complements CRISPR itself, matching a powerful and modular biological system with a flexible online web tool that can be used in a variety of genome editing experimental contexts. Streptococcus pyogenes) is directed to target sites in the genome by a chimeric single guide RNA (sgRNA) [CRISPR  allows researchers to introduce site-specific mutations in a variety of organisms , 2. SpCa (sgRNA) . The sgR (sgRNA) . CustomiThe PAM is essential for Cas9 binding. SpCas9 primarily recognizes NGG PAMs . Other PTo ensure target specificity and guide activity, researchers depend on intelligent guide RNA design tools to predict guide RNA behavior . SeveralTo address these problems, we developed Guide Picker. Guide Picker is a cloud-based tool that allows the user to visualize guide RNA designs plotted according to ten scoring functions using one simple graphical interface. Guide Picker can compare on- and off-target scores, as well as other parameters, for every guide RNA targeting the protein-coding transcripts in a given mouse or human gene. Filtering and selecting guides according to different scores in one interface alleviates the labor involved in testing designs across disparate guide RNA design tools Fig. . Once thGuide Picker is also unique because it is the only online resource that allows guide design around all protein-coding transcripts of a gene. Transcripts are identified using Ensembl database annotations indicating known coding DNA sequences. Some design tools limit guide design to a 250 nucleotide input sequence while others only generate guides for a single transcript. By using all transcripts for a given gene, Guide Picker can offer more guide design options and help the user target as many transcript variants as possible to ensure gene knockout.Guide sequences are determined by performing an exhaustive search throughout all protein-coding regions of the mouse or human genome based solely on available NGG SpCas9 PAM sites. This is accomplished using in-house Python scripts which, along with the scores, are contained in a Python wrapper to facilitate automation. This loading process occurs on a cloud-based web server and not on the user’s computer.MUC4). Guide Picker displays all of these guides in an easily manageable graphical format that can be adjusted to improve visual accessibility.In addition to pre-loading guide sequences, Guide Picker further speeds up the CRISPR design process by pre-computing all scores for every guide RNA targeting coding genomic regions in the mouse and human reference genomes. For any given scoring function and gene, rendering all available guides takes fewer than five seconds , a JavaScript framework, and a data visualization library, D3.js (https://github.com/d3/d3). Interfacing with the open source community assured maintainability and compatibility of the Guide Picker tool with most modern web browsers. Using a powerful D3.js library enabled fast prototyping and development of the graphic component, as well as access to its advanced data visualization algorithms such as quadtree (https://github.com/d3/d3-quadtree) which is used in the “Force Layout” mode to detect collisions between data points  is built on custom in-house components developed using open source libraries Vue.js  and Homo sapiens (GRCh38) provided by Ensembl. This is because the scoring functions provided in the tool were developed with datasets from mammalian models and depended on standard U6 delivery plasmid systems. Therefore, all algorithms displayed by Guide Picker are constructed to make predictions within that context [Guide Picker uses the reference genomes for np (where n is a number of guides and p is a number of properties). The multidimensionality of guide RNA data yields an unwieldy number of variables for an investigator to navigate during the guide RNA design process. It also presents a challenge for creating a simple and efficient web tool UI. Guide Picker arose as a solution to this problem.The number of relationships between guide RNA data points can be explained by the formula https://www.deskgen.com/guidebook/advanced.html), where the user selected guides by manually navigating inside genes with a sequence browser, turned out to be an impractical, labor-intensive solution. In parallel, an internally used algorithm-assisted UI which presented the user with five top scoring guides for a given gene limited user engagement and control. It also stymied the user’s ability to cross-validate guides across multiple parameter thresholds.A previously trialed UI  (where n is the number of properties and k is the number of axes) produced an unworkable UI. To compensate for this loss of dimensionality, we drew two scatter plots side-by-side, each displaying different properties for the same set of guides. The axis values of these two plots were user-selected guide design parameters. This improved readability while maintaining a robust level of customizable dimensionality.However, any gains in clarity over previous UI iterations and peer web tools were outweighed by a reduction in data depth. The two-dimensional x/y view did not present a comprehensive method for selecting optimal guides. Also, a number of relationships between data points We also chose to round score values to the nearest integer. Although floating numbers would provide a more continuous distribution of values, they are harder for users to read and compare. A decimal point is also likely to be statistically insignificant for choosing an optimal guide. Therefore, we decided to round score values as is consistent with other guide RNA design web tools , 8.Due to the volume of guide RNA data and our decision to round score values to the nearest integer, highly dense overlapping regions became common in the scatterplots. In order to explore these dense regions more easily, we implemented a “Force Layout” view and “Fisheye” lensing (advanced Guide Picker features) to allow users to visualize overlapping guides or guides in close proximity to one another Fig. . In concHomo sapiens (GRCh38) or Mus musculus (GRCm38). The user then inputs the gene name into the “Gene” entry field. The system will search for the Ensembl gene name and list it in a drop-down menu. Once the gene is selected, the user clicks “Proceed” to be taken to the main Guide Picker interface , but some are either stepped  or binary .The percent peptide score (PPS) refers to the guide position within the protein-coding portion of the entire gene. In Guide Picker, protein-coding exons for each transcript are concatenated together from the ATG/AUG codon to the STOP codon and multiple transcripts are overlaid to produce one theoretical master coding DNA sequence (MCDS) per gene Fig. . The basRepresentation refers to the proportion of the gene’s protein-coding transcripts a given guide RNA design can target. The stepped axis levels represent the percentage of targeted transcripts versus the total transcripts for that gene. This value is useful for designing guides against highly represented (consensus) exons. A graphical representation of how Transcript Representation is computed can be found in Fig. The Hsu 2013 score predicts the specificity of the guide RNA. Off-target sites are evaluated based on genomic similarity to the guide RNA sequence. This evaluation takes into account mismatch number, position and density . It is iHsu 2013 considers both canonical NGG and non-canonical NAG PAM sites for SpCas9 . This inThe Doench 2014 on-target activity score predicts the ability of the guide RNA to knock out the target gene . This schttps://github.com/MicrosoftResearch/Azimuth).Like the Doench 2014 score, the Doench 2016 on-target activity score also predicts the ability of the guide RNA to knock out the target gene . This scDoench 2016 comes in two forms: Full and Positionless. The Doench 2016 Full score is adjusted based on the target location in the coding DNA sequence while the Doench 2016 Positionless score does not. This adjustment is based on the percent peptide score (PPS) which represents the progression through the CDS of that gene.The reason to consider accounting for position in the CDS is that some studies have suggested that targeting in the 3′ end of the gene is less likely to lead to gene knockout , possiblExtreme GC content (low or high) can lead to poor or depleted guide RNA activity. The percentage of GC content refers specifically to the guide RNA protospacer element (not including the PAM). A recent study concluded that a range of 30–70% GC content yields optimal guide RNA activity . GC contFour or more consecutive repeated nucleotides (homopolymers) in the guide RNA sequence have been shown to be detrimental to guide RNA activity . The homThe presence of a TTT DNA sequence (UUU in the RNA product) is detrimental to guide RNA activity because it is a terminator sequence for RNA Pol III transcription . Like thThe microhomology score predicts the likelihood of creating an out-of-frame mutation via NHEJ-mediated repair . Regionshttps://www.deskgen.com/my-projects/) at DESKGEN.com. The list is also text-selectable and can be copy-pasted by the user into a separate document  gene as an example. Due to the size of MUC4, it has more guides than many other genes and therefore demonstrates these parameter relationships more clearly.Guide Picker can be used to compare scoring functions across all guides targeting a single gene’s MCDS. This can illuminate trends and biases in scoring functions. The visualizations in Fig. The two plots in Fig. The plot on the right of Fig. The difference between Full and Positionless is also made apparent in Fig. Comparing Doench 2016 Full and Positionless in this way helps the user elucidate the difference between these two scores. Practically speaking, some investigators may want to avoid targeting the 3′ end of the gene and therefore will want to use Doench 2016 Full. Others may not rely on generalizations about the 5′ or 3′ end of the gene and instead will want to target 3′ proximal functional domains where appropriate in order to ensure gene knockout .Guide Picker can also be used to demonstrate distributions for various scores by plotting parameters against themselves. In Fig. Guide Picker can be used to design both gene knockout and tiling experiments using SpCas9. For gene knockout, the scoring parameters can be set to maximize guide efficiency. Ideally, this means selecting guides with a high Doench 2016 score, a high Hsu 2013 score, no homopolymers or uracil triplets, high transcript representation and a high microhomology score. Location in the MCDS (as indicated by the percent peptide score) will vary depending on the experiment.Guide Picker can also be used to interrogate specific regions of a gene in what is known as a CRISPR tiling experiment. In principle, this involves systematically designing guides that target along the MCDS to determine regional essentiality in protein function . This caHTR2A, as an example. We use Positionless in this example because we do not want to introduce scoring bias against guides targeting the 3′ end of the gene; 3′ regions may still be functional or essential. The guides can then be filtered according to a Doench 2016 Positionless threshold (e.g. 50)  Fig. . The “FiAll guides above this threshold are then brought over to the right-hand plot and further narrowed down according to, for example, a relatively high Hsu 2013 off-target score  and a GC content range of 30–70%  Fig. 6c6c. The fHTR2A. These guide sequences can then be checked in the Knockout or Knockin tools (https://www.deskgen.com/guidebook/) or a comparable genome browser to determine their exact location in the transcript(s) or MCDS. From there, the user can execute the experiment using a small-scale library generated with the list from Guide Picker to elucidate the essentiality of each protein-coding region.In this example, the user now has 55 guides with convincing GC content, on-target scores and off-target scores that will direct SpCas9 across the entirety of the MCDS of http://www.deskgen.com) cloud platform. It brings together ten literature-based guide RNA scoring parameters and functions. It is unique to other design tools because it can simultaneously visualize all guides for a given gene according to four pre-computed guide RNA design parameters at a time. The tool can be used to support various experimental applications by accelerating and improving the guide design process. As new scores are published, the tool will be updated to accommodate, utilize and compare the latest algorithms.Guide Picker is the newest addition to the DESKGEN  (http://


Potexvirus is one of the eight genera belonging to the family Alphaflexiviridae according to the Virus Taxonomy 2015 released by International Committee on Taxonomy of Viruses (www.ictvonline.org/index.asp). Currently, the genus contains 35 known species including many agricultural important viruses, e.g., Potato virus X (PVX). Members of this genus are characterized by flexuous, filamentous virions of 13 nm in diameter and 470–580 nm in length. A potexvirus has a monopartite positive-strand RNA genome, encoding five open-reading frames (ORFs), with a cap structure at the 5′ end and a poly(A) tail at the 3′ end. Besides PVX, Bamboo mosaic virus (BaMV) is another potexvirus that has received intensive attention due to the wealth of knowledge on the molecular biology of the virus. In this review, we discuss the enzymatic activities associated with each of the functional domains of the BaMV replication protein, a 155-kDa polypeptide encoded by ORF1. The unique cap formation mechanism, which may be conserved across the alphavirus superfamily, is particularly addressed. The recently identified interactions between the replication protein and the plant host factors are also described.The genus  Bamboo mosaic virus (BaMV) primarily infects members of the Bambusoideae in nature; nonetheless, it also replicates in Nicotiana benthamiana, which thereby has been used as the surrogate in laboratories. The RNA genome of BaMV contains 6366 nucleotides (nts) plus a 5′ m7GpppG (cap0) structure and a 3′ poly(A) tail . It is functionally organized into a 94-nt 5′ untranslated region (UTR), five ORFs, and a 142-nt 3′ UTR (BaMV) that has been thought to be essential for replication/transcription of the viral genome and the formation of the 5′ cap based on the presence of signature motifs of Sindbis virus-like methyltransferase , separated by a disordered hydrophilic region, from approximately amino acid residues 406–520, and a proline-rich segment (PRS), residues 895–910, according to a secondary structure prediction using the PHD algorithm  was provided in the reaction buffer. Alternatively, it could be radiolabeled by Ado[methyl-3H]Met when GTP was present. The radiolabeled moiety covalently linked to the domain was subsequently determined to be m7GMP. This [m7GMP-enzyme] adduct was thought to represent an intermediate in the pathway to form the 5′ cap. In other words, this viral domain could be a guanylyltransferase (mRNA capping enzyme) except that it is covalently modified by m7GMP rather than GMP. In addition, this N-terminal domain of REPBaMV was found capable of catalyzing a methyl transfer reaction from AdoMet to GTP, leading to the formation of m7GTP, consistent with the prediction of its function as a methyltransferase. This viral domain was therefore proposed to possess an AdoMet-dependent guanylyltransferase activity, by which the methyl group of AdoMet is transferred to GTP, leading to m7GTP formation, and then the m7GMP moiety of m7GTP was transferred to an active-site residue to form the covalent [m7GMP-enzyme] intermediate. Analogous reactions have been observed also in other members of alphavirus-like superfamily including alphavirus (Brome mosaic virus (Semliki Forest virus (Hepatitis E virus (Tobacco mosaic virus (TMV) (BaMV) are conserved.The enzymatic activity of the N-terminal 442 amino acids of REPrevisiae . The recphavirus , Brome mic virus , Semlikist virus , Hepatit E virus  and Tobaus (TMV) , suggest7GMP-enzyme] intermediate  GTP and AdoMet bind to the capping domain of REPBaMV in proximity (7GTP and S-adenosyl-L-homocysteine (AdoHcy). (3) The nitrogen atom (not determined whether Nδ1 or N𝜀2) of His68 functions as a nucleophile attacking the α-phosphate of m7GTP, under the assistance of Mg2+, to form the covalent [m7GMP-enzyme] intermediate  Finally, the RNA with a 5′ cap0 structure is released from the domain.According to the data aforementioned and others, the cap formation pathway catalyzed by the capping domain of REProximity . The prermediate . This strm m7GTP . (4) TheBaMV (residues 514–895) forms inclusion bodies when it is expressed in E. coli. This domain resumes soluble after denaturation and refolding processes. The purified HLD is able to remove the γ phosphate from nucleoside triphosphates as well as RNA  was determined to be 93 μM, which is close to the Km value of ATP (150 μM) for the NTPase activity (Ki(AMPPNP) and Km(ATP) and the simultaneous inactivation of both activities by mutations at the featured motifs of helicases suggest that a common catalytic site is used for the hydrolysis of both NTP and RNA. Nonetheless, the greater value of Km(ATP) than Km(RNA), which is about 2.5 fold, suggests that more active-site residues are involved in RNA binding. The peptidyl regions employed by the HLD to bind biotinylated RNA were mapped by the reversible formaldehyde crosslinking method followed by tandem mass spectrometry  may be recruited as an accessory subunit of the helicase to confer unwinding activity on the viral protein.TGBp1 of BaMV is also a member of SF1. TGBp1 is capable of hydrolyzing NTPs but not RNA , implyin the HLD . Taken tN. benthamiana identified a strong protein–protein interaction between the HLD of REPBaMV and the viral CP . A230G mutation in FoMV CP, analogous to BaMV A209G, also reduces the viral HLD-CP interaction and restricts the cell-to-cell movement of FoMV in C. quinoa ppp(5′)Np, in eukaryotic mRNAs is a basic structural unit required for mRNA export from the nucleus, prevention of mRNA degradation by 5′-exonucleases, and recognition by eIF4F complex to initiate the translation process . Three consecutive enzymatic reactions are responsible for the cap formation in the nucleus. First, the γ-phosphate of a nascent mRNA is removed by RNA 5′-triphosphatase. The GMP moiety of GTP is then transferred to the 5′ end of the 5′-diphosphorylated mRNA via a covalent enzyme-lysyl-GMP intermediate by GTP:mRNA guanylyltransferase. Finally, the guanine-N7 of G(5′)ppp(5′)Np cap is methylated by RNA (guanine-N7) methyltransferase to produce the cap0 structure  performs another unconventional mRNA 5′ cap formation pathway  methyltransferase of eukaryotic cells and DNA viruses. With the limited genome size, BaMV has evolved an efficient capping enzyme, with merely 442 amino acids, to accomplish the work of forming the 5′ cap.The BaMV enzymes performing the catalytic steps in the pathway are also unique from the viewpoint of protein structures. Apparently, the BaMV RNA 5′-triphosphatase activity has emerged from the helicase motif-containing domain. By contrast, the RNA 5′-triphosphatases of yeast and DNA viruses, e.g., vaccinia virus and baculovirus, belong to a metal-dependent phosphohydrolase family , while tBaMV had been thought to be the key component of the viral replication complex, due to the presence of the hallmark signature of polymerase S/TGX3TX3NS/TX22GDD  tail  tail . A footpleavages . Similarleavages . Reducinleavages . UV-crostem-loop . In summBrome mosaic virus and Red clover necrotic mosaic virus recruit the membrane derived from endoplasmic reticulum to constitute their replication complexes  and PVX are two other potexviruses that have been demonstrated to be associated with chloroplasts in their infection processes. The TGB3 of AltMV preferentially accumulates around the chloroplast membrane and disruption of TGB3 targeting to chloroplast impairs cell-to-cell movement of the virus  UV-induced crosslinking of proteins in leaf cell extract to the 32P-radiolabeled 3′ UTR of BaMV, (2) nuclease digestion, and (3) radiolabeled protein identification using mass spectrometry, has identified several 3′ UTR-interacting proteins including chloroplast phosphoglycerate kinase (PGK), cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heat shock protein 90 homolog (NbHsp90). PGK promotes BaMV accumulation presumably by facilitating transport of the viral genomic RNA to chloroplasts, the plausible replication site for BaMV  was thus isolated from the cDNA library prepared from N. benthamiana leaves  preparation of the P30 fraction from the agroinfiltrated leaves, (3) protein solubilization using anionic detergent Sarkosyl, (4) protein precipitation using anti-HA antiserum, and (5) identification of the co-precipitated proteins by tandem mass spectrometry. Accordingly, dozens of host proteins were identified. To examine the role of the proteins in BaMV replication, each of the genes was transiently silenced in N. benthamiana. Those plants without apparent changes in phenotype were then challenged with a genetically modified BaMV that carries GFP as a reporter gene. Several potential host factors affecting BaMV replication were thus identified based on the effect of gene silencing on GFP expression. A cytoplasmic 5′→3′ exoribonuclease (NbXRN4), a ripening-related protein, S-adenosylmethionine synthetase, and a respiratory burst oxidase homolog were found capable of promoting BaMV replication. By contrast, NADP+-dependent isocitrate dehydrogenase and MAP kinase phosphatase-like protein appeared to suppress BaMV replication. The relevance between the activity of NbXRN4 and BaMV replication was further investigated. In brief, NbXRN4 benefits BaMV replication, probably by removal of the uncapped genomic and subgenomic RNAs produced erroneously during the replication/transcription process.Recently, we found that the expression of REPfraction . This apBaMV may thus not only apply to other members of the Potexvirus but also serve as references for those of other genera that also belong to the alphavirus-like superfamily. Nonetheless, the structural information at the atomic level regarding the functional domains of REPBaMV is still lacking, thanks mostly to the aggregation nature of these viral proteins. Methods that can overcome this obstacle are urgently needed. The search for host proteins, including those either boost or attenuate the enzymatic activity of REPBaMV, should be continued. More importantly, the mechanism underlying the function of host proteins should be elucidated so that the holistic and dynamic interplay between REPBaMV and its host can be understood.Studies on replication-related proteins of plant RNA viruses have long been limited by inefficient protein expression and difficulty in protein purification. The catalytic characteristics of REPMM organized the contents of the text and wrote the manuscript. C-CL participated into the discussion about the text contents and approved the manuscript.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.


Inhalation of nebulised medications is performed in upright sitting to maximise lung volumes. The pattern of deposition is poor for inhaled medications in people with Cystic Fibrosis. The pattern tends to be non-uniform and typically the upper lobes receive a reduced dose compared to the rest of the lung. One strategy that has been proposed as having the potential to improve homogeneity of deposition is to adopt an alternate side lying position for the inhalation procedure. This study sought to determine whether, among adults with Cystic Fibrosis, there is any disadvantage to delivery time of nebulised medications with a strategy of alternate side lying, compared to upright sitting.A randomised crossover trial with concealed allocation, intention-to-treat analysis and blinded assessors was undertaken. The participants were 24 adults with stable Cystic Fibrosis. They inhaled 4 mL of normal saline via an LC Star™ nebuliser twice within 24 h. In random order, participants sat upright throughout nebulisation, or alternated between left and right side lying at each minute during the nebulisation period. The nebuliser was stopped and weighed each minute until the residual volume was reached. The primary outcome was the time required for 3.5 mL to be delivered. The secondary outcomes were: respiratory rate; ratio of the volume delivered on right and left sides; and calculation of how long the periods in side lying can be extended without causing greater than 20% discrepancy in dose delivered in the two positions.2 < 0.4). Increasing side lying duration from 1 to 2 min did not significantly impact the dose delivered on each side. Turning each 3 min however, significantly worsened the disparity .The delivery time did not significantly differ between sitting and side lying  -1.40 to 0.24). There was no significant correlation between delivery time, lung function or subject height  contains supplementary material, which is available to authorized users. Over the past few decades, research has established many nebulised therapies that improve the clinical status and quality of life of people with Cystic Fibrosis, including inhaled tobramycin  recombinThe distribution of ventilation (V), perfusion (Q), and V/Q matching in the lungs are primarily influenced by gravity , 6. VentA strategy of alternate side lying has been proposed as a means to improve upper lobe deposition. In this strategy, patients alternate between right and left side lying at regular intervals during delivery of a single nebulised therapy. The rationale for this approach is that gravity will tend to increase ventilation in the most dependent region of the lungs. Adopting a sidelying position therefore will result in deposition of nebulised medication preferentially throughout the dependent lung, including its upper lobe. Importantly, regular turning is required to dose both sides (including both upper lobes) equally during a single nebulisation period. This is because the dose delivered by an LC Star nebuliser over time is initially rapid and then tapers after 8 to 10 min, rather than constant delivery. This has been demonstrated with its delivery of recombinant human deoxyribonuclease  and withIt remains unclear, however, whether this alternate sidelying strategy to improve homogeneity of deposition has an impact on nebulisation time. If the nebulisation time was significantly increased then any potential benefits in the pattern of drug deposition may be outweighed by poorer patient compliance in completing the drug administration regimen.nebuli- AND deliver- AND (body position OR side lying), which identified no evidence about the side lying strategy.We assert that this is the first study to examine the alternate side lying strategy for nebulised delivery of medication. This assertion is reinforced by a Google Scholar search using the terms Among adults with Cystic Fibrosis, is there any disadvantage to delivery time of nebulised medications with a strategy of alternate side lying, compared to upright sitting?How long can the periods in side lying be extended without causing greater than 20% discrepancy between the dose delivered in the two positions?The aims of the study were to determine:Upright sitting: maintained throughout the nebulisation period, or.Alternate side lying: alternated between left and right at each minute during the nebulisation period. The starting side was also randomly allocated.A randomised, crossover trial with concealed allocation, blinding of assessors and intention-to-treat analysis was undertaken at Royal Prince Alfred Hospital, Sydney. Participants were recruited by personal approach by one of the investigators at the Cystic Fibrosis clinic at the hospital. Once enrolled in the trial, each participant was invited to attend the Department of Respiratory Medicine on two consecutive days. On both days, participants performed spirometry in standing in accordance with the most recent European Respiratory Society criteria . ParticiParticipants were requested to adopt their allocated position and maintain a slightly deeper than normal tidal breathing pattern during the subsequent standard study inhalation. When participants returned for their second study day, they adopted the other positioning regimen.All standard morning medications were inhaled on the study days, with the appointment scheduled at least 4 h after these medications, and not within 1 h of a meal. Participants were requested to keep their medication regimen constant during the two study days. After testing on each study day, participants were free to inhale medications and eat as usual.1) within 10% of the best recorded value for the past 6 months. Potential participants were excluded if they: had received a lung transplant; were colonised with Burkholderia cepacia complex; were not clinically stable; had significant malignant, neurological or musculoskeletal co morbidities; had hepatomegaly, hepatosplenomegaly or current intestinal obstruction; or were pregnant. Research procedures were approved by the Ethics Committees of the Sydney Local Health District (RPAH Zone)  and the University of Sydney. All participants provided written informed consent prior to participating in this study.Participants were required to meet the following criteria to be eligible for the study: aged at least 18 years; a diagnosis of Cystic Fibrosis confirmed with sweat testing or genotyping; and clinically stable with a forced expiratory volume in one second , delivered by an LC Star nebuliser . The nebuliser and tubing were weighed before and after the 4 mL dose was loaded. The nebuliser was driven with 6 L/min of medical air via the hospital wall supply and a calibrated flow meter. This supply was interrupted after one minute of nebulisation time, as timed with an 870A electronic timer . At this time, the nebuliser and tubing were weighed on a 1206MP Scale . The nebulisation was then recommenced for another timed minute and then weighed again. This continued  until the weight indicated that 3.5 mL of the loaded dose had been delivered from the nebuliser. This approach assumed that the residual volume of 0.5 mL has been reached, in accordance with the manufacturer’s specifications.Weighing of the nebuliser was performed by an investigator who was unaware of the position adopted by the participant. This ‘blinding’ was achieved by shielding the investigator from the patient by a curtain and blocking the sound of the participant changing position by padded headphones playing music. The investigator responsible for randomising and positioning the participant (RD) passed the nebuliser and tubing around the curtain to the blinded investigator for weighing each minute. Each weight was recorded until the dead volume has been reached. An Excel file  was used to store the data. The investigator responsible for randomising and positioning the participant recorded the respiratory rate of the participant, counted during the middle 30 s of each minute of nebulisation.The primary outcome was the time required for 3.5 mL of saline to be delivered by the nebuliser as determined by nebuliser weight. The secondary outcomes were respiratory rate, as an explanatory variable; ratio of the volume of saline delivered on the right and left sides; and calculation of how long the periods in side lying can be extended without causing greater than 20% discrepancy between the dose delivered in the two positions, i.e. the ratio exceeding 1:1.2.For the primary outcome was nebulised medication delivery time, we were unable to find an estimate of the smallest effect on inhalation time that adults with Cystic Fibrosis would consider using a particular positioning regimen worthwhile. Clinical experience in our centre would indicate that time alone is not the only consideration used by patient for position selection when following a nebuliser regimen. Many patients prefer to nebulise in side lying due to comfort and convenience. Therefore we postulated that the increase in delivery time would need to be substantial to stop the use of this strategy in clinical practice. We considered that a minimum difference of 5 min in delivery time for the 3.5 mL dose would be large enough to contribute to clinical decision-making about which positioning regimen to use. Pilot data provided a standard deviation (SD) of 3.7 min for this change in delivery time among eight adults with Cystic Fibrosis who met the eligibility criteria. Assuming this SD, 20 participants would provide 80% power, at the 2-sided 5% significance level, to detect a 5-min difference in delivery time as statistically significant between two groups in the study. We increased participant recruitment to 24 to allow for possible drop outs.As an explanatory variable, respiratory rates were compared between sitting and side lying, to see if this variable may have influenced the delivery rate due to breath activation. Respiratory rate in the two delivery positions was compared using a paired t-test. The alternate side lying data were further examined to assess whether the amount of saline delivered while in right side lying differed from that delivered while in left side lying, also using a paired t-test. As noted above, nebuliser delivery rates are not constant, with greater output initially tapering off to low output as the residual volume is approached . To asse1).Twenty-four participants with Cystic Fibrosis were recruited and all completed the study (see Table The delivery time did not significantly differ between the sitting regimen (mean 18.54 ± SD 3.80 min) and the alternate side lying regimen (17.96 ± 3.53 min), a mean difference of 0.58 min (95% CI -1.40 to 0.24)  and the alternate side lying regimen (17.1 ± 3.8 breaths / min), which was a mean difference of 0.8 breaths / min (95% CI -0.4 to 2.0).There was no significant difference in the volume delivered in right (1.63 ± 0.14 mL) and left (1.60 ± 0.19 mL) side lying, which was a mean difference of 0.06 mL (95% CI -0.07 to 0.19). Based on extrapolation of the data, increasing side lying duration from 1 to 2 min would not significantly unbalance the dose delivered on each side .There was no significant correlation between delivery time and any of the following parameters in either position: FEVThe delivery times in this study {sitting regimen (mean 18.54 ± SD 3.80 min) and the alternate side lying regimen (17.96 ± 3.53 min)}are consistent with the anticipated delivery rates stated in the product information for an LC Star and observed in previous work in our centre. Clinicians may note that this is a longer duration than is often observed for the delivery of 4 mL of normal saline in clinical practice. We believe that this is because some patients only nebulise until the first sign of intermittent delivery, rather than continuing until the dead volume has been reached. The time burden of inhalation therapy supports the need to find strategies to improve convenience for individuals with cystic fibrosis.Alternate side lying does not slow the delivery of nebulised medications substantially. The best estimate is that delivery of 3.5 mL of medication is just over half a minute faster, which would make up for the time spent turning, making it time neutral on average. This finding generates implications for further research. The time-neutral nature of the alternative side lying regimen indicates that it is suitable for further investigation of its effect on medication deposition pattern. This research could be pursued not just in people with Cystic Fibrosis but also in the other diseases where poor upper lobe deposition has been identified during delivery of nebulised therapies in upright sitting , 18–21.Turning between right and left side lying each minute delivered an equivalent dose by weight while in each side lying position. However, turning this frequently is likely to be impractical in clinical practice. Based on extrapolated data, increasing side lying duration from 1 to 2 min would not significantly unbalance the dose delivered on each side, but any further extension of time (≥ 3 min) would cause medication delivery imbalance. It is possible that patients with Cystic Fibrosis would choose to adopt a single side to nebulise for a therapy session and then lie on the other side for the next session. Alternatively, they may spend half the delivery side on one side and turn to the other side for the remainder, but alternate the starting side for subsequent doses. However, investigation of the side lying regimen’s effect on the pattern of drug deposition is needed before it can be recommended in clinical practice. Therefore we do not recommend that the results of this study be used to generate any immediate clinical implications about positioning regimens during nebulised delivery of medications for people with Cystic Fibrosis. However, this study will be useful to inform the design of further studies about the effects of body position on nebulised drug delivery. Subsequent future work could then consider the impact of body position in relation to faster delivery systems and other inhaled therapies  with differing viscosities.The use of an alternate side lying positioning strategy during nebulised therapies does appear to be suitable, from the perspective of nebuliser delivery time, as an alternative to upright sitting. However while total nebuliser dosage delivered while in each side lying position does appear to be equitable if the side is changed at least every 2 min, further investigation of the side lying regimen’s specific effect on the pattern of drug deposition is needed before this position can be recommended.


Tetrahymena group I intron. We investigated self-splicing of the intron from spinach RNA, serving as a model exonic sequence with a 3D structure.Group I self-splicing intron constitutes an important class of functional RNA molecules that can promote chemical transformation. Although the fundamental mechanism of the auto-excision from its precursor RNA has been established, convenient assay systems for its splicing activity are still useful for a further understanding of its detailed mechanism and of its application. Because some host RNA sequences, to which group I introns inserted form stable three-dimensional (3D) structures, the effects of the 3D structures of exonic elements on the splicing efficiency of group I introns are important but not a fully investigated issue. We developed an assay system for group I intron self-splicing by employing a fluorescent aptamer RNA (spinach RNA) as a model exonic sequence inserted by the  The promotion of chemical transformation in a substrate-specific manner is one of the most important biological functions of naturally occurring biopolymers. In current cellular systems, protein enzymes are predominantly employed for such tasks. On the other hand, RNA molecules also conduct biologically important chemical transformations . RNA molRNA splicing is an important step in a series of RNA processing events . In RNA 2+ ions /[intensity of precursor RNA + intensity ligated exons].The BODIPY fluorophore-labeled precursor RNA  and P5abc RNA  were incubated separately at 80 °C for 5 min. Precursor RNA and P5abc RNA were then mixed at 37 °C and incubated for 30 min. After incubation of the resulting solution, the reaction was started by adding a 5× folding buffer  were mixed to prepare a 50 μL reaction solution. An appropriate volume (40 μL or 50 μL) of the solution was transferred to a microplate well with a mineral oil overlay. The plate was then incubated at 37 °C for 120 min in a plate reader , which was pre-warmed at 37 °C. Fluorescence intensity was measured every 10 min. In an assay of bimolecular spinach aptamer, the partner RNA fragment  of the splicing product was added to the reaction mixture. To compare the reactions with different solution volumes containing different amounts of RNA and DFHBI, raw values of fluorescence intensity were divided by the solution volume of the reaction mixture. The resulting values were then employed to derive normalized fluorescent intensities shown as vertical lines at the left side  and P5abc RNA  were incubated separately at 80 °C for 5 min. Precursor RNA and P5abc RNA was then mixed at 37 °C and incubated for 30 min. To this solution (45 μL), a 10× reaction buffer , a 10× rNTP solution , a recombinant T7 RNA polymerase, and a recombinant RNase inhibitor were mixed to prepare a 50 μL reaction solution. Aliquots of 40 μL of the solution were quickly transferred to a microplate well, with a mineral oil overlay. The resulting plate was incubated at 37 °C for 120 min in the plate reader  that was pre-warmed at 37 °C. Fluorescence intensity was measured every 10 min.Template DNA (2000 ng), purified P5abc RNA , a 10× transcription/splicing buffer  was added to the solution. The mixture was incubated at 37 °C for 30 min and subsequently incubated at 4 °C for 30 min. After adding 1/6 volume of glycerol containing xylene cyanol (0.1%), the samples were loaded onto a 5% nondenaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide) containing 71.2 mM Tris-borate (pH 8.3) and 5 mM or 15 mM Mg(OAc)2 or 0.10 mM EDTA. Electrophoresis was performed at 4 °C, 200 V for the initial 5 minutes, followed by 75 V for 4.5 h. The resulting gel was analyzed with a FluoroImager Pharos FX (Bio-Rad).Unlabeled precursor RNA (1.0 μM) and BODIPY fluorophore-labeled P5abc RNA (250 nM) were incubated separately at 80 °C for 5 min. The two RNA solutions were mixed, and the resulting solution was incubated at 37 °C for 5 min. The 10× folding buffer Tetrahymena intron ribozyme, we inserted the intron ribozyme at four different positions in the secondary structure of a shortened derivative of spinach RNA  because Mg2+ ions can generally stabilize RNA tertiary structures [2+ ions (<10 mM) due to the insufficient stability of its active tertiary structure [2+ ions. The P5abc module serves as an activator of ∆P5 ribozyme because the P5abc module associates tightly with the active structure of ∆P5 ribozyme [cis-acting module [The parent r module A. The ∆Pructures ,31. On ttructure . The preribozyme ,33,34. TBased on the experimental design using ∆P5 intron ribozyme, the splicing efficiencies of four precursors were analyzed via two methods, which are an analysis of splicing products with denaturing polyacrylamide gel electrophoresis (denaturing PAGE) and a direct observation of fluorescence of DFHBI complexed with spinach RNA as an indicator of the product (ligated exons).2+ ions by denaturing PAGE , the transcription of the precursor of the site-3 ribozyme efficiently showed the fluorescence emission of spinach RNA–DFHBI complex  assay of the ∆P5/P5abc bimolecular complex . As P5abTetrahymena ribozyme) in the P7 element. This C-G pair is completely conserved among all group I introns and serves as the binding site for guanosine (G-site) [To prevent splicing and related reactions of the intron ribozyme during EMS analysis, we introduced mutations to eliminate the catalytic ability of the ribozyme without disturbing its 3D structure A. We sub(G-site) ,14. In t(G-site) . The G-s(G-site) ,10,11,14(G-site) . We prep2+ ions  with Spfr1 RNA and DFHBI. Fluorescence measurement indicated that three precursor RNAs produced fluorescence of DFHBI–spinach RNA complex although their fluorescence varied D. The si2+ ions , exhibit2+ ions D. On the2+ ions B. One po2+ ions D. In the2+ ions D.Based on the observation that the site-3 and site-4 ribozymes spliced from Spfr1 and Spfr2 RNAs and the resulting ligated exons formed the bimolecular spinach aptamer , we inseTetrahymena group I intron, the exonic sequence of which, in the precursor, forms stable secondary and 3D structures in its product form. In this system, the formation of the ligated exons can be monitored readily by the fluorescence of the ligated exons (serving as an aptamer) complexed with the fluorophore, DFHBI. In the precursor RNAs with the full-length spinach aptamer as the exonic sequence, the efficiency of production of the functional ligated exons varied significantly among the four insertion sites of the intron ribozyme tested in this study. This difference was largely resolved in the truncated exonic sequences (Spfr1 and Spfr2), presumably because the truncated exonic sequences no longer formed a stable secondary structure. In the presence of the partner strand to reconstitute the bimolecular spinach aptamer (Spfr1 + Spfr2), the formation of the bimolecular spinach aptamer varied among precursor RNAs. In the site-1 and site-2 RNAs with Spfr2 exons, the partner RNA fragment (Spfr1) may perturb the catalytically active structure of the intron ribozyme or release of the ligated exons from the excised intron may proceed inefficiently. Use of spinach RNA aptamer as an exonic sequence of group I intron yielded a variable phenotype in self-splicing reactions according to the insertion position of the intron. Further studies of this model splicing system, especially from the viewpoint of interplay between two structured RNA elements (spinach aptamer as the exon and group I ribozyme as the intron), would provide valuable information that provides a better understanding of the self-splicing of group I introns.In this study, we constructed a model system to analyze the self-splicing of the The model splicing system constructed in this study is applicable to the development of molecular tools that control the production of functional RNAs in a post-transcriptional manner. In this study, we constructed a bimolecular spinach aptamer with two group I introns A. This sTetrahymena group I intron. The resulting system enables fluorescent monitoring of the self-splicing reaction of the Tetrahymena ribozyme. Although it needs to be further improved mainly through optimization of the insertion site of the intron ribozyme and its P1–P10 elements, this system can be applied in the real-time monitoring of self-splicing reactions not only in test tube but also in living cells. The strategy of this splicing monitoring system would also be applicable not only to other group I intron ribozymes but also to other classes of RNA-based splicing systems such as group II intron ribozymes and pre-mRNA introns excised by spliceosome.In this study, we employed a fluorescent aptamer RNA (spinach RNA) as a model exonic sequence inserted by the


CGG repeats in the 5’UTR of Fragile X Mental Retardation 1 (FMR1) RNA mediate RNA localization and translation in granules. Large expansions of CGG repeats (> 200 repeats) in FMR1, referred to as full mutations, are associated with fragile X syndrome (FXS). Smaller expansions (55–200 repeats), referred to as premutations, are associated with fragile X tremor ataxia syndrome (FXTAS) and fragile X premature ovarian insufficiency (FXPOI). TMPyP4 is a porphyrin ring compound that destabilizes CGG repeat RNA secondary structure. Here we show that exogenous CGG repeat RNA by itself, lacking the FMRP ORF, microinjected into hippocampal neurons is localized in RNA granules and inhibits translation of ARC RNA, which is localized in the same granules. TMPyP4 rescues translation of ARC RNA in granules. We also show that in human premutation fibroblasts with endogenous CGG repeat expansions in the FMR1 gene, translation of ARC RNA is inhibited and calcium homeostasis is disrupted and both phenotypes are rescued by TMPyP4. Inhibition of granule translation by expanded CGG repeats and rescue of granule translation by TMPy4, represent potential pathogenic mechanism and therapeutic strategy, respectively, for FXTAS and FXPOI. CGG repeat sequences have been identified in >200 different RNAs in the human exome . In someMost eukaryotic RNAs undergo conventional translation, which initiates at an AUG start codon at the beginning of the open reading frame (ORF) and terminates at a stop codon at the end of the ORF, resulting in synthesis of the protein encoded by the ORF. RNAs such as FMR1, which contain trinucleotide repeats in the 5’UTR, can also undergo an unconventional type of translation, called repeat associated non-AUG (RAN) translation, which initiates at non-AUG sites in the vicinity of the repeats in the 5’UTR, resulting in synthesis of poly-amino acid RAN translation products encoded by the repeat sequences –11. PathCGG repeats can form secondary structures  by a combination of canonical C(anti)::G(anti) and non-canonical G(syn)::G(anti) base pairing , which mHeterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds to CGG repeats, which are found in multiple different RNAs, including FMR1 RNA –17, and Since FMR1 RNA, and ARC RNA, are both localized and translated in the same RNA granules , and sinSeveral RNAs containing CGG repeats or A2RE sequences, which may be localized in the same granules as FMR1 RNA, encode proteins that regulate calcium homeostasis in the cell . PreviouHere we show that exogenous CGG repeat RNA, microinjected into neurons and endogenous CGG repeat expansions in FMR1 RNA expressed in premutation fibroblasts both inhibit translation of ARC RNA, which is localized in the same granules as FMR1 RNA and serves as a reporter for translation in granules, and that calcium homeostasis is also affected in premutation fibroblasts. Furthermore, TMPyP4 rescues translation of ARC RNA in neurons and in premutation fibroblasts and also rescues calcium homeostasis in premutation fibroblasts.Use of animals as described in this study followed the guidelines of UConn Health and those of the National Institutes of Health for the Care and Use of Laboratory Animals, and was approved under Protocol # 100686–0416 by the Institutional Animal Care and use Committee (IACUC) of UConn Health. UConn Health complies with all applicable provisions of the Animal Welfare Act and other Federal statutes and regulations relating to animals. UConn Health Animal Welfare Assurance number is A3471-01.Skin biopsies were obtained from selected patients at the Waisman Center at the University of Wisconsin—Madison following recruitment, consent and sample collection procedures approved by the University of Wisconsin-Madison Health Sciences Institutional Research Board of the Human Research Protection Program (HRPP). Prior to consent and sample collection potential subjects were screened in person to determine study eligibility. Subjects found to be eligible underwent approved consent procedures, including written informed consent. Skin biopsies were obtained by a physician at the Waisman Center and fibroblasts were isolated from skin biopsies.The University of Connecticut Health Institutional Review Board has specifically exempted the use of human cell lines  in this study from IRB approval because the cell lines are de-identified.Profiles of sequential CGG repeats in target sequences were calculated using a sliding sequence algorithm that compares a query sequence consisting of four sequential CGG repeats to sequential consecutive overlapping 12 nt sequences in the target RNA sequence and calculates homology scores at each position. Perfect homology between the query sequence and the target sequence yields a homology score of 1 at that position. Homology scores < 1 indicate less than perfect homology. Multiple sequential CGG repeats in the target sequence produce a profile of peaks with homology scores of 1 at positions where the CGG repeats in the query and target sequences are aligned, separated by intervening lower scores where the CGG repeats in the query and target sequences are misaligned.http://gwips.ucc.ie).Ribosome profiling is a technique that reveals the probability of ribosomes localized at each position on an RNA molecule . In mostARC RNA and Venus-ARC RNA were prepared by in vitro transcription of linearized plasmid DNA, as previously described ,19. CGG Biotin labeled CGG 0, 30, 62 and 99 RNA ligands prepared by in vitro transcription in the presence of biotin-UTP, were immobilized on a streptavidin Biacore chip, and binding to serial dilutions of TMPyP4 analyte was measured using a Biacore T100 instrument. Sensorgrams were fitted to a heterogenous ligand model to determine binding parameters for specific and non-specific binding of TMPyP4 to each RNA.Hippocampal neurons obtained from C57Bl/6 E19 mouse embryos were grown in culture as described previously . Human fPrimary mouse hippocampal neurons (14 to 21 days in culture) or human fibroblasts, grown in the presence or absence of 1 micromolar TMPyP4  were microinjected using an electronic microinjection system . Injected cells were identified by fluorescence microscopy. Three channel confocal images of injected cells were collected by recording simultaneously in the 488 nm, 560 nm and 633 nm channels using a Zeiss LSM 510 confocal laser scanning microscope with a 63X 1.4 numerical aperture oil immersion objective lens . Images were collected with maximum dynamic range and minimum background in each channel, while avoiding image saturation (pixels >255) or image offset below background (pixels <0). Individual RNA granules were masked and fluorescent intensity for each granule was integrated in each channel after subtracting background fluorescence using Image J. Specific translational activities were calculated by dividing the fluorescence intensity of newly-synthesized Venus-ARC protein, measured in the 488 nm channel, by the fluorescence intensity of Venus-ARC RNA, measured in the 633 nm channel.The numbers of fluorescent RNA molecules in individual granules were determined by FCS ,38. Coun0.Fibroblasts in culture were loaded with Fluo-4 calcium indicator, according to the manufacturer’s protocol (Life Technologies). Bradykinin was bath applied to the cells to induce calcium transients. Fluo-4 fluorescence was recorded by time lapse confocal microscopy after bradykinin addition. Fluo-4 fluorescence in individual cells was quantified by masking each cell and integrating total fluorescence in the cell at each time step. Calcium transients were expressed as F/FFibroblasts were isolated from skin biopsies obtained from selected patients at the Waisman Center at the University of Wisconsin-Madison per Institutional Review Board–approved human subject protocols. The following fibroblast lines were analyzed: FX08-01 (full mutation), FX08-02 (premutation), FX11-01 (full mutation), FX11-02 (premutation), FX13-01 (full mutation), FX13-02 (premutation), WC26 (double premutation), C0603 (control). In addition GM00497 (control) and GM00498 (control) fibroblasts from apparently unaffected individuals were purchased from the Coriell Cell Repository.In the case of FX08-02, FX11-02, FX13-02 and C0603 cell lines the numbers of FMR1 CGG repeats were determined using a PCR-based protocol previously described . The proTCAGGCGCTCAGCTCCGTTTCGGTTTCA) and CGG-R (AAGCGCCATTGGAGCCCCGCACTTCC). Samples were amplified with an initial denaturation step of 98°C for 5 min; 35 cycles of 98°C for 35 s, 64°C for 35 s, and 68°C for 2 min, and a final extension step at 68°C for 5 min. PCR products were resolved on agarose gel electrophoresis. The CGG repeat number was estimated by comparing to DNA sizing ladder visualized on agarose gel electrophoresis.In the case of WC26 and C0603 a different, previously described assay was used to determine the FMR1 CGG repeats . AmplifiExpression of FMRP in fibroblast cell lines was analyzed by Western blotting with antibody to FMRP as described previously ,43.Expansion of CGG repeats in the 5’UTR of FMR1 RNA in premutation cells could potentially increase ribosome density in this region. This has not been tested directly because ribosome profiling has not been performed with premutation cells.KD ~ 0.5 micromolar), presumably corresponding to non-specific binding of TMPyP4 to common nonCGG repeat regions present in each transcript. In addition, CGG 30, 62 and 99 RNAs, exhibit a second set of binding parameters, presumably corresponding to specific binding of TMPyP4 to different number of CGG repeats in each transcript. Apparent on-rates for CGG-specific binding are similar for CGG 30, 62 and 99 RNAs (ka ~ 2 x 104 M-1s-1) but apparent off-rates are slower for CGG99 RNA (kd ~ 2 x 10−6 s-1) compared to CGG30 and CGG62 RNAs (kd ~ 2 x 10−4 s-1). This may reflect tighter binding of TMPyP4 to CGG 99 RNA compared to CGG 30 and CGG 62 RNAs, possibly because of increased numbers of CGG repeats, or alternatively, may reflect rebinding of TMPyP4 to CGG99 RNA during the dissociation phase. In any case, these results indicate that TMPyP4 binds non-specifically to nonCGG repeat sequences and specifically to CGG repeat sequences. Overall the apparent KD for TMPyP4 binding to all RNAs is < 1 micromolar. Accordingly, in subsequent experiments, cells were incubated with TMPyP4 at a final concentration of 1 micromolar to ensure saturation binding to all CGG repeat RNA molecules in the cell.TMPyP4, is a membrane-permeant tetraporphyrin ring compound that binds to, and disrupts, CGG repeat RNA secondary structure ,14. TMPyFMR1 RNA containing CGG repeats is localized and translated in RNA granules . To deteThe experimental protocol outlined To quantify specific translational activities in individual granules, Venus-ARC RNA and newly-synthesized Venus ARC protein were measured in multiple granules, with and without detectable CGG99 RNA  photobleaching  37,38].,38.37,38In the absence of TMPyP4  panel B,In the absence of TMPyP4 the range of values for the ratio of CGG99 RNA to ARC RNA in individual granules 1–10) is narrower than the range of values for translational activities in CGG99 containing granules (0.01–5)  in granules that: 1) regulate translation; 2) are affected by CGG repeats; and 3) whose effects are reversed by TMPyP4. Here we will discuss three potential mechanisms involving components that meet these criteria: hnRNP A2, ribosomes, and EIF2AK2,.The first potential mechanism involves hnRNP A2, which binds to CGG repeat RNA –17 and iThe second potential mechanism involves ribosomes, which are required for translation of ARC RNA and also accumulate in the vicinity of CGG repeats. Ribosome profiling reveals increased ribosome density in the vicinity of CGG repeats in the 5’UTR of FMR1. If the number of CGG repeats is expanded, the ribosome density in this region could be further increased, although this has not been tested experimentally. Increased ribosome density in the CGG repeat region might reduce the number of ribosomes available for translation of ARC RNA in the same granule. The magnitude of such an effect would depend on the relative number of ribosomes associated with expanded CGG repeats in the 5’UTR of premutation FMR1 RNA molecules compared to the total number of ribosomes in each granule. In this regard, single molecule imaging reveals that granules with increased numbers of RNA molecules do not have increased numbers of translational events per granule , which iThe third potential mechanism involves EIF2AK2, eukaryotic translation initiation factor 2 alpha kinase 2, a serine/threonine protein kinase activated by dsRNA that inhibits conventional translation by phosphorylating translation initiation factor EIF2alpha . EIF2AK2The above, and other, potential mechanisms could be distinguished by knocking out or inhibiting specific components that are: localized in the same granules as FMR1 RNA, regulate translation of other RNAs in the same granule, are potentially affected by CGG repeat expansions in FMR1 RNA, and whose effects are potentially reversed by TMPyP4. However, without such experimental validation it is not possible to identify specific molecular mechanism(s) for the observations reported here.The human exome contains multiple different CGG repeat RNAs besides FMR1 RNA and multiple different A2RE RNAs besides ARC RNA, some of which may be localized in the same RNA granules as FMR1 RNA because CGG repeat sequences and A2RE sequences both bind to hnRNP A2, which mediates granule assembly. This may create an intra-granule translational regulatory network, where expansion of CGG repeats in one RNA increases the overall CGG repeat burden in the granule, thereby decreasing translation of other RNAs in the same granule. Since CGG repeat RNAs and A2RE RNAs encode multiple different proteins that mediate multiple different cellular functions, regulating translation of these RNAs in granules could have pleiotropic effects on the cell. In this regard, expression of a CGG repeat transgene is known to affect translation of multiple different RNAs in mice and flies , which cCalcium homeostasis is a complex cellular process mediated by multiple different proteins, some of which are encoded by CGG repeat and/or A2RE RNAs. For example: CGG repeat RNAs such as: CACNA1A  and KCNMA1 , could potentially affect calcium homeostasis and A2RE RNAs such as CAMK2A  and NRGN  could also potentially affect calcium homeostasis. If calcium homeostasis is mediated by proteins encoded by CGG repeat and/or A2RE RNAs that are localized in granules and translationally regulated by CGG repeat RNA this could explain why calcium homeostasis is disrupted in premutation fibroblasts from individuals with CGG repeat expansions in the endogenous FMR1 gene. However it is difficult to identify one specific gene or group of genes that mediates the calcium phenotype in premutation fibroblasts because multiple different CGG repeat and/or A2RE RNAs encode proteins that could potentially affect calcium homeostasis and TMPyP4 can potentially affect multiple different pathways in the cell.TMPyP4 binds to CGG repeat RNA and rescues granule translation and calcium homeostasis. The effect of TMPyP4 on calcium homeostasis may be secondary to the effect on granule translation if translation of RNAs that mediate calcium homeostasis is inhibited by CGG repeat RNA and rescued by TMPyP4. However, it is also possible that TMPyP4 affects calcium homeostasis directly. Previous studies have shown that TMPyP4 can induce calcium release by direct interaction with the Ca2+ release protein from sarcoplasmic reticulum . FurtherThe observation that granule translation and calcium homeostasis are dysregulated in premutation fibroblasts indicates that pathogenic effects of CGG repeat expansions in FMR1 (and possibly other CGG repeat genes) are manifested in non-neuronal cell types. In this study premutation fibroblasts that exhibit dysregulated granule translation and calcium homeostasis were obtained from apparently healthy young female premutation carriers with no apparent neurological or neuromuscular symptoms. This indicates that disrupted granule translation and calcium homeostasis in human fibroblasts may provide early preclinical biomarkers for identifying individuals at increased risk to develop late onset neurological or neuromuscular disorders such as FXTAS, FXPOI or other CGG repeat disorders. Chronic, sustained disruption of granule translation and/or calcium homeostasis may be deleterious to the cell over the long term and may contribute to late onset development of neurological or neuromuscular pathology in these individuals. Restoring normal granule translation and/or calcium homeostasis with TMPyP4 could potentially reduce the risk of subsequent onset of these disorders in premutation carriers. Inhibition of translation in granules by CGG repeat expansions in FMR1 RNA represents a potential pathogenic mechanism for CGG repeat expansion disorders such as FXTAS and FXPOI. Rescue of translation in granules by TMPyP4 could potentially ameliorate pathogenic effects in CGG repeat expansion disorders.


The finding that RNA modules can provide direct IRES-ribosome interaction suggests that linking these motifs to additional sequences able to recruit trans-acting factors could be useful to design synthetic IRESs with novel activities.Internal ribosome entry site (IRES) elements are RNA regions that recruit the translation machinery internally. Here we investigated the conformational changes and RNA dynamics of a picornavirus IRES upon incubation with distinct ribosomal fractions. Differential SHAPE analysis of the free RNA showed that nucleotides reaching the final conformation on long timescales were placed at domains 4 and 5, while candidates for long-range interactions were located in domain 3. Salt-washed ribosomes induced a fast RNA local flexibility modification of domains 2 and 3, while ribosome-associated factors changed domains 4 and 5. Consistent with this, modeling of the three-dimensional RNA structure indicated that incubation of the IRES with native ribosomes induced a local rearrangement of the apical region of domain 3, and a reorientation of domains 4 and 5. Furthermore, specific motifs within domains 2 and 3 showed a decreased flexibility upon incubation with ribosomal subunits  Conformational transitions could be spatially and temporally tuned to achieve distinct functions enabling complex ribonucleoproteins (RNPs) to be assembled in a hierarchical ordered manner. In addition, RNA conformational changes occurring over broad timescales can range from local rearrangements in motifs involved in tertiary interactions to large global remodeling in the orientation of helices3.The function of RNA molecules depends on their three-dimensional (3D) structure5. As such, distinct RNA local nucleotide dynamics can be detected by computing the differential reactivity obtained using slow- and fast-reacting selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagents. For instance, isatoic anhydride (IA) is a slow reagent  while 1-methyl-6-nitroisatoic (1M6) is a fast reagent . Nucleotides with enhanced reactivity toward IA are usually in the rare C2′-endo conformation, experience slow local dynamics, and in some cases, govern the folding of entire RNA domains4. 1M6 is an IA derivative that stacks with RNA nucleobases with one face available for stacking, which occurs in long-range interactions, bulges, turns, or at the termini of some helices4. On the other hand, 1-methyl-7-nitroisatoic (1M7) is a fast-reacting compound6 that, unlike 1M6, does not stack with nucleobases7. The RNA reactivity toward these compounds provides information on nucleotides that undergo local conformational changes on long timescales (IA) and those involved in tertiary interactions (1M6)8, hence time-dependent RNA-ligand interactions.Nucleotides involved in tertiary interactions may have unusual backbone or stacking geometries or undergo conformational changes on different timescales7G(5′)ppp(5′)N structure (designated cap) placed at the 5′ end of most mRNAs9. This process begins with the binding of the translation initiation factor (eIF)-4F complex to the cap. This complex recruits the 40S ribosomal subunit bound to eIF3, eIF2, and the initiator tRNA, and scans the 5′UTR until an AUG triplet is found in the appropriate context to start protein synthesis. Joining of the 60S ribosomal subunit follows this step, producing a translation competent complex. However, specific mRNA regions referred to as internal ribosome entry sites (IRES), can recruit the 40S ribosomal subunit through a cap-independent mechanism (reviewed in10).Translation control is a key step in gene expression regulation in all organisms. In eukaryotes, the vast majority of mRNAs initiates translation by a cap-dependent mechanism that depends on the recognition of the m11. However, different IRES elements perform the same function despite lacking conservation of primary sequence, secondary RNA structure, and host factor requirement to recruit the ribosomal subunits12. Internal initiation of translation can occur either by interaction of the IRES with the 40S ribosomal subunit, or by binding to initiation factors, which mediate the recruitment of the 40S ribosomal subunit. For instance, the dicistrovirus intergenic region (IGR) or the hepatitis C (HCV) IRES physically associate the 40S subunit in vitro14. Nevertheless, there are notable differences among these IRESs. Whereas the IGR assembles a complex with 80S ribosomes in the absence of eIFs16, the HCV IRES, and also those of picornaviruses, require different combinations of eIFs to assemble 48S complexes in vitro18.RNA structure determines the function of viral IRES elements11. Although RNA probing data revealed a stable secondary structure of the FMDV IRES, the nucleotides involved in the dynamic folding and in tertiary interactions of this regulatory element remain unknown. Moreover, although it is well established that domains 4 and 5 provide the binding site for initiation factors, the specific role of domains 2 and 3 is less well understood, despite containing binding sites for proteins such as PTB or Ebp1. Indeed, it was hypothesized that domains 2 and 3 could contribute to promote the specific IRES conformation critical for the interaction with the translation machinery19.The picornavirus IRES elements are prototypes for understanding the mechanism of ribosome recruitment due to their requirement of factors for translation initiation. Previous structural analyses have shown that the IRES of encephalomyocarditis virus (EMCV) and foot-and-mouth disease virus (FMDV) are arranged in structural domains Here we have investigated the RNA conformational flexibility and RNA dynamics of the FMDV IRES by differential SHAPE in the presence of various components of the cellular translation machinery. We show that the ribosomal subunits induced fast structural changes on domains 2 and 3, whereas host factors mostly induced slow structural changes on domains 4 and 5. Consistent with our results, analysis of the mRNA association to ribosomal subunits inside cells indicated that an IRES-containing mRNA remained bound to both subunits more prominently than an mRNA lacking the IRES.4. Hence, the slow reagent IA provided information on nucleotides that undergo local conformational changes on long timescales5, and the fast reagent 1M6 informed of nucleobases that achieve their conformation on short timescales4. The RNA local flexibility of the free IRES was first analyzed using the normalized reactivity (mean ± SD) towards IA (n = 9) and 1M6 (n = 5) 21. The elongation factor eEF2 was present in all fractions, although it was significantly reduced in RSW. Furthermore, the initiation factors eIF2α, eIF4B, eIF4G, and eIF4E were detected in S30, S100, F, and R fractions to different extents, but not in RSW, showing that the high salt washing effectively removed factors from the ribosomes. In addition, we evaluated the presence of the IRES-interacting factors PTB, Ebp1 and Gemin5 in the cellular fractions. The three proteins were detected in S30, S100, F, and R fractions with different intensity. Similarly to the analyzed eIFs, these factors were strongly reduced in RSW.Next, we prepared cellular fractions with distinct content of ribosomes Fig. . Brieflyp < 0.05), and absolute values D > 0.2 were taken into consideration. The differences of SHAPE reactivity with free RNA rendered positive values (more accessible residues), and negative values (less accessible residues). In this study we focused on the nt positions that decrease their reactivity, which could be the result of either a more constrained RNA structure or protein/ribosome interaction. Conversely, analysis of nt with increased reactivity would render positions with more relaxed conformation. The results indicated that incubation of the IRES transcript with S100 did not produce significant differences relative to the free RNA with any of these compounds, IA or 1M6 , using IA (slow reagent) and 1M6 (fast reagent). The effect on the local RNA flexibility was determined computing the SHAPE difference, subtracting the normalized reactivity obtained for the free RNA to that obtained for the IRES incubated with the different fractions. In all cases, data represent the mean ± SD from at least 3 independent experiments, and only the statistically significant SHAPE differences  Fig.  prior trThese results prompted us to analyze if ribosome-associated factors could be responsible for the conformational changes observed on the domains 4 and 5 upon incubation with R extract Fig. . To testCollectively, these results illustrate unique structural features of the IRES element. First, the ribosome free of factors could induce modifications on the conformational flexibility of domains 2 and 3 of the IRES element (the 5´end domains). Second, the cellular factors associated to native ribosomes, as illustrated for eIF4G, eIF4B, PTB, Ebp1 or Gemin5, could be responsible for the changes in domains 4 and 5 (the 3´end domains).22.The observation that the ribosome fraction free of factors (RSW) induced reactivity changes on the IRES altering the local flexibility of domains 2 and 3 Fig.  promptedThe IA and 1M6 reactivity profiles (mean ± SD) obtained for the IRES RNA incubated with 40S and 60S are shown in Supplementary Fig. in vitro, we decided to investigate these results using an independent approach. Hence, we studied if an mRNA that contained a functional IRES element could remain associated to the ribosomal subunits in the cellular context in the absence of associated-ribosome factors in comparison to a cap-mRNA. Thus, ribosomal subunit association was analyzed using two reporter transcripts that only differ in the presence of the IRES element , and also that the IRES-containing mRNA remained bound to ribosomal subunits more efficiently than the cap-mRNA.11, the RNA dynamics of this regulatory element remain to be elucidated. In addition, since we found that ribosomes and ribosome-associated factors interact with different domains of the IRES in a time-dependent manner, we decided to analyze the RNA dynamics for the free IRES and the differences relative to its ligand-bound states (R and RSW). This was performed calculating IA-1M6 differences, as well as 1M6-1M74.Although the secondary structure of the FMDV IRES has been reported7. The statistically significant reactivity differences 1M7-1M6  modified the conformational flexibility of domains 2 and 3 of the IRES element (the 5´end domains)  Fig. . Second,ns) Fig. . Taken t2. Our study reveals that IRES-R and IRES-RSW complexes affect differentially the RNA dynamics of the IRES region. Thus, the ligand-free RNA resembles the RSW RNA dynamics  by the EcoRI-BamHI fragment of pGEM-IRES43. The plasmid pCAP-luc was generated in two steps. First, the BamHI site was substituted by EcoRI site in plasmid Tagged-IRES using the QuikChange mutagenesis procedure (Agilent Technologies) with the primers 5′-TTTTTGGCGTCTTCCATGAATTCTCGAGCTCAGGGTCATT and 5′-AATGACCCTGAGCTCGAGAATTCATGGAAGACGCCAAAAA. Then, ligation of the EcoR1 digested plasmid generated the pCAP-luc construct. The sequences were verified by DNA sequencing (Macrogen).The construct expressing the monocistronic IRES RNA was previously describedin vitro synthesized RNA was folded by heating at 95 °C for 2 min, snap cooling on ice for 2 min, and subsequently incubated in a final volume of 18 μl of folding mix  for 20 min at 37 °C40. Prefolded RNA (2 pmol) was incubated with 6.5 mM 1-methyl-7-nitroisatoic anhydride (1M7), 1-methyl-6-nitroisatoic anhydride (1M6) or isatoic anhydride (IA) (Thermo-Fisher) for 70 s, 3 min or 36 min, respectively44 at 37 °C. Untreated RNA was incubated with DMSO. Treated and untreated RNAs were precipitated and finally resuspended in 10 μl of 0.5x TE.Prior to treatment, 30 or ribosomal subunits during 10 min at 37 °C. RNA-S100 complexes were assembled using 1 µg of total protein. Then, RNA alone or incubated with the indicated fractions was treated with IA, 1M6, or 1M7.For differential SHAPE probing of RNA-ribosome, RNA-40S or RNA-60S subunits, complexes were assembled in folding buffer in the presence of 4-fold ribosome2, 7.5 mM DTT), 10 U RNase OUT (Thermo-Fisher), 1 mM each dNTP and 60 U of Superscript III RT (Thermo-Fisher). Reverse transcriptase reactions were performed during 30 min at 52 °C, followed by 15 min at 70 °C. Primer extension products were resolved by capillary electrophoresis. The 5′-FAM-TAGCCTTATGCAGTTGCTCTCC primer was used for the sequencing ladder, using 2.5 pmol of untreated RNA in the presence of 0.1 mM ddCTP, 30 min at 52 °C RT reaction45.Treated and untreated RNA (2 pmol) was incubated with the fluorescent primer 5′-NED-TAGCCTTATGCAGTTGCTCTCC (0.1 µM) at 65 °C for 5 min, then at 35 °C 5 min, and 4 °C 1 min. Primer extension reactions were conducted in a final volume of 16 μl containing reverse transcriptase (RT) buffer  were subtracted from the corresponding  treated RNA to obtain the net reactivity for each nucleotide for the free RNA, or RNA incubated with S100 fraction, native ribosomes (R), salt-wash ribosomes (RSW), factors disassociated from the native ribosomes (F), or the ribosomal subunits (40S) and (60S). Quantitative SHAPE reactivity for individual datasets were normalized to a scale spanning 0 to 2 in which 0 indicates an unreactive nucleotide and the average intensity at highly reactive nucleotides is set to 1. Data from at least 3 independent assays were used to calculate the mean (±SD) SHAPE reactivity [see Dataset SHAPE electropherograms were analyzed using QuSHAPE softwaret-test. Only nucleotide positions with absolute difference (D) ≥ 0.2 arbitrary units and p value < 0.05 were considered statistically significant. RNA secondary structures were visualized with VARNA.For differential SHAPE analysis, the normalized mean 1M6 reactivity was subtracted to IA or 1M7 mean reactivity. The statistical significance of the SHAPE reactivity data obtained under different conditions from at least 3 independent experiments was determined by the unpaired two-tail Student´s 47 with little modifications. Briefly, HEK293 cells grown to 90% confluence were lysed in buffer 1 ). Cell debris was discarded by spinning at 14000 g 10 min 4 °C, twice. The supernatant of the second spinning is the S30 fraction. S30 centrifugation at 95000 rpm during 1.5 h using the TLA100.3 rotor yielded the S100 fraction (supernatant), and the ribosomes plus associated factors (pellet). The ribosomes pellet was resuspended in buffer 3  to yield the R fraction. To prepare the fraction containing ribosomes free of associated factors, the ribosomal pellet was dissolved in high-salt buffer 2 , loaded in a discontinuous sucrose gradient, centrifuged at 4 °C 95000 rpm 2 h using a TLA100.3 rotor. The pure ribosomes pellet (RSW) was resuspended in buffer 3. The supernatant of the ultracentrifugation was dialyzed against to buffer 3 to prepare the F fraction containing the factors that disassociated from the ribosomes. The total protein content in S30, S100, and F fractions was measured by the Bradford assay; the ribosome concentration in R and RSW fractions was determined as 14 units A260 = 1 mg/ml.HEK293 cells were maintained in standard conditions in Dulbecco’s modified Eagle’s medium (DMEM). The S30, S100, R and RSW fractions were obtained as described48. Appropriate secondary antibodies (Thermo-Fisher) were used according to the manufacturer instructions. Protein signals were visualized with ECL plus (Millipore). Quantification of the signal detected was done in the linear range of the antibodies.Proteins from cell lysates were separated by SDS-PAGE and probed with the indicated antibodies. Commercial antibodies were used to detect eIF4E (Transduction laboratories), RACK1, eIF2α, eIF4G (Santa Cruz), eEF2 , eIF4B, and Gemin5 (Novus). The ribosomal proteins P0, P1 and P2 were detected with the monoclonal antibody 3BH547. A western blot analysis of the 40S and 60S purified subunits is shown in Supplementary Fig. 22.Ribosome dissociation profiles were prepared from HEK293 cells, as described47.HEK293 monolayers (about 70% confluent) were transfected with the plasmid pCAP-luc or pIRES-luc using lipofectamine (Thermo-Fisher). Cell lysates were prepared 24 hpt in lysis buffer  for determination of luciferase activity, or in buffer B for RNA-ribosomal subunits association assays, as describedIRES or CAP activity was quantified as the expression of luciferase normalized to the amount of protein (determined by Bradford assay) in the lysate obtained from transfected HEK293 cell monolayers. Each experiment was repeated independently at least three times. Values represent the mean ± SD.http://bioinfo.ut.ee/primer3-0.4.0/primer3/) and tested for amplification efficiency. qPCR was carried out with GoTaq qPCR Master Mix (Promega) according to the manufacturer’s instructions on an ABI PRISM 7900HT Fast Real-time PCR system (AppliedBiosystems) using primers specific to Luciferase mRNA (5′-TGGCAGAAGCTATGAAACGA and 5′-ATAAATAACGCGCCCAACAC). Values were normalized against an endogenous control mRNA RPL11 (5′-GTGCTGGGTAGGCCAG and 5′- TTCTGCTGGAACCAGCG). The comparative cycle threshold (CT) method49 was used to quantify the results.Total RNA was isolated from transfected cell lysates and from dissociation gradient fractions corresponding to 40S and 60S peaks using TRIzol reagent (Thermo-Fisher). Equal amount of RNAs were used to synthesize cDNA using SuperScript III (Thermo-Fisher) and hexanucleotide mix (Merck) as primer for the reverse transcription reaction. Primers for Quantitative polymerase chain reaction (qPCR) were designed . This approach computes a combined energy score of the interaction as the sum of the free energy of hybridization and the free energy required for making the interaction sites accessible.IntaRNA50 imposing SHAPE reactivity values as pseudo-free energy change constraints together with nearest neighbor thermodynamic parameters using -0.8 kcal/mol and 2.6 for the intercept (b) and slope (m), respectively51. These secondary structures were used as input in RNAComposer52 to model the 3D RNA structure using default parameters. 3D structures graphics were performed with the UCSF Chimera package53.The RNA secondary structure of the IRES element under different conditions was predicted using RNAstructureSupplementary InformationDataset1Dataset 2Dataset 3