S. cerevisiae correctly detected all of the 43 known pseudouridines in yeast 18S and 25S ribosomal RNA with high specificity. Moreover, application of PSI-seq to the yeast transcriptome revealed the presence of site-specific pseudouridylation within dozens of mRNAs, including RPL11a, TEF1, and other genes implicated in translation. To identify the mechanisms responsible for mRNA pseudouridylation, we genetically deleted candidate pseudouridine synthase (Pus) enzymes and reconstituted their activities in vitro. These experiments demonstrated that the Pus1 enzyme was necessary and sufficient for pseudouridylation of RPL11a mRNA, whereas Pus4 modified TEF1 mRNA, and Pus6 pseudouridylated KAR2 mRNA. Finally, we determined that modification of RPL11a at Ψ -68 was observed in RNA from the related yeast S. mikitae, and Ψ -239 in TEF1 mRNA was maintained in S. mikitae as well as S. pombe, indicating that these pseudouridylations are ancient, evolutionarily conserved RNA modifications. This work establishes that site-specific pseudouridylation of eukaryotic mRNAs is a genetically programmed RNA modification that naturally occurs in multiple yeast transcripts via distinct mechanisms, suggesting that mRNA pseudouridylation may provide an important novel regulatory function. The approach and strategies that we report here should be generally applicable to the discovery of pseudouridylation, or other RNA modifications, in diverse biological contexts.We developed a novel technique, called pseudouridine site identification sequencing (PSI-seq), for the transcriptome-wide mapping of pseudouridylation sites with single-base resolution from cellular RNAs based on the induced termination of reverse transcription specifically at pseudouridines following CMCT treatment. PSI-seq analysis of RNA samples from  There is much still to be discovered about the post-transcriptional control of gene expression S. cerevisiaeWhile nucleotide modifications in non-coding RNAs are well known, relatively little is known about possible modifications in mRNAs, and even less is known about what roles these modifications could play. The only established nucleotide modifications in mRNAs, outside of the 5′-cap complex, are N6-methyladenosine Three transcriptome-wide screens for a second covalent modification of mRNA bases - cytosine 5-methylation – have identified about 10,000 such sites in human mRNAs S. cerevisiae, many pseudouridines are present in tRNAs, nearly 50 pseudouridines have been found in rRNA, and 8 have been identified in snRNAs The ability to identify modification of mRNAs at specific sites opens a search for a potential new molecular mechanism by which gene expression could be regulated. Are there other modified based in mRNAs, in addition to N6-methyladenosine and 5-methylcytosine? The single most abundant modified RNA base is pseudouridine (Ψ), a structural isomer of uridine that has been found in tRNAs, rRNA, and snRNA Most of the known RNA modifications are constitutive, but a few pseudouridines can be induced in snRNAs in yeast under certain stress conditions–specifically, in heat shock and in stationary phase In this study, we developed pseudouridine site identification sequencing (PSI-seq), a high-throughput sequencing technique based on the CMCT reverse-transcription stop assay to identify pseudouridines in mRNAs, which we used to systematically characterize the pseudouridine content of the yeast transcriptome.Our strategy for identifying for pseudouridylated RNAs was based on the ability of chemical treatment with CMCT to modify pseudouridines specifically , and thein vitro from amplified copies of all annotated yeast transcripts, 3. A library prepared identically to library 2, but omitting CMCT treatment. For bona fide sites of pseudouridylation, we would expect to see a surplus of sequencing reads representing cDNA products with 5′-ends one base upstream of a pseudouridine in the corresponding yeast RNA molecule, exclusively in the library made from CMCT-treated natural yeast RNA. Because some pseudouridines in noncoding RNAs are specifically induced by heat shock or in stationary phase, we analyzed two samples of RNA isolated from log phase yeast and one each from heat shocked yeast and yeast in stationary phase, and corresponding controls.We isolated RNA from yeast grown in rich media to log phase, and from this RNA, we prepared and sequenced four libraries. The first was made using the pseudouridine-specific CMCT/alkali protocol as described above using natural RNA isolated from yeast; three control libraries were as follows: 1. A library produced by the identical procedure, with the exception that CMCT treatment was omitted (mock control), 2. A library prepared by the procedure described above, starting with RNA transcribed p-values <10−15) and were better correlated than the values from non-pseudouridines, demonstrating significant reproducibility of the results between independent samples  selection, enabled us to use the well-mapped pseudouridines in rRNA as a set of internal positive controls. For each nucleotide in the 18S or 25S ribosomal RNA , we calculated the ratio of reads ending one nucleotide upstream in the library from CMCT-treated yeast RNA, to corresponding reads in the library from the same yeast RNA without CMCT treatment. We would expect sites of pseudouridylation to have the highest values for this ratio, and this is what we found –for log-phase replicate #1 and for the heat shock condition, all 43 of the known pseudouridine sites in rRNA were among the top 60 nucleotides with the highest values for this ratio, whereas 30/43 pseudouridines were included in the top 60 sites for log-phase replicate #2 (38/43 in the top 120 sites) and 25/43 for the stationary phase sample (30/43 in top 120) . These d samples . In the  samples . Due to We next considered the possibility that site-specific pseudouridines may occur in mRNAs. In a genome-wide comparison of relative frequencies of individual nucleotides immediately downstream of sites where reverse transcription of yeast RNAs stopped, we observed outliers represented by many more reads in the libraries from CMCT-treated RNA than in the –CMCT library . Most of2-transformed number of reads terminating at a specific nucleotide in the CMCT-treated sample, as a function of the log2-transformed number of reads terminating at that nucleotide in the mock-treated RNA sample. For each nucleotide, we calculated the difference between the observed and model-predicted values for the +CMCT condition, and divided this quantity by the standard deviation of the observed vs. predicted residuals. We then ranked the nucleotides based on this “score”. We considered a nucleotide to be a candidate pseudouridylation site if its score exceeded a threshold defined as the highest score observed for any rRNA nucleotide that was not identical or adjacent to a known pseudouridylation site, corresponding to a false positive rate of about 0.0002. In the data from log phase yeast, 41/43 (replicate 1) and 20/43 (replicate 2) of the known rRNA pseudouridines scored above this threshold, and in the data from heat-shocked yeast, 38/43 rRNA pseudouridines exceeded this threshold. To further filter the candidate pseudouridylation sites, we excluded nucleotides with scores that were not at least four-fold greater (i.e. a difference in log2 scores of more than 2.0) than the corresponding “score” obtained for the in vitro transcribed RNA control data (where the block to reverse transcription cannot be ascribed to a pseudouridine), and positions derived from non-uridine bases were also eliminated , due to the vast number of possible nucleotides evaluated from throughout the transcriptome (2(+CMCT/−CMCT) scores in other conditions, suggesting that differences in identification of these sites are at least partly due to the precise classification thresholds we adopted (th nucleotide in the RPL11a coding sequence (RPL11a nt 68), and the 239th nt in the coding sequence of TEF1/2. Because the two strongest candidate pseudouridine sites derived from two genes with clear functional roles in translation, we performed gene ontology (GO) analysis to search for functional themes that were enriched among the genes we identified as harboring pseudouridylation sites. Interestingly, significant GO term enrichment for the category “cytoplasmic translation” was observed for the set of genes with candidate sites from log-phase growth  as well as heat-shock , suggesting that pseudouridylation may provide a mechanism for coordinately regulating these functionally related mRNAs. However, it is also worth noting that PSI-seq analysis is inherently biased towards detecting sites in more abundant transcripts (due to sampling effects), which may be a confounding factor for GO analysis.To systematically identify candidate pseudouridylation sites, we calculated a best-fit linear model for the logiminated . After tcriptome , Table 1 adopted . Overall adopted . NotablyThere was no discernable bias in the location of putative pseudouridines relative to canonical features of mRNAs  - matched a six nucleotide sequence containing a known pseudouridine–nt 44 in U2 snRNA, whose synthesis is catalyzed by the pseudouridine synthase Pus1p UCGA, which contains nt 239 (underlined), matches a sequence in the TΨC loop in tRNAs that includes the eponymous, highly conserved pseudouridine. The TΨC loop pseudouridylation is catalyzed by Pus4p We searched the putative pseudouridylation sites for sequences that might direct modification by known tRNA pseudouridine synthases. We focused initially on the two strongest candidates from our sequencing data: nt 68 in RPL11a and nt 239 in TEF1/2. A six nucleotide sequence containing nt 68 in RPL11a - UCUGin vitro with unmodified NTPs, served as negative control templates, while synthetic RNAs containing pseudouridine at the predicted, in vivo sites served as positive controls. We observed the predicted RT stops in both RPL11a and TEF1 RNAs isolated from wild type yeast , pus1Δ or pus4Δ yeast with a CMCT RT-stop assay using fluorescently-labeled gene-specific primers, and evaluating the primer extension products by capillary electrophoresis. The corresponding RNA sequences, transcribed pe yeast . With a Based on standard curves constructed by applying the RT-stop assay to mixtures of the negative and positive controls at known ratios  by nickel affinity chromatography from yeast strains in which each was overexpressed. The partially purified proteins  specifically for CMCT-dependent RT stops at the candidate sites, to try to identify more mRNA pseudouridines , 20 nt (R20), 10 nt (R10) surrounding the in vivo modification site, or just the 6 nt motif that matches the site of modification in U2 snRNA (R6). For TEF1, the sequences we tested contained 55 nt (T55) or 20 nt (T20) from the sequence surrounding the in vivo TEF1 pseudouridine, or just the 6 nt motif that matches the site of modification in tRNAs (T6). Previous work showed that modification by Pus4 requires a stem-loop structure in its target RNA To better define the sequences required to specify pseudouridylation of mRNAs we synthesized 75 nt RNA oligonucleotides and assayed them for UU with UCUGCU , UCUGUA (R55-1A), AGCGUU (R55-AGC), and AGCGUA (R55-AGC1A). In the T6-ds substrate, we replaced the sequence GUUCGA with GUCCGA , GUUUGA (T6-ds-1U), AUUCGA (T6-ds- -2A), and AUUUGA (T6-ds-1U-2A). Apart from the negative controls, each of the tested mutations was synonymous. The negative controls, as expected, showed no evidence of pseudouridylation in our in vitro assay , we compared the rate of CMC-dependent stop at every 7-mer with U in the 4th position. We could use these data to look at enrichment of CMC-dependent stops in specific 5- and 6-mers as well. Compared to all possible 6-mers with U in the 3rd position, GUUCGA sites are somewhat enriched for CMCT dependent stops in the heat shock data . Compared to all possible 5-mers with U in the 4th position, CUGUU are also somewhat enriched for CMCT dependent stops in the heat shock data . However, neither of these oligomers were enriched for CMCT-dependent stops in the log phase data. These data support the idea that these sequences alone do not direct modification of specific sites in the transcriptome, but they may play a part in directing pseudouridylation.The S. cerevisiae were conserved in other species. First, we aligned the sequences for RPL11a mRNA and TEF1 with their available mRNA orthologues in eight fungal species  , the first U is conserved in all seven species, the nucleotides in positions 2-6 are conserved in six species, and the final U is conserved in four species. In TEF1's orthologues, the entire six-nucleotide motif (GUUCGA) is conserved in all seven species, except in C. albicans, where the first G is replaced with an A.Having identified sites of modification in Saccharomyces mRNAs, we investigated whether the mRNA pseudouridines we identified in S. mikitae, S. pombe, and S. cerevisiae  may have escaped detection. The pseudouridines we identified may therefore be only the “tip of the iceberg” in terms of the broader landscape of transcriptome-wide chemical diversity of RNA.Covalent modifications of non-coding RNAs have been recognized for more than 50 years, but have rarely been investigated at the genome-wide level in vivo in knockout mutant strains lacking Pus1, Pus4 and Pus6, respectively. We confirmed the roles of these three enzymes biochemically by recapitulating the reactions in vitro, using partially purified enzymes and synthetic substrates.Because of the versatility of the snoRNA programmed H/ACA snoRNP system, we initially considered it a prime suspect for the mRNA modifications. All of the modifications we characterized in detail, however, were catalyzed by pseudouridine synthases that were previously thought to modify only tRNAs and snRNAs at specific sites. We found three such enzymes that introduce pseudouridines into mRNA in yeast, based on the loss of specific pseudouridine modifications While numerous pseudouridines and other covalent modifications of noncoding RNAs have been appreciated for many years, their specific functions are, in most cases, still undefined. We eagerly sought to uncover the functional roles of the pseudouridine sites we identified in yeast mRNAs, but a clear role for these modifications in regulating gene expression has remained elusive. Given the strong functional coherence of the pseudouridylated mRNAs involved in translation and protein biosynthesis, as well as the extensive evolutionary conservation of the modifications to RPL11a and TEF1, we suspect that the pseudouridylation events we have uncovered are likely adaptive. Although we were unable to detect any clear effects of these pseudouridylation events on mRNA stability, translation rate, or protein coding potential, we nevertheless anticipate that future investigations into the functional consequences of these RNA modifications will shed light on this intriguing possibility. Considering the growing awareness of the previously unappreciated chemical diversity of cellular mRNAs, it is tempting to speculate that extensive combinations of covalent mRNA modifications may represent an additional layer of post-transcriptional regulatory information modulating the transcriptome, analogous to the way that DNA and histone modifications epigenetically control genome function.The PSI-seq method we developed to search for pseudouridines in RNA should be broadly applicable to finding such sites in other species, and for comprehensive identification of the RNA targets of specific pseudouridine synthases. Although we have only so far discovered mRNA pseudouridines in fungi, there is no reason to believe they are unique to these species. Indeed, the presence of pseudouridine modifications in the regulatory non-coding RNA, SRA, in humans 600 of 0.6-0.8 for “log phase” yeast. For heat shock experiments, cells were grown to an OD600 of 0.6-0.8, harvested by centrifugation at 3000 x g for 5 min, resuspended in YPD media preheated to 45°C, and then incubated at 45°C for an additional 30 minutes. For stationary phase experiments, cells were grown to an OD600 of 0.6-0.8 at 30°C, then grown for another ∼24 hours to an OD600 of ∼4-6. TAP-tagged yeast strains were derived from BY4741 (Open Biosystems Cat# YSC1177). Purification of Pus proteins was done using moveable ORF strains (Open Biosystems). Knockout strains were a gift from the Davis lab. Pus knockout, TAP-tag strains were made by genomically inserting TAP tags into the Pus knockout strains, then selecting on his- plates, according to standard yeast genetics techniques.Unless otherwise stated, the strain used in this study was BY4741 grown in YPD at 30°C to an ODAny oligonucleotides used in this study are in 2x1L of yeast were grown to log phase. Half of each liter was harvested immediately, while the other 500 mL of one L was heat shocked, and 500 mL of the other liter was taken to stationary phase. The cells were harvested by centrifugation, and the pellet was washed in 5 mL ice-cold TES buffer , then resuspended in 1–2 mL TES buffer. An equal volume of acid phenol-chloroform was added to each resuspended pellet, then the mixture was heated to 65°C for one hour with vortexing every 10 minutes. The mixtures were spun for 5 min at 4°C, then the aqueous phase was isolated. An equal volume of chloroform was added, the mixtures were vortexed and spun down for 5 min at 4°C. The aqueous phase was isolated, 1/10 volume of 3 M NaOAc and an equal volume of isopropanol were added, and the RNA was precipitated and resuspended in 1–2 mL water. For each condition, 300 µg of RNA was subjected to two rounds of poly(A) purification using the Poly(A) Purist MAG kit (Ambion), giving about 3–6 µg of poly(A) RNA. The poly(A) purified RNA was isopropanol precipitated and resuspended in 20 µL water.in vitro transcribed (IVT) RNA, the following procedure was performed. 1 µg of total RNA was used as input for the MessageAmp II aRNA amplification kit (Ambion) to make antisense RNA (aRNA). A short poly(A) tail was added to the aRNA using ePAP (Invitrogen), then that RNA was used as input for the MessageAmp II aRNA kit to give unmodified sense RNA. 18S and 25S rDNA were amplified from genomic DNA using primers that added at T7 promoter at the 5′ end of the DNA. Unmodified 18S and 25S rRNA were transcribed from these constructs using the MEGAScript in vitro transcription kit (Ambion). Unmodified IVT 18S and 25S rRNA were spiked into the IVT samples.To generate transcriptome-wide samples of unmodified To 3 µg (20 µL) of the poly(A) purified RNA, 20 µL of 2x alkaline hydrolysis buffer  was added, and the RNA was fragmented for 6.5 minutes at 98°C, then isopropanol precipitated and resuspended in 40 µL water. The sample was then split in half–one +CMCT sample and one mock sample. To each sample was added 100 µL BEU buffer  with (+CMCT) or without (mock) 200 mM 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate . These samples were incubated at 37°C for 20 minutes, then precipitated and resuspended in sodium carbonate buffer  and incubated at 37°C for 4 hours. The RNA was then isopropanol precipitated and washed 2x in 70% EtOH, then resuspended in 5 µL water. 5 µL 2x denaturing loading buffer The library preparation protocol is adapted from the ribosome footprinting library preparation Data were aligned to the yeast genome using Bowtie, with no more than 2 mismatches per read, and with reads that mapped to more than one genomic locus randomly assigned to one locus with equal probability. Ribosomal RNA reads at the same loci were combined, and moved to separate rRNA reads files. The number of reads mapping to each site in the genome were counted. For each condition, a scatterplot of ln(number of reads + 1) in the mock condition vs. +CMC condition was made using R. Pseudouridines in rRNA were marked with red squares. For each of these scatterplots, a regression line was calculated. For each site, the y-distance from the regression line  was calculated, and it was divided by the standard deviation of all residuals to get what we referred to as a score. In order to define potential sites of pseudouridylation (hits), we used the site in rRNA with the highest score (other than sites of pseudouridylation or sites one nucleotide downstream of these sites) to determine a cutoff–anything with scores greater than these sites was considered a hit. The cutoffs used were 6.26 for log phase replicate 1, 5.52 for log phase replicate 2, 4.47 for heat shock, and 7.38 for stationary phase. To limit our lists of potential pseudouridines to sites more likely to be pseudouridines, any nts that were not Us were discarded, and any sites with scores for the IVT data within 2 of the sites score for the natural data were discarded. This left 103 sites for log phase replicate 1, 141 sites for replicate 2, 273 sites for heat shock, and 42 sties for stationary phase. Raw files and processed data have been uploaded to GEO with the accession number GSE60445.600 1.2 in 200 mL SC-Ura with 2% raffinose, then 100 mL 3x YP-6% galactose was added, and cells were shaken for 6 hours at 30°C. Cells were pelleted, washed in water, and resuspended in 2 mL lysis buffer . Cell extract was made by bead beating 4x 1 min, then spinning down the lysate to remove debris. Proteins were purified from these extracts using Qiagen Ni-NTA protein purification kits, then resuspended in elution buffer  plus 10% glycerol at 0.5–1 mg/mL total protein. To check purification, samples were run on SDS-PAGE gels stained with GelCode blue (Pierce).Moveable ORF strains (Open Biosystems) were used to overexpress pseudouridine synthase enzymes (Pus1p-Pus8p). Strains were grown at 30°C to ODin vitro transcribed RNA. 2–10 pmol of IVT RNA was added to yeast extract (0.01–1 mg/mL) or purified pseudouridine synthase enzymes (0.05–0.1 mg/mL) in 100 mM Tris-HCl pH 8.0, 100 mM ammonium acetate, 5 mM MgCl2, 2 mM DTT, 0.1 mM EDTA and incubated for 1 hr at 30°C. RNA was isolated by acid phenol-chloroform extraction and isopropanol precipitation, then resuspended in water.TEF1 and RPL11a genes were amplified from genomic DNA using PCR with primers that added a T7 binding site at the start of the 5′-UTR and 20 As at the end of the 3′-UTR. MEGAScript (Ambion) was used according to manufacturers' instructions to make RT-stop capillary sequencing protocols were adapted from the Das lab Mutate-and-Map protocols RT-stop capillary sequencing data were processed using the HiTrace package for Matlab S. cerevisiae, S. mikitae, C. albicans, and S. pombe were grown to an OD600 of 0.8–1.0 in YPD (or YES for S. pombe) at 30°C. The yeast was then pelleted, washed in water, and each pellet was resuspended in 40 mL of TES buffer. For east yeast species, RNA was extracted by hot acid-phenol chloroform extraction (4x10 mL acid phenol-chloroform), and re-extracted with 10 mL chloroform. The RNA was ethanol precipitated and washed with 70% EtOH, and resuspended in 25–30 mL. For each species, total RNA was purified once over a 0.5 g Oligo(dT) celluose column, using the conditions explained above. This poly(A) RNA was then ethanol precipitated and the pellet was washed with 70% EtOH, dried, and resuspended in 100 uL water. These samples were then used in the CMCT-RT-CapSeq assay described above.4 L each of Table S1PSI-seq read counts data for ribosomal RNA.(XLSX)Click here for additional data file.Table S2PSI-seq read counts data for mRNA candidate sites.(XLSX)Click here for additional data file.File S1Supplemental text and methods.(DOCX)Click here for additional data file.File S2Supporting figures S1-S9.(TIFF)Click here for additional data file.File S3List and description of oligonucleotides used in this study.(XLSX)Click here for additional data file.


Leg-length inequality is an extensively studied complication of total hip arthroplasty in normal patients. However, few studies have focused on the pelvic obliquity of coronal pelvic malrotation. We hypothesized that pelvic obliquity with a fixed abduction/adduction contracture deformity of the hip may intraoperatively affect the release of soft tissues, ultimately resulting in a leg-length inequality. This study also investigated whether the femoral and vertical offsets of total hip arthroplasty were correlated with pelvic obliquity. This prospective study divided 98 patients into six groups based on the inclination of pelvic obliquity before total hip arthroplasty. Leg-length inequality, variation of pelvic obliquity, offset, and vertical offset were measured after total hip arthroplasty. Leg-length inequality and vertical offset were not significantly different among groups, whereas the variation of pelvic obliquity was significantly higher in type IIC pelvic obliquity than in other groups. Type IC pelvic obliquity had a significantly shorter offset than did the other groups, which may have been an important factor leading to type IC pelvic obliquity. Pelvic obliquity exhibited no significant effect on leg-length inequality in patients with total hip arthroplasty. A shorter offset may be caused by the higher tension of the abductor in the operated hip, which may result in the formation of type IC pelvic obliquity. Releasing the abductor contracture and restoring femoral offset are important for increasing hip stability and maintaining pelvic balance following total hip arthroplasty. Total hip arthroplasty (THA) can relieve hip pain , correctPelvic obliquity (PO), which here refers to infrapelvic obliquity, is a coronal obliquity of pelvic malrotation that is commonly secondary to abduction or adduction hip contractures and is frequently related to THA , 8. A prThe sensation of LLI after THA has been described in normal patients , but onlSome intraoperative factors may affect LLI after THA, including anesthesia type . TheoretAlthough the main limitations of the hip joint are attributed to the soft tissue structures around the hip, the importance of the bony architecture has recently become of greater interest . FemoralA total of 183 consecutive patients who underwent THA Shanghai Sixth People's Hospital Affiliated with Shanghai JiaoTong University between April 2013 and June 2013 were considered for this study. This experiment was approved by both the institutional research ethics and scientific committees of the hospital, and written informed consent was provided by participants for their clinical records to be used in this study. Eighty-five patients who experienced femoral neck fracture or bilateral one-stage THA that did not reflect the reality of PO were excluded. Thus, 98 patients (35 men and 63 women) were ultimately included. The preoperative diagnoses of these patients were as follows: developmental dysplasia of the hip (35 cases), avascular osteonecrosis of the femoral head (39 cases), ankylosing spondylitis (3 cases), septic arthritis (1 case), primary osteoarthritis (3 cases), and secondary osteoarthritis (17 cases). All patients had non-cemented acetabular and femoral components . Ninety-six patients had ceramic-on-ceramic bearing surfaces, and two had metal-on-polyethylene (PE) bearing surfaces. Patient ages ranged from 20 to 82 years (58.02 ± 10.47 years). Preoperative and postoperative radiographs from all patients were reviewed.THA was performed as described in a previous report ; that isStanding preoperative and postoperative anteroposterior (AP) pelvic and hip radiographs were taken for each patient two days before and two days after the operation during their hospital stay. These radiographs were used as parameters. Both legs were internally rotated 15° to obtain the pelvic radiographs . The autPO angles were measured as described in previous studies, with modifications  8, 12]., 12.8, 1Offset is the perpendicular distance between the long axis of the femur and the center of rotation of the femoral head  Fig 2)..Fig 2).Vertical offset is the distance between the trans-teardrop line and the medial apex of the lesser trochanter  Fig 2)..Fig 2).LLI. LLI is the length of a line perpendicular and connecting to the trans-teardrop line and the most prominent point of the lesser trochanter on AP pelvic and hip radiographs [iographs . The disThe defined anatomical landmarks included the ischial tuberosities, apex  of the lesser trochanter, apex of the teardrop, and longitudinal axis of the femur. The teardrop was selected as a standard reference instead of the ischial tuberosity because the teardrop is a more consistent landmark . The diap < 0.05. Data were presented as means ± standard deviation (SD).ANOVA was performed using SPSS statistical software . The Student-Newman-Keuls multiple comparisons test was used when multiple groups were compared. If equal variances were not assumed, Dunnett’s T3 test was used to compare differences between groups. Differences for all analyses were considered significant at The average actual diameter of the cup that was applied intraoperatively was 50.04 mm (44.00–60.00 mm). The average magnification for the measured value was 13.73% (3.79%–22.65%).p = 0.863) after THA. The leg-length measurement demonstrated that the average LLI was 6.55 ± 5.49 mm (0.24–25.80 mm). Leg shortening and lengthening were observed in 31.63% (31/98) and 68.37% (67/98) of patients, respectively. LLI in three patients  reached or exceeded 20 mm, and only one of these three patients exhibited a shortened leg (25.80 mm). The LLIs were within 0–5 mm in 45.92% (45/98), 0–7 mm in 66.33% (65/98), and 0–20 mm in 96.94% (95/98) of the cases. No significant differences were observed among the groups for operated vertical offset (ov-offset) (p = 0.359), contralateral vertical offset (cv-offset) (p = 0.967), operated offset (o-offset) (p = 0.501), or contralateral offset (c-offset) (p = 0.502) before THA. Meanwhile, vPO was significantly different among the groups (p = 0.001). The vPO for type IIC was significantly increased compared with types IA (p = 0.001), IB (p < 0.001), IC (p = 0.021), and IIA (p = 0.003). An increasing tendency was also observed for type IIC compared with type IIB (p = 0.196). Group type IIC showed the biggest vPO value among all of the groups.As shown in p = 0.633) after THA; importantly, no case of type IIC presented after THA. There were no significant group differences for the ov-offset (p = 0.421), cv-offset (p = 0.873), and c-offset (p = 0.535) after THA. However, significant differences were observed among the groups for the o-offset (p = 0.028). The o-offset for type IC was significantly shorter than those for types IA (p = 0.016), IIA (p = 0.004), and IIB (p = 0.011), and a decreasing trend was observed compared with type IB (p = 0.055). Group type IC showed the smallest o-offset value among all of the groups.As shown in LLI between types I and II PO were not significantly different after THA, suggesting that LLI in patients with THA was not affected by soft tissue tightness or PO. Our results are consistent with those of previous studies . Only 3.The vPO slightly changed in patients with type I PO compared with that in those with type IIC PO. Type IIC PO showed greater variation after THA than did the other types of PO. This result implies that type IIC PO was intraoperatively easier to correct than was type I PO. The deformity of type I PO, especially type IC, was relatively irreversible and fixed. This result was indirectly demonstrated in a previous study, where type IC PO (>6°) was not easily changed intraoperatively .Type IIC PO was not found after THA. This result was likely not due to an insufficient sample but rather because this type is easy to correct intraoperatively, as shown by the highest vPO values for type IIC PO.Meanwhile, the o-offset of type IC was prominently shortened compared with that of types IA, IB, IIA, and IIB based on their postoperative PO . HoweverAs the main connection between the lower limbs and the trunk, the hip joint and its motion between these the parts of body allows for bipedalism. Enormous effort has been expended over the years to define the various forces that contribute to the joint reaction force of the human hip . The freTo examine the biomechanical principles involved in the function of the human hip, the normal anatomy of the proximal femur and pelvis should be considered. The muscles, ligaments, and bony structures all contribute to the equilibrium of forces that allow for controlled motion at the femoral–acetabular articulation. The significance of the local anatomy surrounding the hip should be scrutinized because of its important contribution to hip biomechanics . There aThe moment arms of gravitational force and the abductor muscles can be manipulated during reconstructive hip surgery. Similarly, increasing the femoral offset or lateralizing the greater trochanter during total hip arthroplasty increases the mechanical advantage of the abductor musculature by increasing the abductor moment arm, which correlates with the decrease in the joint reaction force .This finding can partly explain the genetic mechanism of type IC PO; its inclination was mainly caused by the contracture of the ipsilateral abductor (gluteus) and tensor fasciae latae ,30. HoweBased on these observations, type I PO  can be considered irreversible and fixed. If greater tension and contracture of the abductor were present, the adoption of a femoral prosthesis with a normal offset would be difficult intraoperatively. This situation may lead to the shorter offset of the operated hip, which would, in turn, result in the formation of type IC PO. The adequate release of the contracture of the abductor helps to restore the femoral offset intraoperatively, whereas restoration of the normal femoral offset can increase hip stability by preventing impingement and improving soft tissue tension , formattThe main limitation of this study was the absence of follow-up. The monocentric nature of the data also presents the possibility of selection bias. More precisely designed studies that involve the anatomy, biomechanics, and finite element analysis of the hip and pelvis are necessary to determine the mechanism contributing to PO.In summary, PO exhibited no effect on LLI in patients with THA. The results demonstrated that type IC PO was a relatively fixed PO, and type IIC PO was easy to be corrected intraoperatively. Shorter offsets may be caused by the greater tension of the abductor in the operated hip, which may result in the formation of type IC PO. Releasing the abductor’s contracture and restoring femoral offset are important for increasing hip stability and maintaining postoperative pelvic balance following THA.S1 Text(XLS)Click here for additional data file.S2 Text(XLS)Click here for additional data file.


Tomato spotted wilt virus (TSWV) messenger transcripts, and not the (anti)genomic RNAs, are targeted by the RNA silencing machinery. Here, the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) of the TSWV S RNA, and present at the 3′ end of viral mRNAs, was analyzed as a target and inducer for RNA silencing. Virus-derived siRNAs (vsiRNAs) purified from virus infected plants were found to derive from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the IGR from where hardly any vsiRNAs were found. Similar profiles were observed with the distantly related Tomato yellow ring tospovirus (TYRV). Dicer cleavage assays using Drosophila melanogaster (Dm) embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3′ UTR (GFP-HP) did not show more rapid/strong silencing and profiling of the corresponding siRNAs, generated outside the context of a viral infection, still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP is a weak inducer of RNA silencing and only plays a minor role in the amplification of a strong antiviral RNAi response.Earlier work indicated that  RNA silencing, also named post transcriptional gene silencing (PTGS), is a conserved cellular mechanism in plants and animals in which double-stranded (ds)RNA, imperfect hairpin RNAs or highly structured single-stranded (ss)RNA trigger a chain of processes leading to sequence-specific RNA degradation Brome Mosaic virus) and thereby avoiding exposure of viral dsRNA molecules to dicer Plant viruses also induce RNA silencing often referred to as Virus-Induced Gene Silencing (VIGS), as can be observed by the generation of viral specific siRNA molecules during the infection process Cymbidium ring spot tombusvirusIn contrast to the increasing insight into the working mechanisms of plant viral suppressor proteins, information on the origin of dsRNA molecules that induce VIGS still remains limited for many viruses. For RNA viruses it is generally assumed that ds replicative intermediates play a role in this, but nice examples exist, e.g. from In plants silencing requires an amplification step involving a host RNA-dependent RNA polymerase (RDR) and this may occur in two ways. In the first way, primary siRNAs recruit RDR to homologous RNA molecules that serve as template for the generation of complementary RNA, thereby generating dsRNA from which secondary siRNAs are synthesised. In the second way, aberrant RNA molecules that arise as incomplete viral transcripts or resulting from RISC-mediated RNA target cleavage are recognised by RDR independent from primary siRNAs, and used as template to generate dsRNA. The amplification not only results in the production of secondary siRNAs identical to the dsRNA inducer sequence but also to the adjacent regions of target mRNA. This phenomenon of silencing spreading along the entire mRNA target sequence is referred to as transitive RNA silencing Tomato spotted wilt virus (TSWV) as its representative, are the plant-infecting members of the arthropod-borne Bunyaviridae, a family that primarily consists of animal infecting viruses Tospoviruses, with Arenaviridae, the floating genus Tenuivirus and the genus Phlebovirus within the BunyaviridaeAmbisense RNA segments are relatively unique and besides tospoviruses, only found with members of the family in planta. To confirm that the findings where likely generic to all tospoviruses, the S-RNA-derived IGR-HP from tomato yellow ring virus (TYRV), another distinct (Asian) tospovirus, was included in the analysis. Results demonstrate that synthetic IGR-HP transcripts are recognized as dsRNA substrate during dicer-cleavage assays but during tospovirus infection, as well as during transient expression in the absence of NSs, hardly any siRNAs are produced from the IGR-HP.Considering the presence of long stretches (30–40 nts) of almost full complementarity within the predicted IGR encoding HP, and thus within viral mRNA transcripts, here the TSWV S RNA-derived IGR-HP was investigated as a potential target and inducer of RNA silencing N. benthamiana leaf material and, after radiolabeling, probed on total RNA and genomic RNA purified from isolated viral RNPs  While vsiRNAs were found hybridizing to the L, M and S RNA segments, strong hybridization signals were observed with the ambisense M and S RNA segments , lane 3.To test whether the vsiRNAs originating from the ambisense M and S RNA segments predominantly corresponded to the IGR encoded HP, suggestive for the status of HP as strong inducer/target of RNA silencing, the vsiRNAs were further fine mapped on the S RNA segment. To this end, radiolabeled TSWV vsiRNAs were hybridized to similarly sized PCR fragments spanning the entire S RNA segment. Although vsiRNAs hybridized to sequences covering the entire TSWV S RNA segment, and good amounts were obtained from sequences of the NSs and N genes , unexpecTo verify whether a similar vsiRNA distribution profile would be obtained with TYRV, a similar fine mapping study was performed for this virus. Like TSWV, TYRV infections gave rise to S RNA-derived vsiRNAs that mapped to all regions of the S RNA segment , but thoDrosophila melanogaster (Dm) embryo extracts containing Dicer-1 and Dicer-2 While only few vsiRNAs were found mapping to the IGR encoded predicted hairpin-structure, this region was further investigated as potential inducer and target of antiviral RNAi in a dicer cleavage assay. To this end, synthetic radiolabeled transcripts of the TSWV IGR-encoding HP sequence were made and after being allowed to fold into a dsRNA hairpin structure, subsequently offered to RNAi-induced N. benthamiana. As controls, GFP constructs were included that either lacked the entire HP-structure sequence (GFP) or contained part of an antisense N gene sequence that was shown to be well targeted by the silencing machinery during a natural virus infection and predicted to not fold into a stable hairpin structure genomic mRNA molecules in vitro dicer cleavage assays is recognized as dsRNA or as an imperfect hairpin RNA somewhat resembling precursors to miRNAs is not clear yet, since Drosophila embryo extracts contain both DCR1 and DCR2, of which DCR1 is normally resident to the nucleus and involved in miRNA production whereas DCR2 localizes to the cytoplasm and produces siRNA Considering that the ambisense encoded tospoviral N and NSs transcripts contain a 3′ UTR consisting of the IGR-encoding HP structure sequence in planta is strengthened by the observation that transient expression of a GFP construct containing a 3′ IGR-HP structure sequence did not reveal an elevated level of HP-derived siRNAs either and, relative to the 3′ part of the preceding ORF  Nicotiana benthamiana and N. rustica cv. America.The tospovirus strains TSWV BR-01 N.benthamiana leaves) was ground in liquid nitrogen and next mixed with extraction buffer (2% Sarcosyl – 5 M NaCl), followed by phenol extraction. The aqueous phase was collected and subjected to polyethylene glycol (PEG) precipitation 32P]-ATP (Perkin Elmer) by T4 polynucleotide kinase (Promega) according to the manufacturer's instructions.Isolation of small RNAs was performed as previously described N. rustica cv. America as previously described 32P]-labelled siRNAs (see below) purified from healthy and tospovirus-infected N.benthamiana leaves.Tospoviral RNPs were purified from N.benthamiana leafs using Trizol (Life Technologies). The S RNA segment was RT-PCR-amplified, using Superscript RT (Invitrogen), in 6-7 fragments of similar size and spanning the entire S RNA segment from TSWV and TYRV respectively. The products were further cloned in pGem-T Easy (Promega) according to the manufacturer's instructions and verified by sequence analysis. For TYRV S RNA-specific fragments, equimolar amounts of PCR products were resolved on 1% agarose gel. For TSWV S RNA, due to difficulties in obtaining single PCR products, S RNA-specific fragments were excised from pGem-T Easy plasmid DNA and equimolar amounts resolved on 1% agarose gel. DNA was blotted to Hybond-N membrane (Amersham Biosciences) by top-down blotting. Filters were subsequently hybridized (at 48°C) overnight in Church buffer 32P]-labelled siRNAs purified from healthy or tospovirus-infected N.benthamiana leaves. After washing, filters were exposed for two days to phosphor screen (Kodak) and visualized by phosphorimaging . Signal quantification was performed with ImageJ software Total RNA was purified from systemically infected in vitro transcription using T7 RNA polymerase (Promega) in the presence of [α-32P]-rNTP  according manufacture's instruction. Products from the in vitro transcription were resolved on an 8% denaturing acrylamide gel and the radiolabelled A-U rich predicted hairpin transcript was excised from the gel and extracted by diffusion into 20 µl 2x PK buffer  followed by phenol chloroform and ethanol precipitation. Prior to use, purified RNA transcripts were briefly heated for 10 min. at 85°C and gradually cooled down to room temperature to allow RNA folding. 114-nt dsRNA molecules were prepared as previously described DNA templates of the A-U rich predicted hairpin encoding sequence (from TSWV S RNA IGR)  were RT-Drosophila melanogaster (Dm) embryo extract was prepared as previously described Drosophila embryo extract, 5 nM 32P-labeled transcript of the IR hairpin or dsRNA were incubated for 2–3 h at 25°C Agrobacterium tumefaciens transient expression assay (ATTA) system were performed as previously described TSWV and GFP-HPTYRV. As a control, an inverted part of the TSWV N gene sequence  was fused to the 3′ end of the GFP gene, resulting in the GFP-noHP construct. All GFP-HP, GFP-noHP and GFP constructs were cloned in binary vector pK2GW7 NSs and tombusvirus P19 genes were expressed from binary vectors pK2GW7 and pBin19, respectively. To this end, binary vectors were transformed to Agrobacterium tumefaciens strain cor308 2HPO4, 4.5 g/l KH2PO4, 1.0 g/l (NH4)2SO4, 0.5 g/l Sodium Citrate Dihydrate, 0.25 g/l MgSO4, 0.2% (w/v) glucose, 0.5% (v/v) glycerol, 50 mM acetosyringone and 10 mM MES pH 5.6) and grown at 28°C overnight. The induced culture was pelleted and ressuspended in Murashige-Skoog (MS) medium  containing 150 µM acetosyringone and 10 mM MES (pH 5.6) to an OD600 of 0.5. This suspension was used to infiltrate fully expanded leafs of N. benthamiana plants. Silencing of GFP and suppression by NSS and P19 proteins was assessed by UV light and western blot analysis, respectively. To suppress silencing, RNA silencing suppressor constructs were provided in a co-ATTA with GFP constructs. To this end, induced Agrobacterium suspensions were mixed at a final OD600 of 0.5 prior to infiltration. Infiltrated plants were kept at 25°C and monitored for GFP fluorescence during a 5-day period using a GFP fluorescence-stereo-microscope. Pictures were taken at 5 dpi (days post infiltration). Quantification of GFP fluorescence from 1 cm2 leaf disk was performed using Fluorstar Optima (BMG Labtech) as previously described To analyse the IGR hairpin as an inducer of silencing outside the context of a tospoviral infection, leaf patch assays with the Pictures of whole leafs (as shown in Folding predictions were performed at 37°C, using Mfold


Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates.RNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN’s Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats  were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison.Transcriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.The online version of this article (doi:10.1186/s12896-015-0155-7) contains supplementary material, which is available to authorized users. RNA-Seq is quickly becoming the preferred method for comprehensively characterizing global transcriptome activity. This approach has emerged as a powerful tool for determining the link between genotype and phenotype given that the transcriptomes of specific tissue types and individual cells reflect functionality -4. MonitTechnologies are emerging to circumvent these challenges. With whole transcriptome amplification (WTA), RNA can be synthetically enriched for gene profiling experiments with both research and diagnostic applications ,17-19. WAn obvious concern when using WTA for sensitive RNA-Seq is that the replication of transcript abundance occurs without the loss or distortion of transcript expression. Using technologies such as quantitative polymerase chain reaction (qPCR) or microarrays to measure expression levels investigators have compared expression profiles from i.) amplification of varying amounts of starting total RNA ,20,21; iCommercially available kits are becoming increasingly prevalent for seamless incorporation of amplified RNA into the NGS pipeline. These are offered by companies such as Clontech, Sigma, Miltenyi Biotec, and NuGEN, and use a variety of techniques to amplify RNA. When considering WTA for RNA-Seq studies, it is important to carefully consider the differences in chemistry and approaches that each commercial kit utilizes. Variations in priming strategies, cDNA synthesis, and amplification of newly converted cDNA can have impacts on length of cDNA products, 3′ bias, amplification efficiency, and fidelity of maintaining relative transcript abundance . All of As commercially available WTA kits continue to improve, investigators should continually reassess their efficiency and compatibility with new sequencing platforms, to determine the best practices as methods evolve. In previous work, Tariq et al. (2011) performed an exploration of the efficacy of the NuGEN Ovation RNA-Seq system by using two different library preparation protocols to reduce ribosomal RNA contamination (i.e. poly-A enrichment and rRNA depletion), and ran their sequencing experiment on two separate sequencing platforms, the SOLiD platform and the Illumina Genome Analyzer IIX, to assess sequencing fidelity and platform-specific biases. Notably, this study attempted to identify the best combination of approaches (i.e. which combination of library preparation protocols and sequencing platforms) will result in the largest amount of high quality data generated from a WTA sample for RNA-Seq characterization. As comprehensive as this investigation was, the authors neglected to determine how WTA technologies, such as the Ovation kit, influence gene expression profiles. Their investigation was instead limited to evaluating differential gene expression between the two sequencing platforms. In addition, since the publication of that study, NuGEN has released an enhanced version of their Ovation kit and Illumina sequencing technology has become more advanced with the advent of their HiSeq platform. In particular, the HiSeq boasts lower error rates, better precision, and longer reads than the GAIIX . MoreoveRattus norvegicus), building upon and enhancing knowledge regarding current WTA methodologies. To that end, we used the Tuxedo analysis pipeline [R. norvegicus genome, in addition to providing annotation and an exploration of differentially expressed genes. Our study was designed to sequence samples at the greatest depth possible, given the resources at hand, in order to identify any subtle differences between raw RNA and amplified RNA in biological replicates. Ultimately, our goal is to provide foundational insight for future studies to build upon regarding the utility of WTA for clinical and field-based studies that are investigating the functional link between genotype and phenotype via gene expression and RNA-Seq.It remains unknown whether WTA preferentially amplifies specific gene products to the exclusion of others. Therefore, our study integrated the design of matched amplified and unamplified RNA from six biological replicates to empirically test this question using NGS transcriptomic sequencing on the Illumina HiSeq 2000 platform, which has become the gold standard for next-generation sequencing experiments ,13,15,16pipeline ,31 to asR. norvegicus genome . Quality filtering resulted in the retention of approximately 90% and 86% of reads from Lanes 1 and 2, respectively , and this threshold was used to determine the number of expressed genes for each sample. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively  values averaged slightly higher in libraries constructed from raw RNA samples Figure . TranscrAs a complimentary bioinformatic approach to investigate potential differential gene expression, we used HT-Seq  to quantWe next assessed potential biases in transcript representation (i.e. coverage at 3′ and 5′ end) by determining the average coverage at each percentile of length from 3′ to 5′ end of the known transcripts Figure . The covRNA-Seq has enabled the sequencing of transcriptomes to allow both identification and quantification of transcript levels. The digital precision and sensitivity of RNA-Seq is well-suited to the analysis of low-input or degraded samples, especially with respect to older methods such as qPCR or microarrays, yet many available protocols require total RNA around 1.0 microgram, presenting a potential obstacle for clinical or field-based studies. Here, we have tested the efficacy of NuGEN’s Ovation RNA-Seq V2 system for whole transcriptome amplification by comparing matched amplified and unamplified samples to identify if there exists any subtle changes in gene expression after WTA among biological replicates. We were specifically interested in investigating the degree to which accurate representations of gene diversity and transcript abundance levels were achieved when sequenced on an Illumina HiSeq.Hnrnpa0), a DNA binding protein that is thought to influence pre-mRNA processing, mRNA metabolism and transport, was found to be differentially expressed in three out of the six sample pairs. The remaining differentially expressed genes were only found in two or fewer comparisons, and no pairwise comparison shared more than five genes. Our results from DESeq also indicate that there is no consistent biological distortion, as 48% (15 out of 31) of differentially expressed genes were found in five or fewer of our pairwise comparisons. This result is remarkably consistent with the differential expression results produced using Cuffdiff. Moreover, we observe no overlap with the genes that Cuffdiff identified as differentially expressed, indicative of the small proportion of differentially expressed genes being a product of background noise from stochastic events observed with typical RNA-Seq experiments. This result thus lends strong support to our hypothesis that there is no consistent biological distortion when using WTA.We identified consistent patterns of gene presence and absence across each of the six comparisons, with an average overlap of approximately 93%. We found no evidence that one or more specific gene products were differentially expressed in matched unamplified vs. amplified RNA in this study. Using Cuffdiff, the only gene found to be differentially expressed in as many as four of the six sample pairs was classified as a pseudo-gene and one protein-coding gene, heterogeneous nuclear ribonucleoprotein A0  technology for achieving WTA. This technology incorporates a combination of 3’-Ribo-SPIA (i.e. polydT) primers, which prime synthesis of cDNA at the 3’ polyA tail, and whole transcript (WT)-Ribo-SPIA (i.e. random hexamer) primers to prime cDNA synthesis across the full length of the transcripts, providing amplification independent of sample degradation at the 3’ end ,8-10,35.ases, which rapidly degrade RNA molecules at ambient conditions [Studies that investigate the relationship between genotype and phenotype have largely focused on model organisms for which a genome is publicly available, and have been performed using captive populations, or with cell culture methods under controlled laboratory settings for which sample material is relatively easy to obtain ,18,36-46nditions ,23,51,52nditions -26,53,54Our study builds upon and enhances existing knowledge regarding the NuGEN Ovation RNA-Seq V2 system’s performance in studies that use WTA for RNA-Seq experiments. Previous studies have demonstrated that this chemistry performs well across technical replicates of varying starting amounts of input RNA and across multiple laboratories. The novelty of our study is that it empirically tests biological variation, by incorporating the design of matched amplified and unamplified RNA from six biological replicates. The Ovation RNA-Seq V2 kit is able to amplify small quantities of RNA from tissue samples while maintaining transcript levels nearly identical to those from matched unamplified input RNA, demonstrating an overlap of 93% when comparing each matched pair individually. Despite this high fidelity of transcript representation, future studies should anticipate the slight discrepancy revealed here and take measures to account for the potential for small differences in downstream analyses. Additionally, future work could benefit from testing this methodology using more realistic scenarios, such as repeating the experiment with smaller input concentrations, or using partially degraded samples that may be more representative of RNA that may be extracted from clinical or from field-based studies. Nonetheless, this study provides the essential first step into investigations into the putative alteration of gene expression profiles due to WTA among biological replicates.Rattus norvegicus, n = 6) and snap frozen in liquid nitrogen. Samples were transferred to −80°C and stored there until sample processing.Animal handling was carried out in strict accordance with the approval of Duke University’s Institutional Animal Care and Use Committee (IACUC protocol #A096-13-04). Gonadal white adipose tissue was excised from standard laboratory rats . Briefly, frozen tissues were homogenized in 1 ml TRI Reagent  using a hand-held rotor-stator homogenizer to provide efficient disruption and homogenization of samples. BCP (1-bromo-3-chloropropane) extraction was performed using 100 ul of BCP, followed by centrifugation at 12,000 × g for 15 min at 4°C. Aqueous layer containing total RNA was transferred to a fresh 1.5 ml centrifuge tube and one volume of 70% EtOH was added to sample. The entire sample volume was loaded onto Qiagen filter columns and kit protocol was followed according to manufacturer’s instructions, including an on-column DNRNA integrity and concentration was assessed using the 2100 Bioanalyzer  at the Duke Institute for Genome Science and Policy’s Microarray Facility. RNA integrity is provided by a RNA Integrity Number (RIN), which is calculated based on the comparison of the areas of 18S rRNA and 28 s rRNA ,14,55. VTotal RNA extractions were divided into two tubes for each rat, for a total of 12 tubes. Six tubes were normalized to 100 ng/μl in a 5 μl volume, for a total of 500 ng of RNA, and subjected to whole transcriptome amplification using NuGEN’s Ovation RNA-Seq V2 kit  according to manufacturer’s instructions. This kit provides a rapid method for preparing amplified cDNA from total RNA for downstream RNA-Seq applications. It employs a single primer isothermal amplification (SPIA) method to amplify total RNA into double stranded cDNA and depletes rRNA without preselecting mRNA. Amplified cDNA samples were then purified using the MinElute Reaction Cleanup Kit  according to manufacturer’s protocol. After the amplification procedure, cDNA concentrations ranged from 421–575 ng/ul . In summary, 1 μg of total RNA was enriched for mRNA using oligo-dT coated magnetic beads, fragmented, and reverse transcribed into cDNA. The cDNA was fragmented into smaller pieces, blunt-ended, and ligated to indexed (barcoded) adaptors and amplified using PCR. Previously amplified cDNA libraries were prepared for sequencing using Illumina’s TruSeq DNA Sample Preparation Kit according to manufacturer’s protocol. Final library size distribution was determined using Agilent Bioanalyzer 2100. Insert size was between 120–130 base pairs (bp) and average library size was 230 bp. Twelve libraries were multiplexed, pooled and sequenced on two lanes of the Illumina HiSeq2000 platform  using 100 bp paired-end reads. Specifically, six multiplexed non-amplified samples were sequenced on Lane 1, while six multiplexed amplified samples were sequenced on Lane 2. The data set supporting the results of this article are available in the NCBI Short Read Archive repository [accession number SRP049463; R. norvegicus genome  using TopHat2 (v 2.0.5) [RNA-Seq reads were filtered using the Trimmomatic program  and aligv 2.0.5) ,57. The v 2.0.5) . HTSeq wv 2.0.5) . To estiv 2.0.5) . Since wv 2.0.5)  was used


Although numerous approaches have been developed to map RNA-binding sites of individual RNA-binding proteins (RBPs), few methods exist that allow assessment of global RBP–RNA interactions. Here, we describe PIP-seq, a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply PIP-seq to the HeLa transcriptome and compare binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites. RNA–protein interactions are central to all of the post-transcriptional regulatory processes that control gene expression. From the initial processing of a protein-coding transcript in the nucleus to its final translation and decay in the cytoplasm, cellular mRNAs are involved in a complex choreography with various transacting RNA-binding proteins (RBPs) -3. RBPs Eukaryotic genomes encode a large collection of RBPs that interact with mRNAs to form dynamic multi-component ribonucleoprotein complexes (mRNPs) ,7. TheseThe essential nature of RNA–protein interactions in eukaryotic biology has led to numerous biochemical, genetic and computational approaches being utilized, alone and in combination, to identify and validate RBPs and their specific RNA-binding sites ,11,12. Tin vivo. Finally, these studies have focused on poly-adenylated (polyA) transcripts, reducing their ability to monitor RBP binding in non-polyA and nascent RNAs.Recently, several groups have established experimental approaches for interrogating RNA–protein interaction sites on a more global scale. These approaches utilize 4-thiouridine and UV cross-linking to identify RNA–protein interactions by uncovering sites of T > C transversion (representing RNA–protein cross-linking events) ,28. HoweTo address the limitations of the currently available methodologies, we present a ribonuclease (RNase)-mediated protein footprint sequencing approach that we call protein interaction profile sequencing (PIP-seq). This approach identifies RNA–protein interaction sites within both unprocessed and mature RNAs in a mostly unbiased manner and on a transcriptome-wide scale. We describe multiple cross-linking techniques to capture both direct and indirect RNA–protein interactions. We also show that both single-stranded and double-stranded RNases uncover distinct but overlapping sets of RNA–protein interaction sites. Using this approach, we find PIP-seq to be a reproducible approach that reveals both previously known and novel RBP interaction sites. We demonstrate the utility of PIP-seq by uncovering enriched sequence motifs within the complement of identified RBP interaction sites. We also investigate the interactions among protein-binding sites and provide evidence for co-binding of RNAs by specific sets of RBPs, some of which bind to groups of transcripts encoding functionally related proteins. These results reveal novel insights into networks of post-transcriptional gene regulation mediated by specific groups of RBP-bound sequence motifs. Finally, we identify a significant enrichment for disease-associated variants within RBP interaction sites, and demonstrate the effects of some of these single nucleotide polymorphisms (SNPs) on RNA–protein interactions. Overall, our approach provides an RNA-centric global assessment of RNA–RBP interactions that directly identifies RNA–protein interaction sites and is applicable for all organisms and sample types.To obtain an unbiased, genome-wide view of RNA–protein interactions for both unprocessed and mature RNAs in eukaryotic transcriptomes, we developed an RNase-mediated protein footprint sequencing approach, known as PIP-seq, by performing nuclease-sensitivity sequencing assays ,30 on crin vivo. However, for the purposes of this manuscript we focus our analysis specifically on the identification of protein-interaction sites, which we refer to as protein-protected sites (PPSs).We had previously used nuclease-sensitivity sequencing assays on purified RNAs to determine RNA base-pairing probabilities by treating RNA with either single-stranded or double-stranded RNase  and sequencing the resulting populations ,30. We rTo perform PIP-seq, we started with adherent HeLa cells cross-linked by one of the methods described above  or used cells that had not been cross-linked. The resulting cell lysates were then split into experimental and background samples. Due to the structure-specific nature of the RNases used, it was essential to have a background sample to control for RNase insensitive regions. Therefore, a ‘footprint sample’  was directly treated with either a single-stranded RNase (ssRNase), known as RNaseONE, or double-stranded RNase (dsRNase), known as RNaseV1. In contrast, the RNase digestion control sample was first denatured in SDS and treated with proteinase K prior to RNase digestion. In this way, regions that were protein-protected in the footprint sample became sensitive to RNase digestion in the control sample and regions that were unbound but insensitive to one of the nucleases due to their structural status, remained that way. For both samples, cross-links were subsequently reversed , which was followed by strand-specific library preparation  using the Illumina 50-bp single-end sequencing protocol, and obtained approximately 31 to 60 million raw reads per library  of the total variation in PPS counts between transcripts    overlap between PPSs identified in two replicates Figure A. SimilaWe also interrogated the relation between PPSs identified by different RNases. We compared RNaseONE, which preferentially cleaves single-stranded RNA, to RNaseV1, which preferentially cleaves paired bases  enrichment of the previously identified transversion events within our identified PPSs relative to the expressed transcriptome background, suggesting that at least some fraction of binding events are cell-type independent  data from HEK293T cells , in whicIt is also worth noting that PIP-seq identified a total of 428,713 of approximately 40-nucleotide-long protein-protected regions, while gPAR-CLIP yielded 706,586 loci of similar length  relative to their expression levels in the RNase digestion control sample Figure C,D. ThisIn contrast to protein-coding mRNAs, we found that lncRNAs were consistently depleted for protein binding Figure C,D. TherP < 2.2 × 10–16) more evolutionarily conserved than flanking regions , lending further support to the notion that disruption of RNA–protein interactions underlies the disease mechanism of the polymorphisms in question.A growing set of evidence suggests that multiple RNA-level mechanisms, some of which depend upon RNA–protein interactions, are the means by which particular single nucleotide polymorphisms (SNPs) in mRNAs effect human disease phenotypes -41. In sP < 0.001)  , and vaFor these experiments, HeLa cells were seeded in 15-cm standard Corning tissue-culture treated culture dishes , grown to 90% confluence (approximately 18 million cells) in DMEM media  supplemented with L-glutamine, 4.5 g/L D-glucose, 10% fetal bovine serum ) and Pen/Strep .2. The cross-linked cells were collected by scraping, pelleted and then frozen until used.For formaldehyde cross-linking, a 37% formaldehyde solution  was added drop-wise with mixing directly to cell culture dishes containing 90% confluent cells to a final concentration of 1% and incubated at room temperature for 10 minutes. Next, 1 M glycine  was added to a final concentration of 125 mM and incubated for an additional 5 minutes with mixing. Then, cells were washed twice with ice-cold PBS and collected. Finally, cells were pelleted and frozen until the PIP-seq digestions were performed. For UV-cross-linking experiments, 90% confluent cells were washed twice with ice-cold PBS and resuspended in 5 mL of PBS. Cell culture dishes were placed in a UV Stratalinker 2400  with the lid removed and irradiated with UV-C (254 nm) once at 400 mJ/cmTo begin, we lysed the cell pellets in RIP buffer  and ground them manually (850 μl of RIP was used per 10 million cells). The resulting cell lysate was treated with RNase-free DNase . Subsequently, these DNA-depleted lysates were split and treated with either 100 U/mL of a single-stranded RNase (ssRNase) ) with 200 μg/mL BSA in 1× RNaseONE buffer for 1 hour at room temperature, or 2.5 U/mL of a double-stranded RNase (dsRNase) ) in 1× RNA structure buffer for 1 hour at 37°C as previously described ,30  and ethanol-precipitated prior to being treated with the two RNases. In this way, the SDS and proteinase K solubilized and digested the proteins allowing us to deduce PPSs within all detectable RNAs in the cells of interest . Finally, the purified RNA was used as the substrate for strand-specific sequencing library preparation, as previously described ,30, withPIP-seq reads were first trimmed to remove 3′ sequencing adapters using cutadapt (version 1.0 with parameters -e 0.06 –O 6 -m 14). The resulting trimmed sequences were collapsed to unique reads and aligned to the human genome (hg19) using Tophat (version 2.0.9 with parameters --read-mismatches 2 --read-edit-dist 2 --max-multihits 10 --b2-very-sensitive --transcriptome-max-hits 10 --no-coverage-search --no-novel-juncs). PCR duplicates were collapsed to single reads for all subsequent analyses.PPSs were identified using a modified version of the CSAR software package . SpecifiMapped reads from chromosome 9 of formaldehyde-cross-linked ssRNase-treated PIP-seq replicate 1 libraries were randomly subsampled at 10% to 90% by a custom Perl script. CSAR was used to identify PPSs as described and the total number of PPSs was plotted as a function of subsample size.iCLIP, PAR-CLIP, and CLIP-seq datasets were compiled from sources as referenced and overlapped with PPSs. The significance of overlaps with PPSs was assessed using a chi-squared test compared to an expressed transcriptome background. To compute a background distribution for the number of T > C transversions, we generated ten random sets of genomic intervals with the same size distribution as PPSs. These random intervals were selected from a background of actively transcribed regions , and miRBase release 18 was used for the microRNA annotations. PPS annotation was done ‘greedily’, such that all functional annotations that overlapped with a given PPS were counted equally. Conservation was assessed by computing average SiPhy-π log-odds  scores wk-means clustering of the resultant weighted adjacency matrix was used to identify modules of co-occurring motifs. We set k = 5 based on manual inspection of clusters on a multidimensional scaling (MDS) plot of the adjacency matrix. GO analysis was performed using DAVID [MEME  was usedng DAVID .Clinically associated SNPs (snp137Flagged) were downloaded from the UCSC Table Browser. We also downloaded the NHGRI GWAS Catalog  of diseain vitro transcription by annealing T7 sense DNA oligonucleotides (TAATACGACTCACTATAGGG) to antisense probe sequences fused to the antisense T7 (aT7) sequence , rs74315352 disease: CTTGTAAGAATCAGGCCGgCTTTTTCCACACGATTCTC(aT7), rs121918066 normal: CCCAGGTTGGCAATGTAGcGATGTGGTCCAAAGTCATC(aT7), rs121918066 disease: CCCAGGTTGGCAATGTAGtGATGTGGTCCAAAGTCATC(aT7)) . Each hybrid reaction was incubated at 95°C for 5 minutes and cooled to 25°C by step-wise increments of 1°C/minute.We generated asymmetric oligonucleotide hybrids for In vitro transcription reactions were performed by adding 1 μg of the asymmetric oligonucleotide hybrids (see above) to a 25 μL transcription reaction comprising 1× T7 RNA Transcription buffer , 36 μM uridine triphosphate (UTP) (for rs74315352) or 36 μM cytidine triphosphate (CTP) (for rs121918066), 264 μM each of ATP, CTP and guanosine triphosphate (GTP) (for rs74315352) or 264 μM each of ATP, UTP and GTP (for rs121918066), 0.04 mCi 32P UTP (for rs74315352) or 0.04 mCi 32P CTP (for rs121918066), 10 nM DTT, 40 U RNaseOUT , and 75 U of T7 RNA polymerase. The reactions were incubated at 37°C for 2 hours. DNA was digested with four units of Turbo DNase  at 37°C for 20 minutes. RNA probes were chloroform-extracted and precipitated. The amount of a labeled RNA probe was determined by 15% TBE-urea gel electrophoresis followed by phosphor-imaging and densitometry. Normal and disease RNA probes were normalized to equal activities and used for subsequent analysis.in vitro transcription) were added to separate 10.2 μL binding reactions comprising 0.2 mM Tris pH 7.5, 0.02 mM EDTA, 40 mM KCl, 1.3% polyvinyl alcohol, 25 ng/μl tRNA, 3 mM MgCl2, 1 mM ATP, 50 mM creatine phosphate and 1.5 μg/μl HeLa whole cell lysate in RIP buffer  and incubated at 30°C for 20 minutes. The binding reaction was then subjected to UV cross-linking for 20 minutes using a 254-nm UV lamp ). To digest unbound RNA, each reaction was incubated with 20 U RNase T1 and 8 μg RNase A at 37°C for 20 minutes. RNA-bound proteins were denatured in 1× SDS sample buffer and 1 mM β-mercaptoethanol and boiled for 5 minutes. Samples were separated on NuPAGE 3% to 8% Tris-acetate gel  at 130 V for 1.5 hrs. Phosphor-imaging and densitometry were used to visualize and quantify protein-bound RNA, respectively.Equal concentrations of each RNA probe  can also be accessed at . The webbp: Base pair; BSA: Bovine serum albumin; CDS: Coding sequence; CLIP-seq: Cross-linking and immunoprecipitation sequencing; CTP: Cytidine triphosphate; DSN: Duplex-specific nuclease; dsRNA: Double-stranded RNA; dsRNase: Double-stranded RNase; FDR: False discovery rate; GO: Gene ontology; gPAR-CLIP: Global photoactivatable ribonucleoside cross-linking and immunoprecipitation; GTP: Guanosine triphosphate; lncRNA: Long non-coding RNA; MDS: Multidimensional scaling; mRNA: Messenger RNA; ncRNA: Non-coding RNA; NS: Not significant; PAR-CLIP: Photoactivatable ribonucleoside cross-linking and immunoprecipitation; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; PIP-seq: Protein interaction profile sequencing; polyA: Poly-adenylated; PPS: Protein-protected site; RBD: RNA-binding domain; RBP: RNA-binding protein; RNase: Ribonuclease; RNP: Ribonucleoprotein complex; SNP: Single nucleotide polymorphism; ssRNA: Single-stranded RNA; ssRNase: Single-stranded RNase; UTP: Uridine triphosphate; UTR: Untranslated region.The authors declare that they have no competing interests.IS, FL, LG, CT, JLR and BDG conceived the study and designed the experiments. IS, FL, AA and BDG performed the experiments. IS, FL and BDG analyzed the data and wrote the paper with assistance from the other authors. All authors have read and approved the manuscript for publication.PIP-seq library characteristics. Information on PIP-seq libraries, including sequencing reads, processing, mapping and PPS identification.Click here for filePIP-seq reveals protein-binding sites throughout the human transcriptome .  Screenshots show PIP-seq reads for the formaldehyde-cross-linked ssRNase-treated footprint (top sequencing track) and RNase digestion control libraries (bottom sequencing track). Blocks indicate regions identified as PPSs in each of the three replicates. Chromosomal coordinates, UCSC gene tracks , and PhyloP conservation scores are included (as labeled). The screenshots are from our PIP-seq browser [ browser . ExampleClick here for fileSummary statistics for PPSs identified by UV-cross-linked and non-cross-linked experiments . (A) Absolute distribution of PPSs throughout RNA species identified using no cross-linking. (B) Average PPS count per RNA molecule (classified by type (mRNA and lncRNA) and transcript region ) identified using no cross-linking. Percentages indicate the fraction of each RNA type or region that contains PPS information. (C) Average expression (y-axis) of human mRNAs separated by total number of PPSs identified in their sequence (x-axis) for UV-cross-linked experiments. (D) As (C), but for PPSs identified using no cross-linking.Click here for fileCorrelation of PIP-seq read counts . Correlation in read counts between additional formaldehyde- (A and B) and UV-cross-linked (C) PIP-seq replicates as labeled.Click here for filePIP-seq is a reproducible approach . (A) Overlap in PPS calls between additional formaldehyde-cross-linked ssRNase-treated (blue) and dsRNase-treated (green) PIP-seq replicates. (B) Overlap in PPS calls between two replicates of UV-cross-linked ssRNase-treated (blue) and dsRNase-treated (green) PIP-seq replicates. (C) Overlap in PPS calls between formaldehyde-cross-linked ssRNase-treated and dsRNase-treated PIP-seq samples. (D – E) As (C), but for UV-cross-linked replicates (D) and the non-cross-linked experiment (E).Click here for fileValidation of PIP-seq by comparison with previously published RBP binding site datasets. (A) Overlap between PPSs identified from two UV-cross-linked PIP-seq samples and various CLIP datasets. Values are shown as log2 enrichment over shuffled background distributions. (B) As (A), but for a non-cross-linked PIP-seq sample. *** denotes P < 2.2 × 10-16 (chi-squared test). (C) Overlap between UV-cross-linked PPSs and 40-nucleotide T > C transversion event-containing loci from the gPAR-CLIP dataset (T > C transversion events less than 40 bp apart were merged to generate a dataset comparable to PPSs). (D) As (C), but for PPSs identified with no cross-linking. (E) Number of T > C transversion events per PPS identified using UV cross-linking (magenta) versus shuffled regions (gray). Values for the number of events per shuffled region are the average from ten random shuffles. (F) As (E), but for PPSs identified with no cross-linking.Click here for fileCharacterization of PPSs identified by UV-cross-linking and no-cross-linking PIP-seq experiments . (A) Distribution of ssRNase-treated (blue) and dsRNase-treated (green) PPS sizes from UV-cross-linked samples. Dashed lines represent mean PPS sizes . (B) As (A), but for non-cross-linked PPSs. (C) Genomic distribution of UV-cross-linked PPS density, measured as PPS base coverage normalized to RNase digestion control read counts per genomic region. Proximal intron refers to 500 nucleotides at the 5′ and 3′ ends of introns. (D) As (C), but for non-cross-linked PPSs. (E) Fraction of base pairs covered by PPSs in 100 most highly expressed lncRNAs (orange bars) and expression-matched control mRNA 3′ UTRs (purple bars) for PIP-seq libraries made with ssRNase (ss) or dsRNase (ds) under the three different cross-linking conditions (as specified).Click here for filePPSs identified by UV-cross-linking and no-cross-linking PIP-seq experiments are evolutionarily conserved .  Cumulative distribution of average SiPhy-π scores in PPSs identified by UV cross-linking (A) and no cross-linking (B). PPSs (red) are compared to flanking sequences (gray).  Comparison of average SiPhy-π scores between PPSs identified by UV cross-linking (C) and no cross-linking (D). PPSs (red) are compared to flanking sequences (gray) for various genomic regions.  Average SiPhy-π score profiles across the first and last 25 nucleotides of PPSs identified by UV cross-linking (E) and no cross-linking (F), as well as 50 nucleotides upstream and downstream of exonic (green line), intronic (blue line) and lncRNA (orange line) PPSs. *** denotes P < 2.2 × 10–16 (chi-squared test). NS, not significant.Click here for fileGenomic distribution of PPSs identified by UV-cross-linking and no-cross-linking PIP-seq experiments . (A) Average PPS density for no-cross-linking PIP-seq across 100 equally spaced bins in various genic regions. Values are normalized separately for each genic region . (B) Average PPS density for no-cross-linking PIP-seq within 50 nucleotides of CDS ends. (C) Average PPS density for no-cross-linking PIP-seq within the first and last 50 nucleotides of introns. Dotted lines in  represent the remaining  length of each element.Click here for fileMotif and co-occurrence analyses. Motifs identified by MEME analyses of PPS sequences from specific subregions of protein-coding mRNAs. Those motifs used in the co-occurrence analyses (Figure s Figure  are listClick here for fileTable of disease-associated SNPs that overlap with PPSs. Information on all flagged and GWAS SNPs overlapping PPSs identified using formaldehyde-cross-linked PIP-seq.Click here for file


The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells. The ribosome is a large molecular machine, universally composed of two subunits, that decodes non-overlapping triplet codons in mRNAs for the encoded polymerization of amino acids into proteins.E.coli.5To address the challenge of creating an evolvable ribosome, we have previously created orthogonal (O)-ribosome-O-mRNA pairs Figure  in E.coMany key ribosomal functions, including interactions with tRNAs and elongation factors, peptide bond formation in the peptidyl-transferase center (PTC), and the folding and release of the nascent chain through the exit tunnel,2, and these interactions are dynamically regulated through the translation cycle.The large and small ribosomal subunits interact through non-covalent RNA–RNA interactions between 16S rRNA and 23S rRNA that bury approximately 6000 ÅrrnB operon such that a 23S rRNA would be nested within the 16S rRNA as a large insertion  via site-directed mutagenesis.We used high-resolution structures of n Figure , an RNA E.coli expressing O-ribo(h44H101) by northern blot using a biotinylated probe specific to the O-ASD sequence of the orthogonal ribosome  rRNA could complicate ribosome folding and assembly pathways,e Figure . We detee Figure , and thee Figure . These dTo test the activity of O-riboh44H101) in protein translation we co-transformed pRSF O-ribo(h44H101) and an O-cat reporter in which a chloramphenicol acetyltransferase gene (cat) is downstream of an O-SD site for ribosome binding.4H101 in −1 Cm . We further demonstrated the activity of O-ribo(h44H101) in an independent assay by measuring its ability to translate O-luciferase ,Cells bearing the O-cat reporter alone, or provided with pRSF-O-ribo but not induced with IPTG, do not grow on 10 μg mL−1 Cm after 20 h  is dependent on the stapled 23S rRNA Figure  we introm Figure  and cont1 Figure . These dAlthough G2252A and G2553C in 23S rRNA are reported to be dominant-negative when expressed in cells,These data indicate that the mutations do not have a substantial dominant-negative effect on cellular translation in the stapled ribosome, consistent with the large subunit of O-ribo(h44H101) being functionally insulated from the endogenous small subunit Figure .In conclusion, we have described the rational, structure-based design of a stapled orthogonal ribosome. Our design inserts a circularly permuted 23S rDNA into the 16S rDNA at sites determined by structural and phylogenetic analysis, and uses an RNA hinge to staple the two subunits and facilitate subunit association and disassembly. Our results indicate that the stapled orthogonal ribosome allows the effects of mutations in 23S rRNA to be specifically coupled to translation of an orthogonal message and insulated from endogenous translation. Future work will focus on optimizing the activity of our rationally designed stapled ribosome, and fully characterizing the extent to which orthogonality in subunit association (Figure


We identified a protein kinase RIO kinase 3 (RIOK3) targeting MDA5, and here discuss its possible relationship to autoimmune diseases.RIG-I and MDA5 are cytoplasmic RNA sensors that recognize dsRNA patterns and activate MAVS to induce innate antiviral gene program. RNA sensing is regulated by ubiquitination in RIG-I while by phosphorylation in MDA5. Thus, RNA pattern-sensing is a pivotal event in host defense against virus infections, which induces innate immune response, inflammation, and augmentation of lymphocyte functions. These are rooted in RNA sensor-mediated dendritic cell (DC) maturation. TLR3, 7 and 8 in endosome and RIG-I and MDA5 RNA helicases in cytoplasm are involved in RNA sensing in DCs. MDA5 recognizes relatively long double-stranded RNA yielded as a virus replication intermediate, leading to the formation of MDA5 filament required for activating the adaptor MAVS, then inducing IRF3 activation followed by type I IFN production.  Poliovirde novo MDA5 is a prerequisite for keeping MDA5 inactive. We identified that RIO kinase 3 (RIOK3) phosphorylates MDA5 to be inactivated , B cells producing anti-DNA/RNA Ab, and other lymphocytes are involved in the process of autoimmunity. We notice here that innate immune response to RNA may trigger autoimmune disorder. Recent reports further suggest that Regnase-1 vs Loquin recognize 3′-stem-structured mRNA and regulate cytokine production such as IL-6 and TNF−α, which may suppress autoimmune disorder . RIG-I aWe have elucidated the process of MDA5 activation, by which it recognizes cytoplasmic RNA. Excess RNA production via viral infections or tumor growth allows the cells to liberate a large amounts of structured RNA and facilitate autoimmune disorders Figure . We may


Arctium lappa and has anti-inflammation, anti-microbial, and anti-carcinogenic effects. In the present study, we found that arctiin exerts anti-oxidative effects on human hair dermal papilla cells (HHDPCs).Accumulating evidence indicates that reactive oxygen species (ROS) are an important etiological factor for the induction of dermal papilla cell senescence and hair loss, which is also known alopecia. Arctiin is an active lignin isolated from 2O2)-induced cytotoxicity, cell death, ROS production and senescence after arctiin pretreatment of HHDPCs. The results showed that arctiin pretreatment significantly inhibited the H2O2-induced reduction in cell viability. Moreover, H2O2-induced sub-G1 phase accumulation and G2 cell cycle arrest were also downregulated by arctiin pretreatment. Interestingly, the increase in intracellular ROS mediated by H2O2 was drastically decreased in HHDPCs cultured in the presence of arctiin. This effect was confirmed by senescence associated-beta galactosidase  assay results; we found that arctiin pretreatment impaired H2O2-induced senescence in HHDPCs. Using microRNA (miRNA) microarray and bioinformatic analysis, we showed that this anti-oxidative effect of arctiin in HHDPCs was related with mitogen-activated protein kinase (MAPK) and Wnt signaling pathways.To better understand the mechanism, we analyzed the level of hydrogen peroxide (HTaken together, our data suggest that arctiin has a protective effect on ROS-induced cell dysfunction in HHDPCs and may therefore be useful for alopecia prevention and treatment strategies. Under sncreased , 4. In sncreased . One groncreased . Furtheralopecia –9.Alopecia is a hair loss skin disorder that typically causes baldness . It was Arctium lappa. This chemical reagent has been known to exert anti-inflammatory, anti-proliferative, and anti-microbial effects [2O2 and performed cell-based assays to determine whether H2O2-induced cell dysfunction could be inhibited by arctiin treatment. We also investigated the role of miRNA-mediated mechanisms in using microarrays and bioinformatic analysis.Arctiin is a lignin chemical reagent isolated from  effects –21. We r effects , 23. We  effects , 23. How2O2-induced cell dysfunction, we preferentially sought to evaluate the effect of arctiin on HHDPC growth. First, HHDPCs were exposed to different concentrations of arctiin for 24 h, and cell viability was analyzed with WST-1 assays. As shown in Figure Before we analyzed the inhibitory effect of arctiin against H2O2-induced cell dysfunction. We first performed WST-1 assays to analyze the inhibitory effect of H2O2 on HHDPC viability. Cells were pretreated with 0 to 30 μM arctiin for 8 h and then stimulated with 750 μM H2O2. Cell viability was analyzed after 24 h. As expected, arctiin pretreatment attenuated the H2O2-induced decrease in cell viability in a dose-dependent manner  cells as compared with untreated control cells  assays, we evaluated cellular senescence by counting the SA-β-gal-positive blue-stained senescent cells after arctiin and/or H2O2 treatment under the same experimental conditions shown in Figure 2O2 treatment increased the percentage of senescent cells by 24.89% compared with untreated control cells, however, 20 μM arctiin pretreatment the percentage of senescent cells by 10.89% compared with H2O2-treated cells , and then determined the biological functions associated with the target genes by Gene Ontology (GO) analysis using AmiGO bioinformatic tools. Our data show that arctiin has a protective effect against H2O2-induced alterations in HHDPCs may be regulated by arctiin-specific miRNAs and pathways that are possibly affected by miRNAs.Previous studies have indicated that arctiin-mediated alterations in miRNAs may be involved in regulated the four cellular mechanisms mentioned above. Also, as a matter of fact, the GO term contains bi-directional processes for each term. The term of ‘apoptosis’ includes both anti-apoptotic and pro-apoptotic processes. Therefore, GO analysis is not sufficient for understanding the biological functions of altered miRNAs in our study. To improve the accuracy of the biological meaning of the target genes, the targets were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based enrichment analysis program in the bioinformatic database DAVID. As shown in Tables 2O2-induced decreases in viability in HHDPCs. Flow cytometry demonstrated that arctiin pretreatment also increased HHDPC resistance against cell death and G2 cell cycle arrest mediated by H2O2. Furthermore, those effects of arctiin were related to its anti-oxidative effect. Upregulation of intracellular ROS levels, which was mediated by H2O2, was dramatically inhibited following arctiin pretreatment. Recent reports have demonstrated that the loss of proliferative capacity in balding dermal papilla cells is associated with expression changes SA-β-gal and markers of oxidative stress [2O2-mediated upregulation of SA-β-gal activity in HHDPCs. These findings suggest that arctiin is a novel potent therapeutic agent for oxidative stress-induced cell dysfunction in HHDPCs.The identification of novel anti-oxidant chemo-reagents is required to effectively treat or prevent alopecia due to androgen dysregulation or oxidative stress. Here, we used cell-based approaches, to demonstrate the potent anti-oxidant activity of the lignin derivative arctiin. Interestingly, pretreatment with arctiin drastically inhibited He stress . We also2O2 in HHDPCs. Although further experiments are needed to validate miRNA expression levels to confirm the microarray data, we focused on the biological meaning of the altered miRNAs in our study because the cellular functions of miRNA are dependent on their target mRNAs. Therefore, we used several bioinformatic tools to understand the biological meaning of the altered miRNAs. Our bioinformatical analysis showed that the miRNAs altered in response to arctiin pretreatment before H2O2 stimulation are commonly involved in MAPK and Wnt signaling pathways. TAK1/MAP3K7 is a member of the MAP3K family, and it has been reported that impaired hair follicle morphogenesis and hair loss are mediated by TAK1/MAP3K7 deletion in mice [Using miRNA microarrays, we identified 30 miRNAs that may have important roles in the arctiin-mediated protective effect against H in mice . Also, T in mice . Extrace in mice . In addi in mice . Althouget al. demonstrated that H2O2-induced ROS can regulate Wnt/β-catenin signaling pathways [Our bioinformatic analysis also revealed that WNT signaling pathway is putatively targeted by the miRNAs altered following arctiin treatment. Wnt signaling has also been implicated in alopecia. A recent study demonstrated that Wnt10A, which is a member of the Wnt family, is involved in the etiology of androgenetic alopecia . A Wnt10pathways . Also, ipathways . Althoug2O2-induced cell death, cell cycle arrest, and ROS production in HHDPCs. Arctiin also inhibits H2O2-induced cell senescence. We identified 30 miRNAs that were significantly expressed following arctiin treatment, indicating that they may be involved in arctiin-mediated anti-oxidative processes. Taken together, our results provide evidence that the novel putative chemoreagent arctiin can prevent HHDP cell damage mediated by oxidative stress.In summary, our results demonstrate that arctiin regulates H2. Arctiin, propidium iodide (PI) for cell cycle analysis and 2′7′-dichlorofluorescein diacetate (DCF-DA) for intracellular ROS analysis were purchased from Sigma-Aldrich .HHDPCs provided by Innoprot  were purchased and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37°C and 5% COTo analyze cell viability, HHDPCs were plated on 96-well culture dishes. After overnight growth, the cells were treated with various concentrations of arctiin (0–60 μM) for 24 h. WST-1 assay solution  was added for 40 min after the 24-h incubation. Cell viability was measured using an iMark microplate reader  at 490 nm with a reference filter of 620 nm. The results are presented as mean percentage ± standard deviation (S.D.) of three independent experiments.3) were gathered by trypsinization and fixed by adding cold 70% ethanol at −20°C for 1 h. After fixation, cells were stained by incubating with PI staining solution  at 37°C for 1 h. The distribution of each cell cycle phase was determined by evaluating the intensity of fluorescence PI staining using the FL2-H channel of a FACSCalibur .To analyze cells in different phases of the cell cycle, treated HHDPCs  and incubated at room temperature for 1 h in the dark. After incubation, cells were washed once with PBS and subjected to flow cytometer-based fluorescence analysis using a BD FACSCalibur flow cytometer (BD Biosciences).2O2 treatment, treated cells were gathered and fixed by the addition of 2% formaldehyde/0.2% glutaraldehyde. After fixation, senescence-associated β-galactosidase  staining solution  was added to the fixed cells and incubated at 37°C overnight. Senescent cells (positive blue color) were observed and counted using a bright-field microscope at × 200 magnification, and the percentages were determined.To analyze the level of cellular senescence in HHDPCs after arctiin and HTo investigate which miRNAs are altered in our study, treated HHDPCs were gathered and lysed using TRIzol reagent (Life Technologies) for total RNA purification. Total RNA was extracted from the lysed cells according to the manufacturer’s protocol and estimated its integrity and purity was estimated using an Agilent 2100 Bioanalyzer®  and a MaestroNano® microvolume spectrophotometer . We confirmed that the RNA samples had values integrity values higher than 8.0 and A260/280 and A260/230 values greater than 1.8. The qualified RNA samples were subjected to miRNA microarray analysis as described previously . Brieflyhttp://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/), AmiGo 2 GO analysis tool and DAVID  bioinformatics resources v6.7 [To investigate the biological meaning of miRNAs with significantly altered expression, we used three kinds of bioinformatical tools: MicroCosm Targets Version 5 (ces v6.7 . Using t