Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus–host interactions.Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide ‘slippery’ sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both −2 and −1 PRF and in its requirement for a  First dpol ORFs , relatedpol ORFs . PRF haspol ORFs ,7.Central to almost all examples of −1 PRF is the interaction of the ribosome with a stimulatory mRNA structure (a stem-loop or RNA pseudoknot) which promotes frameshifting on a stretch of homopolymeric bases known as the slippery sequence. How these RNA structures act is incompletely understood, but accumulating evidence supports the view that by presenting an unusual topology ,8–11 theArteriviridae in the order Nidovirales . Nsin vitro, we show here that in addition to the viral transactivator nsp1β, efficient frameshifting requires the participation of cellular poly(C) binding proteins (PCBPs). In mammalian cells, two PCBP subsets have been described, hnRNPs K/J  binding proteins and prototypes a new class of virus–host interactions.Through reconstitution of the PRRSV −2/−1 PRF signal RNPs K/J  and the RNPs K/J ,23. The RNPs K/J . PCBPs aRNPs K/J ,26. Usinin vitro frameshift efficiencies employed pDluc/PRRSV wt and mutant derivatives. This vector contains the GG_GUU_UUU shift site, 5 upstream nucleotides (nt) and 66 downstream nt  inserted between Renilla and firefly luciferase reporter genes of plasmid pDluc  and continued for 2 h at 37°C (or overnight at 22°C) after which cells were pelleted and resuspended in lysis buffer . Cells were incubated on ice for 30 min and sonicated to complete lysis. Protein purification was performed using Ni-nitrilotriacetic acid  according to standard procedures  by the addition of 10 volumes of 2× Laemmli's sample buffer, boiled for 4 min and resolved by SDS-PAGE. Dried gels were exposed to a Cyclone Plus Storage Phosphor Screen (PerkinElmer), the screen scanned using a Typhoon TRIO Variable Mode Imager  in storage phosphor autoradiography mode and bands were quantified using ImageQuant™TL software . The calculations of frameshifting efficiency (%FS) took into account the differential methionine content of the various products and %FS was calculated as % −1FS = 100 × [IFS1/MetFS1) / [IS/MetS + IFS1/MetFS1) + IFS2/MetFS2)] and similarly for % −2FS. In the formula, the number of methionines in the stop, −1FS and −2FS products are denoted by MetS, MetFS1 and MetFS2 respectively; while the densitometry values for the same products are denoted by IS, IFS1 and IFS2, respectively. All frameshift assays were carried out a minimum of three times and the measured frameshift efficiencies (±SEM) are provided in the Supplementary Data.Frameshift reporter plasmids were linearized with FspI and capped run-off transcripts generated using T7 RNA polymerase as described . Messengin vitro translation reactions (30 μl) were supplemented with 1 μM nsp1β, PCBP2 (or KH mutant derivative) or both proteins and programmed with mRNAs derived from AvaII-cut pPS0/PRRSV WT or pPS0/PRRSV CC2. Reactions were incubated at 18°C for 5 min prior to addition of edeine to 5 μM. Aliquots (1.5 μl) were subsequently withdrawn at intervals post-edeine addition, mixed with an equal volume of 10mM EDTA, 100 μg/ml RNase A and placed on ice. At the end of the time-course, samples were analysed by SDS-PAGE.WG 32P-labelled template RNAs (58 nt) containing the PRRSV PRF region (with slippery sequence precisely at the 5′ end and C-rich region towards the centre) were prepared by T7 transcription of a PCR product generated using primers flanking the frameshift region, with the 5′ primer containing the T7 polymerase promoter sequence. Test proteins were diluted where necessary in dilution buffer (DB)  and added to reactions  alongside electrophoretic mobility shift assay (EMSA) buffer , after which the radiolabelled probe was introduced. After incubation at 30°C for 10 min, samples were loaded promptly onto 4% acrylamide non-denaturing gels (acrylamide:bisacrylamide ratio 10:1) and run at 175V at room temperature until free and bound RNA species were resolved. Gels were fixed for 15 min in 10% acetic acid, 10% methanol, dried and exposed to X-ray film.Short, g, 5 min), the supernatant removed and the beads washed sequentially with 200 μl water and thrice with GST wash buffer . Following incubation with nsp1β (6 μg) for 1 h at 4°C on a rotating wheel, the protein was removed and the beads washed three times with GST wash buffer. Test protein (2 μg) was added to the beads and the incubation and washing steps repeated, with the GST wash buffer supplemented with 0.1% NP-40. After removal of the final wash, 25 μl 4 × Laemmli's sample buffer was added, the beads boiled for 5 min and supernatants analysed by SDS-PAGE.For each protein tested, 40 μl of a 50% suspension of glutathione-agarose beads was centrifuged  such that expression of the downstream cistron (fluc) was dependent upon −2 PRF within the inserted PRRSV sequences. In RRL in vitro translations, mRNAs transcribed from pDluc/PRRSV specified the synthesis of only the product of the 5′ cistron of the reporter mRNA (Rluc) and no frameshifting was evident . These experiments indicate that the 79-nt region cloned into pDluc likely contains all of the cis-acting elements required for PRRSV −2/−1 frameshifting and that supplementation of the RRL translations with nsp1β alone is sufficient to trans-activate frameshifting to levels similar to those seen in infected cells . The stimulation of PRF by nsp1β was specific to the PRRSV signal in that the protein had no effect on frameshifting at a variety of well-established sites of viral −1 PRF . Furthermore, the protein was unable to promote stop codon readthrough at variants of the PRRSV signal with an in-frame stop codon placed within the slippery sequence at the position that would be decoded in the peptidyl- (P) or aminoacyl- (A) site of the ribosome .l genome ,19. To st Figure . However123GKYLQRRLQ131  or within the putative nsp1β RBM  and the et al., . In thiset al., , where the effect was more modest. As an explanation for these observations, we speculated that the RNA downstream of the slippery sequence may interact with PCBPs. The ‘minimal’ region of PRRSV RNA required for full activity in PRF (21 nt) has only two C-rich patches, whereas previously established PCBP-binding consensus sequences more commonly have three (or more) C-triplets, potentially with each triplet binding one KH domain . To invet al., . It was  domain  32P-labelled transcript encompassing the minimal PRRSV frameshift-stimulatory region was incubated with nsp1β, PCBP1 (or PCBP2) or both proteins, prior to electrophoresis on a 4% non-denaturing polyacrylamide gel. As shown in Figure Kd of the interaction was found to be 130 nM . In the presence of PCBP2, the PR mutant of nsp1β was able to bind the target RNA indistinguishably from the WT protein, but the RBM mutant gave a smeary pattern, indicative of weak, non-specific, RNA binding . As a control, a version of the RNA probe mutated in the C-rich region . Consistent with expectation, a dose-dependent reduction in frameshifting efficiency was observed upon addition of short, uncapped transcripts containing the WT PRRSV PRF signal or IRES stem-loop IV RNAs, but not with the control mRNA with the C-rich stretch mutated.We carried out competition experiments to assess whether the WT PRRSV mRNA, the PRF-defective CC2 variant or a known PCBP2-binding RNA from poliovirus  was observed  close to the N-terminus and a third (KH3) towards the C-terminus, separated from KH1 and KH2 by an intervening sequence of variable length Figure . To testd Figure . Note thd Figure .KH1m, abolished with PCBP2KH3m and largely unaffected for PCBP2KH2  and that mutations within the GxxG loop of KH2 do not compromise the specific interaction with nsp1β. However, the KH1 and KH3 mutations likely modify the interaction of the protein with RNA, and conceivably, with nsp1β. EMSA analysis confirmed the predicted effects on RNA binding, which was reduced in the case of PCBP22 Figure . SimilarA hallmark of RNA structure-dependent PRF signals is the necessity to maintain a precise spacing between slippery sequence and stimulatory RNA to position the ribosome appropriately at the slip-site ,39. The in vitro derived mRNAs in the WG system. The extent of pausing was estimated by comparing the levels of a translational intermediate corresponding to pausing at a bound nsp1β/PCBP2 complex with that of the full-length polypeptide produced during a time course in which translation was synchronized by the addition of edeine, a potent inhibitor of initiation, 5 min after the start of the reaction. A control mRNA was prepared from plasmid template linearized at the position of the inserted PRRSV signal to serve as a pause marker. As can be seen in Figure KH1m, close to WT levels for PCBP2KH2m and much less pausing for PCBP2KH3m.Ribosome pausing at structured RNA stimulators of PRF has been widely documented and may play a mechanistic role . We testtrans-activator of this process  within the C-rich region. The KH2 domain itself is not associated with the RNA, serving rather to bind nsp1β, with the complex poised to subvert the elongating ribosome. In its capacity to stall ribosomes, the nsp1β–PCBP complex joins a growing list proteins that have been shown to modulate the elongation step of protein synthesis. These include Stm1 of ormation , the fraormation  by bindiormation , the HIFormation  and the ormation . StallinKH1m and PCBP2KH1m bound to the PRRSV RNA with similar affinity and in an nsp1β-dependent manner, although binding was noticeably reduced in comparison to the WT proteins. This suggests that PCBP1KH1m and PCBP2KH1m still associate with the RNA, but the stability, conformation or position of the complex may be changed such that only in the case of PCBP2KH1m is PRF possible, and only −1 PRF . As noted in the ‘Results’ section, compensatory activity in PRF was detected upon mixing the individually inactive PCBP1KH1m and PCBP1KH3m, suggesting that two (or more) PCBPs can cooperate to stimulate frameshifting. The differential activity of PCBP1KH1m and PCBP2KH1m could reflect an altered capacity to homo-oligomerize.Not all PCBPs were capable of stimulating PRF. The lack of activity of PCBP4 and hnRNP K/J is potentially a consequence of differences in overall size and domain organization. PCBP1, 2 and 3 are globally similar, with KH domains located at analogous positions in the protein, whereas the linker separating KH2 and KH3 is shorter in PCBP4 and much longer in hnRNP K/J Figure . These dtrans-activation of −2/−1 PRF by nsp1β/PCBP has intriguing parallels with a recently described translational readthrough event in human vascular endothelial growth factor A (VEGFA) mRNA (The FA) mRNA . Here, lFA) mRNA . How it trans-frame  products at various stages of the replication cycle.Our description of a role for PCBPs in ribosomal frameshifting adds to a growing number of examples of viral subversion of PCBP function. In the best characterized system, poliovirus, the capacity of PCBPs to associate at two discrete genomic RNA locations and in concert with different protein partners is critical to almost all aspects of the virus replication cycle (reviewed in Ref. Presently, stop codon readthrough in VEGFA  and PRF


KITLG 3′-UTR through DNA sequencing. The three SNP loci were closely linked in Guanzhong dairy (GD) goats. Two alleles of the c.1457A > C SNP introduced two miRNA sites (chi-miR-204-5p and chi-miR-211). Individuals with combined genotype TT-CC-AA had a higher litter size compared with those with combined genotypes CC-AA-GG, TC-CC-GA and CC-AC-GG (P < 0.05). Luciferase assays showed that chi-miR-204-5p and chi-miR-211 suppressed luciferase expression in the presence of allele 1457A compared with negative control (NC) and allele 1457C (P < 0.05). Western blot revealed that KITLG significantly decreased in the granulosa cells (GCs) of genotype AA compared with that in the GCs of genotype CC and NC (P < 0.05). The KITLG mRNA levels of the CC-AA-GG carriers significantly decreased compared with those of the TT-CC-AA, TC-CC-GA and CC-AC-GG carriers. In addition, cell proliferation was reduced in haplotype C-A-G GCs compared with that in haplotype T-C-A GCs. These results suggest that SNPs c.1389C > T, c.1457A > C and c.1520G > A account for differences in the litter size of GD goats because chi-miR-204-5p and chi-miR-211 could change the expression levels of the KITLG gene and reduce GC proliferation.This study identified three novel single nucleotide polymorphisms (SNPs)  in the caprine  KITLG gene contains ten exons and nine intronsKITLG mRNA is expressed in the GCs of many species; it can be expressed as either a membrane-bound (KL-1) or a soluble protein (KL-2) depending on the mRNA processing after transcriptionKITLG remains high in early antral folliclesKITLG gene may be an excellent candidate for reproductive traits in humans and livestock.KIT ligand (KITLG), also known as stem cell factor, steel factor and mast cell growth factor, has multiple biological functions during the development of goat, rats and humans by triggering its receptor tyrosine kinase (KIT)12et al.FOXO1 mRNA. Zhang et al.et al.PON1 gene, thereby increasing the risk of ischemic stroke and carotid atherosclerosis. Clop et al.GDF8 allele of Texel sheep is characterised by a G → A transition in the 3′-UTR that creates a target site for miR-1 and miR-206, which are highly expressed in skeletal muscle; consequently, translational inhibition of the myostatin gene occurs and contributes to muscular hypertrophy in Texel sheep. Given the regulatory role of miRNAs in gene expression, miR-SNPs may function as promising markers for reproductive traits.MicroRNAs (miRNAs) are small non-coding RNA molecules (each containing about 22 nucleotides) that post-transcriptionally regulate the expression of their target genes via either translational repression or mRNA degradation by binding to the complementary seed sites within the 3′-untranslated region (3′-UTR) of the target mRNA1214KITLG gene expression and the role of KITLG gene in litter size in GD goats. The following parameters were investigated: 1) the association of combined genotypes of the KITLG gene with litter size in the GD goats; (2) the potential target miRNAs of the KITLG gene; 3) the effect of target miRNAs on KITLG gene expression by the functional SNP of the caprine KITLG gene; and 4) the relationships among functional SNP, target miRNAs and litter size in GD goats.The present study aims to elucidate the potential molecular mechanism which regulates the caprine KITLG3′-UTR by DNA sequencing (KITLG 3′-UTR was submitted to NCBI (GenBank Accession No. KR869087). Two alleles of the c.1457A > C SNP introduced two miRNA sites (P < 0.05). The genotypic distribution and allele frequencies of three SNPs are shown in Figure S222s CC-AA-GG, TC-CC-GA and CC-AC-GG  were identified in the caprine quencing . The mutNA sites . The SNPNA sites . The c.1P < 0.05). GCs of different genotypes were transfected with chi-miR-204-5p mimics to determine whether chi-miR-204-5p affects the protein level of KITLG. In consideration that the 16 bases in the 5′ region sequences of chi-miR-204-5p and chi-miR-211 are the same, chi-miR-204-5p was used in Western blot analysis. Western blot analysis revealed that KITLG significantly decreased in the GCs of genotype AA compared with that in the GCs of NC and genotype CC -2, 5-diphenyl-2H-tetrazolium bromide (MTT) at 36 h after transfection in GCs to evaluate the effects of chi-miR-204-5p on cell proliferation. Cell proliferation was reduced in haplotype C-A-G GCs compared with that in haplotype T-C-A GCs (P < 0.001). Thus, the population analysed may be subject to evolutionary forces, such as selection, mutation or migration. Genome-wide bioinformatics analysis predicted that approximately 64% of transcribed SNPs as target SNPs of miRNAs can modify (increase/decrease) the binding energy of putative miRNA–mRNA duplexes by >90%17KITLG gene.In the present study, the three SNPs showed Hardy–Weinberg disequilibrium in GD goats . The Institutional Animal Care and Use Ethics Committee of Northwest A&F University approved this study.Blood samples were obtained from 385 Guanzhong dairy goats reared in Longxian County of Shaanxi Province. All diets were based on alfalfa, corn silage and a combination of concentrates, such as corn, soya meal and bone meal. Health, fertility and production records were maintained by the dairy producers and veterinarians. The litter size from the first to fourth parity was obtained from production records. Blood samples (5 mL) were collected aseptically from the jugular vein of each individual and stored in a tube containing anticoagulant ACD (citric acid : sodium citrate : dextrose = 10:27:38). All samples were delivered to the laboratory in an ice box. The genomic DNA was extracted from white blood cells by using a standard phenol–chloroform extraction protocol.KITLG genes (GenBank Accession Nos. AC_000162 and NM_001285670) were used to design five pairs of primers to amplify the 3′-UTR of the caprine KITLG gene. Their optimal annealing temperatures are shown in KITLG gene through pooled DNA sequencingKITLG gene. A PCR Master Mix (2×) Kit  was used for PCR amplification. The 25 μL PCR reactions contained 50 ng of pooled genomic DNA, 12.5 μL of 2× reaction mix , 0.5 μM of each primer and 0.5 U of Taq DNA polymerase . The cycling protocol involved initial denaturation at 95 °C for 5 min;35 cycles of denaturation at 94 °C for 30 s, annealing at 50–53 °C for 30 s and extension at 72 °C for 35 s; and a final extension at 72 °C for 10 min. Ten PCR products amplified with pooled DNA as a template were sent to the Beijing Genomics Institute  for Sanger sequencing in both directions. SNP discovery was conducted using the Chromas version 2.31 and DNAstar version 7.0 software. SNP nomenclature and numbering followed the recommendations of http://www.hgvs.org/mutnomen/recs-DNA.html#number. If SNPs were found in the 3′-UTR of the caprine KITLG gene, each sample was amplified independently. Then, the PCR products of each sample were sequenced independently to detect different genotypes by sequencing.Bovine and caprine http://www.ualberta.ca/~fyeh/popgene _download.html). Association analysis between combined genotypes and litter size was performed using repeated measures in the general linear model procedure of SPSS version 16.0 statistical software (http://en.softonic.com/s/spss-16-software). The mixed linear model was described by Yijkl = μ + Gi + Pj + Sk + Eijkl, where Yijkl is the trait measured in each of the ijklth female goat, μ is the overall population mean, Gi is the fixed effect associated with the ith genotype , Pj is the fixed effect associated with the jth parity (from first to fourth parity), Sk is the fixed effect associated with the kth sire  and Eijkl is the random error. Multiple comparisons of the means for different genotypes were performed via Fisher’s least significant difference test by SPSS version 16.0. The effects associated with the farm, birth year and birth season were not included in the linear model because preliminary statistical analyses indicated that these effects did not significantly influence the variability of traits in the analysed populations.The allele frequencies, heterozygosity (He) and PIC were calculated using POPGENE ,MicroInspector (http://www.targetscan.org/),TargetScan , RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html) and Segal Lab (http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html). According to a minimal free energy hybridisation of RNA sequence and miRNA , these five tools were used to predict miRNA binding sites. The lower (more negative) the value of the energetic score ΔΔG, the stronger the binding of the microRNA to the given site is expected to be. The combination of these approaches was considered to reduce the possibility of false positives.miRWalk . The recombinant pMD19-T vectors with different genotypes were digested by the XhoI and NotI endonuclease enzymes. Finally, the digested products with different genotypes were inserted between the Renilla and firefly luciferase genes in a psiCHECK-2 vector .The plasmids containing genotypes AA and CCwere obtained (5 cells per well) and then co-transfected with psiCHECK-2 vectors containing the 3′-UTR genotypes AA and CC. The mimics of miRNAs (chi-miR-211 and chi-miR-204-5p) and their negative controls  were co-transfected with the reporter plasmids to a final concentration of 20 nmol/μL. For GCs, the Renilla luciferase activity in lysates was measured 36 h after transfection with the Dual-Luciferase Reporter Assay System . The results were normalised against firefly luciferase activity. Assays were performed in accordance with the manufacturer’s suggestions. Each experiment was independently performed three times, and each sample was evaluated in triplicate. The Mann–Whitney test was used to compare data, and a two-sided P value of 0.05 was considered significant.Fragments of the obtained  and thenKITLG mRNA and SNPs in vivo, ovarian GCs were directly isolated from 30 ovarian tissues with different genotypes  for RNA extraction. Total RNA was extracted from these samples using TRIzol Reagent . The extracted RNA was homogenised for RT-qPCR. The quantity and integrity of each RNA sample were assessed with an Agilent 2100 Bioanalyser . A total of 30 RNA samples, which included the genotypes CC-AA-GG, TT-CC-AA, TC-CC-GA and CC-AC-GG, were subjected to reverse transcription using a cDNA High Capacity Kit . RT-qPCR analysis with the SYBR Green PCR Master Mix  was performed on a CFX Connect Real-time PCR Detection System . The relative expression levels of objective mRNAs were calculated using the ∆∆Ct method. The primers of the KITLG, β-actin and GAPDH genes are described in β-actin and GAPDH, and each assay was performed in triplicateTo detect the correlation between the expression levels of 4/well. After 24 h, the cells were treated with chi-miR-204-5p mimic for 36 h. At the end of culture, 50 μL of MTT (Amresco) was added to each well, including controls. After 4 h, the liquid in the wells was drawn out, 150 μL of DMSO was added to each well (including controls), and then the cells were incubated for 10 min at 37 °C. The OD at 570 nm was determined using an Epoch microplate spectrophotometer.The GCs’ different genotypes were seeded in 96-well culture plates at a density of 5 × 10How to cite this article: An, X. et al. Association of polymorphisms at the microRNA binding site of the caprine KITLG 3′-UTR with litter size. Sci. Rep.6, 25691; doi: 10.1038/srep25691 (2016).


Chronic obstructive pulmonary disease (COPD) is associated with cognitive impairment, but consequences of this association on a person’s functional limitations are unclear. We examined the association between COPD and increased confusion and memory loss (ICML) and functional limitations among adults with COPD. We studied adults aged 45 years or older in 21 states who participated in the 2011 Behavioral Risk Factor Surveillance System . Presence of COPD was based on self-reported physician diagnosis. ICML was based on self-report that confusion or memory loss occurred more often or worsened during the prior year. ICML-associated difficulties were defined as giving up household chores and former activities, decreased ability to work or engage in social activities, or needing help from family or friends during the prior year due to ICML. General limitations were defined as needing special equipment as a result of a health condition, having had activity limitations for 2 weeks or more in the prior month, or being unable to work. Multivariable models were adjusted for demographics, health behaviors or conditions, and frequent mental distress.COPD was reported by 9.3% of adults. ICML was greater among those with COPD than among those without COPD . People with COPD, either with or without ICML, were more likely than those without COPD to report general functional limitations. Among people reporting ICML, those with COPD were more likely to report interference with work or social activities than those without COPD .Functional limitations were greater among those with COPD than among those without, and ICML may further affect these limitations. Results from our study can inform future studies of self- management and functional limitations for people with COPD. Cognitive impairment among people with chronic obstructive pulmonary disease (COPD) has been observed in community populations –4. DetaiCognitive health issues may range from mild impairment to some type of dementia, including Alzheimer’s disease. People with COPD may have overall cognitive impairment or impairment in specific cognitive domains that affect information processing, attention, concentration, memory, executive functioning, and self-control . Memory Cognitive impairment in conjunction with COPD may be related to hypoxia or hypercapnia ,13 or toThe 2011 BRFSS collected self-reported information on both physician-diagnosed COPD  and on cwww.cdc.gov/brfss). The survey includes core questions asked in all states and optional modules on specific topics, which states select based on their programmatic needs. Optional modules may include the full state sample or split samples to allow states to obtain information on more public health topics of interest to the state programs. In 2011, COPD status was part of the core BRFSS survey. An optional module asked 138,874 people in 21 states about increased confusion or memory loss (ICML) and associated functional difficulties. The 21 states were Arkansas, California, Florida, Hawaii, Illinois, Iowa, Louisiana, Maryland, Michigan, Nebraska, New Hampshire, New York, North Carolina, Oklahoma, South Carolina, Tennessee, Texas, Utah, Washington, West Virginia, and Wisconsin. The median response rate was 53.4% using response rate standards of the American Association of Public Opinion Research . BRFSS procedures were reviewed by the CDC Human Research Protections Office and determined to be exempt research. All analyses were performed using data from the BRFSS public use files.BRFSS is a state-based telephone survey of adults aged 18 years or older, which is conducted by state health departments with assistance from the Centers for Disease Control and Prevention (CDC). BRFSS samples people with landline and people with cellular telephones to obtain representative samples of the noninstitutionalized adult population. Data are weighted to state population estimates using iterative proportional fitting or “raking.” Details on BRFSS design, methodology, sampling procedures, reliability, and validity are available  and general physical limitations . BRFSS assessed ICML-associated difficulties by asking respondents who reported ICML how often in the previous year 1) they gave up household activities or chores they formerly engaged in because of ICML or 2) how often in the previous year ICML interfered with their ability to work, volunteer, or engage in social activities. We grouped responses for these 2 questions as “always,” “usually,” or “sometimes” versus “rarely” or “never.” Respondents also noted in which areas they needed most help because of ICML . We determined whether a respondent needed help in any category of help compared with needing no help. Additionally, respondents noted if they received help from family or friends in the previous 30 days because of ICML; we categorized responses as “always,” “usually,” or “sometimes” versus “rarely” or “never.”). We examined 3 general areas of physical or mental limitations. First we determined whether a respondent needed special equipment because of a health condition. Second we determined whether a respondent had 2 or more weeks of limited physical activity in the prior month; such determination was based on responses to questions asking whether respondents had any days when physical health was not good, had days when mental health was not good, and the number of days that poor physical or mental health kept them from their usual activities . Third, we determined respondents’ employment status from reports of “unable to work.” Specific health reasons for these general limitations were not ascertained. 2), normal weight (BMI 18.5–24.9 kg/m2), overweight (BMI 25.0–29.9 kg/m2), and obese (BMI ≥30 kg/m2). Respondents were asked if they engaged in any leisure time physical activity in the prior month (yes/no). Chronic disease risk factors and conditions were determined by asking, “Have you ever been told by a doctor or health professional that you have . . . high blood pressure, high cholesterol, heart disease , stroke, diabetes, or current asthma?” Finally, frequent mental distress  was assessed with the question, “Now thinking about your mental health, which includes stress, depression, and problems with emotions, for how many days during the past 30 days was your mental health not good?” , education , number of adults in the household , health care insurance coverage, and state of residence. Health behaviors used were smoking status, obesity status, and physical inactivity. Current smokers were those who smoked at least 100 cigarettes in their lifetime and currently smoked every day or some days. Former smokers were those who smoked at least 100 cigarettes in their lifetime but did not currently smoke. Never smokers were those who had never smoked at least 100 cigarettes in their lifetime. Body mass index (BMI), calculated from self-reported height and weight, was categorized as underweight , because the sample sizes for those with COPD younger than 45 were too small for reliable comparisons. We used appropriate population weights for full and split samples for the ICML module. SAS (SAS Institute) and SAS-callable SUDAAN (Research Triangle Institute) were used to account for the complex sampling design and for variance estimation. We first examined differences in demographic and health characteristics by COPD status. We then examined differences in ICML by COPD status. Finally, we compared functional limitations by ICML and COPD status. Adjusted prevalence ratios were calculated by using 3 models. Model 1 was adjusted for sex, race/ethnicity, education, number of adults in the household, health care coverage, state of residence, and age (as a continuous variable). Model 2 was adjusted for model 1 covariables plus health behaviors and risk factors  and presence of at least 1 other chronic condition . Model 3 was adjusted for models 1 and 2 plus frequent mental distress. For descriptive analyses, sample sizes were allowed to vary. Multivariable analyses were restricted to respondents with no missing data for all covariables so we could compare prevalence ratios across models for the relevant outcome. Significance was determined as Among people aged 45 years or older in the 21 states studied, the weighted percentage with COPD was 9.3% . Compared with those without COPD, a higher percentage of people with COPD were aged 65 to 84 years, were women, were white, had a high school education or less, were current smokers, were physically inactive, were obese, had other chronic disease risk factors and conditions, and had frequent mental distress . People Overall, 12.4%  of people reported ICML during the previous year, and ICML was greater among people with COPD  than among those without COPD . Overall, the percentage reporting ICML was higher among those reporting stroke  than it was among those with COPD (25.8%), but was lower for those reporting current asthma , heart disease  or diabetes .Within each demographic, health behavior, or health condition group, the percentage with ICML was greater among those with COPD than without COPD . The onlP < .001). However, this association was not significant after we adjusted for frequent mental distress . Similar patterns were observed when we examined age grouped into 5-year intervals.We used logistic regression to further examine the inverse association of age and ICML among people with COPD. When age was examined as a continuous variable, the inverse association of age with ICML among people with COPD was significant  adjusting for demographics, the prevalence ratio of ICML for people with COPD versus those without COPD was 2.25 . When further adjusted for health risk behaviors and conditions, the prevalence ratio was 1.69 . After additional adjustment for frequent mental distress, the prevalence ratio was 1.48 .P < .05). People with both ICML and COPD were more likely than those with ICML but without COPD to report having ICML-associated limitations or having received assistance because of ICML. ICML-associated limitations among those with COPD ranged from almost one-third reporting having received assistance from family or friends because of ICML to almost two-thirds reporting needing assistance in at least 1 of 5 domains .The When adjusted for all covariables , people Associations between COPD and cognitive impairment are complex and may be due to numerous factors, including hypoxia or hypercapnia, combined effects of co-occurring chronic conditions with COPD, or effects of tobacco smoking. Although we did not have information on severity of either COPD or ICML, our results in a large sample of community dwelling adults aged 45 years or older suggested that people with COPD were more likely than those without COPD to report confusion or memory loss that worsened over the previous year, even after controlling for numerous potential confounders, such as demographic variables, co-occurring health risk factors and conditions, and frequent mental distress. General limitations were greater among those with COPD than those without regardless of ICML status, and the percentages reporting ICML-associated difficulties were substantial in this community-dwelling sample.Subjective cognitive decline has received considerable attention in the literature as interest has grown in identifying early markers of cognitive decline. Previous studies suggest that subjective memory complaint is associated with psychological distress, yet is also independently related to development of dementia –11. We oOur observation of an inverse association of ICML with age among people with COPD was at least partly explained by frequent mental distress, confirming potential confounding between psychological distress and ICML –24. SeveSeveral limitations should be noted ,26. FirsAlthough further research is needed, our results can inform treatment and self-management of people living with COPD. Because even middle-aged adults (age 45–64 y) with COPD were more likely to report ICML than were middle-aged people without COPD, health care providers should consider assessing the cognitive functioning of their patients with COPD because impairments may affect treatment and management outcomes. Furthermore, although the associations between ICML and COPD status was attenuated with adjustment for frequent mental distress, these associations remained, suggesting that both cognitive impairment and psychological distress should be assessed and monitored for effective treatment and self-management. Finally, specific functional limitations that may be of particular concern to people with COPD can be addressed.


RNA sequencing (RNA-Seq) is a powerful tool for analyzing the identity of cellular RNAs but is often limited by the amount of material available for analysis. In spite of extensive efforts employing existing protocols, we observed that it was not possible to obtain useful sequencing libraries from nuclear RNA derived from cultured human cells after crosslinking and immunoprecipitation (CLIP). Here, we report a method for obtaining strand-specific small RNA libraries for RNA sequencing that requires picograms of RNA. We employ an intramolecular circularization step that increases the efficiency of library preparation and avoids the need for intermolecular ligations of adaptor sequences. Other key features include random priming for full-length cDNA synthesis and gel-free library purification. Using our method, we generated CLIP-Seq libraries from nuclear RNA that had been UV-crosslinked and immunoprecipitated with anti-Argonaute 2 (Ago2) antibody. Computational protocols were developed to enable analysis of raw sequencing data and we observe substantial differences between recognition by Ago2 of RNA species in the nucleus relative to the cytoplasm. This RNA self-circularization approach to RNA sequencing (RC-Seq) allows data to be obtained using small amounts of input RNA that cannot be sequenced by standard methods. RNA sequencing (RNA-Seq) has become a widely used tool for investigating gene expression . MillionRNA-Seq can be used to analyze both long RNA and small RNAs. For the sequencing of long RNA fragments >200 bases), the most sensitive methods may allow researchers to study single cell transcriptome and require as little as 10–100 pg of total RNA as input 00 bases,. Standarhttp://support.illumina.com/sequencing). When total RNA is used as input for miRNA sequencing, 1 μg of total RNA is required (http://support.illumina.com/sequencing). Intermolecular ligations are also sensitive to sequences close to the RNA termini  extracellular RNA ; (ii) reTo accomplish this goal, we exploited the principle that intramolecular reactions are more favorable than intermolecular reactions by developing a sequencing methodology that uses RNA self-circularization (RC-Seq) Figure . A basicIn our protocol, we circularize the RNA template through an intramolecular ligation. This circularization allows us to prime cDNA synthesis with tagged random primers that bind the RNA template by base-pairing. These steps avoid the need to attach adaptor oligonucleotides to the RNA through intermolecular ligations. By substituting intra- for intermolecular ligation, we increased sensitivity of RNA sequencing, simplified the library purification steps and enable applications that could not be accomplished otherwise. Using this method, we show that we can obtain RNA sequencing data from nuclear samples after UV-crosslinking to protein and immunoprecipitation for samples where the use of standard methods did not yield data.2. All synthetic RNAs, primers for generating cDNA and PCR primers were obtained from Integrated DNA Technologies (IDT) and purified by polyacrylamide gel electrophoresis (PAGE).T47D cells  were maintained in RPMI-1640 media supplemented with 10% (v/v) fetal bovine serum (FBS), 0.5% (w/v) nonessential amino acids, 0.4 units/ml bovine insulin . Cells were cultured at 37°C and 5% (v/v) CO2 on ice. Nuclei were isolated by first incubating the cells in hypotonic lysis buffer , 0.5 mM dithiothreitol (DTT) and 50 U/ml Promega RNase-In) for 5 min on ice. The supernatant was removed following centrifugation at 500 × g for 5 min at 4°C. This process was repeated once. The crude nuclei were further washed twice with this hypotonic buffer to yield pure nuclei. The pure nuclei were then suspended in nuclear lysis buffer  for 10 min on ice. After vigorous vortexing and pipetting, nuclei were subjected to three freeze-thaw cycles using liquid nitrogen and a 22°C water bath. The mixture was then subjected to short pulse sonication using an Ultrasonic Homogenizer , followed by DNase I (Worthington) treatment at 37°C for 15 min. Insoluble material was removed by centrifugation at maximum speed for 15 min at 4°C. Nuclear extracts were quickly frozen in liquid nitrogen and stored at −80°C.T47D cells were incubated in fresh media containing 4-thiouridine (Sigma) at 100 μM. Medium was removed 14 h later and cells were washed once with Dulbecco's phosphate buffered saline (Sigma) and UV-irradiated at 365 nm with an energy of 300 mJ/cmThe Ago2 immunoprecipitation and clipped RNA isolation were carried out based on the original PAR-CLIP protocol  with two2 (Epicentre), 4 μl 5 M Betaine (Epicentre) and 1 μl ligase. To remove the remaining linear RNA, 2.3 μl of 10× RNase R buffer (Epicentre) and 1 μl of RNase R  was added to the reaction mixture. The RNase R digestion was carried out at 37°C for 10 min. After the digestion, an oligo purification column  was used to isolate the circularized RNA following the manufacturer's instructions. Purified RNA was eluted with nuclease-free water.RNA, including synthetic RNA or clipped RNA were circularized with CircLigase™ II ssDNA Ligase (Epicentre) at 60°C for 1 h in a 20 μl reaction volume containing 2 μl 10× reaction buffer, 1 μl 50 mM MnCl2O to a total of 12 μl. Following heating of the solution at 65°C for 5 min on a thermal cycler, it was cooled directly on ice for at least 1 min. To this solution was added 4 μl 5× Superscript II reaction buffer, 2 μl 0.1M DTT, 1 μl RNase-Out and 1 μl Superscript II (Life Technologies). The solution was mixed gently and placed on a thermal cycler at 25°C for 15 min, then 42°C for 30 min. Next, 2μl of 1 M KOH was added solution and heated to 95°C for 15 min after which the solution was cooled to room temperature and neutralized by 2 μl 1 M HCl.The first step in library preparation is generation of the complementary DNA (cDNA) strand from the circularized RNA. To generate the cDNA strand, we mixed a circularized RNA solution with 0.5 μl cDNA primer , 1 μl 10 mM dNTP solution  and H2O. To this cDNA solution was added 0.5 μl 0.1 M DTT, 1 μl 10 mM dNTP, 0.5 μl 100 μM 3′-end extension primer , 5 μl 5× Buffer 2  and 0.5 μl Klenow DNA polymerase I (NEB). The extension reaction was carried out at 25°C for 15 min, and then at 95°C for 3 min to deactivate the enzyme. The tagged cDNA was purified again with a column  and eluted with 23.5 μl H2O. To the purified cDNA solution, 25 μl Failsafe PCR Premix E (Epicentre), 0.5 μl forward PCR primer , 0.5 μl reverse PCR primer , and 1 μl Failsafe PCR enzyme (Epicentre) were added. The mixture was first heated at 95°C for 1min, followed by 12–24 cycles (depending on the quantity of RNA input) of 95°C for 30 s, 55°C for 30 s and 68°C for 3 min with a final extension step at 68°C for 7 min. The crude PCR product was purified by Agencourt AMPure XP magnetic beads (Beckman Coulter) using a 1:1 volume ratio. Final PCR product was eluted with H2O and analyzed by Bioanalyzer for library size distribution. The library was then quantitated by PicoGreen Assay and sequenced with Illumina HiSeq2000 in a paired-end mode (2 × 100nt).The cDNA was column-purified  and eluted with 17.5 μl HEach pair of the raw reads was merged to obtain the full-length sequence of the original cDNA molecules using the program FLASH. The minimum overlapping length was set at 10nt. The merged paired-end reads were kept in one file, while the unmerged paired-end reads were kept in two different files. Next, each merged paired-end read as well as each first read in the unmerged reads file underwent repeating unit extraction using a Perl script (part of the developed package). In this script, maximum error number is set at 10% of the length of a repeating unit. After the repeating unit is identified for each read, a read expansion script is used to expand the repeating unit by moving one base at a time from its 5′-end to its 3′-end so the number of reads generated in the group is equal to the number of bases of the repeating unit. Each read in the group was then aligned to hg19 using TopHat2 with the default parameters (maximum two errors). For CLIP-Seq data, each read was aligned to a reference transcriptome (hg19) generated within the TopHat2 program. A SAM output file for accepted alignments was obtained. For a group of expanded reads, only uniquely aligned derivatized reads were kept. Next, an algorism to examine if all the unique alignments were converged to the same genomic/transcriptome location was executed. If aligned, the best alignment  is chosen as the read representing the original RNA sequence.http://www4.utsouthwestern.edu/coreylab/protocols.html. Script-1 uses the raw read file containing merged paired-end reads as the input. The output file of script-1 is the input file of script-2. Script-3 uses alignment output SAM file as the input and produces sorted BAM file for downstream analysis.Three separate perl scripts used for read repeating unit extraction (script-1), repeating unit expansion (script-2) and finding the original RNA sequence (script-3) will be made available at: in silico simulation was carried out. In the simulation, five groups of reads of different lengths were generated, 20 nt, 40 nt, 60 nt, 80 nt and 100 nt. For each group, 5000 reads were randomly selected computationally. The original genomic location of each read was recorded during generation. For the traditional alignment method, these 5000 reads were aligned to hg19 using TopHat2 and only uniquely aligned reads and their alignments were retained. By comparison with their original genomic locations, the percentages of correctly uniquely aligned reads were calculated. For the expanded alignment approach, each of the 5000 reads was circularized to mimic the repeating pattern of the RC-Seq sequencing data. Then the expanded alignment method was employed to align the generated reads to the genome. Similarly, the percentages of correctly and uniquely aligned reads were calculated.To examine whether the expanded alignment approach is valid in aligning the sequencing data to the correct positions in the genome, an http://cran.r-project.org) (We used MiClip to find the RNA binding sites of the Ago2 protein. The MiClip package has been reported and is publicly available at CRAN (ect.org) . We firsOur sequencing method is based on circularizing input RNA to avoid the need to introduce primer binding sites using an intermolecular ligation step. We chose Circligase II for the intramolecular ligation Figure , step 1 We optimized ligation conditions using a synthetic 21 base oligomer. We added Circligase II to circularize the RNA followed by RNase R to remove residual linear RNA Figure  was chosThe presence or absence of terminal phosphate groups can affect the activity of nucleases and the efficiency of template preparation. miRNAs have 5′ phosphate groups while other input RNAs could have 3′ phosphate groups. It is important, therefore, to understand the capacity of RNase R to degrade RNAs with differing phosphate groups. To ensure that RNase R would be capable of fully digesting all the RNA in our input samples, we examined digestion of RNA with phosphate groups at either or both the 3′ and 5′ termini. RNase R efficiently degraded RNA regardless of phosphate location Figure , suggestWe then tested the conversion of linear to circularized RNA by Circligase II in the presence or absence of RNase R. We found that we could achieve a conversion rate to a single circularized product of >80% Figure . AdditioBy contrast, using the best reagents available for RNA adaptor ligation , we carried out the ligation reaction at 25°C for 2 h and observed a conversion rate of only ∼49%, with at least one by-product formed, likely a concatamer   in which multiple copies of the template are transcribed ,25. To gAfter obtaining linear cDNA, we hybridized a tagged random primer and added DNA polymerase to extend the DNA strand. This polymerization creates a product with two primer recognition sites that could be used for PCR Figure , step 3.We then performed one round of PCR on the purified cDNA with one primer binding the 3′ tag, and a second primer (indexed) binding the 5′ tag. The resultant library was subjected to single-end or paired-end sequencing Figure , step 4.After library preparation we performed RNA-Seq and analyzed the resulting reads. Existing software was not able to efficiently locate the original sequences because our template is circular and subject to rolling circular amplification (RCA) ,25. We aAll the reads contained repeating units, and our first step was to identify the repeating unit as a single sequence Figure , step 1.Because the method requires sequencing tandem RNAs, the output sequences are repetitive and cannot be mapped to reference sequences using the standard, direct alignment approach. We used RC-Seq data obtained from partially randomized synthetic 40 nt RNA mixture (with 10 randomized sequence at each terminus) to test the efficiency of standard direct alignment approach and found that only ∼3% of the reads could be aligned Figure . Using eWe carried out a computational simulation to validate our approach to regenerate the original RNA sequence and subsequently identify its origin within an actual genome. In the simulation, five groups of linear RNA with different sizes  were randomly chosen from human genome hg19 and their original locations were recorded. We then used standard alignment program TopHat2  to identWe then computationally circularized each of these RNAs to mimic the output of RC-Seq and applied the expanded alignment approach. We found that circular random sequences can be uniquely and correctly aligned to their original locations with percentages close or equal to those from the linear RNA of analogous size Figure . This fiSince miRNAs are an important biological small RNA, we also carried out a simulation for every human miRNA following the strategy described above for both linear and circularized miRNAs Figure . As we hWe first tested our experimental and computational protocols using a synthetic forty base RNA template containing ten randomized bases at each terminus. This RNA template was commercially synthesized and it is possible that at certain randomized positions four RNA bases  are not equally represented. To gauge the sequencing sensitivity of RC-Seq relative to standard methods we prepared a series of RNA-Seq libraries in parallel using commercially available Tru-Seq small RNA preparation kit and RC-Seq with RNA template quantities varying from 100 to 0.1 ng.We consistently observed a significant increase in unique read numbers for RC-Seq over Tru-Seq when the same amount of starting RNA was used Figure . For exaTo confirm that cDNA library was indeed generated from original RNA input, not from possibly contaminated reagents used during the RC-Seq library preparation, we prepared libraries with no input RNA and RNA of 10 ng of 40 nt randomized sequence side by side and resolved them on an agarose gel. We observed a single band at low molecular weight region (<100 bp) in the "no input RNA" control library, likely a primer–dimer by-product . The lane of the library made with 10 ng RNA input displayed expected high molecular weight product. These data confirm the specificity of RC-Seq in generating useful reads from original RNA input molecules.Ligations may differ in efficiency depending on the identity of terminal bases, creating a bias during library preparation. To investigate possible sequencing biases caused by using CircLigase II, we analyzed the base profile of the first or the last base in each RC-Seq library. At 5′-end, we found that G was the most prevalent base, followed by A, C and U. G was also the major base at 5′-end of Tru-Seq libraries Figure . At 3′-eTo test our methodology in a demanding application, we applied it to the analysis of human nuclear Argonaute 2 (Ago2)-associated RNA obtained from photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) . In PAR-CLIP-Seq can be used to analyze fragmented cellular RNA or miRNAs. Unlike miRNAs, which contain a 5′ phosphate and a 3′ hydroxyl group, RNA fragments may lack these features, preventing their amplification. To ensure that all RNA could be amplified regardless of origin, we used T4 polynucleotide kinase because the enzyme removes 3′ phosphates while adding 5′ phosphates.A successful PAR-CLIP experiment requires a robust and specific antibody. We used 11A9, a well characterized anti-Ago2 antibody and used by others for CLIP-Seq ,30. NeveUsing the same cellulose membrane, we analyzed the presence of Ago2 by western blot and observed a strong band at 100 kDa in both 11A9 and 4G8 lanes, corresponding to free-form Ago2 . Additionally, when RNA and Ago2 were not crosslinked with UV light, no bound RNA complex was detected . These data suggested that this band (∼110 kDa) contained Ago2 and was the Ago2-RNA crosslinked complex. Sequencing of the isolated RNA revealed substantial miRNA reads in the dataset (data not shown), further confirming that the 110 kDa band is Ago2-associated.PAR-CLIP of these nuclear samples is particularly challenging because much less RNA is associated with Ago2 in nucleus than that in cytoplasm Figure . PAR-CLIUpon obtaining the RNA from nuclear Ago2 PAR-CLIP, we made repeated efforts to prepare sequencing libraries using standard Tru-Seq protocols . In seven independent ligation experiments, we did not detect ligated products between input RNA and adaptor oligonucleotide. Including more stringent treatment with polynucleotide kinase to remove 3′ phosphates or tripling the amount of input nuclear lysate for immunoprecipitation did not afford detectable product . Even though no ligated product was apparent, we attempted to prepare cDNA libraries. We observed no cDNA upon gel electrophoresis after six attempts using the Tru-Seq protocol for small RNA library preparation . Bioanalyzer examination showed no visible product peaks. These outcomes emphasized the need for a new approach for library preparation.After observing the poor outcome of library generation based on intermolecular ligation between RNA and adaptors, we applied our RC-Seq method to prepare libraries. After reverse transcription, 3′ end extension, and PCR amplification we obtained products ranging in size from 200 to 450 bp for replicate libraries Figure . The sizThe 200–450 bp range is typical for long RNA-Seq library and allowed the use of paired-end sequencing. Another advantage of using 200–450 bp fragments is that the crude RC-Seq library of CLIP-Seq RNA can be purified with AMPure XP magnetic beads. Use of these beads avoids the laborious and low efficiency PAGE purification step required for standard small RNA-Seq methods and further improves the efficiency of the protocol.We sequenced the nuclear Ago2 PAR-CLIP library derived from our RC-Seq protocols and analyzed the results. The aligned sequencing data showed a dominant T to C mutation, consistent with incorporation of 4-SU and successful adaptation of the PAR-CLIP protocol to RC-Seq Figure  14). We. We14). R2 = 0.94. Subsequent genomic annotation shows that >50% of clusters are localized within intronic regions .The sequencing data were reproducible between duplicate determinations with a strong concordance between grouped aligned reads Figure . Of 7839s Figure . This das Figure , likely s Figure ,14. The It has become clear that background in PAR-CLIP experiments needs to be taken into account for proper interpretation of data . To filtTo further evaluate the quality of our data, we examined the overlap between the nuclear Ago2 binding sites detected by RC-Seq and those determined by other methods using Ago2 PAR-CLIP. There are no publically available data specifically on nuclear Ago2 PAR-CLIP for comparison, but we found six publically accessible Ago2 PAR-CLIP datasets generated from using whole cell lysate ,32–36. TNotwithstanding these differences, we carried out a comparison to determine overlap in potential binding sites between these previous studies and our own , and the previous studies with one another . We found that ≤10% of the clusters overlapped between the individual whole cell datasets and our nuclear data . The overlapping percentage among any two of the six datasets ranged from 15 to 45% . When comparing our cytoplasmic Ago2 datasets to those six published datasets from whole cell lysate, similar overlapping cluster percentage (15–50%) was obtained (data not shown). These comparisons suggest that our RC-Seq method identifies many of the same cytoplasmic signifcant clusters as had been observed previously, but that the identities of cytoplasmic and nuclear clusters are substantially different.Ongoing studies have analyzed data from ten additional RC-Seq experiments and have shown similar sensitivity and reproducibility from nuclear, chromatin, and cytoplasmic fractions . Analysis of duplicate read clusters from independent experiments has shown high reproducibility both in the breadth and shape of the clustered reads relative to the transcript targets. For example, at the intron-exon junction for the gene EEF1D we observed almost perfect overlap in both read location and distribution between two independent replicate experiments Figure .RNA-Seq is a valuable technology and is becoming increasingly important as costs decrease and computational analysis becomes more accessible. Challenges to even more widespread use include the need to analyze small sample quantities with greater sensitivity. Meeting these challenges will require innovative approaches to library preparation.http://support.illumina.com/sequencing). Insufficient amounts of available RNA may be a significant obstacle for some investigations of small RNAs including miRNAs, piRNAs, snoRNAs, extracellular small RNAs, and RNAs fragmented generated by approaches like CLIP.Standard methods for sequencing small RNAs (<200 bases) are limited by the need to employ sequential intermolecular RNA and adaptor ligations  and sequWe have shown that, by combining intramolecular RNA circularization and RNA-based rolling circle amplification (RCA), small RNA sequencing sensitivity can be greatly increased. In addition, using RCA  makes it feasible to exploit a library purification approach that uses magnetic beads—an approach commonly used to purify long mRNA-Seq libraries. Finally, the first step is the RNA self-circularization carried out at 60°C by a thermostable Circligase II. The higher temperature for circularization reaction reduces interactions among RNA template molecules, making more sequences accessible to analysis.RNA circularization followed by RCA has been used to detect specific miRNAs with quantitative PCR . This meTo achieve strand-specific sequencing, RC-Seq first uses tagged random primers to generate the first strand cDNA during RCA, rather than random hexamers used in CirSeq or other traditional long RNA-Seq methods. This first strand cDNA is further extended with a DNA polymerase and 3′-end blocked tagged random primers so that a PCR can be applied directly on the first strand of cDNA and original RNA strand information may be inferred from the sequencing reads. Tagged random priming has been used in efficient long RNA sequencing  and genoCapture and Amplification by Tailing and Switching) which can sequence as low as 100 pg of small (or long) RNA or DNA  and those that represent distinct cDNAs.When using less than 1ng of small RNA input for RC-Seq, extensive PCR amplification (more than 20 cycles) is often needed to generate sufficient DNA for sequencing. Thus, non-uniform amplification of cDNA molecules could become an issue. A few publications have explored the use of random sequences to correct for PCR-generated artifacts –43. In tOur laboratory has a longstanding interest in the nuclear interactions between RNA and RNAi factors like argonaute and GW182. Relative to RNA from whole cells, nuclear RNA is scarce and difficult to obtain in quantities typically used for RNA-Seq. For CLIP-Seq, the RNA being analyzed is also limited by the efficiency of the crosslinking and immunoprecipitation steps. When attempting CLIP-Seq using isolated nuclear RNA and standard ligation-based methods, we failed to prepare a library that could serve as a basis for sequencing. We concluded that improving library preparation methods would be necessary to accomplish the investigation. Using RC-Seq, we were able to identify more than 7000 significant Ago2 binding clusters in cell nucleus, further highlighting the high sensitivity of RC-Seq.R2 = 0.94 for two nuclear replicates. For three cytoplasmic sequencing datasets a 0.82 < R2 < 0.94 was determined between any two replicates. By carrying out an Argonaute HITS-CLIP using whole-cell lysate and a ligation based library preparation approach, Chi et al. reported a 0.80 < R2 < 0.83 between datasets (et al. reported a R2 = 0.88 (For a sequencing method to be used widely, it must show good reproducibility in generating sequencing libraries. We obtained a coefficient of determination datasets . In anot2 = 0.88 . Our resIt is not surprising to see limited overlap between our nuclear Ago2 binding sites with those published datasets. The published Ago2 PAR-CLIP were carried out using whole-cell lysate for the immunoprecipitation, likely with an emphasis on cell soluble fraction. When preparing nuclear samples, we purified nuclei first, followed by sonication and DNase I treatment to remove chromosome DNA, so that maximum amount of Ago2-associated RNA could be recovered. These steps were not applied by prior publications when harvesting the whole cell lysate. In addition, the difference in cell line being studied and different types of anti-Ago2 antibodies being used may also contribute to the observed difference on cluster locations among these samples.In summary, our RC-Seq data on nuclear Ago2 associations suggest previously unknown interactions between RNA and RNAi factors in cell nuclei and will guide detailed investigations into the natural function of nuclear RNAi and its potential impact on splicing and transcription. More generally, successful application of RC-Seq to PAR-CLIP indicates that intramolecular circularization during library preparation can facilitate access to sequencing data from samples where amounts of RNA are limiting and structures of RNA hinder intermolecular ligation. Rare RNA sequences can be analyzed with better coverage, allowing more accurate characterization of transcripts that may be present in only a few copies per cell.Supplementary Data are available at NAR Online.


The major focus of ALS research has recently moved to RNA control of motor neuron functions, as most of the newly identified genes, that alone account for more than half of ALS familial cases, are clearly associated to RNA regulation. These include FUS and TDP43, two DNA/RNA binding proteins with a role in the regulation of RNA transcription, splicing, transport and translation, and C9orf72, a gene that is marked by the presence, in carriers, of an highly expanded GGGGCC repeat that is believed to provide the mutant gene of an acquired, toxic feature by an RNA-dependent gain of function mechanism. Thus, RNA dys-metabolism is likely to represent a central issue in ALS pathogenesis. Yet, whether a specific step of RNA processing is particularly affected in ALS motor neurons is unclear. We have recently obtained evidence that a prominent effect of FUS and C9orf72 expression is the induction of stress granules-associated translational repression. In this presentation I will discuss our recent work on the molecular mechanisms underlying these effects and their potential relevance in ALS pathogenesis.


The synthesis of ribosomal RNA (rRNA) is tightly coupled to nutrient conditions and growth factors. Murayama et al. described a novel nucleolar silencing complex eNoSC involved in rRNA transcription regulation in response to glucose deprivation . This coin vitro. The RNA and SirT1 binding sites on NML overlap partially. Thus, RNA and SirT1 compete for interacting with NML. The NML complex purified from cells contains predominantly ribosomal rRNA , presumably due to its nucleolar localization. The interaction of NML with rRNA in vivo was inhibited by glucose deprivation, which is known to down-regulate pre-rRNA transcription through the AMPK-mTOR-RNA Pol I signaling pathway. Therefore, glucose deprivation strongly stimulates NML-SirT1 binding, resulting in the recruitment of a minor fraction of SirT1 to the nucleolus. Assembly of eNoSC contributes to the down regulation of rRNA transcription in the absence of glucose. In fact, various stresses that down regulate rRNA transcription, such as DNA damage, inhibition of mTOR using rapamycin, or direct inhibition of RNA Pol I using actinomycin D or CX5461, all promote SirT1-NML binding.Unlike yeast Sir2, which localizes to the nucleolus and telomeres, mammalian SirT1 is predominantly located in the nucleoplasm. Therefore, SirT1 function in the nucleolus may require recruitment by the nucleolar NML. In a recent study, we investigated the dynamic regulation of eNoSC assembly by nutrient conditions and found that the interaction of SirT1 and NML was regulated by rRNA [These findings led to a positive feedback model that coordinates SirT1 function in the nucleolus with nutrient signaling: Under nutrient-rich condition, mTOR and other factors stimulate RNA Pol I and Pol III synthesis of rRNA. Nascent rRNA is processed and assembled into pre-ribosomal particles at the nucleolus. The abundant nascent rRNA also interacts with NML and prevents the recruitment of SirT1. This mechanism provides a positive feedback loop to nutrient signaling by protecting the active nucleolar rDNA from repression by the NML- SirT1 complex Figure . During SirT1 is well known for its regulation by both the substrate and the reaction products. NAD+ is required for SirT1-mediated deacetylation reaction, whereas the deacetylation byproduct nicotinamide is an inhibitor of SirT1 activity . Assemblswi6 and Chp1 from fission yeast [swi6 binds RNA through a positively charged flexible region, whereas Chp1 binds RNA through a flexible sequence in its chromodomain. RNA binding has diverse functional effects on these proteins. RNA competes with the HP1swi6 chromodomain for binding to H3K9me, stimulates Chp1 chromodomain binding to H3K9me, and inhibits NML binding to SirT1. It is possible that sequence non-specific RNA binding through unstructured region is an important feature of heterochromatin proteins, which allow them to be regulated at many chromosomal loci by locally produced coding and non-coding RNA.The new findings on NML provides another example of sequence non-specific RNA binding similar to several heterochromatin proteins such as HP1on yeast , 6. HP1sIn addition to regulation of NML-SirT1 binding, it is possible that RNA may regulate other biochemical activities of NML. NML has a methyltransferase- like domain at the C-terminus; but its substrate is still unknown. Murayama et al. has excluded histone proteins as the target of NML . Recentl


Neurovascular inflammation is associated with a number of neurological diseases including vascular dementia and Alzheimer’s disease, which are increasingly important causes of morbidity and mortality around the world. Lipotoxicity is a metabolic disorder that results from accumulation of lipids, particularly fatty acids, in non-adipose tissue leading to cellular dysfunction, lipid droplet formation, and cell death.Our studies indicate for the first time that the neurovascular circulation also can manifest lipotoxicity, which could have major effects on cognitive function. The penetration of integrative systems biology approaches is limited in this area of research, which reduces our capacity to gain an objective insight into the signal transduction and regulation dynamics at a systems level. To address this question, we treated human microvascular endothelial cells with triglyceride-rich lipoprotein (TGRL) lipolysis products and then we used genome-wide transcriptional profiling to obtain transcript abundances over four conditions. We then identified regulatory genes and their targets that have been differentially expressed through analysis of the datasets with various statistical methods. We created a functional gene network by exploiting co-expression observations through a guilt-by-association assumption. Concomitantly, we used various network inference algorithms to identify putative regulatory interactions and we integrated all predictions to construct a consensus gene regulatory network that is TGRL lipolysis product specific.System biology analysis has led to the validation of putative lipid-related targets and the discovery of several genes that may be implicated in lipotoxic-related brain microvascular endothelial cell responses. Here, we report that activating transcription factors 3 (ATF3) is a principal regulator of TGRL lipolysis products-induced gene expression in human brain microvascular endothelial cell. The estimated prevalence of dementia of persons greater than 70 years of age is 14.7% [One of the potential inducers of neurovascular inflammation is triglyceride-rich lipoprotein (TGRL) particles and their lipolysis products . Lipopro-4), over all techniques. Consistent with our previous results in endothelial cells [As shown in Figure al cells , ATF3 shThe 79 DE genes that have been in parallel identified from all methods form a functional gene network with 54 functional categories that are significantly over-represented in this gene set, which also implicates 12 KEGG pathways Figure A. We intDifferential analysis of gene expression data showed that HBMVEC treated with either LpL or TGRL altered the expression of a small percentage of genes . TGRL + LpL (TGRL lipolysis products) treatment affected the expression of 5.61% of all genes detected . Functional classification of genes modulated by lipolysis product (TGRL + LpL) treatment identified multiple functional classes including transcription factors, inflammatory responses, apoptosis, cell cycle, cell proliferation, ion metabolism, lipid metabolism, kinase activity, signalling pathways, DNA binding, protein binding, protein folding, and proteins of unknown function.TGRL lipolysis products activated genes encoding transcription factors including activating transcription factor (ATF3), ATF4, ATF2, DNA damage-inducible transcript 3 (DDIT3), CREB binding protein (CREBBP), Krueppel-like factor 4 (KLF4), KLF5, aryl hydrocarbon receptor (AHR), peroxisome proliferator-activated receptor delta (PPARD). ATF3 is a member of the mammalian activation transcription factor/cAMP responsive element-binding (CREB) protein and a member of the ATF family of transcription factors. This gene is induced by a variety of signals including many of those encountered by cancer cells, and is involved in the complex process of cellular stress response  and postCREB binding protein (CREBBP) is ubiquitously expressed and is involved in the transcriptional coactivation of many different transcription factors and known to play critical roles in embryonic development, growth control, and homeostasis by coupling chromatin remodeling to transcription factor recognition. Transforming growth factor-β signalling pathways mediate epithelial-mesenchymal transition are dependent on the transcriptional co-activator CREBBP . CREBBP Krueppel-like factor 4  was up-regulated 1.7-fold by TGRL lipolysis Figure C. HMGB2 UDP-glucose 6-dehydrogenase ; Figure The A-kinase anchor protein 5 (AKAP5), a member of the AKAP family and also known as AKAP75 or AKAP79. AKAP5 is predominantly expressed in cerebral cortex and may anchor the PKA protein at postsynaptic densities (PSD) and be involved in the regulation of postsynaptic events. It can bind to the RII-beta regulatory subunit of cAMP-dependent protein kinase (PKA), and also to protein kinase C and the phosphatase calcineurin . AKAP5/ATo gain a better insight on the biological processes, cellular components and molecular functions that are affected by the differentially expressed genes, we performed a functional analysis by using the DAVID toolbox (see Methods) ,36. As sThe most significant discovery of this study is the robust and rapid induction of genes encoding transcription factors, inflammation, and apoptosis. More specifically, HBMVEC treated with TGRL lipolysis products encode transcription factor ATF3-related genes in the MAP Kinase pathway associated with AP-1 signalling. This, then, stimulates an array of pro-inflammatory and pro-apoptotic genes that effect endothelial cell injury. ATF3 can be induced by stress and growth factors in mammalian cells, and is thought to play an important role in the cardiovascular and nervous system. However, little is currently known about how the induction of ATF3 is regulated, except that the JNK pathway is involved. Previous studies have shown up-regulation of ATF3 to act as either pro-inflammatory  or anti-TGRL lipolysis products-induced signalling pathways involve in mitogen-activated protein (MAP) Kinase, TGF-β and p53 signalling. The four major MAP Kinase pathways, ERK, JNK, P38, and BMK/ERK5, perform activating phosphorylations of nuclear transcription factors. Of these, ERK has been shown to inhibit ATF3 expression while JNK activates ATF3 through transcriptional regulation . JNK actOur study has shown that increased expression of ATF3 occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the apoptosis-inducing protein DDIT3 transcriptional factor in response to TGRL lipolysis products at 3 hr. DDIT3, CHOP, or GADD153 protein functions as a dominant-negative inhibitor by forming heterodimers with other C/EBP members, such as C/EBP and is linked to diabetes ,46,47. EPrevious studies have shown up regulation of ATF3 to act as either pro-inflammatory  or anti-TGRL lipolysis products-induced up regulation of HDAC9 suggested vascular inflammation in brain microvascular endothelial cells. Several studies elucidated that the effects of individual HDACs in vasculature. HDAC9 promotes angiogenesis and determines the angiogenic gene expression pattern of endothelial cells . AlthougLipolysis of TGRL produces an array of remnant lipoprotein particles, fatty acids, phospholipids, diglycerides, and monoglycerides . Any andThe peak elevation in blood TGRL is about 3–4 hours after a meal and blood triglycerides can rise 0.3-fold to many-fold higher during the postprandial period. Lipolysis of TGRL by endothelial cell-associated lipoprotein lipase likewise peaks in the postprandial period thus producing more endothelial cell injury or lipotoxicity as a result of the release of TGRL lipolysis products directly on the endothelial cells. Rather than low-grade, chronic injury, endothelial cells are injured repetitively over the course of a day, which could be a more pathological form of injury.The precise pathogenetic mechanisms associated with lipotoxic neurovasclular inflammation remain to be elucidated. These experiments confirm the results of our previous work showing that high physiological to pathophysiological concentrations of TGRL lipolysis products induce brain microvascular lipotoxicity resulting in endothelial cell injury and apoptosis. These effects could alter blood–brain barrier integrity allowing not only potentially injurious lipids access to the brain, but also enabling thrombosis to occur on the injured brain endothelium causing brain microinfarctions. Thus, neurovascular lipotoxicity could induce brain injury and resulting decline in cognitive function.Our microarray data and system biology analysis give us possible clues involving cell signalling pathways in HBMVEC treated with TGRL lipolysis products. The systems biology approach that was taken in this study allows to identify statistically significant associations by using a consensus analysis of 4 different methods, as well as looking simultaneously at the transcriptional, protein-protein interaction and gene ontology networks. Using a supervised interaction inference methodology that exploits the experimentally identified interactions that are known so far, is likely to increase the true positive rate of our analysis when compared to unsupervised methods. An extension to this work is looking at how information propagates through the combined gene regulatory and signal transduction networks by applied information-based methodologies -60. Our Our systems biology approach demonstrated a robust and rapid induction of genes encoding transcription factors, inflammation, and apoptosis. Specifically, HBMVEC treated with TGRL lipolysis products encode transcription factor ATF3-related genes in the MAP kinase pathway associated with AP-1 signalling, which stimulates an array of pro-inflammatory and pro-apoptotic genes that effect endothelial cell injury. These experiments confirm the results of our previous work and demonstrate the complex, parallel, and multifaceted aspects of cell signalling that high physiological to pathophysiological concentrations of TGRL lipolysis products induce in brain microvascular lipotoxicity resulting in endothelial cell injury and apoptosis. Future studies are needed to show if these neurovascular pathophysiological processes are associated with the development of cognitive impairment.Human TGRL isolation: Postprandial blood samples were obtained 3.5 h after consumption of a moderately high-fat meal, which generally corresponds to the peak elevation in plasma triglyceride concentrations. Triglyceride-rich lipoproteins (TGRL) were isolated from human plasma at a density of less than 1.0063 g/mL following an 18 h centrifugation at 40,000 rpm in a SW41 Ti swinging bucket rotor  held at 14°C within a Beckman L8-70 M (Beckman) ultracentrifuge. The top fraction (TGRL) was collected and dialyzed in Spectrapor membrane tubing  at 4°C overnight against a saline solution containing 0.01% EDTA. This protocol was approved by the Human Subjects Review Committee at the University of California Davis.Human brain microvascular endothelial cells (HBMVEC)  were cultured in CSC Complete Medium which includes 10% serum (4Z0-500)  under an atmosphere of 5% CO2: 95% air at 37°C. Lipoprotein lipase (LpL) (L2254) was purchased from Sigma, St. Louis, MO. 3´ IVT Express Kit was purchased from Affymetrix, Santa Clara, CA. Cells were exposed for 3 hr to the following conditions, media, TGRL (150 mg/dL =1.5 mg/mL), lipoprotein lipase (LpL) (2 U/mL), and TGRL lipolysis product (TGRL (150 mg/dL) + LpL (2 U/mL)). The final concentration of TGRL, LpL and TGRL lipolysis products were diluted in media and pre-incubated for 30 minute at 37°C prior to application. After incubation, cells were washed with cold PBS and further process for RNA extraction.Total RNA was extracted from Media, LpL, TGRL, and TGRL lipolysis products in HBMVEC in 6-well plate using RNeasy Mini Kit (Qiagen) including the DNA digestion step as described by the manufacturer. Microarray experiments were performed with pooled RNA isolates from each treatment group.GeneChip analyses of the pooled total RNA samples  were performed as previously described ,61. A 20Since GeneChip analysis was done on pooled samples from each experimental group, confirming qRT-PCR analyses were performed on individual aliquots of total RNA samples from each treatment replicate. The purpose of these analyses was to evaluate the reliability of GeneChip data and develop statistical data to validate the changes suggested by the GeneChip assay of pooled RNA samples as our previous study as two assays may not match quantitatively but they always match qualitatively. Two analytical procedures may contribute to these discrepancies. 1) Total RNA samples used for the PCR assay are different from those used for the microarray assays. 2) The method of hybridization and detection for the two assays are different; the microarray assay utilizes hybridization of fragmented cRNA generated by Affymetrix protocols, whereas the RT-PCR utilizes amplification of DNA fragments using specific primer sets designed by a different method.An aliquot equivalent to 5 μg of total RNA extracted from each sample was reverse-transcribed to obtain cDNA in a final volume of 20 μl solution consisting of buffer, oligo-dT primer, DTT, dNTPs, and Superscript-III reverse transcriptase (Invitrogen). RT-PCR with SYBR as fluorescent reporter was used to quantify the expression of selected genes identified by GeneChip analysis. Specific primers  and KEGG annotations to create a functional gene network from microarray expression profiling data . We inteAfter literature curation, we created a dataset of all known transcription factor (TF)-gene interactions in human cells, with more than 4,000 experimentally validated interactions. Microvascular endothelial cell specific experiments -75 accouNo human subjects were used for this study.The authors declare that they have no competing interests.JCR and HHA initiated the study. HHA prepared the samples for analysis of microarray and the quantitative RT-PCR. AT performed the bioinformatics analysis. IT supervised all aspects of the computational analysis. HHA, NT, JCR and IT wrote the manuscript. All authors read and approved the final manuscript.Supporting material. Table S1. Primer Sequences.Click here for file


The paternal heredity of obesity and diabetes induced by a high-fat and/or high-sugar diet (Western-like diet) has been demonstrated through epidemiological analysis of human cohorts and experimental analysis, but the nature of the hereditary vector inducing this newly acquired phenotype is not yet well defined. Here, we show that microinjection of either testis or sperm RNA of male mice fed a Western-like diet into naive one-cell embryos leads to the establishment of the Western-like diet-induced metabolic phenotype in the resulting progenies, whereas RNAs prepared from healthy controls did not. Among multiple sequence differences between the testis transcriptomes of the sick and healthy fathers, we noted that several microRNAs had increased expression, which was of interest because this class of noncoding RNA is known to be involved in epigenetic control of gene expression. When microinjected into naive one-cell embryos, one of these small RNA, i.e., the microRNA miR19b, induced metabolic alterations that are similar to the diet-induced phenotype. Furthermore, this pathological phenotype was inherited by the offspring after crosses with healthy partners. Our results indicate that acquired food-induced trait inheritance might be enacted by RNA signalling. A fundamental law of genetics states that progenies do not inherit adaptive, pathological or neural features acquired in response to environmental conditions. However, recent studies seem to contradict this dogma. Paternal inheritance of diet-induced obesity, diabetes and its associated metabolic disorders, which is a worldwide epidemic and an acute societal problem26Caenorhabditis elegans to humans reviewed in ref.512C. elegans, small non-coding RNAs have been shown to induce heritable modifications of several morphological, physiological and behavioral phenotypes1617192122The frequent occurrence and heritability of metabolic disorders preclude Mendelian transmission of mutational events and are reminiscent of epigenetic heredity documented in various organisms, ranging from To determine whether RNA molecules can act as epigenetic signals in the hereditary transfer of the disease, a series of experiments was performed according to the general scheme outlined in Then, the F0-WD and control F0-SD males were mated at the age of four months with 8-week-old healthy standard-diet fed females. The males were removed after mating, and both the mothers and progenies were fed a standard diet. As shown in Based on our previous data212222Of two comparable RNA populations isolated from the same tissue, one efficiently induced an epigenetic change (RNA from WD mice), whereas the other RNA population did not (RNA from SD mice). We inferred that available sequence analysis technologies would pinpoint critical differences between the two RNA preparations. First, we performed a comparison of the WD and SD testis mRNA expression profiles by oligonucleotide microarray hybridisation. The analysis was performed on three randomly chosen animals of each group, however those who exhibited the strongest effects of diet were excluded from the selection group. Differentially expressed transcripts, either up- or downregulated, were detected in the WD males through Volcano and MA plot representations . The resvalue ≤ 0. 05; padjustvalue ≤ 0.05; fold change ≤ ±1.5, 1026As small noncoding RNAs of mice and non-mammalian model organisms were found in several instances to act as hereditary epigenetic determinants , microRNAs (miRNA) and piwi-interacting RNAs (piRNA)), reviewed inOur differential RNA-Seq analysis identified miR-19b and miR-29a as the two most abundant deregulated microRNAs in WD mice . These tThe degree of glucose intolerance and insulin resistance was more variable, as only half of the obese R1-miR19b animals showed impaired glucose tolerance and insulin sensitivity . The norIdentifying the nature of the vector involved in the inheritance of obesity and type II diabetes is essential to understand the progression of these major biological and medical problems. Our work provided the first evidence that perturbation of the zygote’s RNA content by microinjection of specific RNA induced zygote’s reprogramming which led to the development of the pathological traits in the adult. Given that the effect was specifically observed when the incoming RNA was prepared from the affected individual’s germ cells, it can be reasonably inferred that the transmission of the disease is initiated by sperm RNA transfer at the time of fertilisation. One caveat of our experiments is that microinjection differs from fertilization in many respects, such as the higher number of injected molecules compared to that present in sperm. This difference may be compounded by the observation in comparable experiments that a high amount of injected RNA is excluded from the nucleus and eliminated within the first minutes, after injectionThe clear difference between the R1-WD and R1-SD animals indicates that RNA carried by the sperm into the oocyte is able to modify gene expression in the resulting animals. However, although F1-WD and R1-WD mice were clearly different from the F1-SD and R1-SD controls, they showed a significant degree of individual variation compared with the more uniform F0-WD parent phenotypes. This individual variation was also evident in R1-miR19 mice. Indeed, some R1-miR19 displayed a relatively low body weight and a normal glucose regulation, whereas their respective R2-miR19 progeny displayed the full disorder . Theses results suggest that upon RNA injection, epigenetic changes take place which can remain phenotypically silent but are later transmitted to the progeny  . In a prWe have established that RNA is a vector of epigenetic information; however, the mechanism that is initiated in the early embryo and leads to the establishment of a complex inheritable pathology remains to be determined. Only partial indications can be gathered from previous studies. In three instances2122However, considering the high number of molecules micro-injected . Mice were maintained with the C57BL/6 genetic background . Male 3-week-old mice were divided into two groups. One group was kept on standard chow and the other was fed a western diet containing 17% casein, 0.3% DL-methionine, 34% sucrose, 14.5% cornstarch, 0.2% cholesterol, 5% cellulose, 7% CM 205B, 1% vit200 and 21% butter  glucose injection (2 mg/g body weight) using the OneTouch Vita  system. Blood glucose measurements were taken using tail blood at 0 min, 15 min, 30 min, 60 min, 90 min and 120 min after injection. Areas under the curve during the insulin tolerance test (AUC-ITT) were calculated using the trapezoidal rule.Bagsværd, Denmark) diluted to 0.08 mU/μl in sterile saline for a final delivery of 0.8 mU/g body weight. Blood glucose measurements were taken using tail blood at 0 min, 15 min, 30 min, 45 min, 60 min and 90 min after injection. Areas under the curve during the glucose tolerance test (AUC-GTT) were calculated using the trapezoidal ruleMice were fasted for 12 h before the experiment. Baseline glucose measurements were analysed using the OneTouch Vita  system from tail blood before ip insulin injection  on dry icemiRNA quantitation was performed using miScript Primer Assays (Qiagen), following the manufacturer-recommended protocols. Real-time PCR was performed with a Light Cycler Instrument  using the miScript SYBR Green PCR kit (Qiagen).t-test implemented in the Bioconductor limma package of the statistical software R (http://www.r-project.org/)http://usegalaxy.orghttp://www.smallrnagroup.uni-mainz.de36Microarray analysis of the expression of 45281 transcripts was performed by the Microarray Service CS3021 Illumina Gene Exp Process , and small RNA high-throughput sequencing and analyses were performed by the Functional Genomics Platform, Institut de Pharmacologie Moléculaire du CNRS, Sophia Antipolis, France. The supplied samples have been sequenced on SOLiD 5500XLHigh Throughput Sequencer. The obtained libraries of sequences (reads) were aligned with the mouse genome (mm9) using Bowtie aligner with the following parameters: Reads trimming at the adaptator position (trimming at 31 bases otherwise) and 2 allowed mismatches over the total read length. The following public databases were used for the read annotation process : miRBase miRNAs data base mirbase18, Rfam database and ncRNA.orgdatabase. The normalised, subtracted background and expression  data were further analysed using Bayesian model moderated Because of the rheostat-type  distributions and the limited number of animalsHow to cite this article: Grandjean, V. et al. RNA-mediated paternal heredity of diet-induced obesity and metabolic disorders. Sci. Rep.5, 18193; doi: 10.1038/srep18193 (2015).Data availability. All microarray and deep sequencing data used in this study have been deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE44301 and GSE43555.