DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript.We investigated the molecular mechanisms for in-frame skipping of  Based on their position and function, SREs can be classified as exonic splicing enhancers (ESEs) and silencers (ESSs), intronic splicing enhancers (ISEs) and silencers (ISSs). These elements are bound by RNA-binding proteins that have either positive (primarily SR proteins)   or negatroteins) ,7 effectentified .DMD gene that result in the absence of the muscle protein dystrophin. Conversely, the mutations that allow the production of reduced levels of normal or truncated and partially functional dystrophin in muscle are associated with Becker muscular dystrophy , a milder allelic variant of DMD. BMD is mostly caused by large deletions in the DMD gene that maintain an open reading frame (DMD in-frame exons (from exon 23 to exon 42) that encode the central rod domain of dystrophin, which is largely dispensable. Several studies reported phenotype attenuation via partial exclusion of the exon harboring the nonsense mutation. In these cases the preservation of an open reading frame allows the production of a partially functional protein  is caused by loss-of-function mutations in the ng frame . Unexpecng frame . BMD non protein . Based oE motifs , but verDMD gene is required. Here we investigated the molecular basis of splicing alteration caused by a nonsense mutation in exon 39  identified in a patient with BMD. Exon 39 is part of the set of in-frame exons prone to exon skipping when mutated. We were particularly interested in characterizing the regulatory cis-elements and cellular trans-acting factors involved in this mutation-induced alternative splicing event. By using biochemical and functional approaches, we have established that the mutation affects RNA-protein complex formation by creating a splicing factor-dependent silencer. We also report that normal DMD exon 39 splicing in muscle cells is dependent on FUPB1, a largely unknown splicing factor.Antisense-mediated exon skipping to by-pass protein-truncating mutations from the dystrophin pre-mRNA is a promising therapeutic approach for DMD patients , which iDMD cDNA sequence (NM_004006.2), with the A of the translation start codon considered as nucleotide number 1.The muscular dystrophin mRNA analysis was performed in the patient in the course of the diagnostic procedure at the Hospital Laboratory. All tests have been approved by the local ethical committee and informed consent obtained. Amplification of dystrophin transcripts was carried out as described before  using prDMD exon 39 and its flanking intronic sequences (272 nucleotides upstream and 262 nucleotides downstream of exon 39) were polymerase chain reaction (PCR)-amplified from genomic DNA isolated from a control individual and from the patient harboring the c.5480T>A mutation using High Fidelity Phusion Polymerase (Finnzymes). Amplicons were cloned in the XhoI and NheI sites of the pSPL3 vector  to obtain the E39-WT and the E39-5480A splicing reporter minigenes, respectively. The same procedure was followed to generate the truncated (Δ1 and Δ2) minigenes. The Δ3 minigene was constructed by overlap extension PCR. Exon 39 minigenes carrying the deletion of the site O (del-O), the two U>A mutations in site O (O-mut) and/or the mutation of the UGU (UGU-mut) were obtained by mutagenesis of the E39-WT construct using the QuikChange II site directed-mutagenesis kit (Agilent Technologies). To generate expression plasmids, the FUBP1 (NM_003902.3), DAZAP1 (NM_170711.1) and hnRNPA1 (NM_031157.2) sequences were PCR-amplified with specific Flag-tagged primer pairs by using cDNA from HeLa cells. The obtained amplicons were cloned in the HindIII and XbaI (FBP and DAZAP1) or the EcoRI and XhoI (hnRNPA1) sites of pcDNA3.1+ (Life Technologies) and then sequenced. The pFlag CMV-4 expression vector for hnRNPA2 was provided by M. Baralle . The list of the primers used in this study is available upon request.2 in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX and 199 medium (4:1 ratio) (Life Technologies) supplemented with 20% calf serum (Eurobio), 5 ng/mL hEGF, 0.5 ng/ml bFGF, 50 μg/ml bovine fetuin (Life Technologies), 5 μg/ml Insulin and 0.2 μg/ml dexamethasone (Sigma–Aldrich). C25cl48 myoblasts were plated in 6-well plates (1.8 × 105 cells/well) and 1 μg of each splicing reporter minigene was transfected the following day using JetPEI® (Polyplus Transfection) according to the manufacturer's instructions. For overexpression studies, the minigene was co-transfected with 100 ng of plasmid coding for the splicing factors or empty vector (negative control). Cells were harvested 48 h after transfection for RNA isolation or protein extraction.The human immortalized myoblast cell line C25cl48 established from a muscle biopsy of a healthy individual was used for all transfection experiments . Cells w5 cells/well in six-well/plates were reverse transfected with 35 nM siRNA (Thermo Scientific Dharmacon) using 7.5 μl Lipofectamine® RNAiMax (Life technologies) following the manufacturer's instructions. After 48 h, a forward transfection was performed using the same amounts of siRNA and reagent. Six hours after the second round of siRNA transfection, C25cl48 cells were transfected with 2.5 μg of minigene using Lipofectamine® LTX (Life Technologies). Cells were harvested 48 h after minigene transfection. Previously described siRNAs were used for silencing hnRNPA1, hnRNPA2B1, DAZAP1 . The Si® II (Life Technologies) and random primers were used to reverse transcribe 900 ng of total RNA. An aliquot (1/20th) was then PCR-amplified with the pSPL3 specific primers as previously described  to the sum of the intensities of the upper and lower bands.Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Superscriptliquot 1/th was thescribed . After eProteins were separated on 10% sodium dodecyl sulphate (SDS)-polyacrylamide gels, transferred to Polyvinylidene fluoride (PVDF) membranes and probed using specific antibodies: polyclonal anti-DAZAP1 , anti-hnRNPA1 (4B10), anti-hnRNPA2B1 (DP3B3) (Santa Cruz Biotechnologies) and anti-FUBP1 (Sigma–Aldrich). Anti-ß-Tubulin  was used as loading control.®-3Z vector (Promega). HindIII linearized templates were then in vitro transcribed using RiboMAX™ Large Scale RNA Production System-T7 (Promega). The control non-specific GST-80 RNA probe was previously described  on a rotator in the dark for 1 h. RNA probes were purified with the RNeasy Plus Mini Kit (Qiagen) using the ‘Purification of total RNA containing miRNA’ Qiagen supplementary protocol to retain small RNAs. The standard RNA pull down experiments were performed as previously described  and 12 μg bovine serum albumin (NEB), at 30°C for 30 min. The beads were then pelleted by centrifugation at 100 g for 2 min and washed four times with buffer D containing 1.5 mM MgCl2 before addition of Laemmli buffer and loading on a 10% SDS-PAGE gel. Pulled-down proteins were analyzed by western blotting using specific antibodies. The RNase-assisted RNA pull down assay was conducted using the same procedure except that 1500 pmol of RNA probes were used. After the fourth wash with 1.5 mM MgCl2–buffer D, beads were additionally washed twice with Diethylpyrocarbonate-treated-water and proteins specifically bound to RNAs were eluted by using a RNase A/T1 treatment as previously described  corresponding to the different RNA probes were cloned into the EcoRI–PstI-digested pGEMescribed . RNA proescribed  with somescribed . RNA-bouIn vitro transcribed RNAs were biotinylated using the Pierce RNA 3′ End Biotinylation Kit (Thermo Scientific) according to the manufacturer's instructions. The gel shift assay was carried out using the LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific). Biotinylated wild-type or mutant RNA probe (20 fmol) were incubated 30 min at room temperature with recombinant FUBP1 (Origene) in a 20 μl binding reaction containing 1× binding buffer, 5% glycerol and 0.1 mg/ml tRNA. The samples were electrophoresed on a 5% native PAGE in 0.5× Tris Borate EDTA, transferred to a positively charged nylon membrane  and crosslinked in a UV Stratalinker 1800 (Stratagene). Detection of biotin-labeled RNA probes was performed using the chemiluminescent nucleic acid detection module (Thermo Scientific).® (Active Motif) according to the manufacturers’ instructions. Briefly, C25cl48 cells were cross-linked for 5 min with 1% formaldehyde. Chromatin was sheared using the Diagenode Bioruptor (3 cycles of 30 s ‘on’ and 30 s ‘off’ in ice-cooled water). Immunoprecipitation was performed with 14 μg chromatin and 4 μg polyclonal anti-FUBP1 antibody (GeneTex), or normal rabbit IgG (Santa Cruz Biotechnology). After purification, RNA was treated with DNase I. Superscript III reverse transcriptase (Life Technologies) and random hexamers were used to convert one-third of the recovered RNA. Products were amplified for 35 cycles from cDNA using the following primers forward 5′-AAGGCTATGAGCACAGTATC-3′ and reverse 5′- AAAGCTGTACATTGTTAACAGAG-3′.RNA chromatin immunoprecipitation (RNA-ChIP) was performed using RNA ChIP-IT® Human Gene 2.0 ST Array, scanned, and quantified at the Affymetrix Service Provider and Core Facility, ‘KFB – Center of Excellence for Fluorescent Bioanalytics’ . A heat-map image of microarray data was generated from log2 values using the Matrix2png web interface  and from C25cl48 cells were processed, hybridized on a GeneChipDMD exon 39 was identified in a 27-year-old patient having a Becker-like phenotype. The milder than expected phenotype arises because of partial skipping of exon 39 in muscle, which removes the nonsense mutation and reframes the dystrophin transcripts  or harboring the c.5480T>A was cloned in the pSPL3 splicing reporter vector  mutation in s Figure . To recar Figure . The twor Figure  and the r Figure . The exotrans-acting factors that differently bind to the wild-type (WT) and the mutated (5480A) RNA probes . RT-PCR analysis of the transcripts derived from the E39-5480A minigene showed that overexpression of all three proteins, but especially hnRNPA1 and hnRNPA2 proteins, led to a marked increase in exon 39 skipping (82% for hnRNPA1 and 79% for hnRNPA2) as compared to the control (53%) , whereas hnRNPA2 was not affected. SiRNA-mediated depletion of each individual protein resulted in a significant decrease in mutated exon 39 skipping only in hnRNPA2-, and DAZAP1-silenced cells  compared to control siRNA (54% exon skipping). Double (hnRNPA1/hnRNPA2) or triple knockdown (hnRNPA1/hnRNPA2/DAZAP1) further reduced the percentage of exon 39 skipping . Altogether, these data provide strong support to the hypothesis that the c.5480T>A mutation leads to the creation of a hnRNPA1/A2-dependent ESS that plays a significant role in exon 39 skipping and that DAZAP1 is also associated with this silencer complex.To address the functional role of hnRNPA1, hnRNPA2 and DAZAP1 in the recognition of mutated ) Figure . Overexpn Figure , bottom.n Figure , hnRNPA1In order to normalize the amount of pulled-down RNAs, the RNase-assisted RNA pull down experiment was also conducted with modified RNA probes containing a TDP43 binding (ug)6-repeat linked by an u5 spacer as previously described . The ideDMD exon 39 splicing. To this aim, C25cl48 myoblasts treated with either siRNAs targeting FUBP1 or firefly luciferase as a control were transfected with the E39-WT or E39-5480A minigene .We sought to clarify whether FUBP1 may have a functional role on e Figure . Depletis Figure . As expeDMD genomic sequence cloned in the minigenes. We looked for tetrads perfectly matching with the previously described ssDNA FUBP1 binding motif   . Besidesgure S3) . To assegure S3)  and B. Tgure S3)  increaseThen, we assessed the ability of FUBP1 to bind to the identified intronic RNA motif by standard RNA pull down assays. As shown by Western blot analysis, the wild-type site O-containing probe was strongly bound by FUBP1, and disruption of the four predicted binding motifs in the double mutant probe (O-mut) was shown to decrease this interaction Figure  and D. WWe next carried out RNA EMSA experiments using 3′-end biotinylated wild-type and mutated RNA probes to confirm direct RNA–protein interaction of FUBP1 Figure . The recOverall, the RNA pull down and the RNA EMSA experiments consistently show on the one hand the ability of FUBP1 to bind to the wild-type RNA sequence efficiently, and on the other hand a marked decrease in binding to RNA probes mutated in the site O or in the sequence UGU. Depending on the technique, we noticed that this decrease was stronger either for the O-mut or the UGU-mut probe. We assumed that these variations can be due to the technical differences between the two methods.We finally investigated the functionality of the UGU sequence. Therefore, we analyzed the splicing pattern of the minigene with the mutated UGU sequence  nonsense mutation in a patient with an attenuated phenotype. We showed that loss of exon identity is caused by the creation of an ESS that recruits splicing-repressor proteins. This study also yielded important insights into how normal splicing of DMD exon 39 is regulated. Notably, we uncovered that FUBP1, a single-stranded DNA- and RNA-binding protein, positively regulates DMD exon 39 splicing, and we identified an ISE in intron 38 that promotes exon recognition.Here, we thoroughly investigated the molecular basis for partial in-frame exon skipping of DMD exons 23–42, where mono- or multi-exon skipping maintains an open reading frame  is resembling but does not match perfectly any of the previously described DAZAP1 binding motifs in human genes   ,26,37. ODMD exon 39 is most likely a result of the cooperative as well as the antagonistic effects of multiple splicing factors. In this study, we could establish the role of one protein (FUBP1) that contributes to the recognition of endogenous DMD exon 39, thus partly counteracting the effect of the ESS.The signaling network to specify splicing is particularly dense within and around exons  and the Myc mRNA levels by binding to FUSE, an A/T-rich element located 1.7 kb upstream of the c-myc promoter. FUBP1 also has functions in RNA metabolism as a member of the AU-rich element (ARE) binding protein family  . Then, the siRNA-mediated depletion of FUBP1 in C25cl48 muscular cells also confirmed its physiological function for proper splicing of endogenous DMD exon 39.FUBP1 was originally identified as a single stranded DNA-binding protein that modulates c-eview in ). Thus,  context , or convpre-mRNA . Still, triadin exon 10) and intronic (MDM2) SREs are AU-rich sequences and display characteristics of binding sites for (ARE)-binding proteins in 3′UTR of transcripts. Here, a functional screening based on systematic deletion analysis and site-directed mutagenesis in the DMD exon 39 minigenes allowed us to delineate an intronic region located about 80 bp upstream of DMD exon 39 that acts as an ISE on exon 39 splicing. The identified ISE contains an UG-rich RNA sequence (site O) that is similar to the previously identified optimal DNA binding motif for the KH domains of FUBP1  contain four KH domains connected by a flexible Gly-rich linker . KH domaDMD gene for inclusion of constitutive exons. A better knowledge of SREs in the DMD gene may prove useful in the context of the ongoing therapies for DMD targeting mRNA splicing such as the antisense-mediated exon skipping and the trans-splicing approach (In conclusion, this study illustrates how in-depth investigation of disease-causing mutations in human genes contribute to basic knowledge of fundamental cellular processes. We characterized the splicing factors that are recruited to the ESS created by the c.5480T>A mutation in exon 39, and repress its inclusion in the mature transcripts. Moreover, we identified a splicing factor required for efficient normal exon 39 inclusion, and we localized one new UG-rich ISE that can recruit this factor. This data further extends the repertoire of known ISEs, which are not as well characterized as the other SREs . It is aapproach ,43.Supplementary Data are available at NAR Online.


RNA editing encompasses a post-transcriptional process in which the genomically templated sequence is enzymatically altered and introduces a modified base into the edited transcript. Mammalian C-to-U RNA editing represents a distinct subtype of base modification, whose prototype is intestinal apolipoprotein B mRNA, mediated by the catalytic deaminase Apobec-1. However, the genome-wide identification, tissue-specificity and functional implications of Apobec-1-mediated C-to-U RNA editing remain incompletely explored.Apobec-1-deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs, all within 3′ untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites are unique to liver. Changes in RNA editing lead to corresponding changes in intestinal mRNA and protein levels for 11 genes. Analysis of RNA editing in vivo following tissue-specific Apobec-1 adenoviral or transgenic Apobec-1 overexpression reveals that a subset of targets identified in wild-type mice are restored in Apobec-1-deficient mouse intestine and liver following Apobec-1 rescue. We find distinctive polysome profiles for several RNA editing targets and demonstrate novel exonic editing sites in nuclear preparations from intestine but not hepatic apolipoprotein B RNA. RNA editing is validated using cell-free extracts from wild-type but not Apobec-1-deficient mice, demonstrating that Apobec-1 is required.Deep sequencing, data filtering and Sanger-sequence validation of intestinal and hepatic RNA from wild-type and These studies define selective, tissue-specific targets of Apobec-1-dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific. CAA codon introduces a translational termination (UAA) codon in the edited transcript. ApoB mRNA editing is a critical adaptive pathway for lipid transport in both the mouse intestine and liver, and exhibits distinctive developmental and metabolic regulation . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  via the Apobec-1/-- and Apobec-1Int/O mice and from livers isolated from WT, Apobec-1/--, Apobec-1/-- + ad-LacZ and Apobec-1/-- + ad-Apobec-1 mice (three mice per genotype), using TRIZol reagent . DNAse-free RNAs were used for cDNA preparation as previously described [Total RNA was extracted from intestinal mucosa from WT, escribed . Pooled Apobec-1 knockout genotypes. Known SNPs from dbSNP128 that were not annotated as based on cDNA and sites lying outside of the 5′ and 3′ gene boundaries were set aside, and the remaining sites were corrected for strand sense. These sites were then annotated using ANNOVAR [RNA-seq reads for each genotype were mapped to the mouse reference genome (NCBI37/mm9) and single nucleotide variants were called using a modified version from . Reads f ANNOVAR  with a sApobec-1/--, Apobec-1Int/O and Apobec-1/-- + ad-Apobec-1 mice. Genomic DNA was prepared as follows: 100 ng of liver tissue was incubated at 55°C overnight in 600 μl cell lysis solution . After protein removal, DNA was precipitated and resuspended. Total RNA was TRIzol-extracted and subjected to cDNA synthesis using random hexamers and MultiScribe Reverse Transcriptase from High Capacity cDNA Reverse Transcription kit . Both isolated genomic DNA and cDNA were used as templates to amplify sequences containing RNA-seq-identified Apobec-1-dependent putative editing sites. PCR amplifications were performed using Pfultra II DNA polymerase . Primer sequences are listed in Tables S13 and S14 in Additional file Genomic DNA and total RNA were isolated from intestine and liver of WT, 2, 0.5% NP-40, 1 mM DTT, 20 units/μl RNAse inhibitor  and 1× protease inhibitor) homogenized and centrifuged at 7,000 g for 10 minutes at 4°C. Supernatant was saved as cytoplasmic fraction. Nuclear pellets were resuspended in 2 volumes of buffer B supplemented with one- tenth volume detergent (3.5% sodium deoxycholate (w/v) and 6.6% Tween 20 (v/v)) incubated for 30 minutes at 4°C and centrifuged at 1,000 g for 5 minutes. Supernatant was combined with the previous cytoplasmic fraction and nuclear pellet was rinsed once in buffer B. Cytoplasmic and nuclear RNAs were extracted using TRiZol (Invitrogen) following the manufacturer’s protocol, treated with DNAse  and subjected to cDNA synthesis as described above. Targets of interest  were then PCR amplified using specific primers , 0.15 M NaCl, 2 mM EDTA, 1 mM sodium vanadate, 0.1 M sodium fluoride, 50 mM β-glycerophosphate, 5% glycerol, 2× protease inhibitor , 1% Triton, and 0.1% SDS. Aliquots of homogenate (60 μg protein) were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with goat anti-CD36 antibody , mouse anti-IDO1 . Equal loading was verified using a rabbit anti-α-actin antibody .2, 0.5 mM PMSF, 200 μg/ml heparin (Sigma), 5 mM dithiothreitol, 20 U/ml RNAsin, 100 μg/ml cycloheximide, 1% Triton X-100, 1× protease inhibitor). Scraped mucosa was homogenized and centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was loaded onto a 10 to 50% sucrose gradient and centrifuged at 40,000 rpm for 2.25 h at 4°C using an SWT41i rotor . Fractions (900 μl) were collected from the bottom of the gradient and 260 nm absorbance monitored by spectrophotometry. RNA was phenol/chloroform extracted from each fraction, precipitated, resuspended in 20 μl H2O and used for cDNA synthesis followed by PCR amplification of specific targets . PCR products were cloned and sequenced as described above.Each polysome isolation used three to four mice with two to five isolations per genotype. Intestinal mucosa was prepared in ice-cold phosphate-buffered saline supplemented with 100 μg/ml cyclohexamine  was incubated in 1 ml lysis buffer . Resulting RNA (1 μg) was incubated for 3 h at 30°C with variable amount of hepatic S100 extract prepared from either WT or Apobec-1/-- mice liver [Total hepatic RNA was isolated from ce liver . Followice liver . ExtensiApobec-1-/- intestine samples using a buffer containing 2% SDS, 30 mM Tris pH 8, supplemented with protease inhibitors , phosphatase inhibitors  and benzonase . Proteins were methanol/chloroform precipitated and resuspended in urea/thiourea buffer . Protein concentration was estimated using Bradford. Unlabeled samples were mixed with a lys6-labeled SILAC standard  at a ratio of 1:1. Samples were in-solution digested [18 StageTips according to [Mus musculus UniProtKB protein sequence database (as of 8 May 2013) consisting of 79,342 entries, including canonical and isoform sequences. Search parameters were set as follows. Lys-C was selected with a maximum of two missed cleavages. Precursor mass tolerance was set to 20 ppm for the first search and to 6 ppm for the main search. Oxidized methionines and amino-terminal protein acetylation were allowed as variable, carbamidomethylation as a fixed modification. The false discovery rate for peptide and protein identification was set to 1%. Minimum peptide length was set to 7 with no maximum. Peptide identification by chromatography alignment and ID transfer ('match between runs') was enabled and led to identification of 3,210 proteins. Differentially expressed genes were identified by t-test (significance cutoff of 0.1) in R, a language and environment for statistical computing and graphics [Total proteins were isolated from three WT and three digested  using Lyrding to . The frarding to . After tgraphics .Analysis of bases flanking the editing sites was performed by aligning 10 nucleotides surrounding the editing sites (5 nucleotides immediately upstream and 5 nucleotides immediately downstream). Frequency plots and logos were generated using the WebLogo application ,52. IdenDifferential gene expression analysis was performed using the Tuxedo suite of tools . RNA-seqP value <0.05. All analyses were performed using Graphpad Prism 4.0 .Degree of enrichment of the Apobec-1 targets was represented by the difference in hypergeometric distribution using one-tailed Fisher’s exact test. Correlation between editing frequency and fold protein expression is reflected by Spearman’s rho (ρ) rank correlation coefficient. Statistical significance was set at a ACF: Apobec-1 complementation factor; ADAR: adenosine deaminase acting on RNA; ApoB: apolipoprotein B; FPKM: fragments per kilobase of exon per million; miRNA: microRNA; PCR: polymerase chain reaction; SNP: single-nucleotide polymorphism; UTR: untranslated region; WT: wild type.The authors declare that they have no competing interests.VB, NOD, JHN and AM designed the experiments. VB, EP, SS, MM, YL, SK, AMB, HBH and JG conducted the experiments. EP and AM undertook the bioinformatics analysis. VB, EP, NOD, JHN, SS, MM and AM analyzed the results. SS, AMB, HBH and JG undertook the proteomic analyses. VB undertook the RNA editing assays and polysome isolation and analyses. VB, NOD, JHN, SS and AM wrote the manuscript with input from all the authors. All authors read and approved the final manuscript.Ali Mortazavi, Joseph H Nadeau and Nicholas O Davidson shared supervision of this work.The following supplemental data are available with the online version of this paper. Table S1A. lists WT intestinal Apobec-1 exonic targets. Table S1B-D. list WT intestine discordant RNA targets. Table S1E. lists the WT liver Apobec-1 exonic targets. Table S2. shows alignment of mooring sequence-like motifs of WT intestine Apobec-1 RNA targets. Table S3A. lists intestinal RNA targets with increased editing efficiency correlating with increased Apobec-1 expression. Table S3B. lists hepatic C-to-U RNA editing targets shared in WT and Apobec-1/-- mice following ad-Apobec-1 rescue. Table S3C. lists C-to-U editing targets shared between WT and Apobec-1/-- + ad-Apobec-1 showing hyperediting following ad-Apobec-1 rescue. Table S4. shows alignment of mooring sequence-like motifs of WT liver Apobec-1 RNA targets. Tables S5 and S6. show, respectively, RNA expression of intestinal and hepatic Apobec-1 targets. Tables S7 and S8. show, respectively, intestinal and hepatic Apobec-1 editing sites in miRNA seed sequences. Table S9. lists the proteins differentially expressed between WT and Apobec-1/-- intestine. Table S10. shows intestinal Apobec-1 RNA editing targets with altered protein expression. Table S11. lists miRNA seed sequences in Apobec-1 C-to-U RNA editing targets with altered RNA and protein expression. Tables S12 and S13. list primer sequences for PCR amplification of, respectively, intestine and hepatic 3' UTR Apobec-1 RNA targets. Figure S1. shows frequency plot analysis of nearest nucleotides flanking Apobec-1 3' UTR RNA editing sites. Figure S2A,B. shows that the extent of differential mRNA expression for each edited transcript is unrelated to the percent C-to-U editing.Click here for file


Athetis lepigone (Möschler) (Lepidoptera: Noctuidae) is an important insect pest of corn crops in China. To determine the effect of temperature on A. lepigone growth, and to provide a forecasting model for this pest, the development and fecundity of A. lepigone under five different temperatures  was investigated, and an experimental population life table was constructed based on the obtained results. The results showed that the duration of development of A. lepigone decreased as the temperature increased from 18 to 30° C. Approximately 95% of mature larvae stopped pupating at 18° C, and about 70% of mature larvae stopped pupating at 21° C. When the growth chamber temperature was above 24° C, no growth arrest was observed. The results indicated that the optimum growth temperature of A. lepigone was about 26.47° C. In this study, the highest survival rate, fecundity per female, and population index trend were observed when the temperature was set at 27° C. The percentages of larvae that could spin cocoons after the 5th or 6th instar differed at the different temperatures. The developmental threshold temperatures for A. lepigone eggs, larvae, pre-pupae, pupae, preoviposition females, and the whole generation  were 11.03, 9.04, 15.08, 11.79, 11.63, and 10.84° C, respectively, and their effective accumulative temperatures were 63.51, 339.42, 30.04, 118.41, 35.06 and 574.08 degree-days, respectively. Based on the effective accumulative temperature law, this pest insect can have four generations in most of the Huang-Huai region of China, and two to three generations annually in some cold regions. Athetislepigone may have four generations in the mid-southern part of Hebei Province. This prediction matches the field survey results. Athetis lepigone  (Möschler 1860) is known to occur in many European countries, east-central parts of Russia, Japan, North Korea, and Mongolia  annually in the Hebei Province. The earliest emergence time for adult A. lepigone was in mid-April  set at 18, 21, 24, 27 and 30 ± 0.5° C, respectively, and at 80 ± 5% RH and 14:10 L:D photoperiod. The total number of eggs hatched under each temperature was recorded, and the developmental duration of each egg  was recorded.Over 150 newly-laid eggs were collected and treated with 5% formaldehyde for disinfection. After air-drying, the eggs were placed in plastic containers and incubated in growth chambers  of 6-well, flat-bottomed, tissue culture plates and were fed with fresh maize leaves until larvae matured. If the newly-emerged larvae had a high mortality rate at a temperature, the 1st instar larvae reared at 27° C were supplied to determine the development and survival of immature stages at this temperature. The headcapsule popping off was used to determine larval molting, and the complete formation of a cocoon was considered to be pre-pupae. In this study, the survival rate and the developmental duration of each growth stage were recorded. The survival rate was calculated as:The eggs, larvae, pre-pupae, pupae, and adults were examined and recorded at three different times  each day for their development progresses. The experiment was repeated three times.In the adult stage, one newly emerged female and one newly emerged male (less than 24 hr old) were paired in an insect cage at each treatment temperature, fed with 10% honey solution, and this was replicated at least five times. Male and female adults were determined based on the appearance of the hair clusters at the ends of their bodies. The female hair cluster is open, and its gonopore is visible. The pleon end of an adult male is semitriangular, and its hair cluster is closed. The number of eggs laid under each temperature condition by each female was counted, and the pre-oviposition period, oviposition period, and longevity of adults were also recorded.3 + bT2 + cT + d.Temperature requirements and the rates of each developmental stage were calculated by using the results from the different treatments. The least squares method was used to calculate the developmental threshold temperature (C) and the effective accumulative temperature (K) for instars at various stages as described previously . The folA. 1epigone survival, growth, development, and reproduction. The index of population trend was calculated by using the data shown in the life tables as described previously (p < 0.05) between different treatments was determined by using the single-factor analysis (one-way ANOVA), Duncan's multiple range test (p < 0.05), and correlation analysis .The experimental population life tables were formulated based on the effects of temperature on eviously . Means aeviously , and theA. lepigone adults were trapped by using the automatic pest forecasting lamps . The lamps were set up in fields in Guantao County. The observation period was from 1 April to 31 October 2011. The number of trapped adults was recorded daily, and the annual occurrence figure of A. lepigone adults was then prepared.A. lepigone. For the same developmental stages, the development duration significantly decreased as the temperature was increased from 18 to 30° C .The results of this study showed that temperature had significant effects on the developmental durations of to 30° C   and showed approximately 17% reduction when the temperatures were reduced to 24 and 21° C, and 12.5% reduction when the temperature was increased to 30° C. The highest population trend index of A. lepigone was observed at 27° C, suggesting that under this condition A. lepigone can propagate rapidly and may result in an outbreak if food sources are available.The experimental population life tables were produced based on the survival rates of the different developmental stages and the adult fecundity data obtained from the different temperature treatments. The number of live eggs under different temperatures was assumed to be 100 . The numA. lepigone begins to develop when the temperature is above 10.84° C. Assuming the regional effective accumulative temperature is the sum of temperature above 10.84° C each month and throughout the year, the total effectiveaccumulative temperature in Guantao, Hebei Province, in 2011 was 2183.34 degree days. Because A. lepigone requires approximately 574.08 degree days to complete a whole generation cycle, it was proposed that A. lepigone could occur 3.80 generations in Guantao in 2011. Using the same method, it was propose that the estimated generations per year for Zhengding, Zhengzhou, Jinan, Bozhou, and Xuzhou would be 4.14, 4.01, 4.04, 4.20, and 4.16, respectively. For Shenyang, Chengde, Tangshan, and Jincheng, the estimated generation numbers are 2.67, 2.57, 3.51, and 3.23, respectively (The data suggest that ectively .A. lepigone (overwintered adults) appeared in early April 2011 in the fields of Guantao, and its population remained small until May. A small emergence peak was observed on 30 April. Three obvious emergence peaks were observed in 2011 in Guantao. During the first emergence peak period (mid-June), the number of moths captured by using the insect trapping lamps ranged from 100 to 523 per day. The second peak period was observed between mid and late July, and the largest number of moths captured was 806 per day.As shown in A. lepigone can have four generations per year in Guantao.The third peak period occurred in late August until early September, with less than 125 moths captured per day. This observation indicates that A. lepigone of each generation, the effective accumulative temperature was calculated using the local average temperature provided by the meteorological department. The laboratory observations indicated that, after overwintering, mature larvae could start their pupation just 1 day after incubation at room temperature (data not published). The meteorological data collected in 2011 showed that the daily mean temperatures of 6 days in March were above the developmental threshold temperature for pre-pupae, and 4 of the 6 days had temperatures above 20° C . If the emergence peak of the winter generation adults was predicted using the pupae developmental threshold temperature and the average daily temperature, the number of theoretical A. lepigone generations in Guantao per year would be 3.88 , and which larval instar is the most susceptible to the external influence require further investigation.It was reported that ae had 5  or 6 insae had 5 . Howeverure. Fan  and othe instars . In our A. lepigone, causing some larvae to stop pupating in their cocoons during maturation at 18° C and 21° C. Low temperature was the inducing factor for A. lepigone dormancy (or diapause). In addition to the detailed developmental responses to temperature, the influence of photoperiod on A. lepigone development is currently under the investigation.Many insects alter their developmental rate, or even suppress a particular developmental stage, to cope with environmental conditions differing from place to place and/or time to time . SeveralA. lepigone. In addition, newly-planted summer corn seedlings provide A. lepigone with sufficient food sources. Under suitable temperature conditions, A. lepigone larvae grow well and mature quickly, and the adults are often more fertile, leading to rapid reproduction and then an outbreak of the pest within a short period of time in June, the month with an average temperature of 26° C, in the south-central part of the Hebei Province (Each year, during the overlapping growing season between the wheat and corn crops in the Huang-Huaihai area of China, large amounts of wheat straw and stubble are left in the fields, and these leftover materials create a favorable environment for Province .A. lepigone larvae do not produce cocoons, and some pupae survive during the warm winters in some regions. These surviving mature larvae and pupae often represent part of the first generation for the following year (A. lepigone would occur in late May. The actual field survey showed, however, that the overwintered A. lepigone adults first appeared in early April and reached the emergence peak in late April in 2011. The possible explanation for this earlier field emergence peak is that the accumulative temperature of the overwintered generation met the requirement sooner than had been predicted. The weather information obtained indicated that the daily highest temperatures in late May often exceeded 15.08° C. Consequently, a short period of warmer weather had led A. lepigone larvae to pupate sooner than had been predicted. Because the developmental threshold temperature for pupae is lower than that for the other developmental stages, the adult A. lepigone captured in April might have emerged from the overwintered pupae. Therefore, the prediction of the emergence peak of the overwintered adults, based on the developmental threshold temperature for pupae, agreed with the actual field survey results.Due to irregular developmental patterns, some mature ing year . The devA. lepigone emergence peaks are still useful. It is noteworthy A. lepigone often survives inside the leftover plant materials in the field, which provide environmental conditions that are quite different from the conditions reported by meteorological data. In addition, the law of effective temperature emphasizes only the effect of temperature on development. Other factors, including food sources, can also have a significant impact on insect development duration, longevity, and fecundity (A. lepigone using this method (A. lepigone have different food sources. The impacts of different food sources on A. lepigone growth remain unknown and require further investigation.The predicted number of generations per year using the developmental threshold temperature and the effective accumulative temperature was similar to that observed in the field using insect trapping lamps. Although the developmental threshold temperature and the effective accumulative temperature presented in this paper were obtained under constant temperature conditions, and the variable temperatures in the field were reported to be more favorable for insect development and reproduction , the preecundity . Thus, fs method . DiffereA. lepigone generation number, the emergence peaks in a given year, and the duration of individual developmental stages could be predicted using the developmental threshold temperature, effective accumulative temperature, and temperature data provided by the local meteorological departments. This prediction provides useful information on A. lepigone occurrence dynamics and control strategies. According to the field surveys made in recent years, A. lepigone occurred in four generations annually in the south-central part of Hebei Province and often caused severe damage to the summer maize crops in late June until July. Overlapping between A. lepigone generations was seen frequently in field. The overwintered A. lepigone larvae could develop irregularly, resulting in small numbers of adult A. lepigone observed in the field in April of the following year (A. lepigone was distributed unevenly in the field. For example, adults and larvae often stayed in shade to avoid significant temperature changes caused by direct sun light (A. lepigone damage to the maize crops in the northern part of Hebei Province, this insect was captured previously in Shenyang area using insect killer traps (A. lepigone larvae are known to be omnivorous. Indoor feeding surveys showed that this insect could eat leaves, stems, fruits, and even dry plant materials of more than 30 plant species belonging to 13 different families. These plant species include maize, wheat, soybean, peanut, sweet potato, cabbage, tomato, spinach, amaranth, goosefoot, purslane, and more (A. lepigone larvae were able to feed on maize, wheat, sweet potato, soybean, peanut, and several vegetable species (A. lepigone damage to these field crops, it is obvious that favorable environments for this pest are widely present. Close attention should be paid to the population dynamics of this pest in the field.The results showed that the ing year . The firun light . Althouger traps . A. lepiand more . Field i species . Althoug


Specifications tableValue of the data•6A) is one of the most abundant RNA methylations and plays very important roles in many biological processes N6-methyladenosine , a web-server predictor for identifying m6A sites in the S. cerevisiae genome. By means of the m6A-seq technique, Schwartz et al. 6A) sites in the S. cerevisiae genome. They have observed that most of the m6A sites share a consensus motif GAC where its center base may be methylated S. cerevisiae genome, and obtained 33,280 RNA segments with exactly the same GAC consensus motif that, however, were not detected by the m6A-seq technique as methylated sites. Furthermore, it had been observed via preliminary tests that when the length of the RNA segments thus derived was 51 bp, the corresponding outcomes were most promising http://lin.uestc.edu.cn/server/iRNAMethyl/data.The data presented here are the benchmark dataset for training and testing iRNA-Methyl None of the authors claims conflicting interest.


All-in-one assemblies of separator, electrode and current collector (SECA) for lithium ion batteries are presented by using 1D nanowires of Si and Cu (nwSi and nwCu). Even without binders, integrity of SECA is secured via structural joints based on ductility of Cu as well as entanglement of nwSi and nwCu. By controlling the ratio of the nanowires, the number of contact points and voids accommodating volume expansion of Si active material are tunable. Zero volume expansion and high energy density are simultaneously achievable by the architecture. We undo entangled lines of our earphone before connecting it to cell phones. The inter-tangled bundles of hairs are easily found on mesh drain traps or at the corners of dirty rooms. High-aspect-ratio structures are easily inter-tangled with high probability, forming 3D network occupying a volumetric space with voids within its structure as well as keeping integrity of the entangled object bodies. Here, we demonstrate 3D network electrodes of binary phases as anodes for lithium ion batteries (LIBs). The architecture was built by inter-tangling silicon nanowires (nwSi) as an active material with copper nanowires (nwCu) as a conductive pathway. Polymeric binders used in conventional LIBs were not used in this architecture because nwCu plays a role as the binder due to its ductile properties. Structural joints are developed by pressure in an inter-locking way that nwSi is fitted into concaved parts of nwCu. As the second point to distinguish it from conventional electrodes, the 3D architecture was built not on current collectors but on separators. Additional layer of nwCu stacked on bare face of the 3D architecture worked as an ultra-light porous current collector. The separator-electrode-current collector assembly (SECA) showed very superior performances in terms of cyclability and rate capability.+/Li, have been used as the chemistry for anodes of LIBs, including intercalation (graphites−1 at room temperature that is ten times as large as that of widely used graphites (372 mAh g−1). However, serious drawbacks difficult to overcome come with the silicon anode materials, including not only low electric conductivity of silicon but also large volume change during lithiation up to ~400 % leading to pulverization of particles. Resultantly, serious decay of capacity with repeated cycles of charging/discharging follows with silicon8910121314151617181917192122232426272930313233Electrochemical reactions, the potential of which is relatively negative enough to be close to that of Ligraphites2, convergraphites4 and allgraphites and tin6aphites 32 mAh g−1Based on the merits of 1D structures of nwSi and inter-tangled networking between nwSi and nwCu, we developed all-in-one architectures assembling electrodes and current collectors onto separators (SECA = separator-electrode-current collector assembly) . An etha−2) are used. The value of thickness occupies 53 % of overall thickness of electrode and 94 % of total mass of electrode, when considering that electrode mass consisting of silicon, binder and conducting agents at 6:2:2 wt. ratio on current collector are 34 um thick with 17 mg cm−2. In our SECA of 30 % nwSi as a representative case, however, only 44 % of electrode thickness (14 um/32 um) and 39 % of total mass (1.3 mg cm−2/3.3 mg cm−2) were occupied by the current collectors of inter-tangled nwCu. Resultantly, for example, lighter electrodes including current collector can be possible by the SECA with only 30 % to 70 % of mass of a conventional tri-component electrode at the same silicon loading of 0.6 mg cm−2  electrode of this work, copper foils of micrometer thickness (e.g. 18 um thick with 16 mg cm mg cm−2 .c) as well as the contact-to-contact distance (Lcc) totally depends on the nwSi contents (xSi) in the nwSi/nwCu composites. Based on the lines-on-a-plate model in which nanowires of the minor component is evenly distributed on the plate consisting of nanowires of the major component, the maximum values of nc (ncmax) and the minimum values of Lcc (Lccmin) were calculated as functions of xSi  was maximized at the optimized composition xSiopti (open circles in e and f) where the contactable area is matched between nwSi and nwCu. The increase in nwSi contents directly leads to the increase in ncmax/mtot up to the optimized composition because nwCu can provide enough contactable faces to nwSi. At nwSi concentrations higher than xSiopti, the lack of nwCu is experienced by nwSi so that the contact number decreases. From the viewpoint of the contact-to-contact distance, the values of Lccmin are equivalent to the diameter of nwCu, the smallest value at which a contact point can approach its nearest contact point, at compositions lower than xSiopti. After the composition, the contact points move further away from their neighborhoods as silicon contents increase and the lack of nwCu become more serious. The composition at xSiopti would be the best choice when considering that higher nwSi contents are required with a closer contact. The xSiopti can be traced with the radius ratio of rCu/rSi as an independent variable (dashed red lines with red arrows indicating the increasing direction of rCu/rSi in Siopti = 1/(1 + (dCu/dSi)(rCu/rSi)) with d = density. Relatively thinner nwCu leading to smaller values of rCu/rSi extends the region of the smallest Lccmin to higher nwSi composition: e.g. xSiopti was shifted from 9.3 % at rCu/rSi = 2.5 to 20 % at rCu/rSi = 1.0. Also, the thinner nwCu provides more number of contacts with shorter distance between contacts at the optimized composition.The number of contact points between nwSi and nwCu  of the SECA decreased after pressing: RCT from 30 ohm to 9 ohm; Rsheet from the very high value that is not measured to 50 ohm sq−1.By using nwCu as a conducting agent as well as a binder, the inter-tangled SECA was successfully developed, showing a stable physical integrity in terms of cohesion between particles and adhesion to separator . Even wh−2 for all samples for fair comparison. In terms of specific capacity normalized by silicon weight (Q/mSi with Q = capacity and mSi = nwSi mass), as expected, the SECA of higher nwCu contents showed higher capacities with better rate capability due to the improvement of electric conduction by nwCu  were calculated based on the mass of silicon. Electrochemical performances were characterized in terms of rate capability and cyclability. Coin half cells were constructed with the SECA and lithium metal. For comparison, a conventional electrode consisting of nwSi, a binder (a mixture of poly(acrylic acid) and sodium carboxymethyl cellulose or PAA/CMC) and carbon black (Super P) at weight ratio of 6:2:2 was tested at the same condition by nwCu . In the  by nwCu  was comp by nwCu . The SEC by nwCu . The inf by nwCu , both ofEd + CC with mEd + CC = the mass of electrode (Ed) + current collector (CC)) ) . By savior (CC)) .−3. On the contrary, the SECA of 30 % nwSi had 64.2 % void volume without additional gap required, showing capability of accommodating 280 % volume expansion . The density of silicon loading was estimated at 0.52 g cm−3, which is significantly higher than that of the conventional electrode with enough void fraction (0.12 g cm−3).The electrochemical performances of the SECA showing improved capacities results from the combination of its structural merits. Two points have been discussed above. Inter-locking joints between nwSi and nwCu (or nwCu and nwCu) possibly improve physical integrity without binders, accelerate charge transfer between electroactive mass (nwSi) and electric pathways (nwCu), percolate networking of electric conduction pathways and provide mechanical framework to suppress volume expansion of silicon. Ultra-light porous current collector enables high energy density cells by reducing total mass with keeping the electroactive mass. As the third benefit of the SECA structure, in addition, volume expansion of silicon was successfully accommodated by voids of the inter-tangled network of nwSi and nwCu . The con15C = 67.3 % of Q0.5C for SECA with Q#C = capacity at indicated C rate; cf. Q15C = 14.1 % of Q0.5C for a conventional electrode system) and cyclability , the specific capacity normalized by the weight including electrode and current collector was estimated at 350 to 570 mAh g−1 which is incomparable with that of the conventional electrode system (45 mAh g−1). Also, we believe that the 3D structure can be used as a platform to estimate the volume expansion of silicon material as shown in structure-related modeling.In summary, 3D structures based on inter-tangling of two different nanowires (nwSi and nwCu) was developed in this work to achieve high-energy-density lithium ion batteries. Based on the nwSi/nwCu composites, uniquely designed electrode assemblies (SECA) unifying separator, active electrode part and current collector into one compartment were constructed . The thr10 mg of nwSi (diameter (D) = 40 nm, length (L) = 1 to 20 um; Sigma; w = 3,000,000, Sigma) and sodium carboxymethyl cellulose  at a ratio of 1:1 by weight was used as the binder. Carbon black  was used as the conducting agent. The loading amount of the active material was around 0.6 mg cm−2.An aqueous mixture slurry of nwSi, a binder and a conducting agent at 6:2:2 by weight was pasted in 15 um thickness on a current collector (18 um thick Cu foil) by doctor blade. The electrode was dried in vacuum at 150°C for 2 h. A mixture of poly(acrylic acid) (PAA, MC.H. and H.-K.S. conceived the idea and designed experiments. C.H. fabricated electrodes. C.H., T.-H.K., Y.-G.C. and J.K. carried out electrochemical characterization. C.H., T.-H.K. and J.K. performed morphological characterization. C.H. and H.-K.S. analyzed data and co-wrote the paper. H.-K.S. led overall project.supporting information


The RNAi machinery is a mighty regulator in a myriad of life events. Despite lines of evidence that small RNAs and components of the RNAi pathway may be associated with structure and behavior of mitotic chromosomes in diverse organisms, a direct role of the RNAi pathway in mammalian mitotic chromosome segregation remains elusive. Here we report that Dicer and AGO2, two central components of the mammalian RNAi pathway, participate in the chromosome segregation. Knockdown of Dicer or AGO2 results in a higher incidence of chromosome lagging, and this effect is independent from microRNAs as examined with DGCR8 knockout cells. Further investigation has revealed that α-satellite RNA, a noncoding RNA derived from centromeric repeat region, is managed by AGO2 under the guidance of endogenous small interference RNAs (ASAT siRNAs) generated by Dicer. Furthermore, the slicer activity of AGO2 is essential for the chromosome segregation. Level and distribution of chromosome-associated α-satellite RNA have crucial regulatory effect on the localization of centromeric proteins such as centromere protein C1 (CENPC1). With these results, we also provide a paradigm in which the RNAi pathway participates in vital cellular events through the maintenance of level and distribution of noncoding RNAs in cells. The centromere is a special region of chromosome needed for successful assembly of kinetochore and chromosome attachment to spindle . CentromThe core centromere of mammalian chromosome is the region for the assembly of kinetochore, and it consists of α-satellite repeats in human and minor satellite repeats in mice. Centromere protein B (CENP-B) is a highly conserved centromeric DNA-binding protein recognizing and binding to a 17-bp sequence (CENP-B box) in the centromeric α-satellite DNA , 6. In vThe RNA interference (RNAi)/microRNA (miRNA) pathway in mammalian cells is mediated by a cascade of enzymatic reactions . First, Previous results showed that small RNAs and components of the RNAi pathway might be associated with mitotic chromosome segregation , 24, 25,We started to investigate roles of the RNAi pathway in chromosome segregation by knocking down Dicer or AGO2 with siRNAs. Dicer or AGO2 knockdown resulted in chromosome lagging in mitotic human RPE-1 and HeLa cells . As a coFunctions of Dicer and AGO2 are largely associated with two classes of small RNA, miRNA and siRNA . To see Interestingly, knocking down the other Argonaute family members including AGO1, AGO3 and AGO4 did not induce chromosome lagging . It seemTaken together, our results demonstrated that AGO2 and Dicer acted in mammalian mitotic chromosome segregation. Their roles in this cellular event may be independent from miRNAs.in situ hybridization (FISH) in this study, unless specified. The immunostaining assay verified that there was AGO2 but almost no Dicer on metaphase mitotic chromosomes  into nuclei of human HeLa cells resulted in higher incidence of lagging chromosomes during mitosis  revealization .Taken together, these results showed that α-satellite RNAs were component of metaphase chromosomes. Too many of these RNAs would be deteriorative to the formation and function of centromere/kinetochore as shown by CENPC1 dislocation and further chromosome lagging in mammalian cells.Considering that both AGO2 or Dicer knocking down and transfection of α-satellite RNA led to a similar phenotype of CENPC1 mislocation and chromosome lagging, we suspected that the RNAi pathway might function to keep the levels of α-satellite RNA in check. AGO2 RNA immunoprecipitation (RIP) assays with isolated metaphase chromosomes revealed that AGO2 interacted with α-satellite RNA on chromosomes . FurtherCollectively, our data demonstrated that AGO2 and Dicer regulated the amount and distribution of α-satellite RNA and further CENPC1 distribution on mitotic chromosomes. The total amount of CENPC1 was not altered , but ratHow does the RNAi pathway manage level and distribution of chromosomal α-satellite RNA? Considering that AGO2 needs the guidance from small RNAs, we sequenced HEK293 cells for small RNAs and analyzed the sequencing data along with small RNA reads from other human cells such as MCF7 and ESC , 36. We in vitro synthesized ASAT siRNAs could reverse the abnormal increase in α-satellite RNA seen under Dicer knockdown  resulted in accumulation of abnormal mitotic cells, but the localization of CENP-C was normal . The reaCaenorhabditis elegans Argonaute proteins, only CSR-1 is known to be required for chromosome segregation [Another point of particular interest is the localization of AGO2 on mitotic chromosomes . Locatioregation .Drosophila, both Dicer-2 and Ago-2 are required for chromosomal localization of the RNA helicase Belle, and Dicer-2 and Ago-2 are related to chromosome segregation by promoting proper localization of proteins such as Belle [Drosophila [Drosophila RNAi machinery may have a role in chromosome segregation by directly managing SAT III RNAs, just similar to what we report here for the mammalian RNAi pathway. It is also known that centromere-encoded RNAs are components of the kinetochore in maize [In as Belle . This reas Belle . We susposophila , 42. It in maize . Whetherin trans to regulate telomerase via interacting with Aurora Kinase B in mouse embryonic stem cells [trans effect of ncRNAs transcribed from centromeric repeats.Murine cells with abnormal accumulation of minor satellite RNAs showed chromosome lagging , in a waem cells . It woulThe canonical RNAi pathway mediated by miRNAs or siRNAs is viewed mainly as a post-transcriptional regulatory mechanism of gene expression in mammalian cells , 23, 39.−1) for 15 h, resulting in a mitotic index of 60–70%. Mitotic chromosomes were isolated following the protocol from Hayashihara et al. [Cells were incubated with colchicine  glycerol, 4% sodium dodecyl sulfate (SDS), 10% β-mercaptoethanol). Western blots were performed with samples from the same number of cells containing the corresponding fraction. Only one-sixth of the WCL (whole-cell lyses) and CYTO (cytoplasmic) was loaded to avoid saturation of the corresponding bands  following the protocol provided. For whole-cell lysis preparation, we collected cells and added with sample buffer (125 mng bands .−1) for 4 h and collected after phosphate-buffered saline washing. Collected cells were treated with hypotonic solution (75 mM KCl) for 30 min at 37 °C. Then, hypotonic cells were collected and fixed with methanol: glacial acetic acid (3:1) solution twice for 1 h. Fixed cells were spun down and resuspended with methanol: glacial acetic acid solution. A small quantity of cell suspension was held vertically ~1 m above the slide (cold). Then a single drop was released from this height on the slide. Slides were washed with phosphate-buffered saline twice and then used for immunostaining and FISH.Cells were synchronized with colchicine  containing RNase inhibitors  and bovine serum albumin carried out as previously described with some modifications. RIP was performed with the relevant antibodies  and captured with protein G Sepharose 4 Fast Flow suspension . The beads were performed with proteinase K  digestion for 1 h followed by RNA purification by TRIzol reagent . Real-time quantitative (qPCR) or semi-quantitative reverse-transcription PCR (RT-PCR) was used to examine the RNA yielded, and primers used for these PCR reactions are listed in RIP was performed following the protocol from Li al. 2015 . Cells wFor HeLa chromosomal RNA high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina GAIIx system for 80-nt single-end sequencing by ABlife . For chromosomal RNA repeats dividing, we filtered the reads to the reference genome downloaded from UCSC (HG18). Small RNA high-throughput sequencing was performed with Illumina GAII 2000  with total RNA isolated from HEK293 cells. Small RNA reads for MCF7 cell (GEO Accession: GSM402329) and ES cell (GEO Accession: GSM450239) were also analyzed for ASAT siRNAs , 36. In −1) at 62 °C overnight. Membranes were stringently washed twice in wash buffer  at 62 °C for 30 min, respectively, and detection was performed following the protocol provided . Images were taken with LAS-4000mini Image Reader . Information for oligos used is provided in Digoxin-labeled RNA probes were transcribed with the corresponding insertion in the T vector as a template following the manual of DIG Northern Starter Kit . Total RNA and chromosomal RNA (both were 7.5 μg) were loaded on a precast gel  containing 15% urea and were ran for 3 h in 1× TBE buffer. RNA was transferred onto Hybond-N+ membrane  by electrophoretic transfer. Hybridization was performed with probes . Flow cytometry was performed with FACSCalibur  and FlowJo 7.6 software .The separation profiles of cleared mitotic chromosome fraction and nucleus fraction were confirmed using flow cytometry after staining with propidium iodide , AGO3 mRNA (TRCN0000009636) and AGO4 mRNA (TRCN0000009640) with negative shRNA control (SHC002) were obtained from the MISSION shRNA Library . Information for oligos used is provided in −1) were denatured at 80 °C for 10 min, and then incubated at 37 °C for 15–17 h with yeast tRNA blocking. Slides were washed twice with 2× SSC at 45 °C for 10 min. Images were taken with Zeiss Imager A2 microscope . Information for oligos used is provided in RNA probes were prepared by SP6 (Thermo Scientific) with the corresponding insertion in the T vector as a template, and were labeled with Alexa Fluor546 by using the ULYSIS Nucleic Acid Labeling Kit  following the manual provided. The fixed cells and RNA probes , CENP-B , Phospho-Histone H3 , AGO2 , Dicer  or ACTB  followed with incubation with Alexa Fluor488 or 546 labeled secondary antibody .2. MEF (DGCR8 null) cells were cultured under standard conditions with DMEM plus 10% fetal bovine serum and 1% NEAA at 37 °C and 5% CO2. RPE-1 cells were used in essentially all the FISH and IF experiments because they are diploid. Plasmids and siRNA transfection was performed with Lipofectamine 2000 (Invitrogen) according to the supplier’s instructions . α-satellite RNA and Line 1 repeat-derived RNA  transfections were performed with electroporation using Nucleofection  according to the attached instructions. Information for oligos used is provided in HEK293, HeLa and RPE-1 were cultured under standard conditions with DMEM plus 10% fetal bovine serum at 37 °C and 5% COFor western blots, samples were separated on SDS-polyacrylamide gel electrophoresis gels and then transferred to Polyvinylidene fluoride membranes . Membranes were processed following the ECL western blotting protocol . These antibodies were used in western blots: anti-CENPC1 , anti-Phospho-Histone H3 , anti-ACTB , anti-Dicer , anti-GAPDH , anti-AGO2  and anti-HDAC2 . Images were taken with LAS-4000mini Image Reader .Total and chromosomal RNAs were purified from cells and chromosomes by using the TRIzol reagent (Invitrogen) with the attached instructions. Complementary DNA for qRT-PCR was synthesized from total and chromosomal RNA using the GoScript Reverse Transcription System  according to the attached procedures using random hexamer primer or oligo dT. qPCR was performed with Platinum SYBR Green qPCR Supermix UDG (Invitrogen) on PikoReal Real-Time PCR System (Thermo Scientific) according to the standard procedures. For semi-quantitative PCR and semi-quantitative RT-PCR, PCR cycle numbers were set between 20 and 25 to avoid saturation of PCR reactions. Information for oligos used is provided in Cq method. The values reported in the graphs represent the average of three independent experiments, with error bars showing s.d. After analysis of variance by F-test, the statistical significance and P-value were evaluated by Student’s t-test.Relative mRNA level was calculated by ∆∆


This results in toxic gain-of-function effects mediated through altered functions of RNA-binding proteins . In this study we demonstrate the potential of a new acceptor photobleaching assay to measure FRET (AP-FRET) between RNA and protein. We chose to focus on the interaction between MBNL1 and mutant DMPK mRNA in cells from DM1 patients due to the strong microscopic evidence of their co-localization. Using this technique we have direct evidence of intracellular interaction between MBNL1 and the DMPK RNA. Furthermore using the AP-FRET assay and MBNL1 mutants, we show that all four zinc-finger motifs in MBNL1 are crucial for MBNL1-RNA foci interactions. The data derived using this new assay provides compelling evidence for the interaction between RNA binding proteins and RNA foci, and mechanistic insights into MBNL1-RNA foci interaction demonstrating the power of AP-FRET in examining RNA-Protein interactions in DM1.Förster Resonance Energy Transfer (FRET) microscopy is a powerful tool used to identify molecular interactions in live or fixed cells using a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity. FRET can be a very sensitive tool to study protein-protein and/or protein-nucleic acids interactions. RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy. Myotonic dystrophy (DM1) is caused by a (CTG) Förster Resonance Energy Transfer (FRET) microscopy is a powerful tool widely used to identify molecular interactions in live or fixed cells. FRET is a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity DMPK gene DMPK mRNA is retained in the nucleus as discrete foci, or RNA foci Myotonic Dystrophy type 1 (DM1), a dominantly inherited multisystemic neuromuscular disorder is the first example of RNA-mediated disease amongst genetic disorders DMPK mRNA foci in cells derived from DM1 patients. We have corroborated our findings with EGFP-fused MBNL1 and have used RNA-IP with anti-MBNL1 antibodies to biochemically validate the FRET analysis. Further, we have used deletion mutagenesis to provide mechanistic insights by identifying functional domains in MBNL1 involved in this interaction and in regulation of alternative splicing. Taken together these results demonstrate the power of AP-FRET in not only identifying interactions between RNA and proteins but also in determining the functional domains involved in that interaction.In this study, we have developed and used an acceptor photobleaching FRET assay to identify RNA-protein interactions. Using this technique we provide the first direct evidence of intracellular interaction between endogenous MBNL1 and mutant 10 probe at 0.1 ng/uL for 2 h at 37°C in the hybridization buffer . After the hybridization, the cells were washed in 30% formamide/2xSSC at 45°C for 30 min. Next the cells were blocked in 1% BSA/PBS for 1 h at 37°C. Primary antibody, either MBNL1 rabbit polyclonal antibody (1∶2000) DM1 cells were grown on a glass coverslip. When the desired cell density was reached the cells were washed in PBS three times then fixed in 4% paraformaldehyde/PBS for 10 min at room temperature. Following fixation they were permeabilized in cold 2% acetone/PBS for 5 min at room temperature. The cells were then washed with PBS three times and incubated with 30% formamide/2x SSC buffer at 37°C for 10 min. Hybridization was then carried out with either a CY3 or FITC labeled (CAG)Acceptor photobleaching FRET (AP-FRET) was performed on a Leica SP5X white light laser (WLL) confocal microscope using the Leica AP-FRET wizard. Images were acquired using a 63x NA 1.4 oil objective on a 512x512 resolution format. In order to minimize photobleaching during image acquisition, low percentages (5–25%) of the full power of WLL 488 nm and 552 nm with PMT gain of 650 v were used. A region of interest (ROI) was drawn over the selected foci and either 100% of WLL 552 nm for 8 iterations was used for photobleaching the acceptor/CY3-RNA foci or 40% of WLL 552 nm for 6 iterations for photobleaching acceptor/Alexa555-MBNL1 or RNA Pol II to background levels. The “n” in the figure legend denotes number of RNA foci (ROIs) analyzed in DM1 cells. Prebleach and Postbleach images were acquired using identical imaging settings. A 2x2 pixel ROI was drawn over bleached foci and efficiency of energy transfer (E%) was calculated using the equation: E%  =  (Donor Postbleach-Donor Prebleach)* 100/(Donor Postbleach). Representative images were processed using Adobe Photoshop CS3 and Minitab 16 was used for E% distribution graphs.DMPK gene 6 cells were trypsinized, pelleted, PBS washed and processed for nuclear-cytoplasmic fractionation using the PARIS kit . The nuclear lysate was diluted 1∶3 in Sanford IP-RT PCR buffer  supplemented with, RNase inhibitor to a final concentration of 100 units/mL, Protease inhibitor to a final concentration of 1x, Phosphatase inhibitor to a final concentration of 1x, and DTT to a final concentration of 1 mM. The DM1 fibroblasts' nuclear lysate and DM200 skeletal muscle homogenate were immunoprecipitated either with anti-MBNL1 or isotype control using magnetic beads . RNA was extracted with Trizol , followed by EGFP RT-PCR for the mutant transgene and DMPK RT-PCR/BpmI RFLP for mutant and wildtype transcripts. DMPK RT-PCR primer for cDNA synthesis: 5′ ACT GGA GCT GGG CGG AGA CCC A 3′ and DMPK RT-PCR primers: FP- 5′ GGCTCACTGCCATGGTGAG 3′, RP- 5′ CTCGGCCTCAGCCTCTGC 3′.DM200 mouse skeletal muscle MBNL1 backbone was used for generating the MBNL1 deletion mutants. The Quick exchange mutagenesis kit  was used for generating mutants: Del-C′, Del-Linker and Del-ZF4-C′; whereas, a Pst1 restriction site within the MBNL1 cDNA was used for generating Del-N′ followed by conventional self ligation and transformation. All deletion mutants were sequence confirmed and were in frame with the N′ terminus EGFP tag. Primers for quick exchange mutagenesis are as follows. Primer set 1: FP-Del-C′: 5′ gccaccgcagctgccatgtagccacaagtatggatcc 3′; RP-Del- C′: 5′ ggatccatacttgtggctacatggcagctgcggtggc 3′. Primer set 2: FP-Del-ZF4-C′: 5′ gcacaatgattgacaccaattagccacaagtatggatcc 3′; RP-Del- ZF4-C′: 5′ ggatccatacttgtggctaattggtgtcaatcattgtgc 3′. Primer set 3: FP-Del-Linker: 5′ gccccattacaacccgtgacagacagacttgaggtatg 3′; RP-Del-Linker: 5′ catacctcaagtctgtctgtcacgggttgtaatggggc 3′The pEGFP-SERCA1 exon 22 with primers forward: 5′ ATCTTCAAGCTCCGGGCCCT 3′ and reverse: 5′ CAGCTCTGCCTGAAGATGTG 3′; IR exon 11 with primers forward: 5′ CCAAAGACAGACTCTCAGAT 3′ and reverse: 5′ AACATCGCCAAGGGACCTGC 3′ and NFIX exon 7 with primers forward: 5′ AGCCCTGTTGATGACGTGTT 3′ and reverse: 5′ AGTGCAGGGCTGATGCTGT 3′. Minitab 16 was used for one-way ANOVA with Tukey's multiple comparison method for statistical significance.Total RNA was extracted using the lysis buffer 1000.The immortalized DM1 fibroblasts/myoblasts inducible for the Transient transfections in DM1 fibroblasts/myoblasts were done using NucleofectorTM , and in HEK293T using Lipofectamine 2000 . Cells were either fixed for RNA FISH or processed for RNA extraction 48h post transfection or 24 h for HEK293T. For AP-FRET experiments (1 µg) and for splicing assays (4 µg) of plasmids were used. For the EGFP-tagged RNA binding proteins and for MBNL1-full length (MBNL1- FL) and its deletions, the coverslips were mounted after RNA-FISH and subsequently analyzed by AP-FRET. Plasmids pEGFP-MBNL1, pEGFP-hnRNP-C and pEGFP-CUGBP1 were gifts from Dr. David Brook.Protein expression of FL-MBNL1 and its deletion mutants and their molecular sizes were analyzed and confirmed by western blotting. The blot was probed with anti-EGFP and anti-Dynein antibodies .Standard statistical methods were employed using the software Minitab 16.1.0, produced by Minitab, Inc. Two-sample one-sided T-tests were used to determine differences in E% between donor-acceptor and donor only FRET. A one-sided Fisher's exact test was used to determine differences in positive E% between groups. A one-way ANOVA with Tukey's multiple comparison was used to analyze the alternative splicing results. Statistical significance was set at a p-value of <0.05.Though conventional or laser scanning confocal microscopy (LSCM) can be used to detect the co-localization of molecules, its optical resolution  limits the ability to make any conclusive statements about molecular interactions. However, FRET microscopy typically detects interactions in the 1–10 nm range. FRET can be analyzed by a number of different methods DMPK RNA, which have been extensively studied in myotonic dystrophy. Using fibroblasts from DM1 patients we performed a combined RNA-FISH potocol to simultaneously detect RNA foci with a FITC labeled (CAG)10 antisense probe (the donor), and MBNL1 (the RNA-binding protein) with an Alexa555 labeled secondary antibody (the acceptor). To optimize of photobleaching for the AP-FRET experiment we avoided any saturation in our Regions of Interest (ROIs) by using low power of the exciting laser below saturation for signal acquisition. These conditions result in images of MBNL1 foci without excessive nucleoplasmic/cytoplasmic MBNL1 staining. After optimizing photobleaching conditions, AP-FRET was performed using a LSCM (Leica SP5X white light laser (WLL)). Dequenched signal from the donor was seen after photobleaching the acceptor, demonstrating FRET and an interaction between MBNL1 and RNA foci >200 transgene (termed DM200) DMPK gene DMPK transcripts in DM1 fibroblast extracts .We next looked at another RNA binding factor with much less evidence for RNA foci binding, hnRNPH, using our AP-FRET assay. There is evidence that hnRNPH is both increased in DM1 but also is pulled down with a modified UV crosslinking assay using Identifying and characterizing RNA-protein interaction can be a difficult undertaking, especially as many techniques to study the process do not provide evidence of direct interaction. FRET is a distance dependent phenomenon 10 antisense probe, AP-FRET was performed using EGFP (donor) and CY3 (acceptor) as the FRET pair. As a negative control, plasmid expressing EGFP alone was used to measure background noise in AP-FRET experiments. Similar experiments were done using EGFP fused versions of CUGBP1 and hnRNP-C, two other RNA-binding proteins thought to have in vitro binding with the DMPK mRNA EGFP-MBNL1 corresponds to an isoform of MBNL1  that has previously been shown to co-localize with the RNA foci The domains in a protein that are responsible for binding and the domains in a protein that have other biological functions can be difficult to study independent of each other. Utilizing AP-FRET in the context of the DM1 MBNL1-RNA interaction we examined the different domains present in MBNL1 and how they relate to its RNA binding and mRNA splicing activity.21 and (CCUG)22 RNAs in vivo data exists defining the domains of MBNL1 necessary for interactions with the mutant DMPK mRNA. To address this we generated a series of MBNL1 deletion mutants using the EGFP-MBNL1 backbone , showed strong dequenched signal from the donor after photobleaching the acceptor . Their EIR), muscle-specific chloride channel (CLCN1) and sarcoplasmic/endoplasmic reticulum Ca+2 ATPase  SERCA1 exon 22, IR exon 11 and NFIX exon 7 SERCA1 without exon 22, IR without exon 11 and NFIX with exon 7. Western bloting and qPCR were used to verify that the transfected cells were over-expressing the relevant MBNL1 constructs , resulted in significant loss in splicing regulation in both HEK293T and DM1 myoblasts for endogenous SERCA1 and NFIX, analogous to what was observed using IR and cTNT minigenes in COSM6 cells RT-PCR showed that over-expression of FL-MBNL1 resulted in 17.82 fold increase in nclusion  and 10.7xclusion . The delgulation . A simil exon 22 . The NFIIR showed that over-expression of FL-MBNL1 resulted in 2.4 fold increase in IR exon 11 inclusion in HEK293T >200 transgene  and exon 4 . Exon 3 encodes a “linker” domain connecting the two pairs of zinc fingers. It has been reported that this “linker” region was essential for the interaction of MBNL1 with CUG repeats based on three-hybrid assays Our AP-FRET data also provides insights into the molecular basis of the RNA foci-MBNL1 interaction. Using deletion mutants of MBNL1, it is evident that all four zinc finger domains are necessary for this interaction . This isTNT and IR minigenes using co-transfection assays SERCA1 exon 22 and also increased exon 22 inclusion in HEK293T cells (MBNL1-exon 3 (Del-Linker) or deletion of MBNL1-exon 6 (Del-C′) dramatically reduced the efficiency of MBNL1 in rescuing splicing defects in both DM1 myoblasts and the HEK293T cells , Huntington Disease Like-2 (HDL-2), Spinocerebellar Ataxias  and more recently in Amyotrophic Lateral Sclerosis/Frontotemporal Dementia (ALS/FTD). Our study opens up new means of identifying and characterizing RNA-protein interactions in RNA foci complexes in such disease states and has the potential utility as an assay screening for compounds capable of disrupting deleterious RNA-protein interactions in DM1 and other disorders where these interactions play a key role in disease pathogenesis.FRET is one of the most sensitive techniques available for studying molecular interactions in cells and AP-FRET is an ideal method to investigate protein interactions in fixed cells. We have now developed and validated a novel FRET based assay for studying intracellular RNA-protein interactions. Furthermore, in validating this assay we have provided the first intracellular evidence of interaction between the mutant


Over 98% of our genes code for RNA transcripts that will never become translated into protein. Numerous non-coding RNA (ncRNA) transcripts are structurally and functionally diverse. In particular, micro RNAs (miRNAs), piwi-interacting RNAs (piRNAs), and, more recently, transfer RNAs (tRNAs) are implicated as regulators of key genes and processes that are involved in various human diseases, including cancer. Here, we summarize the recent findings and perspectives in the small RNA and cancer research. Breakthroughs in non-coding RNA biology, including the sequencing of the human genome, reveal that over 98% of our genes code for RNA transcripts that will never become translated into protein , 2. ThesThese ncRNAs have all been found to be deregulated in cancer, with miRNA being profoundly better characterized than the other subclasses. Genetic alterations caused by chromosomal abnormalities leading to deletion, amplification, or rearrangements are especially prevalent in the more than 50% of miRNAs located near fragile genome sites . Recent As their name suggests, piRNAs complex with PIWI proteins to form piRISC as the effector complex in retrotransposon silencing within the germline . PiRNAs et al. was able to show the direct correlation between overexpression of the initiator tRNA (iMET) and increased cell proliferation, which leads to oncogenic tranformation of fibroblasts and thus tumour formation in mice; this finding demonstrates that tRNA deregulation was not just a byproduct of cancer but a driving force in carcinogenesis [Increased cell proliferation requires elevated protein synthesis levels, which makes the correlation between tumour cells and deregulated tRNAs, a component in translation, probable. With limited research, a few studies have demonstrated that the components involved in translation are shown to be dysregulated in cancer, including elevation of tRNA transcripts in ovarian cancer . With feogenesis . The undogenesis . Therefoogenesis . The samogenesis . These f