Science 2009); however, only a handful of these sites have been independently confirmed. Here, we systematically and quantitatively examined 109 putative coding region A-to-I RNA editing sites in three sets of normal human brain samples by ultra-high-throughput sequencing (uHTS). Forty of 109 putative sites, including 25 previously confirmed sites, were validated as truly edited in our brain samples, suggesting an overestimation of A-to-I RNA editing in these putative sites by Li et al (2009). To evaluate RNA editing in human disease, we analyzed 29 of the confirmed sites in subjects with major depressive disorder and schizophrenia using uHTS. In striking contrast to many prior studies, we did not find significant alterations in the frequency of RNA editing at any of the editing sites in samples from these patients, including within the 5HT2C serotonin receptor (HTR2C). Our results indicate that uHTS is a fast, quantitative and high-throughput method to assess RNA editing in human physiology and disease and that many prior studies of RNA editing may overestimate both the extent and disease-related variability of RNA editing at the sites we examined in the human brain.A-to-I RNA editing is a post-transcriptional modification of single nucleotides in RNA by adenosine deamination, which thereby diversifies the gene products encoded in the genome. Thousands of potential RNA editing sites have been identified by recent studies (e.g. see Li et al,  Adenosine Deaminases Acting on RNA). A-to-I RNA editing converts genomically encoded adenosine to inosine, which is recognized as guanosine by the translational apparatus Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification of RNA transcripts catalyzed by ADARs .Based on the consistency between the samples and the background A-to-G error rate in the system, three categories of editing sites were identified. Category I consists of sites with an editing frequency greater than 1%, which included 11 of the ‘new’ sites identified by Li et al Previous studies have shown differences in the frequency of RNA editing in different brain regions with as few as 10 total reads per tissue were considered to have adequate sequence coverage in their study 4 reads and only 1.9% sites had less than 1,000 reads in three sets of brain samples. One general problem of all current next generation sequencing (NGS) reads is their higher error rate compared with Sanger sequencing Taq DNA polymerase . Thirdly, some SNPs (single nucleotide polymorphisms) appear to be mis-annotated as RNA editing sites, e.g., ATXN7 (GenBank ID: rs1053338), Bin1 (GenBank ID: rs138047593) (Of 109 putative RNA editing sites 8047593) , C1ORF178047593) . Thus, t2C receptor 2C receptor have been implicated in schizophrenia, major depressive disorder and in suicide. However, the results have been highly inconsistent among studies and, given the methodology used, inconclusive 2C receptor) were selected from Category I (vide supra) and examined in five groups of a total of 72 subjects: age-, sex-, tissue pH- and post-mortem interval (PMI)-matched controls (n = 15), major depressive disorder , MDD with suicide (n = 15), schizophrenia (n = 15) and schizophrenia(7)/schizoaffective disorder(5) with suicide (n = 12). The right prefrontal cortex (Brodmann Areas 8/9) was sampled. There were no significant differences in age, gender, PMI and tissue pH among the groups  p<0.01, GRIA2 (R/G) p<0.05, GRIA3 p<0.05, GRIK2 (Y/C) p<0.01, GRIK2 (Q/R) p<0.05, KCNMA1 p<0.05) compared to the normal pH subgroup, and there was a statistically significant difference between the two subgroups   . Therefoanalysis . After ranalysis  and any he data. .2C receptor transcripts, also in contrast with many previous studies. Our findings, however, do indicate that uHTS is a fast, powerful and quantitative method for assessing RNA editing in human tissues provided adequate attention is paid to experimental variables including tissue pH, estimations of sequencing and amplification errors, and statistical power considerations. uHTS of known RNA editing sites on multiple samples, as done in the present study, provides a more robust testing of focused hypotheses than more-or-less random whole-genome sequencing of one or even a few samples, with its attendant low coverage at each site. The results neither support many previous findings that large-scale alterations in RNA editing occur in psychiatric disorders nor the recent proposals that large numbers of genes contain coding regions is RNA edited.In summary, we have systematically and quantitatively examined 109 putative coding region A-to-I RNA editing sites by ultra-high-throughput sequencing (uHTS) in human brain. Only 40 of 109 sites identified by Li’s (ref 24) study were confirmed in our brain samples, which may suggest an overestimation of these RNA editing sites in the human brain. After analyzing the prefrontal cortex samples from MDD and schizophrenia in our study, we did not find any significant alterations in the frequency of RNA editing at any RNA editing sites we evaluated, including the sites of the 5HTHuman cortex and cerebellum total RNA from pooled normal human control samples (n = 10) were obtained from Clontech. Tissue samples from a normal control subject containing 5 different brain areas (premotor cortex (Brodmann areas 6), motor cortex (Brodmann area 4), parietal cortex (Brodmann area 7), occipital cortex (Brodmann area 19), and cerebellum) were obtained from the Stanley Medical Research Institute . The age, PMI, and average tissue pH of this subject was 37 years, 50 hours, and pH 6.61. Tissue samples from a normal control cohort containing 5 subjects, two brain regions ) were obtained from the Human Brain Collection Core . The average age, PMI, and tissue pH of the cohort was 48.4±12.15 years, 20.49±6.63 hours, and pH 6.57±0.3. Samples from a 72 patient cohort ) was obtained from the Human Brain Collection Core and consisted of five groups: normal controls (n = 15), major depressive disorder (MDD) (n = 15), major depressive disorder who committed suicide (MDD suicide) (n = 15), schizophrenia (SCZ) (n = 15), schizophrenia (7)/schizoaffective disorder (5) who committed suicide (SCZ suicide) (n = 12). Detailed information for this cohort is shown in th ed, 1994). All depressed subjects met criteria for a major depressive episode within the last two weeks of life except for three non-suicides: two depressed subjects were in partial remission and one was in full remission. Twenty two and five subjects met DSM-IV criteria for schizophrenia and schizoaffective disorder, respectively. Among depressed subjects committing suicide, the following comorbid diagnoses were noted: alcohol dependence (2), alcohol abuse (2), alcohol and cannabis abuse (1). Among depressed subjects not committing suicide, the following comorbid diagnoses were noted: opiate dependence (1), cannabis dependence (1), alcohol dependence and polysubstance abuse (1), polysubstance abuse (2). Among subjects with a psychotic disorder committing suicide, the following comorbid diagnoses were noted: cannabis dependence (1), polysubstance abuse (1) and alcohol dependence and cannabis abuse (1). Among subjects with a psychotic disorder not committing suicide, the following comorbid diagnoses were noted: alcohol dependence and cannabis abuse (2) alcohol dependence (1) and cannabis abuse (1).All tissues from the Human Brain Collection Core were collected at autopsy at the Cuyahoga County Coroner’s Office, Cleveland, OH, using an ethical protocol approved by the Institutional Review Board of the University Hospitals of Cleveland and the University of Mississippi Medical Center. Informed written consent was obtained from the next-of-kin for all subjects. Informed consent for tissue donation at autopsy and a subsequent psychiatric assessment was sought from the legally-defined, adult next-of-kin of the deceased. The next-of-kin of the deceased were contacted by telephone, informed of the broad goals of the study and asked to participate by permitting sampling of tissue as well as agreeing to an interview held about three months later. If the family declines to participate, they are not re-contacted and no tissue is collected. If the family indicates a willingness to participate, written consent is obtained shortly thereafter by a research coordinator, after which the coroner releases the tissues for dissection, freezing and storage. Included with the informed consent form is a request to obtain medical records of the deceased. An appropriate, IRB-approved HIPAA Authorization Form is used in requesting relevant personal health information from doctors and/or hospitals identified by the next-of-kin or coroner’s records. If written consent is not received, no tissue is sampled nor are the families contacted again. All potential participants who do not consent to the study are not disadvantaged in any way by not participating in the study. After an initial contact, those who decline to participate are not contacted again. Cortical tissues were frozen in isopentane cooled by dry ice and stored at −80°C. The causes of death – natural, accidental or suicide – were determined by the coroner. Toxicology screening of postmortem blood and urine was performed by the coroner’s office. An antidepressant medication was present in four subjects with MDD and five subjects with schizophrenia. An antipsychotic medication was present in four subjects with schizophrenia. A trained interviewer assessed Axis I psychopathology for each subject by a structured clinical interview with knowledgeable next-of-kin, as previously described Trizol (Invitrogen) was used to extract RNA from ∼123 mg of each human brain sample. Ten µg of RNA was treated with DNAse , and 2 µg of the DNase-treated RNA was added to a reverse transcription reaction performed using the SuperscriptTM III RNase H Reverse Transcriptase kit (Invitrogen) with random hexamer primers (Invitrogen). The cDNA product was used as template to generate a double stranded DNA fragment by PCR for use in high throughput sequencing experiments.−7 (http://www.finnzymes.fi/pcr/phusion_products.html). PCR fragments (106–233 base pairs in length) were diluted 6X, and 5 µL were used as template for a second round of PCR (50 µL reaction) (the conditions for this 2nd round were the same as for the 1st round). PCR fragments were purified using the QIAquick96 PCR Purification kit (Qiagen) and 5 ul of product was run on a 1.5% agarose gel to determine quality and quantity of amplification. In order to confirm amplification of the correct gene with the correct tag, 5 ul of each PCR product was submitted for Sanger sequencing. The primers used for amplification are listed in One µL of cDNA (of 20 µL) was used as template for a 20 µL PCR reaction  to amplify a fragment containing the edited region of interest using Phusion Hot Start High-Fidelity DNA Polymerase (New England BioLabs). The error rate of Phusion DNA Polymerase is 4.4×10For sequencing of the Clontech samples, two “barcodes” were used (cortex and cerebellum); PCR products of 96 genes from two samples were mixed in equal parts and designated for one lane in the Genome Analyzer II flow cell. Five “barcodes” were used for the five-patient, two-brain area normal control cohort samples from the Human Brain Collection Core, as well as the one-patient, five brain-area normal control samples from the Stanley Medical Research Institute (SMRI) to differentiate a similar number of genes and editing sites as above for each tissue sample. PCR products from five samples were then mixed in equal parts creating three mixed sample sets, each set designated for one lane in the Genome Analyzer II flow cell. For the cohort of the 72 human psychiatric and control samples, 12 different “barcodes” were used to differentiate 19 genes and 29 edited sites for each sample, which were then mixed in equal parts to create 6 sample sets, each set designated for one lane in the Genome Analyzer II flow cell. The process of Ultra high throughput sequencing was similar to our previous publication. Briefly, purified PCR products were diluted to a concentration of 15 nM. 2 µL of the diluted PCR product was used for denaturation . 4 µL of the denaturation mixture was diluted in 996 µL of hybridization solution. The hybridization mixture  was loaded into the Cluster Station for cluster generation. Primer hybridization was performed on the Cluster Station using 6.6 µL of 500 nM sequencing primer (Primer sequence: 5′-acactctttccctacacgacgctcttccgatct-3′) diluted in 1313 µL of hybridization buffer. Cluster generation was performed for 76 cycles followed by base-by-base sequencing initiated by the sequencing primer on the Genome Analyzer II. The Genome Analyzer II uses two different lasers to excite the dye attached to each nucleotide. Since the emission spectra of these four dyes overlap, the four images thus obtained are not independent. As in Sanger sequencing, the frequency cross-talk is deconvolved using a frequency cross-talk matrix. Therefore, the crosstalk matrix calculation requires control lanes for samples with skewed base compositions. Thus, a control human genomic DNA sample was run in parallel on the same flowcell concurrently with the human RNA editing samples.http://pdsp-temp.pha-med.unc.edu/Download/code.php) written by us to filter the data through two rounds of filters and sort the data that passed through the filters. Reads containing “Ns”, or short reads mapped to the target region shorter than 30 bp, were filtered out. The mapped reads equal or large than 30 bp were counted. The percentage of RNA editing was calculated by the number of reads containing “G” at the editing site divided by the total number of reads containing “A” or “G” at the editing site. The human genomic reference sequence for analysis was downloaded from UCSC . The A-to-G error rate for each editing site investigated was determined by measuring the rate of A-to-G misreads within a few base pairs of each editing site. Further data analysis was performed in Microsoft Excel and Graphpad Prism 5.0. All statistical analyses were performed in Graphpad Prism 5.0. For purposes of making statistical comparisons in the cohort of 72 human psychiatric and control samples, all reads generated from one sample were treated as one experiment (N = 12−15 for each sub cohort). The data were analyzed by two-tailed t-test and subsequent Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as criterion of statistical significance.Sequences were analyzed using a Perl 5 script Click here for additional data file.Figure S22C receptor produced by RNA editing in psychiatric patients and normal controls, including 8 samples with pH<6.1.Expression pattern of 24 isoforms of the 5HT The RNA editing frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. The data were analyzed by t-test with Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as criterion of statistical significance.(TIF)Click here for additional data file.Figure S3Validation of RNA editing sites by Sanger sequencing. 15 RNA editing sites were verified by Sanger sequencing with the matched genomic DNA and cDNA samples from the same individuals. RNA editing was indicated by a trace of guanosine in cDNA sequence, while the genomic DNA sequence shown only adenosine signals. The RNA editing sites with the frequency above 10% measured by our approach have shown a clear signal of guanosine.(TIF)Click here for additional data file.Table S1Category I RNA editing sites with >1% A-to-I RNA editing in three sets of normal human samples. RNA editing frequency is presented as the percentage of the total population of transcripts. Three independent sets of normal human samples were used in this study. The first is the cortex and cerebellum pooled from 10 normal humans; the second comprises one subject (ID: S343) with 5 different brain regions sampled  and the third is of 5 normal humans  and two brain regions, anterior temporal cortex (ATC) and right frontal cortex  (Human Brain Collection Core). 11 new RNA editing sites are shown in red. The values of RNA editing frequency in the cerebellum, which were more than 1.5 fold different from the cortex, were highlighted.(XLSX)Click here for additional data file.Table S2Category II RNA editing sites with 0.08%–1% A-to-I RNA editing in three sets of normal human samples. RNA editing frequency is presented as the percentage of the total population of transcripts. Three independent sets of normal human samples were used in this study. The firstt is the cortex and cerebellum pooled from 10 normal humans; the second comprises one subject (ID: S343) with 5 different brain regions sampled  and the third is of 5 normal humans  and two brain regions, anterior temporal cortex (ATC) and right frontal cortex  (Human Brain Collection Core). The values of RNA eidting frequency above 0.08% were highlighted.(XLSX)Click here for additional data file.Table S3RNA editing frequency (%) in category III sites with <0.08% A-to-I RNA editing in three sets of normal human samples. RNA editing frequency is presented as the percentage of the total population of transcripts. Three independent sets of normal human samples were used in this study. The first is the cortex and cerebellum pooled from 10 normal humans; the second comprises one subject (ID: S343) with 5 different brain regions sampled  and the third is of 5 normal humans  and two brain regions, anterior temporal cortex (ATC) and right frontal cortex  (Human Brain Collection Core).(XLSX)Click here for additional data file.Table S4Four single nucleotide polymorphisms (SNP) were misannotated as RNA editing in three cohorts of normal human samples. RNA editing frequency is presented as the percentage of the total population of transcripts. Three independent sets of normal human samples were used in this study. The first is the cortex and cerebellum pooled from 10 normal humans; the second comprises one subject (ID: S343) with 5 different brain regions sampled  and the third is of 5 normal humans  and two brain regions, anterior temporal cortex (ATC) and right frontal cortex  (Human Brain Collection Core). The values of A-to-G rate greater than 0.08% were highlighted.(XLSX)Click here for additional data file.Table S5Characterization of 72 human psychiatric and control samples. The values are presented as mean ± SEM. PMI: post-mortem interval.(XLSX)Click here for additional data file.Table S6A-to-I RNA editing frequency (%) of 29 editing sites from 19 genes in normal and psychiatric disease patients. RNA editing frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. Data were analyzed by t-test with Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as criterion of statistical significance.(XLSX)Click here for additional data file.Table S7A-to-I RNA editing frequency (%) of 29 editing sites from 19 genes in normal and psychiatric diseases patients excluding 8 low pH samples. RNA editing frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. The data were analyzed by t-test with Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as criterion of statistical significance.(XLSX)Click here for additional data file.Table S8Distribution of the frequency (%) of 24 isoforms of 5HT2C receptors produced by RNA editing in normal and psychiatric disorder patients. 5-HT2C receptor isoform frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. The data were analyzed t-test with Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as criterion of statistical significance.(XLSX)Click here for additional data file.Table S9Distribution of the frequency (%) of 24 isoforms of 5HT2C receptors produced by RNA editing in normal and psychiatric disorder patients (excluding 8 low pH samples). 5-HT2C receptor isoform frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. The data were analyzed by t-test with Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as criterion of statistical significance.(XLSX)Click here for additional data file.Table S10A-to-I RNA editing frequency (%) of 29 editing sites from 19 genes in normal pH (pH >6.1) and low pH (pH <6.1) samples. RNA editing frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. Data were analyzed by t-test with Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as criterion of statistical significance. The significant differences between the control group and other groups are shown by asterisks .(XLSX)Click here for additional data file.Table S11The primers used in the study. All primers are in the 5′-to-3′ direction from left to right. One “barcode” (CAGCTA) was used to illustrate this set of primers. See (XLSX)Click here for additional data file.Table S12The “barcodes” used in the study.(XLSX)Click here for additional data file.


The vast majority of the mammalian genome is transcribed giving rise to many different types of noncoding RNAs. Among them, long noncoding RNAs are the most numerous and functionally versatile class. Indeed, the lncRNA repertoire might be as rich as the proteome. LncRNAs have emerged as key regulators of gene expression at multiple levels. They play important roles in the regulation of development, differentiation and maintenance of cell identity and they also contribute to disease. In this review, we present recent advances in the biology of lncRNAs in muscle development and differentiation. We will also discuss the contribution of lncRNAs to muscle disease with a particular focus on Duchenne and facioscapulohumeral muscular dystrophies. In mammals, the vast majority of the transcriptional output is noncoding . While 7LncRNAs loci are often in close association with protein-coding genes as they are encoded from exonic or intronic sequences in both sense and antisense orientation or even from gene regulatory regions . LncRNAsIn terms of transcriptional profile, lncRNAs are generally expressed at lower levels than protein-coding transcripts and, compared to the latter, their pattern of expression is more developmental stage- and cell type-specific ,16. The LncRNAs can be divided into multiple functional categories based on the site of action and the level of gene expression at which they act. However, as our knowledge of lncRNAs increases, new functional groups emerge and the distinction between classes is not always adequate. Here, we present a very brief classification to provide a framework for the examples of lncRNAs acting in muscle differentiation and disease later described.cis-acting RNAs that work in proximity to their site of transcription, and trans-acting RNAs that operate at distant loci. Both cis- and trans-acting lncRNAs can activate or repress transcription through the recruitment of chromatin remodelers and modifiers, thus shaping the chromatin status of a particular locus or even of an entire chromosome  and the Myocyte Enhancer Factor-2 (MEF2A-D) families of transcription factors (reviewed in  and 52]52]). In in cis or in trans  regulate the expression of MyoD and MyoG  and acetylated H3K27 (H3K27ac). Intriguingly, RNA polymerase II (RNAPII) is also enriched at active enhancers where it drives localized transcription of lncRNAs called enhancer-derived RNAs (eRNAs) ,66. Many and acetYY1-associated muscle lincRNAs or Yams) have been recently identified -8882-88]tastasis . Malat1 ein Cbx4  and withein Cbx4 . Malat1 G1 phase , suggestyostatin , an impoyostatin . Future Dlk1-Dio3 region is a very complex, imprinted locus involved in tissue growth regulation and human cancers . Bo. BoCdc6 ogenesis . Importaogenesis , lncRNAsGrowing evidence shows that the vast majority of disease-associated genetic variations occur in the noncoding portion of the genome. In fact, whereas only 7% of disease-associated SNPs localize in protein-coding exons, the remaining 93% arise in noncoding areas of the genome, of which 43% fall in intergenic regions . Considedystrophin (DMD) gene encoded on the X chromosome (Xp21.2) resulting in the lack of a functional dystrophin protein in skeletal muscle. With 79 exons and 2.4 Mb in size, DMD is the largest gene of our genome and up to 2,900 types of mutations have been reported in DMD patients so far [dystrophin is in part unknown and appears much more complex than previously thought. Moreover, the inter-individual variability in the severity and the disease progression is only partially explained by the types of mutations and the occurrence of female patients remains enigmatic. Therefore, the regulation of DMD is an area of intense research that in the last few years has led to the characterization of miRNA and lncRNA involvement [Duchenne muscular dystrophy (DMD) is the most common and severe myopathy affecting 1:3,500 males. It is inherited in an X-linked recessive manner but, in very rare cases, heterozygous females can be mildly affected . DMD is s so far ,117. Desolvement .DMD gene both in sense and antisense orientations were recently identified [dystrophin in at least one of the tissues that normally express it: skeletal muscle, heart and brain [in trans by downregulating the expression of specific dystrophin isoforms targeting their promoters  [Linc-MD1 is a muscle-specific lncRNA required to activate late stages of the myogenic program. Linc-MD1 is a cytoplasmic ceRNA that acts as a molecular sponge for miR-133 and miRNA-135  and mastermind-like protein 1 (MAML1), two transcription factors with an important role in muscle differentiation  . Linc-MDG and MHC. Linc-MD for DMD .DBE-T. DBE-T is mainly produced in FSHD patients and mediates the aberrant activation of the FSHD locus [DBE-T is a nuclear transcript that acts in cis as it remains associated with the chromatin of the FSHD locus  protein ASHL1 recruiting it to the FSHD locus where it mediates the accumulation of H3K36me2 [Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy 1:14,000). FSHD is transmitted in an autosomal dominant manner and affects both sexes but presents a gender bias, as males are usually more severely affected . FSHD di,000. FSHHD locus . DBE-T iH3K36me2 , a histoH3K36me2 -134. ConH3K36me2 .It is increasingly evident that the functional repertoire of metazoan genomes extends far beyond protein-coding genes. A growing body of genetic and biochemical work indicates that long noncoding RNAs are important members of the complex muscle regulatory network, being engaged in diverse activities crucial for myogenesis. However, there is still a substantial gap between the expanding list of muscle lncRNAs and the precise molecular tasks they fulfill in the control of muscle differentiation. Moreover, the functional characterization of lncRNAs in muscular dystrophy is still in its infancy. Nevertheless, there is little doubt that results from such studies will significantly contribute to the formulation of specific and complementary diagnostic and therapeutic strategies for muscle wasting diseases.core enhancer RNA; ChIP-seq: chromatin immunoprecipitation sequencing; DBE-T: D4Z4 binding element transcript; DMD: Duchenne muscular dystrophy; DMD: dystrophin gene; DRRRNA: distal regulatory region RNA; dsRNA: double-stranded RNA; eRNA: enhancer RNA; FSHD: facioscapulohumeral muscular dystrophy; Gtl2/Meg3: gene trap locus 2/maternally expressed gene 3; H3K27ac: acetylated histone H3 lysine 27; H3K4me1: monomethyl histone H3 lysine 4; IMP: Igf2 mRNA binding-protein; iPSC: induced pluripotent stem cell; IRES: internal ribosomal entry site; linc-MD1: long intergenic noncoding RNA-muscle differentiation 1; lincRNA: long intergenic noncoding RNA; lncRNA: long noncoding RNA; Malat1: metastasis associated lung adenocarcinoma transcript 1; MAML1: mastermind-like protein 1; MEF2: myocyte-specific enhancer factor-2; miRNA: microRNA; MRF4: myogenic regulatory factors; MyoD: myogenic differentiation; MyoG: myogenin; ncRNA: non protein-coding RNA; Nctc1: noncoding transcript 1; Neat1: nuclear enriched abundant transcript 1; NMD: nonsense-mediated mRNA decay; PcG: Polycomb group; piRNA: piwi-interacting RNA; PRC1 and PRC2: Polycomb Repressive Complex 1 and 2; RNAPII: RNA polymerase II; rRNA: ribosomal RNA; SINE: short interspersed element; siRNA: small interference RNA; SMD: Staufen-mediated mRNA decay; snoRNA: small nucleolar RNA; SNP: single nucleotide; snRNA: small nuclear RNA; SRA: steroid receptor RNA activator; SRAP: SRA protein; STAU1 and 2: Staufen 1 and 2; tRNA: transfer RNA; TrxG: Trithorax group; UTR: untranslated region; Yam: YY1-associated muscle; YY1: Yin Yang 1.ceRNA: competing endogenous RNA; CERNA: The authors declare that they have no competing interests.MVN: manuscript writing, final approval of the manuscript. MJ: manuscript writing, final approval of the manuscript. DG: manuscript writing, final approval of the manuscript. All authors read and approved the final manuscript.


Muscleblind-like 1 (MBNL1) is an alternative splicing factor containing four CCCH Zinc fingers (ZnFs). The sequestration of MBNL1 by expanded CUG and CCUG repeats is a major component in causing myotonic dystrophy. In addition to binding the structured expanded CUG and CCUG repeats; previous results suggested that MBNL1 binds single-stranded RNAs containing GC dinucleotides.We performed a systematic analysis of MBNL1 binding to single-stranded RNAs. These studies revealed that a single GC dinucleotide in poly-uridine is sufficient for MBNL1 binding and that a second GC dinucleotide confers higher affinity MBNL1 binding. However additional GC dinucleotides do not enhance RNA binding. We also showed that the RNA sequences adjacent to the GC dinucleotides play an important role in MBNL1 binding with the following preference: uridines >cytidines >adenosines >guanosines. For high affinity binding by MBNL1, the distance between the two GC dinucleotides can vary from 1 to 17 nucleotides.These results suggest that MBNL1 is highly flexible and able to adopt different conformations to recognize RNAs with varying sequence configurations. Although MBNL1 contains four ZnFs, only two ZnF - GC dinucleotide interactions are necessary for high affinity binding. Alternative pre-mRNA splicing significantly increases genome diversity with recent measurements suggesting that greater than 90% of genes undergo alternative splicing -3. The dMuscleblind-like 1 (MBNL1) is an alternative splicing factor that is associated with the disease myotonic dystrophy (DM). Patients with DM have a CTG or CCTG repeat expansion in an untranslated region of their genome. For DM type 1 (DM1), the CTG repeats are found in the 3' UTR of the DMPK gene, and for DM type 2 (DM2), the CCTG repeats are found within intron 1 of the CCHC-type zinc finger (ZnF), nucleic acid binding protein (CNBP) gene ,8. TheseSeveral MBNL1 binding sites have been identified, and a few of these have been characterized ,14-17. CTo better understand the RNA binding specificity of human MBNL1, we used a version of the protein that contains amino acids 2-260. Previous studies have shown that MBNL1 2-260, which contains all four ZnFs, binds RNA as well as the corresponding full-length protein . This veAccording to the crystal structure of the MBNL1 ZnFs3/4 - RNA complex , the pri4 RNA which was previously shown to be structured and bind MBNL1 ATP. RNAs were then purified on a 10% denaturing polyacrylamide gel (19:1) and eluted into TE Buffer . RNA was then ethanol precipitated with glycogen and resuspended in TE Buffer. Finally a Bio-spin 6 column (Bio-Rad) equilibrated in TE Buffer was used.All RNAs were synthesized by Integrated DNA Technologies and 5' end labeled with [γ-2. RNA was then incubated on ice for 30 minutes. The thermal denaturation analysis was performed on a Carey UV/VIS Spectrophotometer. The absorbance of the RNA was monitored at 260 nm while the temperature was increased from 15-90°C. Melting temperatures were determined by calculating the derivative of the absorbance versus temperature using the Cary program "Thermal". All RNAs were tested three times and Tm values compared. The Tm of all the RNAs used in these studies were measured at below 40°C while the (CUG)4 RNA was measured with a Tm of 60.6 +/- 0.6°C.1.5 μM RNA was incubated at 95°C for two minutes in 100 mM NaCl, 21 mM Tris 7.5, and 5 mM MgCl2, 10% glycerol, 0.02% bromophenol blue, and 0.02 nM RNA. The samples were incubated at room temperature for 30 minutes and then loaded onto a pre-chilled 6% polyacrylamide (19:1) gel running at 50 volts. Once the samples were loaded the voltage was increased to 150 volts and the gel was run for 30 minutes. After running, the gel was dried and exposed overnight.RNA was initially heated to 95 °C for two minutes and then placed directly onto ice. RNA was then mixed with MBNL1 at concentrations indicated in the figures. The final reaction conditions were 100 mM NaCl, 21 mM Tris pH 7.5, 0.1 mM EDTA, 2 mg/mL BSA, 0.1 mg/mL Heparin, 0.5 mM BME, 5 mM DTT, 0.02% Triton X-100, 5 mM MgCl1 = a fitting factor, m0 = nM protein concentration, and m2 = nM Kd.Quantitation of the binding curve was described previously . The datCompetition assays were run under the same conditions as the gel shift assays except that the protein and radioactive RNA were incubated for 20 minutes and then the competitive RNA was added to the reaction and allowed to equilibrate for 30 minutes.2 = competitive RNA Kd, m1 = Kd to radiolabeled RNA, Pt = total protein added in the reaction, and m0 = amount of competitive RNA. When the competitive RNA and the radiolabeled RNA were the same, the equation was simplified to 2 and m1 are the same.Quantitation of the competition assays was done using Kaleidagraph. The data was fit to the equation3D2N and 3D2S were used to create Figure PDB files DC performed most of the experiments, data analysis, and drafted the manuscript. RH, PB, and KJ performed many of the replicate binding assays. DPG performed the thermal melt analysis. JAB conceived of the project, analyzed data and edited the manuscript. All authors read and approved the final manuscript.


The emulsification of oil at the Deepwater Horizon (DWH) well head relegated a large proportion of resultant hydrocarbon plumes to the deep sea, facilitated the incorporation of oil droplets into microbial and planktonic food web, and limited the severity of direct, wetland oiling to coastal Louisiana. Nevertheless, many transient fish and invertebrate species rely on offshore surface waters for egg and larval transport before settling in coastal habitats, thereby potentially impacting the recruitment of transient species to coastal nursery habitats quite distant from the well site. We compared the utilization of salt-marsh habitats by transient and resident nekton before and after the DWH accident using data obtained from an oyster reef restoration project in coastal Alabama. Our sampling activities began in the summer preceding the DWH spill and continued almost two years following the accident. Overall, we did not find significant differences in the recruitment of marsh-associated resident and transient nekton in coastal Alabama following the DWH accident. Our results, therefore, provide little evidence for severe acute or persistent oil-induced impacts on organisms that complete their life cycle within the estuary and those that spent portions of their life history in potentially contaminated offshore surface waters prior to their recruitment to nearshore habitats. Our negative findings are consistent with other assessments of nekton in coastal vegetated habitats and bolster the notion that, despite the presence of localized hydrocarbon enrichments in coastal habitats outside of Louisiana the most severe oil impacts were relegated to coastal Louisiana and the deep sea. Analyzing all the information learned from this accident will undoubtedly provide a synthesis of what has or has not been affected in the Northern Gulf of Mexico, which when put in context with oil spill studies elsewhere should improve our ability to avert and manage the negative consequences of such accidents. The Deepwater Horizon oil flow began on April 20, 2010 and released an estimated 4.9 million barrels of crude oil into the Gulf of Mexico over a period of 84 days Coastal impacts from the DWH spill were most severe in the marshes of coastal Louisiana, resulting in the loss of fringing marsh grasses and enhanced shoreline erosion Callinectes sapidus) also exhibits a spring recruitment period, although peak recruitment occurs in the summer and fall Spring is a key spawning and recruitment period for many fishes and invertebrates, including commercially important penaeid shrimp and many finfish species, which can spend weeks in offshore surface waters as larvae An oyster-reef restoration project conducted from August 2009 to February 2012 in coastal Alabama provided a unique opportunity to assess the potential impacts on marsh-associated nekton assemblages before and after the DWH spill. Alabama beaches received weathered crude oil in June 2010, primarily in the form of sheens and tar balls Callinectes sapidus and penaeid shrimp) relative to estuarine species that complete their life cycles in coastal waters  accounted for 45.15% and 36.04% of the total catch, respectively, after removing P. pugio. All blue crabs and penaeid shrimp were juveniles or subadults, and all transient fishes fell within juvenile size classes.We collected 41,510 individual organisms representing more than 42 taxa in coastal Alabama between the summer of 2009 and winter of 2012. The resident daggerblade grass shrimp, P. pugio differed among years  but not between reef treatments , declined in the first post-spill year (2010), and returned to pre-spill levels in the second post-spill year (2011). Livebearer  abundances did not differ among years  but did differ among reef treatments , exhibiting a post-spill decline in 2010 followed by a return to pre-spill abundances in 2011. Goby abundance did not differ among reef treatments  or between reef treatments  did not differ among years , but significant differences were detected for C. sapidus and penaeids . Annual maxima of C. sapidus did not differ between 2009 and 2011, but maxima in both years were higher than in 2010. Penaeid annual maxima were higher in 2011 than in 2009 and 2010, with the annual maxima in these two latter years not differing significantly from each other.Whenever we did not find significant differences among the yearly means, we tested for differences in peak annual recruitment by comparing sample dates with the highest mean abundances among years. Annual maxima for livebearers and blackcheek tonguefish  but not between reef treatments  or between reef treatments  or reef treatments , but significant variability was detected for transient taxa (p = 0.013). Namely, transient maximum abundance was higher in 2011 than in 2010 although no other significant differences were found between years.Total nekton abundance differed significantly among years  and gobies , but not livebearers . Interannual biomass maxima did not differ significantly among years for livebearers, C. sapidus, penaeids or S. plagiusa .Significant among-year variability in biomass was detected for the residents 2,120 = 1.31; p = 0.298) or between reef treatments .Total nekton biomass did not differ significantly among years  for biomass) and between reef treatments  ordination did not change among years  was present along most of the shoreline and, given the narrow tidal range of the area, constituted the primary habitat of the low intertidal. The adjacent, subtidal mudflat consisted of a patchwork of bare mud, wigeon grass (Ruppia martima), and shoalgrass .We sampled marsh-associated nekton within a fringing salt-marsh on the northeast shore of Point-aux-Pins, AL . A contiFour experimental blocks, each containing one reef and one control plot, were established along 1.2-km of marsh-dominated shoreline. The placement of reef and control plots were randomized within each block, and reefs were constructed of loose oyster shell and reinforcing metal fence. Each reef was composed of three, 25-m long reef modules to produce a structure enveloping 65 m of shoreline. Reef and control plots were separated by 75 m and blocks were separated by a minimum of 100 m. Reefs were located 111 m offshore so that the height of the reef crest coincided with mean low water to facilitate recruitment and growth of oysters.n = 8 nets per sampling period) during low water and left in place for 24 hr. The counts obtained in the traps are indicative of the abundance of organisms that utilized 6 m of linear marsh fringe during a single tidal cycle.Tidal fyke nets were used to sample marsh-associated nekton during flood tides. Each net was constructed of seine netting with 0.64-cm mesh, and enveloped 6 m of fringing marsh and stood 0.75 m tall. The back edge of the net extended 3.25 m seaward from the vegetation edge and all organisms were trapped into a 1 m×1 m seine bag staked to the adjacent flat. The traps were designed to permit lateral movement of organisms within the marsh vegetation as the marsh surface flooded and to capture all organisms as they exit the marsh vegetation on the subsequent falling tide . This study was carried out in accordance with the recommendations in the Guide for the care and Use of Laboratory Animals of the National Institutes of Health. All necessary permits were obtained for the described field studies by the Alabama Department of Conservation and Natural Resources and our sampling protocol was approved by the United States Army Corp of Engineers (Permit Number SAM-2009-00203-JEB).Samples were collected once every one to three months for three years. Most of the studied organisms recruit to the marsh from the open waters of the Gulf, or return to the intertidal zone from deeper estuarine habitats, during the early spring months  among years and between reef treatments. Sampling date was nested within year to account for within-year  variability. Year and reef treatment were defined as fixed factors and sampling date and block were defined as random factors. Separate analyses were conducted for transient and resident organisms. Data transformations were applied as necessary to meet the assumptions of parametric statistics.Palaemonetes pugio, accounted for 84.9% of our total catch and were highly variable among sampling days. Seven species or groups of species contributed >1% to the total catch (residents and transients) when P. pugio was removed from the data set (Table S1 in P. pugio): Callinectes sapidus (45.15%), penaeid shrimp (36.04%), and the blackcheeck tonguefish, Symphurus plagiusa (1.16%); and for the following groups of residents: Palaemonetes pugio, livebearers (7.63%) and gobies (4.56%). Livebearers, which included members of the families fundulidae, poeciliidae and cyprinodontidae, were pooled to assess potential declines in abundance resulting from reproductive and genetic impacts, which have been reported by Whitehead et al. Mugil curema, constituted 4.85% of the total catch, but were not considered for among-year comparisons since 79.9% of all specimens caught were from a single trap in August 2009.We focused our analysis on numerically dominant taxa. Catches of the resident grass shrimp, P. pugio) were compared among years and between reef treatments. We then compared the abundance and biomass of transient and resident species with grass shrimp removed from the catch data to discern variability in habitat use by other species that might otherwise have been masked by the disproportionate contribution made by P. pugio. Separate analyses were performed for transient and resident communities.Total measures of community abundance and biomass  on a fourth-root transformed Bray-Curtis similarity matrix of species abundance and biomass. Multidimensional scaling (MDS) ordination was used to visualize differences among factor levels using PRIMER v6.1.5.File S1Supporting Tables S1–S7.(DOCX)Click here for additional data file.


Structural change in both the habitat and reef-associated fish assemblages within spatially managed coral reefs can provide key insights into the benefits and limitations of Marine Protected Areas (MPAs). While MPA zoning effects on particular target species are well reported, we are yet to fully resolve the various affects of spatial management on the structure of coral reef communities over decadal time scales. Here, we document mixed affects of MPA zoning on fish density, biomass and species richness over the 21 years since establishment of the Saba Marine Park (SMP). Although we found significantly greater biomass and species richness of reef-associated fishes within shallow habitats (5 meters depth) closed to fishing, this did not hold for deeper (15 m) habitats, and there was a widespread decline (38% decrease) in live hard coral cover and a 68% loss of carnivorous reef fishes across all zones of the SMP from the 1990s to 2008. Given the importance of live coral for the maintenance and replenishment of reef fishes, and the likely role of chronic disturbance in driving coral decline across the region, we explore how local spatial management can help protect coral reef ecosystems within the context of large-scale environmental pressures and disturbances outside the purview of local MPA management. Marine Protected Areas (MPAs) are often used as a spatial management tool to balance harvesting pressures against the need to conserve biodiversity and maintain key ecosystem processes Effects of spatial management on coral reef ecosystems can often take considerable time to be fully realised. For species targeted by fishing, up to six-fold increases in fish density and biomass have been documented within no-take areas, but in many cases this has taken 10 or more years to occur Understanding the critical role that certain reef fishes play in ecosystem function and how they respond to changing habitat quality can provide key insights into the causes and consequences of long-term change in coral reef communities In this study we examined the structure of a coral reef community following 21 years since the establishment of an isolated Caribbean MPA on Saba Island, Netherland Antilles. Combining published data with new recordings via the same methodology in a meta-analysis, our aims were threefold: (1) examine whether spatial protection has had an overall effect on the coral reef community since inception of the SMP, (2) explore temporal consistency in the affects of zoning (if any) on various components of the SMP coral reef community, and (3) examine the present distribution and abundance of herbivorous and carnivorous fish families and species across zones open and closed to fishing. In our discussion, we explore both the benefits and limitations of MPA-based local management of coral reefs in this region, and highlight the importance of effective monitoring for identifying and acting on coral reef vulnerability.2, with a no-take area of 4.29 km2 (approximately 33% of the SMP) Underwater visual censuses (UVCs) of fish abundance and habitat variables were conducted within the Saba Marine Park (SMP) surrounding Saba island , Netherland Antilles  using nob described by Bonsack and Harper 2) following Higgins and Thompson 2 and large χ2) suggests that mean differences varied more across years than expected by random error alone (i.e. there was significant change in the magnitude and/or direction of the mean difference among zones from survey to survey). Further graphical examination of temporal trends in the density and biomass of “carnivorous” and “herbivorous” fishes (by merging family-level data according to the prior classification of Roberts 10(x+1) transformed to minimize departures from normality and homoscedasticity. Statistical analyses and presentations were made with SPSS , RevMan  and Sigmaplot .UVCs were made with the stationary point-count method  and temporal trends suggesting changes have occurred among survey years , Fig. 3.us 15 m,  and Sparide 5 m, , and theum 15 m,  and H. fum 15 m, . By contlineatum , carnivois fulva  and piscmahogoni .Despite effective spatial protection of the Saba Marine Park (SMP) by local managers for 21 years, we found no significant difference in overall fish density between open (fished) and closed (no-take) zones. While we did find marginally higher biomass of certain fish species in zones closed to fishing, this was generally restricted to shallow habitats and was tempered by an apparent decline in live hard coral cover between 1991–1995 and 2008. Notably, we found historically low carnivorous fish density across all zones in 2008, which was offset by marginal increases in their biomass, and slight increases in the density and biomass of herbivorous fishes. Such shifts in the habitat composition and trophic structure of the coral reef communities around Saba are cause for concern, as even subtle changes in community composition may have significant ecological consequences, and may indicate altered ecosystem resistance and resilience Evaluating whether spatial management of coral reefs is effective in meeting the intended goals of an MPA must be done in the context of time since establishment and zoning compliance. Overfishing has been a key explanation for regional declines in carnivorous fishes on Caribbean coral reefs with and without MPAs Shifts in the availability of live coral habitat could be a major driver of change in the reef-associated fish communities of Saba. Historical losses of live hard coral cover, such as the 38% decrease from 1995 to 2008 recorded for Saba, have been documented for coral reefs throughout the world and have often been attributed to factors such as thermal bleaching, hurricanes, pests and disease e.g. . Saba anSparisoma viride, which plays a pivotal role in the bioerosion and sculpturing of reefs to facilitate the removal of dead coral skeleton and prime the reef for new coral recruits Sparisoma viride, to bolster reef resistance to regime shifts in the face of large-scale disturbances.Losses in live coral alongside changes in fish community structure can have serious consequences for the health of coral reefs and their capacity to resist and rebound from disturbance events. Around Saba, we found 68% less carnivorous fish across all sites and zones of the SMP in 2008 relative to the 1990s, offset by only marginal increases in their biomass, and slight increases in herbivorous fish across the same period. There was no clear evidence that zoning has played a role in these trophic-level trends, as we found that all families tended to display greater biomass in closed zones across all survey years, with relatively light variability across survey years. The notable exceptions, however, was that in 2008 a substantially greater biomass of herbivorous scarids were recorded in closed zones, while there was markedly lower biomass of carnivorous haemulids. Such dynamic shifts in community trophic structure can produce wider ecosystem effects through trophic cascades, such as the urchin overgrazing of the benthos on reefs bereft of predatory fishes e.g. , which eLong-term change within the coral reef communities of Saba and other parts of the Caribbean highlight the effective scope and limitations of local-scale spatial management, and point to the need for targeted strategies that bolster coral reefs against large-scale threats


Living reef fishes are one of the most diverse vertebrate assemblages on Earth. Despite its prominence and ecological importance, the origins and assembly of the reef fish fauna is poorly described. A patchy fossil record suggests that the major colonization of reef habitats must have occurred in the Late Cretaceous and early Palaeogene, with the earliest known modern fossil coral reef fish assemblage dated to 50 Ma. Using a phylogenetic approach, we analysed the early evolutionary dynamics of modern reef fishes. We find that reef lineages successively colonized reef habitats throughout the Late Cretaceous and early Palaeogene. Two waves of invasion were accompanied by increasing morphological convergence: one in the Late Cretaceous from 90 to 72 Ma and the other immediately following the end-Cretaceous mass extinction. The surge in reef invasions after the Cretaceous–Palaeogene boundary continued for 10 Myr, after which the pace of transitions to reef habitats slowed. Combined, these patterns match a classic niche-filling scenario: early transitions to reefs were made rapidly by morphologically distinct lineages and were followed by a decrease in the rate of invasions and eventual saturation of morphospace. Major alterations in reef composition, distribution and abundance, along with shifts in climate and oceanic currents, occurred during the Late Cretaceous and early Palaeogene interval. A causal mechanism between these changes and concurrent episodes of reef invasion remains obscure, but what is clear is that the broad framework of the modern reef fish fauna was in place within 10 Myr of the end-Cretaceous extinction. The endbivalves ,14: rudibivalves . Althougbivalves , coral dbivalves  and corabivalves ,16. A subivalves  during teef type  and spreeef type .ca 100 Ma), are known from a variety of depositional settings, some of which are interpreted as being located proximal to well-developed benthic communities, although perhaps not true reefs [ca 50 Ma) contains eight of the 10 most common extant coral reef families [The historical influence of changing reef composition and the K–Pg extinction upon the assembly of the reef fish fauna has been a topic of considerable speculation but remains uncertain. Reef systems are known drivers of diversification and net exporters of biodiversity to other marine habitats ; severalue reefs . The earfamilies , all of families ,25 have families , and whefamilies . Indeed,families  and earlfamilies  have beefamilies . Most exfamilies  and it ifamilies .Figure Apart from these broad and often speculative outlines, key details of the evolutionary assembly of the unique reef fish fauna remain unknown. In the absence of a dense fossil record of reef fishes, we used a recent time-calibrated phylogeny of all major lineages of living acanthomorphs  in order2.(a)We generated a morphological dataset of 12 traits that summarize body shape and size and which reflect several prominent functional properties of the feeding and locomotor systems. Measurements were made on adults of one species from 191 of the 228 acanthomorphs families in the phylogeny, each exemplar species was carefully selected to represent the most common head–body shape and size within the family. Ten linear traits were measured in millimetres with hand-held dial callipers from three adult specimens of each species, including standard length, lower jaw length, length of the dentigerous arm of the premaxilla, length of the ascending process of the premaxilla, diameter of the eye, maximum body depth, maximum body width, depth of the caudal peduncle, span of the caudal fin, length of the dorsal fin, along with body mass weighed to the nearest 0.01 g on a scale. Finally, suction index, a metric of the relative capacity to generate suction pressure during suction feeding ,33 was cishBase [We used a binary habitat variable: ‘Reef’ (1) or ‘Non-Reef’ (0). To avoid an arbitrary threshold for assigning a habitat to each family, we used the percentage of extant species in each family that are reef-associated as given by FishBase   generated by Near et al. . Brieflyet al. ) with anet al. ,36. AnalWithin these trees, taxonomic sampling is sparse at the species-level as they contain approximately 3% of extant acanthomorph species. However, family-level sampling is far more complete, including 228 out of a total of 322 extant acanthomorph families . We ther(b)α = 1 and κ = 101) and a branch length prior on the rate parameter, to generate a total of 50 000 stochastically mapped trees. For each of the 100 habitat datasets, we sampled 50 trees from the BPDT and generated 10 stochastic character maps on each tree . We wrote an R script [figure 2a and non-reef in figure 2b. For further details, see the electronic supplementary material, Results and methods.To estimate the number of transitions to reef-living through time, we used stochastic character mapping  in SIMMAR script  to conve(c)Phylogenetic clustering can be inferred from our SIMMAP analyses which compare the empirical number of transitions in our data to a null generated by shuffling the data on the tips of the tree (sometimes referred to as a non-phylogenetic null). This is the test for phylogenetic inertia in discrete data suggested by Maddison & Slatkin  using st(d)et al. [We sampled 10 trees from the BPDT for each of the 100 habitat datasets and pruned the trees into reef and non-reef species. With the morphological PCA axes, we estimated DTT on the 1000 reef and non-reef trees separately using the methods of Harmon et al.  implemenet al. . This meet al.  implemen3.(a)a). Additionally, there is no evidence of elevated rates of taxonomic diversification in acanthomorphs following the K–Pg [b).The stochastic character maps show that reefs were successively invaded by multiple acanthomorph lineages and indicate two clear periods of elevated reef transitions. The first in the Late Cretaceous from 90 to 72 Ma and the second immediately following the K–Pg extinction (65–56 Myr ago) a. Additithe K–Pg . Beginnithe K–Pg . For the(b)a) [p-values confirming that reef lineages are more closely related than expected by chance . By contrast, transitions away from reefs follow the null expectation suggesting that the lineages which move away from reefs are not phylogenetically clustered (figure 3b).In total, we infer about 50 independent transitions to reef-dwelling generated the acanthomorph reef fish fauna we see today , confirmfigure 3a) , indicat(c)b) and non-reef (c) lineages show different patterns of morphospace occupation through time. Early in the evolutionary history of acanthomorphs, non-reef lineages exhibit substantial phenotypic overlap, but lineages that invaded reef habitats early occupy distinct regions of morphospace. Beginning in the Late Cretaceous (ca 85 Ma), there is a shift to greater morphological convergence within sub-clades in both reef and non-reef lineages. By 80 Ma, the Brownian motion model is rejected for non-reef lineages as all results exceed the 97.5th percentile, indicating that all independent non-reef lineages occupy similar regions of morphospace. By contrast, reef lineages do not start to exhibit such strong morphological convergence until the early Eocene (50 Ma), well after the K–Pg mass extinction.High average sub-clade disparity relative to the null indicates that reef lineages independently converge on similar morphologies, while low average sub-clade disparity indicates that lineages occupy distinct regions of morphospace. We find that reef b and nonnon-reef c lineage4.b). However, we stress that these results are at the family-level, most reef families contain both reef and non-reef species, so transitions on and off reefs will have been far more dynamic over the history of acanthomorphs than we can infer. Reefs have clearly been important drivers of both lineage and morphological diversification within fishes [Our results reveal that reef lineages of acanthomorphs successively colonized reef habitats throughout the Late Cretaceous and early Palaeogene. Thus, in contrast to marine benthic invertebrate genera , reefs afigure 3n fishes ,20,47 anc) and the number of reef transitions begins to plateau. For the last 30 Myr, we estimate far fewer transitions to reef habitats than expected under the null model (a). This sequence of events indicates that early transitions to reefs were by phylogenetically over-dispersed lineages but from 30 Ma onwards, reef lineages are phylogenetically clustered. As most modern acanthomorph families had evolved by this time [a) and the NRI results.There is remarkable congruence between the temporal pattern of changes in the number of transitions onto reefs, morphospace occupation by reef lineages and reef history. Around 90 Ma, the number of transitions to reef habitats first exceeds the null expectation and a little later (approx. 85 Ma) morphospace occupation in reef lineages shifts from sub-clades occupying primarily distinct areas to overlapping regions. Later, following the K–Pg mass extinction, we identify a second rapid rise in the number of reef transitions accompanied by a move towards even greater overlap in morphospace occupation by reef sub-clades. By approximately 50 Ma, reef lineages occupied entirely convergent areas of morphospace c and thell model a. This shis time ,25 this Our pattern of early reef colonization by morphologically distinct lineages, later pulses of invasion accompanied by increasingly convergent morphospace occupation culminating with morphospace saturation and a plateau in reef invasions, is consistent with a macroevolutionary niche-filling scenario . Niche-fca 93 Ma) [The two waves of niche filling on either side of the K–Pg boundary coincide with, and might be linked to, changes in reef structure and climate. The Late Cretaceous is a ‘greenhouse’ interval lacking permanent icecaps at the poles . Elevatea 93 Ma)  was folla 93 Ma) . It is pa 93 Ma)  and refeca 50 Ma) suggests the saturation of ecomorphological niches on reefs and the origination of most functional groups within reef-dwelling acanthomorphs. The onset of ecomorphological exhaustion corresponds in time to the exceptional palaeontological window provided by the Bolca Lagerstätte, which confirms the existence of an ecologically diverse reef fish fauna including the earliest herbivorous acanthomorphs [The second wave of colonization following the K–Pg mass extinction is associated with the recovery and expansion of scleractinian coral reefs in the early Palaeocene ,30. Corahomorphs ,51.Reef fishes are one of the most diverse vertebrate assemblages on Earth, and their complex evolutionary history is rapidly being illuminated by improved phylogenetic resolution and continued palaeontological efforts. The correspondence between our phylogenetically inferred results and the existing fossil record suggests that the dynamic history of reefs had a profound impact on the origin and early assembly of the modern reef fish fauna. The colonization of reefs by the ancestors of modern reef fishes may have been promoted by ecological opportunity associated with unstable Late Cretaceous reefs that was amplified by the rapid rise of structurally complex scleractinian coral reefs after the K–Pg mass extinction. The influence of reefs may continue into the present as previous work has found high rates of speciation  and morp


As the orthologue of HSV-1 VP22, ORF9 is believed to be a major constituent of the VZV virion tegument. HSV-1 VP22 has been extensively studied; however, the functional properties of ORF9 are less well understood, despite the fact that its transcript is the most abundant viral message expressed during VZV lytic infection. As an important step toward understanding the detailed functions of ORF9 Transient expression of ORF9 fused to enhanced yellow fluorescent protein (EYFP) in COS-7 cells showed the predominantly cytoplasmic localization in the absence of other viral proteins. Time-lapse examination of ORF9-EYFP demonstrated that the ORF9 was found to possess a pronounced cytoplasmic stage early post transfection, followed by translocation to the nucleus at a later time, which was consistent with the fact that VP22 is predominantly localized in the cytoplasm early in infection and accumulates in the nucleus late in infection, and the nuclear targeting of VP22 is independent of other viral factors.By sequence analysis and constructing a series of deletion derivatives of ORF9 fused to EYFP and fluorescence microscopy analysis in live cells, a bona fide bipartite NLS of ORF9 was for the first time determined and mapped to amino acids (aa) 16 to 32 (RRKTTPSYSGQYRTARR), which containing two arginine-rich motifs RRK and RTARR.Additionally, the NES was identified to locate between the leucine-rich region at amino acids 103 to 117 (LRHELVEDAVYENPL). Besides, ORF9 protein was demonstrated to target to the cytoplasm through the functional NES by Ran and chromosomal region maintenance 1 (CRM1)-dependent pathway, and to the nucleus through importin β-dependent mechanism that does not require importin α5.


LysCUU into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNALysCUU and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms.In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNA Mitochondria are essential organelles of almost all eukaryotic cells and take part in several critical cellular processes. They contain their own genome and perform transcription and translation of their genetic material. However, the vast majority of biological macromolecules found in mitochondria are imported from the cytosol. For instance, the total number of mitochondrial protein species is about 850–900 whereas the mitochondrial genome codes for only 8 proteins in yeast and 13 ones in human cells, so all other proteins are imported from the cytosol. The mechanisms of protein import into mitochondria are described in detail and appear as universal for all eukaryotes Saccharomyces cerevisiae, the cytosolic tRNALysCUU (further referred to as tRK1) is transcribed from a nuclear gene and then unequally redistributed between the cytosol (97–98%) and mitochondria (2–3%) LysUUU becomes undermodified at the wobble position of the anticodon and loses its capacity to recognize the lysine AAG codon in vitro and in vivo was shown to depend on the cytosolic precursor of mitochondrial lysyl-tRNA synthetase (preMSK1p), which serves as a carrier in-gel FRET approach permitted to demonstrate that binding to the protein factors and the subsequent RNA import require formation of an alternative structure, different from the classic L-form tRNA model. In the complex with Eno2p, tRK1 adopts a particular conformation characterized by bringing together the 3′-end and the TΨC loop and forming a structure referred to as F-hairpin , we first analysed the interaction of the recombinant preKARS2 with a T7-transcript of tRK1 by EMSA  in the mitochondrial import of the yeast cytosolic tRNAA, B) and characterized by a high efficiency of mitochondrial targeting dK of 400+/−50 nM, indicating a lower but still important affinity to preKARS2 , thus indicating their import into the mitochondria. The amount of the imported RNA was determined by comparison of the band density of the protected full-size RNA isolated from the mitoplasts after the import assay with that of an aliquot of the input labelled RNA, as shown in in vivo situation in yeast Purified human mitochondria were not able to internalize the external tRK1 in the absence of protein factors Fig. 3. in vitro as the rabbit one (not shown).The amount of the imported RNA increased upon addition of rabbit enolase to the import mixture in combination with preKARS2, however, the effect of enolase was dependent on the RNA structure. tRK1 was very poorly imported with preKARS2 alone but its import has been significantly improved upon addition of either rabbit or yeast enolase Fig. 3A,In contrast to the situation with tRK1, the level of mitochondrial import of the FD-L and FD-H RNA molecules was rather high in the presence of preKARS2 alone and has only been slightly improved upon rabbit enolase addition Fig. 3B.As expected, the truncated RNA molecules HF and HD, which cannot interact with preKARS2, have not been directed into human mitochondria by this protein, independently of the presence of rabbit enolase Fig. 3C.in vitro and in vivo import requirements, the role of preKARS2 in the mitochondrial RNA targeting was studied in cultured human cells. For this, we used the in vivo import assay on the cells transfected with RNA molecules, as described 6 RNA molecules per cell, which number is in the range of most abundant cellular RNAs, for example, 5S rRNA, estimated previously as 3.6±0.5×106 RNA molecules per cell 4 RNA molecules per cell, giving 2.5±0.3% of the molecules imported into mitochondria from the cellular pool, which perfectly correlates with our in vitro data.To compare the We observed a clear difference in the mitochondrial RNA import between the control and preKARS2-downregulated cells: the tRK1 import decreased 2-fold, and a 2.5-3-fold reduction was observed for the small artificial FD-L and FD-H RNAs import Fig. 4B.To confirm the role of preKARS2 as a mitochondrial targeting factor for tRK1 and its derivatives, we tested the RNA mitochondrial import in cells overexpressing preKARS2. For this, we used HeLa Tet-Off cells transiently transfected with a plasmid expressing preKARS2 . In 48 h after transfection, a 2- to 3-fold increase of the preKARS2 protein amount in the cell extract was detected Fig.4C,All the data presented above clearly indicate the role of the human mitochondrial lysyl-tRNA synthetase preKARS2 in the mitochondrial targeting of yeast tRK1 and the artificial RNA molecules containing two structural elements of the tRK1 alternative “import-active” fold, the D-arm and the F-hairpin.invitro, were internalized by mitochondria in vivo  folding of the short truncated RNA molecules. Nevertheless, the same RNAs internalized by cells, were able to be folded and imported into mitochondria.Surprisingly, the small artificial RNA molecules containing either the D-arm or the F-hairpin . This control RNA was not able to interact with the recombinant preKARS2 and to be imported into isolated human mitochondria in the presence of the purified proteins, preKARS2 and rabbit enolase . Contrary to HD and HF RNAs, the control RNA was not detected in mitochondria of transfected HepG2 cells  but unrelated to yeast tRK1 and containing a short G-C stem and a long unstructured loop Fig. 5C). In agreement with these data, the in vivo import of these RNAs was not dependent on preKARS2, since no change in the amount of the RNA molecules transported into mitochondria was observed when preKARS2 had been transiently downregulated or overexpressed .LysCUU into human mitochondria, performed in the present study demonstrated a similarity between the tRK1 import mechanisms in yeast and human cells. In yeast cells, preMSK1p and Eno2p were identified as the tRK1 mitochondrial targeting factors in vitro and in vivo, in a manner clearly dependent on the preKARS2 protein  in yeast MSK1 and can additionally facilitate the import of tRK1 into isolated yeast mitochondria in vivo.Aminoacyl-tRNA-synthetases is a group of enzymes responsible for the specific attachment of amino acids to their cognate tRNAs, thus performing a key step of translation  Lys into mitochondria had been observed Lys3 or the importable tRK1. This is in agreement with our hypothesis that only the alternative fold of tRNA can be recognized by the precursor of mitochondrial lysyl-tRNA-synthetase functioning as an RNA mitochondrial carrier. Thus, only yeast tRK1 and some specially designed RNA molecules capable to adopt the alternative conformation can interact with preKARS2 and be targeted into human mitochondria.Recently and rather surprisingly, the mature mitochondrial enzyme KARS2 was shown to interact with the human cytosolic tRNAIn general, each known case of RNA mitochondrial import appears somewhat special and thus not sufficient to establish a common RNA import mechanism Firstly, to be imported into mitochondria, an RNA should escape from the cytosolic channelling. According to this model, no free diffusion of macromolecules inside the cell is normally possible since all its components are well arranged in space and their movements are strictly regularized (channelled). Channelling was studied in detail on the example of tRNAs The next step of the pathway is a rapid discharge of the chaperone by another mitochondrial import factor. Examples of such a cascade were described in the yeast import mechanism where tRK1 is quickly transferred from enolase to the precursor of lysyl-tRNA synthetase E. coli, enolase is an integral component of the RNA degradosome; in yeast, it was identified as Hsp48 and participates in formation of vacuoles; enolase is found in the eye lens of many organisms and as a plasminogen-binding receptor expressed on the surface of a variety of eukaryotic cells. Thus, the tRNA import into mitochondria seems to be one of many different functions of this enzyme. The mitochondrial enzyme rhodanese is less studied, in fact, even its function is still not clear. We can hypothesize that this protein may also have multiple functions which can be switched by its cellular re-localization.Thus, for all RNA import systems in which the pre-mitochondrial (targeting) step of RNA import has been investigated, several universally present features can be outlined. Namely, in order to direct a cytosolic RNA to mitochondria one needs necessarily two protein factors, the first with a chaperone activity to withdraw the RNA from the cytosolic channeling, the second possessing the signal of mitochondrial localisation to target the RNA into the mitochondria. One of these proteins should be cognate, interacting with the imported RNA in a specific way and thus determining the selectivity of the RNA import . The other protein factor may be unrelated to RNA metabolism and hardly expected to participate in RNA transport, performing thereafter a “second job”, as enolase and rhodanese. Concerning enolase, many non-glycolytic “moonlighting” functions of this protein are known .The general rules of RNA import formulated above presume certain flexibility of the pathway. Indeed, various RNA molecules able to interact with import factors can be targeted into mitochondria even in organisms naturally importing only a very restricted number of RNA species, as we see here for the RNAs FD-H and FD-L. On the other hand, various proteins might perform the function of RNA import factors in certain conditions. This possibility is clearly demonstrated in the present work since the short truncated RNA molecules HD and HF, which have lost the capacity to interact with preKARS2, apparently can be targeted into human mitochondria with the help of other, still unidentified protein(s). This hypothesis is also in agreement with a recent publication claiming that preMSK1p may be dispensable for the tRK1 import into yeast mitochondria Recently, a subset of microRNAs, small non-coding RNAs that associate with Argonaute proteins to regulate gene expression at the post-transcriptional level, has been localized to human mitochondria Plasmid pDEST17 expressing human mitochondrial lysyl-tRNA synthetase (KARS2) was kindly provided by M. Sissler . To produce the precursor of mitochondrial KARS2 (preKARS2) protein, the Quick change mutagenesis kit (Stratagene) was used to insert the mitochondrial targeting sequence at the N-terminus of the mitochondrial KARS2 protein. A thrombin cleavage site and a 6× histidine tag at the N-terminus were deleted and a 6× histidine tag was inserted at the C-terminus using the same approach. For this, the following oligonucleotides were used:5′GCCACGCGGTTCTTTGACGCAAGCTGCTGTAAGGCTTGTTAGGGGGTCCCTGCGCAAAACCTCCTGGGCAG 3′5′CTGCCCAGGAGGTTTTGCGCAGGGACCCCCTAACAAGCCTTACAGCAGCTTGCGTCAAAGAACCGCGTGGC 3′5′ CTTTAAGAAGGAGATATACATATGTTGACGCAAGCTGCTGTAAGG3′CCTTACAGCAGCTTGCGTCAACATATGTATATCTCCTTCTTAAAG 3′5′ CAACAGTTGGCAGTTCTGTCCACCATCACCATCACCATTGAGACCCAGCTTTCTTGTAC 3′5′ GTACAAGAAAGCTGGGTCTCAATGGTGATGGTGATGGTGGACAGAACTGCCAACTGTTG 3′5′ pTRE2hyg plasmid expressing preKARS2 and antibodies directed against the residues 25 to 42 of the preKARS2 protein described in Escherichia coli strain BL21 codon plus (DE3)-RIL cells (Stratagene) were transformed with the pDEST17 plasmid. The transformed cells were grown in 500 ml of LB medium to a cell density corresponding to OD600 = 0.6, then the protein expression was induced for 2 h at 37°C by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to the bacterial culture. The cells were harvested by centrifugation at 6000 g for 10 min, lysed with 1 mg/ml of lysozyme on ice for 30 min and then sonicated thrice for 20 sec in the buffer consisting of 50 mM NaH2PO4, 300 mM NaCl and 20 mM imidazole. The cell lysate was centrifuged at 10,000 g for 15 min and the pellet was solubilized in the denaturing buffer consisting of 100 mM Tris-HCl (pH 8), 100 mM NaH2PO4, 10 mM imidazole and 8 M urea. This was followed by centrifugation at 12000 g for 15 min and the supernatant was applied to a Ni-NTA column (Qiagen) for 2 h at 4°C. After binding, the column was washed three times with the denaturing buffer containing 20 mM imidazole to eliminate weakly bound bacterial proteins. The recombinant preKARS2 protein was eluted from the column with 200 mM imidazole, refolded by stepwise elimination of urea and finally dialyzed against 50 mM Tris-HCl (pH 8), 300 mM NaCl and 40% glycerol and stored at –20°C. The purity of the protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining.To obtain the recombinant KARS2 and preKARS2 proteins, The recombinant yeast enolase Eno2p was isolated as described previously dG), the Mfold program To predict secondary structures of recombinant RNA molecules and estimate their free energies (The yeast tRK1 T7-transcript was obtained as described TAATACGACTCACTATAGCGCAATCGGTAGCGCCTCTTTACAGTGCTTAGTTCTCGAGCCCCCTACAGGGCTCTTFD-L TAATACGACTCACTATAGCGCAATCGGTAGCGCAGTAAGCACTGTAAATGAGCCCCCTACAGGGCTCTTFD-H TAATACGACTCACTATAGCGCAATCGGTAGCGCCTCTTTACAGTGCTTAGTTCTCHD: TAATACGACTCACTATAGGTCTTTACAGTGCTTACTTCTCGAGCCCCCTACAGGGCTCCAHF: in vitro using the Ribomax kit (Promega). Following transcription, the DNA template was removed by digestion with RQ1 RNase-Free DNase (Promega). RNAs were purified by 12% PAGE with 8 M urea and eluted from the gel with the RNA extraction buffer containing 0.5 M CH3COONH4, 10 mM Mg(CH3COO)2, 0.1 mM EDTA and 0.1% SDS. The eluted RNA was precipitated with ethanol.RNA transcripts were obtained 32P-ATP using T4 polynucleotide kinase (Promega). The labeled RNA was denatured at 100°C and then slowly cooled down to the room temperature. For RNA binding assays, the appropriate amount of protein and labeled RNA were mixed in 20 µl of the buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5 mM DTT, 10% glycerol, 0.1 mg/ml BSA and incubated at 30°C for 15 min. The mixture was separated by native 8% PAGE in 0.5×Tris-borate buffer (pH 8.3) and 5% glycerol Purified RNA was dephosphorylated with alkaline phosphatase (Boehringer Mannheim) and labeled at the 5′-end with γ-2, 0,1% Nonidet P40, pH7.5, at 4°C for 1 h with stirring, then washed several times with the same solution and blocked in 10 mM Tris-HCl, pH7.5, 5 mM Mg(CH3COO)2, 2 mM dithiothreitol, 2% BSA, 0,01% Triton X-100 for 5 min at 25°C. Then, the membrane was incubated for 2 h at 4°C in the import buffer without sorbitol, containing 1 nM [32P]-labelled RNA, as in Recombinant preKARS2 was loaded on 10% SDS-PAAG and blotted to nitocellulose membrane. The membrane was incubated in 0,1 M Tris-HCl, 20 mM KCl, 2,5 mM MgClin vitro import assay into isolated mitochondria was performed as in 2, 5 mM DDT and 2 mM ATP. For a standard in vitro assay, we add 3 pmoles of labelled RNA per 0.1 ml of the reaction mixture containing 0.1 mg of mitochondria . This corresponds to the 100% RNA input. After incubation for 15 min at 34°C, 50 µg/ml of RNase A (Sigma) was added and the reaction was incubated for additional 15 min to digest all unimported RNA. The mitochondria were washed three times with the buffer containing 0.6 M sorbitol, 10 mM HEPES-KOH (pH 6.7) and 4 mM EDTA, then resuspended in 100 µl of the same buffer and treated with an equal volume of 0.2% digitonin (Sigma) solution to disrupt the mitochondrial outer membrane, followed by purification of mitoplasts. The mitoplast pellet was resuspended in the solution containing 100 mM 3COONa, 10 mM MgCl2, 1% SDS and 0.05% diethylpyrocarbonate (DEPC), boiled for 1 min and RNA was extracted at 50°C with water-saturated phenol. RNA was precipitated with ethanol andCH separated by 12% PAGE containing 8 M urea, followed by quantification with the Typhoon-Trio scanner using the Image Quant-Tools software . The amount of the imported RNA was determined by comparison of the band density of the protected full-sized RNA isolated from the mitoplasts after the import assay with an aliquot (2–5%) of the RNA input.Mitochondria were isolated and verified for intactness as described 2.HeLa Tet-Off cells stably expressing the tetracycline-controlled transactivator (tTA) were purchased from Clontech Laboratories Inc. The HepG2 and HeLa Tet-Off cells were maintained in the Dulbecco modified Eagle’s medium  with high glucose (4.5 g/l) supplemented with 10% fetal calf serum, 100 µg/ml of streptomycin and 100 µg/ml of penicillin (Gibco). For induction of protein expression in HeLa Tet-Off cells, the Tet system approved fetal bovine serum from Clontech was used. The cells were cultivated in a humidified atmosphere at 37°C and 5% of COFor overexpression of preKARS2, HeLa Tet-Off cells were grown to the 60% confluency and transfected with the pTRE2hyg plasmid expressing preKARS2 CAACTTGCTCCTTTCACAGCG 3′ and siRNA2∶5′ AAGGACAAGTCATTTTCTGAT 3′. As a negative control, a non-silencing siRNA  was used. Our optimized protocol consisted of two subsequent transfections: firstly, HepG2 cells were transfected in suspension with 40 nM of each siRNA using the RNAiMax transfection reagent (Invitrogen), according to the manufacturer’s protocol. 24 h later, the cells formed a monolayer and were transfected again with 40 nM of each siRNA using Lipofectamine 2000 (Invitrogen). The cells were grown for 40 h after the second siRNA transfection and then transfected with one of mitochondrially importable RNAs. In 3 days after the second siRNA transfection, the downregulation was analysed by Western blotting, and the RNA import by Northern hybridization.To downregulate preKARS2, two 21-mer siRNAs corresponding to the mitochondrial targeting sequence of the human preKARS2 mRNA were synthesized. The sequences of the sense strands of these siRNAs are as follows: siRNA1∶5′ 2 flask were used. Transfection was performed with the Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s protocol. After 48 h, the cells were detached, mitochondria were isolated and purified as described above. The total and mitochondrial RNA were isolated with the TRIzol reagent (Invitrogen), separated by 12% PAGE containing 8 M urea and analysed by Northern blot hybridization with 5′-32P-labelled oligonucleotide probes:For transfection of HepG2 and HeLa Tet-Off cells, 3 µg of RNA per 75 cmGAGTCATACGCGCTACCGATTGCGCCAACAAGGC to detect tRK1, FD-L, FD-H and HD RNA;anti-tRK1 (1–34): TGGAGCCCTGTAGGG;anti-HF RNA probe: Val probe: GTTGAAATCTCCTAAGTGanti-mt tRNAAAGTGACGCTCAGACAGGCA.and anti-cyt 5.8S rRNA probe: Val, as described previously Val as a loading control. Thus, we take into account not the absolute intensity of hybridization signals but the ratios between the signals corresponding to the imported into mitochondria tRK1 (or FD-RNAs) and the host mitochondrial valine tRNA’s gene transcript. To calculate the absolute import efficiencies for various RNAs, the total level of the RNAs in the transfected cells was taken into account. For this the relative import efficiencies were divided by the ratios calculated in the same way but for the total RNA preparations.After quantification with the Typhoon-Trio scanner, the relative efficiency of the RNA import into mitochondria was calculated as a ratio between the signal obtained with the anti-tRK1 probe and that obtained with the probe against the host mitochondrial tRNAFor Western immunodecoration, cells were lysed in the Laemmli buffer  for 10 min at 90°C, and 30 µg of protein was separated by 10% SDS-PAGE. The proteins were electroblotted onto a nitrocellulose membrane and probed with a primary polyclonal antibody against preKARS2 and a commercially available polyclonal antibody against actin . Bands were visualized with anti-rabbit or anti-goat secondary antibodies conjugated with horseradish peroxidase using the ECL Plus Western Blotting detection reagent .


Methanothermobacter thermautotrophicus, was identified as a putative nuclease that might contribute to RNA processing. The crystal structure of MTH1203 reveals that, in addition to the metallo-β-lactamase nuclease and the β-CASP domains, it contains two contiguous KH domains that are unique to MTH1203 and its orthologs. RNA-binding experiments indicate that MTH1203 preferentially binds U-rich sequences with a dissociation constant in the micromolar range. In vitro nuclease activity assays demonstrated that MTH1203 is a zinc-dependent nuclease. MTH1203 is also shown to be a dimer and, significantly, this dimerization enhances the nuclease activity. Transcription termination in archaea produces mRNA transcripts with U-rich 3′ ends that could be degraded by MTH1203 considering its RNA-binding specificity. We hypothesize that this nuclease degrades mRNAs of proteins targeted for degradation and so regulates archaeal RNA turnover, possibly in concert with the exosome.MTH1203, a β-CASP metallo-β-lactamase family nuclease from the archaeon  d in the micromolar range ► We propose MTH1203 acts with the proteasome degrading mRNAs of aberrant proteins► MTH1203 combines RNA-processing and binding domains, unique to archaea ► MTH1203 is a Zn-dependent β-CASP RNase that functions as a dimer ► The MBL domain of MTH1203 binds U-rich sequences with a K RNA processing in archaea shares more functional features with the respective processes in bacteria, in contrast to archaeal transcription that, in many respects, resembles transcription in eukaryotes . The macMethanothermobacter thermautotrophicus.The genes encoding exosome subunits in several archaeal genomes have been predicted in a comparative genomic study using the genomic context and in-depth protein sequence analysis . This stMTH1203 belongs to the metallo-β-lactamase (MBL) superfamily . This suβ-lactamase-associated CPSF-73 Artemis SNM1/PSO2) family, a group within the MBL superfamily that comprises enzymes involved in DNA and RNA processing. The signature β-CASP or clamp domain is inserted into the MBL domain and may be involved in substrate recognition . These structures all contain the MBL and β-CASP domains but no distinct RNA-binding modules.Five nonarchaeal β-CASP protein structures have been determined to date: TTHA0252 and RNase J, both functional counterparts of RNase E from mophilus ; the hummophilus ; and EF2Methanothermobacter themoautotrophicus. We show that MTH1203 contains not one but two KH domains associated with the MBL and the β-CASP domains, as recently reported for orthologs from Pyrococcus horikoshii , the central MBL domain and the C-terminal β-CASP domain A. The twThis α/β fold is highly conserved in MBL proteins including MTH1203. It consists of the central region (residues 180–383) between the N-terminal KH2 domain and the β-CASP domain, and the C-terminal region (residues 578–636), which folds back to contribute three β strands and two α helices to the MBL domain  if the glycine-rich RNA-binding segment of KHb (112–120) is excluded from the comparison. The major structural differences between the two domains are in the glycine-rich segment A. Both K2py9) -Binding Protein-2 (PCBP2) complexed with AACCCU RNA  (PDB ID 2py9) B. AlthouThe KHa domain was not identified by sequence analysis. A model of the KHa/RNA complex based on homology to KHb shows that the predicted RNA-binding region of KHa is accessible to RNA C. Howeve2X4G2, a pool of RNAs containing all 256 possible sequences, indicated that MTH1203 binds ssRNA . These domains were all shown to be folded in solution by CD spectroscopy A. An inionly 25% B. This r4C4 and C4U4 than for U7 suggests that the protein binds U-rich sequences tighter than poly(U) sequences. Additionally, as MTH1203 appears to bind A4G4 but not A7 or G7, the protein might recognize the sequence AG. However, it is likely that these interactions involve the MBL domain because the KH domains do not bind the defined sequences. Taken together, the results of these assays suggest that there are two distinct modes of RNA binding by MTH1203; one through the MBL domain and the other through the KHb domain.These results demonstrate that MTH1203 binds ssRNA with affinities in the micromolar range. The lower dissociation constant for UEscherichia coli RNA extract by monitoring the degradation of rRNAs. MTH1203 preferentially degraded the E. coli 23S rRNA (2904 bases) and to a lesser extent the 16S (1541 bases) rRNA, but not the 5S rRNA (120 bases) or tRNAs (75 bases)  A and 6B.in vitro . These ein vitro C. The poZinc was observed in the active site of the MTH1203 structure and other β-CASP proteins require zinc ions for activity . To test+ levels in Mth vary between 0.62 and 0.78 M through different growth phases  and the bacterial protein TTHA0252 (30% sequence identity and an rmsd of 2.4 Å over 427 Cα atoms) C. The mo2, involves a symmetrical interaction between the C terminus region of each monomer, a loop and a β strand from the MBL domain. The amino acid sequence of the C terminus that forms the interaction interface is highly conserved (P. horikoshii and M. mazei orthologs with rmsds of 1.36 (over 610 Cα atoms) and 1.42 Å (over 590 Cα atoms), respectively , and the exosome core, formed by the MTH682 and MTH683 proteins , were miM. thermautotrophicus cells. The pull-down experiment -binding protein 1, SF1 and hnRNP K bind their cognate RNAs with dissociation constants of 4.37, 1, and 1.8 μM, respectively . The preStructural analysis revealed that MTH1203 contains two KH domains instead of the single KH domain predicted by sequence analysis. The presence of the RNA-binding domains suggests that MTH1203 could more specifically target RNA substrates than other β-CASP proteins that only contain the MBL and β-CASP domains. The KHa domain of MTH1203 and its orthologs does not contain the RNA-binding motif GXXG. The evolutionary conservation of the KHa domain suggests a distinct function, either binding RNA in a noncanonical fashion or an alternative structural or regulatory role.4C4 and C4U4 then for U7. These experiments show that MTH1203 binds ssRNA but the precise sequences targeted by the KH domains remain to be determined.Fluorescence polarization anisotropy experiments demonstrated that the full-length MTH1203 has the ability to bind ssRNAs with at least a micromolar affinity. The individual KH domains did not bind to the specific RNA sequences tested, but a pull-down assay showed that the KHb domain binds to a degenerate RNA sequence, whereas the KHa-b and KHa RNA binding was reduced, relative to KHb binding, by 56% and 75%, respectively. Of the sequences tested, the full-length MTH1203 interacted primarily with U-rich sequences. Because the specific RNA sequences did not interact with the KH domains, this interaction is likely to be mediated by the MBL domain. However, the MTH1203 MBL domain seems to be specific for U-rich rather than poly(U) sequences because the dissociation constant was lower for UThe model of the MTH1203-KHb domain complex with RNA does not provide insight into the orientation of the RNA substrate in the catalytic site of the same subunit of the dimer because of the substantial ∼42 Å spatial separation, at least ∼10 nucleotides apart if both sites are bound to an extended RNA molecule. However, the indications that MTH1203 functions as a dimer raise the possibility that an RNA molecule binds to the KH domain from one subunit of the dimer and is cleaved by the MBL-β-CASP of the other subunit . While tThe MTH1203 MBL and β-CASP domains, including the active site, are highly similar to those already characterized in CPSF-73 and TTHA0252, suggesting that the mechanisms of RNA hydrolysis could be the same. Although there is no structure of a β-CASP protein in complex with nucleic acids, a mechanism of RNA cleavage has been proposed for RNase Z, an MBL-family but non-β-CASP endoribonuclease , based oE. coli rRNAs confirmed that MTH1203 has a metal-dependent nuclease activity. In vitro MTH1203 completely degraded the 23S and 16S rRNAs. However, no activity was observed against the short E. coli 5S rRNA or tRNAs. As rRNAs possess extensive secondary structure, this result suggests that MTH1203 degrades substrates independent of their structure. It remains to be determined whether MTH1203 has an endo- and/or exonuclease activity. Additionally, characterization of the sequence specificity of MTH1203, both of the nuclease and RNA binding functions, will provide insight into its in vivo function.Preliminary nuclease activity assays on the degradation of In summary, we present experimental evidence in support of the hypothesis that MTH1203 could function alongside the exosome in RNA degradation . The posmth1203 gene from the delta H strain of Methanothermobacter thermautotrophicus was amplified by PCR from genomic DNA and cloned using a ligase-independent reaction into the vector pETYSBLIC-3C (E. coli BL21 (DE3) Rosetta expression cells grown in Terrific Broth at 37°C. The seleno-methionine (SeMet) derivative of MTH1203 was expressed in the same cells grown at 37°C in autoinduction media (The SBLIC-3C . The KH on media  with ove2PO4 (pH 7.5), 1 M NaCl, 2 mM MgCl2, and 10 mM imidazole and lysed by sonication and then the His-tagged protein was purified from the clarified lysate using a 5 ml HiTrap nickel-chelating column . The protein was then purified using a Superdex 200 gel filtration column  after removal of the His-tag with 3C protease. The purified protein was concentrated to 10 mg ml-1 for crystallization trials.The MTH1203 proteins were purified at room temperature. Cell pellets were resuspended in 50 mM KHFor complete details of the cloning, expression, and purification see the 2PO4 [pH 5.2], 2% PEG 2000 MME, 7% ethylene glycol, 0.5 mM ZnCl2, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 10 mM D-Ribose-5-phosphate (Fluka). This crystal was flash-cooled in liquid nitrogen without further cryoprotection. SeMet protein crystals were grown in 24-well plates in identical condition to the native crystals, containing 5 mM DTT.Preliminary crystallization screening was performed using a Mosquito nanoliter pipetting robot (TTP Labtech) to set up 96-well sitting drop plates at room temperature, with 150 nl of MTH1203 mixed with 150 nl of reservoir solution. Crystals were initially optimized robotically then scaled up using hanging-drop vapor diffusion in 24-well plates with a 1:1 mixture of protein solution and reservoir solution (1 μl each). For diffraction data collection, a single brick-shaped crystal was grown over 24 hr in 50 mM KHDENZO and SCALEPACK (1212 using the program BALBES (2i7v) as the search model. Two molecules were located in the asymmetric unit, corresponding to a solvent content of 72%. SHARP  and refinement using REFMAC  data were collected to 3.1 Å at the zinc peak (1.2820 Å), at the ID29 station at the ESRF. These data were processed with CALEPACK  and solvm BALBES  with the%. SHARP  and DM (HARP (DM  were useUCCANEER  and imprg REFMAC  with TLSPROCHECK  was usedE. coli Nova Blue cells were used as the substrate for nuclease activity experiments. The reaction buffer contained 10 mM Tris (pH 8.0), 0.2 mM DTT, 2 mM MgCl2, 0.1 mM EDTA, and 0.5 M NaCl except to test the inhibition of the protein nuclease activity when MTH1203 was incubated in 10 mM Tris (pH 8.0), 0.2 mM DTT, 0.1 mM EDTA, 0.5 M NaCl, and N,N,N′,N′-Tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). Before each reaction, the protein was incubated in the reaction buffer for 10 min at 37°C prior to RNA addition and subsequently incubated for 90 min at 37°C or at the indicated temperatures/times. For the nuclease inhibition assays MTH1203 was incubated prior to RNA addition for 30 min at 37°C. Bovine serum albumin (BSA) was always used in the same conditions as MTH1203 as a control. Unless otherwise indicated, RNA from the samples was then isolated by phenol/chloroform extraction and loaded on a 1% agarose gel stained using SYBRsafe (Invitrogen). The relative activities of the monomer and dimer were estimated by analyzing the gel using ImageJ .Total RNAs purified from 7, G7, C7, U7, C4U4, U4C4, A4G4, and a degenerate A2X4G2). The fluorescence anisotropy experiments were performed in a Spex Fluoromax-3 (Horiba Jobin Yvon), with excitation and emission wavelengths of 485 and 520 nm, respectively. Experiments were performed in 20 mM Tris (pH 8.0) and 1 M NaCl. In each titration, His-tagged constructs of MTH1203 at 1 mg ml-1 were added to RNA (5 nM). After mixing for 1 min, three measurements were read with a 5 s integration. Control experiments were also performed in the same conditions where BSA was used instead of MTH1203. Titration curves were fit using Scientist (Micromath).Eight different single-stranded RNA fragments labeled with a 5′ fluoresceine were synthesized by Dharmacon , lysed by sonication and then cleared by centrifugation at 15,000 × g for 15 min. MTH1203 (0.15 mg with hexahistidine tag) was bound to 15 μl of chelating Sepharose fast flow resin  charged with Ni2+ equilibrated in the lysis buffer. The resin was then washed and resuspended in buffer to give a total volume of 100 μl. Ten microliters of the resin was added to 400 μl of either the cell lysate supernatant  or buffer (binding control). A control using resin without bound MTH1203 was also performed. After 30 min each sample was washed with 2 × 400 μl of buffer, resuspended in 10 μl of 4 × SDS-PAGE loading buffer, boiled, and run on a 12% SDS-PAGE gel stained with Coomassie blue.