ADAR substrates, and both the sequence context and the expression profile of ADARs are relevant factors in determining the quantitative variance of RNA editing across different sites and tissue types. In support of the functional relevance of some of these editing sites, substitution valley of decreased divergence was detected around the editing site, suggesting the evolutionary constraint in maintaining some of these editing substrates with their double-stranded structure. These findings thus complement the “continuous probing” model that postulates tinkering-based origination of a small proportion of functional editing sites. In conclusion, the macaque editome reported here highlights RNA editing as a widespread functional regulation in primate evolution, and provides an informative framework for further understanding RNA editing in human.Understanding of the RNA editing process has been broadened considerably by the next generation sequencing technology; however, several issues regarding this regulatory step remain unresolved – the strategies to accurately delineate the editome, the mechanism by which its profile is maintained, and its evolutionary and functional relevance. Here we report an accurate and quantitative profile of the RNA editome for rhesus macaque, a close relative of human. By combining genome and transcriptome sequencing of multiple tissues from the same animal, we identified 31,250 editing sites, of which 99.8% are A-to-G transitions. We verified 96.6% of editing sites in coding regions and 97.5% of randomly selected sites in non-coding regions, as well as the corresponding levels of editing by multiple independent means, demonstrating the feasibility of our experimental paradigm. Several lines of evidence supported the notion that the adenosine deamination is associated with the macaque editome – A-to-G editing sites were flanked by sequences with the attributes of  ADARs are relevant factors in determining the quantitative variance of RNA editing across different sites and tissue types. Importantly, some of these RNA-editing events represent functional regulation, rather than neutral signals, as suggested by substitution valley of decreased divergence detected around the editing sites, an indication of selective constraint in maintaining some of these editing substrates with their double-stranded structure. The macaque editome thus provides an informative evolutionary context for an in-depth understanding of RNA editing regulation.RNA editing is a co-transcriptional process that introduces differences between RNA and its corresponding DNA sequence. Currently, the next generation sequencing have allowed study of the editome in a comprehensive and efficient manner. However, fundamental issues involving accurate mapping of the editome as well as its regulation and functional outcome remain unresolved. To further unveil the underlying mechanisms from the evolutionary perspective, we report here the editome profile in rhesus macaque, one of our closest evolutionary relatives. We identified a list of 31,250 RNA-editing sites and deciphered an accurate and informative editome across multiple tissues and animals. We found that the adenosine deamination is associated with the macaque editome, in that both the sequence context and the expression profile of  Since its discovery in 1986 Although the identification of RNA-editing sites by discerning sequence discrepancies between RNA and DNA derived from the same specimen seems to be a straightforward approach, it is error-prone when the RNA/DNA sequences are compiled by short reads generated from NGS technology Overall, NGS technology has helped open the Pandora's box of the editome and so has raised more questions than it answers. Key issues, including experimental and computational strategies to accurately identify the editome, the mechanism by which its profile is maintained, and its functional significance, are presently not well addressed Figure S1). As a reference, genomic DNA derived from prefrontal cortex of the same animal was sequenced; a total of 1,763.3 million 90-bp paired-end reads were uniquely mapped to the macaque genome, with 92.2% of the genomic regions successfully sequenced and 91.6% covered by at least ten DNA reads . Such deep coverage of the genome and transcriptome in multiple tissues of the same animal provided an ideal dataset to profile the RNA editome in rhesus macaque .We performed a genome-wide study to identify RNA-editing sites in rhesus macaque  in coding regions (& S2), as well as 77 of the 79 randomly selected sites (97.5%) in untranslated, intronic and intergenic regions (Table S2) were experimentally verified by PCR amplification and Sanger sequencing of both DNA  and the corresponding cDNA . The validation rates for both coding and non-coding regions suggested that most of the sites identified in this genome-wide study should be verifiable.We next set out to confirm that these sites represent Figure S5).In addition to the traditional approach of PCR amplification and Sanger sequencing, we also performed a medium-throughput study to quantify the levels of coding region-associated RNA editing in the seven tissues using a mass array-based genotyping platform . However, only six additional RNA-editing sites were identified using this targeted genomic reference, but were subsequently discarded by Sanger validation. These false-positives might have arisen largely due to biased capture efficiency in the exome sequencing assay favoring the wild-type allele , suggesting the high calling sensitivity of editing sites in coding region.As stringent cutoffs for the sequencing depth of genome were instituted to distinguish RNA editing from systematic sequencing errors, allele-specific expression and duplication-related polymorphisms, we evaluated whether such a rigorous approach may have hampered the site-calling sensitivity in this study. Focusing on coding regions, we increased the coverage of genomic DNA sequences to 115-fold through an established whole-exome capturing and sequencing strategy Supplementary Table 1 in Reference , However, considering the coding regions are less repetitive and well-annotated than other genomic regions, it is not straightforward to generalize the high calling sensitivity in coding region to other genomic regions. Notably, the overall number of macaque editing sites we identified is lower than that in human, in which 84,750 editing sites were identified from poly(A)-positive RNA-Seq data . We noted that the fraction of A-to-G transitions increased when more stringent filters were incorporated, from 65.6% in the initial list to 99.8% in the final list , suggesting that most of the nucleotide changes of the transversion type may have been due to technical artifacts First, contrary to the previous study reporting all twelve possible forms for RNA-editing sites in human with a large proportion of transversions (43%) ; , Table S4). As the editing levels were estimated according to RNA-Seq data where the estimation might be less accurate for sites with lower sequencing coverage, we further used a mass array-based genotyping platform to quantify the levels of editing in coding regions of RNA from the seven original macaque tissues and nine additional samples . In addition, the variability across macaque animals is significantly lower than that across tissues, as indicated by the pair-wise CV comparisons . However, quantitative analyses with a Triplet model as previously described Figure S4), a proportion comparable to a previous study using fixed RNA substrate and peak-based editing level estimation We next investigated the relevant molecular factors underlying the macaque editome, and subsequently made the following observations. First, the sequence context of the overwhelmingly-represented A-to-G editing sites verified the known attributes of ADARs, although previous studies in rodents did not detect a significant correlation ADARs (Spearman's rank correlation coefficient ≥0.5), such an observation represents a statistically significant excess of editing sites with positive correlations  with ADAR2, and 31 sites (1.6%) with both ADARs .Overall, our qualitative and quantitative data demonstrated that the intra-population variability of editing levels is significantly lower than that across tissues, and that both the Table S4). For the 1,111 macaque editing sites with homology in both of these transcriptomes with adequate cDNA coverage . Some macaque editing sites  were found with the edited forms encoded in human or chimpanzee genome . Further analysis revealed little effect of expression levels of the host genes on the signatures of substitution valley of decreased divergence .On the basis of these clues, we further tested whether the cross-species similarities in RNA editome were maintained by purifying selection due to the functional implications of these regulations, or simply due to the relatively lower sequence divergences among these closely related primate species. As shown above, the local sequence context of the editing site was important in RNA-editing regulation, as it may be implicated in the formation of a suitable ; Hypergeometric test, p-value = 1.7e-3). Among these sites, three were located in orthologous proteins in human with solved crystal structures – the nucleotide/codon re-assignment by RNA editing reportedly regulate the activity of the voltage-gated potassium channel For RNA-editing sites in coding regions, we also examined the distribution of synonymous divergent sites between human and rhesus macaque surrounding the editing sites. Although synonymous sites have been considered to be largely neutral Taken together, the dampened divergence rate or synonymous substitution rate around the editing sites reflects the evolutionary necessity of retaining some of these editing substrates with their double-stranded structure Figure 5Despite extensive efforts and discussion, accurate definition of the human editome by using NGS data alone remains technically challenging cis-natural anti-sense expression In this study, beyond the filters established previously to remove potentially erroneous editing sites with i) low read coverage  & Supplementary Table 1 in Reference e.g., the inherent difference in genetic makeup for the inverted Alu pairs, may also contribute to this human-macaque difference However, the stringent criteria we used to control for the false-positives would cause some false-negatives in RNA-editing detection, although the calling sensitivity in coding region was proved to be good  & 2. To eViewed together, although our rigorous experimental and computational paradigm would cause some false-negatives, it would be a necessary compensation for an accurate and quantitative catalog of RNA-editing sites in rhesus macaque, considering the poor genome annotations and error-prone gene structures in rhesus macaque , S8). Second, the global attributes of editing were further verified and quantified to show that the occurrence of RNA editing is correlated with the flanking sequence signatures, as well as the levels of ADARs expression , a notion that complements the “continuous probing” model by illustrating a clearer process for the tinkering-based origination of functional RNA editing sites Interestingly, when investigating the dampened divergence rate for editing sites across different genomic regions, stronger selective constraint was detected on coding regions, while sites in other regions also showed some degrees of weaker evolutionary constraints  animal facility at the Institute of Molecular Medicine in Peking University. Experiments with animals were done in accordance with protocols approved by the Institutional Animal Care and Use Committee of Peking University and followed good practice.Rhesus macaque tissue samples were obtained from the AAALAC-accredited , unbiased read distribution on transcripts , correct strand information , little DNA contamination , and a low and evenly-distributed mismatch rate across the reads . The quality of genome resequencing and exome-Seq were also evaluated following the strategy reported in a previous study Figure S2). Deep sequencing data in this study are available at NCBI Gene Expression Omnibus and SRA under accession numbers GSE34426, GSE42857 and SRP039366.Strand-specific RNA-Seq libraries were prepared from seven rhesus macaque tissues derived from the same animal as previously reported Exome-Seq or genome resequencing reads were aligned to the rhesus macaque genome (rheMac2) with BWA (v0.5.9-r16), from which only uniquely-mapped reads were retained. RNA-Seq reads were firstly mapped to the macaque genome (rheMac2) by BWA (v0.5.9-r16). Meanwhile, the RNA-Seq reads were also mapped to the transcriptome (Ensembl v65) by BWA (v0.5.9-r16) to address the technical issue of gapped alignment, as well as to control for false-positives derived from pseudogene- or paralog-related misalignments in short-read processing. Unique mapping of a RNA-Seq read was then defined as i) no more than four mismatches detected in the alignment to the genome or transcriptome reference; and ii) with no second-best hit or the second-best mapping model including no less than three additional alignment mismatches than the best model. For reads aligned to both the genome and the transcriptome, unique mapping of RNA-Seq reads was defined when combining the genomic mapping with the transcriptome mapping results, while other reads uniquely aligned only to the genome or the transcriptome were kept. Unique alignments were further examined to remove cases with incorrect read-pairing information Uniquely-mapped reads were first divided into two groups: reads transcribed from the plus-strand and those from the minus-strand. After removing redundant reads with identical start and end positions, single-nucleotide variation (SNV) calling was separately performed for the two groups of reads using Samtools (v0.1.16), with the parameter “samtools mpileup -C 50 -E –Q 25 –ug”. The special parameter “bcftools view -p 1” was set to keep SNVs significantly deviating from heterozygous allele distribution. Reads harboring SNVs within 5 bp of both ends were discarded in SNV calling due to read end-biased sequencing errors et alDNA Filter, i) The RNA SNP Filter, in which at least five RNA-Seq reads, with ≥3 nucleotides sequenced with high PHRED base quality (≥25), were required to support the variant form, thus eliminating false-positives due to amplification bias or sequencing error  A Strand Bias Filter was also introduced as previously proposed Fisher's exact test, p-value<0.05), or supported by <2 reads on either of the two strands, were excluded  The RhesusBase Filter, in which we further checked the RNA-editing sites by mapping the raw reads containing these sites to refined transcript structures in the RhesusBase Whole genome sequencing data were used to determine the genotype by adopting criteria similar to those of Li  & S2). The sequence coverage of these sites ranges from 12 to more than 100 RNA-Seq reads, with the estimated editing levels from 3% to 100%. For editing sites in coding regions, we also performed mass array-based genotyping on all cDNA and the matched DNA samples on an iPLEX Gold MassARRAY system (Sequnom Inc.) to independently verify the RNA-editing sites and the corresponding editing levels. Primers were designed with MassARRAY assay design software. Amplification reactions, digestion of unincorporated dNTPs and MALDI-TOF mass spectrometry were performed in accordance with the manufacturer's instructions. Signal intensities for two alleles were automatically assigned followed by manual confirmation. Briefly, the genotype was assigned as the ratio of the area of ‘G’ signal to the area of both ‘G’ and ‘A’ signals if the editing form was A-to-G, and ideally a ratio of 0 represented homozygous A/A while 1 represented homozygous G/G. Considering the noise in the Sequenom mass array platform All candidate RNA-editing sites in coding regions that passed the above protocol, as well seventy-nine randomly selected RNA-editing sites in untranslated, intronic and intergenic regions, were further verified by PCR amplification and Sanger sequencing of both DNA and the corresponding RNA -positive RNA-Seq data The levels of RNA editing were estimated separately for high-throughput, medium-throughput and low-scale data on the basis of read numbers YAS , a ‘5′ matched’ motif of YAW [W = A/T], a ‘3′ matched’ motif of RAS [R = A/G], and a ‘not matched’ motif of RAW. We further performed a quantitative study to estimate how much site-to-site variances could be explained by the nearby sequence motif. We fitted the relationship between editing level and the local sequence context by controlling for cross-tissue and intra-population variations, using a Triplet model as previously described i th editing site; A, T, C and G were denoted by 1, 2, 3 and 4; i th editing site; 1{A} was characteristic function ; 2R values obtained under the regression model was used to indicate the prediction power of the local sequence context on the editing levels We evaluated the dependence of editing levels on sequence motif. The RNA-editing sites were divided into four categories according to the nearby sequence preferences ( & S8).Public high-throughput datasets of multiple tissues from human Table S4). Only those editing sites covered with at least 30 RNA-Seq reads and at least 5 observations in each group were included. A CV score less than one indicates a smaller standard deviation than the mean, and thus a small intra-population variation for RNA editing levels.Besides the qualitative clustering data, we further measured the coefficients of variation (CV) of editing level across different animals, as well as across tissues. RNA-Seq data in brain samples from seven animals and seven tissues from the same animal were integrated and analyzed in standard pipelines for estimation of editing levels and CVs .To examine whether the editing sites we identified in rhesus macaque could also be detected in human and chimpanzee orthologous regions, we re-analyzed public available RNA-Seq data in human or chimpanzee to trace the orthologous regions of macaque editing sites. Cross-species comparisons of RNA-editing regulation was analyzed in five tissues  in the genomic context of sequence multiple alignments among human, chimpanzee and rhesus macaque ).We then tested whether the cross-species similarities in RNA editome were maintained by purifying selection due to the functional implications of these regulations. The genomic sequences nearby all of these macaque editing sites were compiled and the percentage of sequence divergence between human and macaque was calculated for each 6-nucleotide window. To investigate whether higher expression may be linked with higher conservation, we performed the analysis again using three subsets of these editing sites, divided according to their abundance of expression tags, estimated on the basis of the sum of normalized abundance of expression tags in seven tissues and (dS and dN scores for each window nearby the editing sites were estimated using DnaSP (v5). The average dS and dN scores between human and rhesus macaque were estimated using 2,929 coding sequences with high-quality pairwise alignment Monte Carlo simulations with random sampling of coding regions across the macaque genome were performed to assess whether the detected divergence rate drop was a consequence of sampling bias on limited observations. A series of Perl and R scripts (v2.13.1) were implemented to perform these statistical analyses.For editing sites in coding regions, the Figure S1A) PHRED quality scores across all bases of reads. (B) Distribution of RNA-Seq reads across transcripts. Transcripts were binned into even intervals from 5′ to 3′, with the percentage of all short reads aligned to each interval shown as mean ± SD. (C) Efficiency of strand-specific sequencing strategy. PFC: prefrontal cortex. (D) Average read distribution in exonic, intronic and intergenic regions as measured by RPKM. (E) Mismatch frequency at each position of reads summarized as mean ± SD.High-quality strand-specific RNA-Seq was performed for seven rhesus macaque tissues. ((TIF)Click here for additional data file.Figure S2Statistics for whole-genome sequencing (WGS) and parallel exome capture and sequencing (Exome-Seq) in rhesus macaque. The reads coverage in WGS (upper panel) and Exome-Seq (lower panel) is summarized and shown in cumulative frequency plots.(TIF)Click here for additional data file.Figure S3S3-1 to S3-27).Results of Sanger sequencing validation for all 29 candidate macaque editing sites in coding regions. For each candidate editing site (indicated by genome coordinates and red arrows), raw chromatograms of sequences derived from seven cDNA and the matched DNA (gDNA) samples are shown ((PDF)Click here for additional data file.Figure S4S4-1), untranslated regions (S4-2), or intergenic regions (S4-3), raw chromatograms of sequences derived from one cDNA and the matched DNA (gDNA) samples are shown. *Sites used in regression analysis.Results of Sanger sequencing validation for 79 candidate macaque editing sites in non-coding regions. For each candidate editing site (indicated by genome coordinates and red underlines) in intronic regions ((PDF)Click here for additional data file.Figure S5A). The basic information on these public datasets is summarized in (B).The levels of RNA editing could be accurately estimated using RNA-Seq data. For each editing site with adequate read coverage, the levels of editing were estimated using different RNA-Seq datasets of macaque brain. Pair-wise Pearson correlation coefficients are shown in ((TIF)Click here for additional data file.Figure S6Enrichment of the A-to-G editing sites by the multi-filter strategy. Initial: initial list of macaque editing sites identified by high-throughput sequencing. Relative representation of macaque editing types for sites that progressively passed the DNA filter, the RNA SNP filter, the strand-bias and RhesusBase filter, as described in (TIF)Click here for additional data file.Figure S7A), as well as across different tissues (B) are shown.Quantitative analysis of intra-population and cross-tissue variations of editing levels. The distributions of coefficient of variance (CV) values within macaque animals ((TIF)Click here for additional data file.Figure S8Alu (A), non-Alu repeat (B), non-repetitive (C), un-translated (D), intronic (E) and intergenic (F) regions were analyzed separately.Hierarchical clustering of editing levels for subsets of editing sites. Editing levels were estimated on the basis of RNA-Seq data in this study  and other public RNA-Seq data , with missing data in dark cyan. Six subsets of editing sites, including sites in (TIF)Click here for additional data file.Figure S92R values. Distributions of 2R values in models assuming association of editing level of Alu (A) or non-Alu sites (B) with ADARs expression are shown.Distributions of (TIF)Click here for additional data file.Figure S10A), as well as for the subsets of editing sites in different genomic context (B).Cross-species comparisons of different subsets of editing sites. The percentages of macaque editing sites that had corresponding editing sites in human and/or chimpanzee are denoted by red bars. Comparisons were done for the background using the adjacent non-edited sites to indicate the degrees of RNA-Seq sequence errors ((TIF)Click here for additional data file.Figure S11A, B, D, E, F & G) or human-chimpanzee (C) divergent sites is shown for different subsets of macaque editing sites. A: all macaque editing sites, B: conserved sites between human and macaque, C: non-edited sites in both human and chimpanzee, D: editing sites in Alu regions, E–G: editing sites with low, medium and high expression levels, respectively. The nearby regions are highlighted in red and the average divergence rate of the distal regions is indicated by blue dashed lines.Signatures of purifying selection for editing sites in different subsets. The genomic sequences nearby the macaque editing sites were compiled according to the distances to the editing sites. For each 6-nucleotide window, the proportion of human-macaque ((TIF)Click here for additional data file.Figure S12Distribution of levels of RNA editing in different genomic regions. For editing sites in untranslated, CDS, intronic and intergenic regions, the distribution of the levels of RNA editing is summarized in boxplot.(TIF)Click here for additional data file.Table S1Basic information of RNA-editing sites identified in this study.(XLSX)Click here for additional data file.Table S2Primers used in Sanger sequencing.(PDF)Click here for additional data file.Table S3#Ce: cerebellum, Te: testis, Pr: prefrontal cortex, Ki: kidney, Lu: lung, Mu: muscle, He: heart.Known human RNA-editing sites in coding regions. The percentage of “G” reads, together with the number of total reads (“A” and “G”) in each tissue sample is shown. *Yes: included in final list; DNA Depth: failed to pass inclusion criteria for read coverage; Filter: failed to pass the filter; (PDF)Click here for additional data file.Table S4$PFC: prefrontal cortex; &C: chimpanzee, H: human, R: rhesus macaque; #M: million reads; *Samples for cross-species comparison in @Samples for intra-species editing level comparison in %Samples for calculating intra-population CV values in  & S7.Statistics of public RNA-Seq data integrated in this study. (PDF)Click here for additional data file.Table S5P-values of one-tail Wilcoxon tests for four classes of motif. *M: matched motif; 5′ M: 5′ matched; 3′ M: 3′ Matched; N: Not matched.(PDF)Click here for additional data file.Table S6ADARs.Quantitative correspondence of the tissue-biased profile of the RNA editome to the tissue expression profile of (XLSX)Click here for additional data file.


MYC-IGH@ fusion subsequently deregulating and overexpressing MYC on der 14q32. In CLL, atypical rearrangements resulting in its gain or loss, within or outside of IGH@ or MYC locus, have been reported, but their clinical significance remains uncertain. Herein, we report a 67 year-old male with complex cytogenetic findings of apparently balanced t and unreported complex rearrangements of IGH@ and MYC loci. His clinical, morphological and immunophenotypic features were consistent with the diagnosis of CLL.The t, the cytogenetic hallmark of Burkitt’s lymphoma, is also found, but rarely, in cases of chronic lymphocytic leukemia (CLL). Such translocation typically results in a IGH@, indicative of rearrangement. Karyotype analysis showed an apparently balanced t. Sequential GPG-metaphase FISH studies revealed abnormal signal patterns: rearrangement of IGH break apart probe with the 5’-IGH@ on derivative 8q24.1 and the 3’-IGH@ retained on der 14q; absence of MYC break apart-specific signal on der 8q; and, the presence of unsplit 5’-MYC-3’ break apart probe signals on der 14q. The breakpoint on 8q24.1 was found to be at least 400 Kb upstream of 5’ of MYC. In addition, FISH studies revealed two abnormal clones; one with 13q14.3 deletion, and the other, with concurrent 11q deletion and atypical rearrangements. Chromosome microarray analysis (CMA) detected a 7.1 Mb deletion on 11q22.3-q23.3 including ATM, a finding consistent with FISH results. While no significant copy number gain or loss observed on chromosomes 8, 12 and 13, a 455 Kb microdeletion of uncertain clinical significance was detected on 14q32.33. Immunohistochemistry showed co-expression of CD19, CD5, and CD23, positive ZAP-70 expression and absence of MYC expression. Overall findings reveal an apparently balanced t and atypical complex rearrangements involving 3’-IGH@ and a breakpoint at least 400 Kb upstream of MYC, resulting in the relocation of the intact 5’-MYC-3’ from der 8q, and apposition to 3’-IGH@ at der 14q. This case report provides unique and additional cytogenetic data that may be of clinical significance in such a rare finding in CLL. It also highlights the utility of conventional and sequential metaphase FISH in understanding complex chromosome anomalies and their association with other clinical findings in patients with CLL. To the best of our knowledge, this is the first CLL reported case with such an atypical rearrangement in a patient with a negative MYC expression.Interphase FISH studies revealed deletions of 11q22.3 and 13q14.3, and an extra copy of  IGH@ rearrangement [Chronic lymphocyctic leukemia (CLL) is the most common leukemia in the elderly with clinical presentation of lymphocytosis, bone marrow involvement, lymphadenopathy, hepatosplenomegaly, complex cytogenetics and heterogeneous clinical course . Immunopangement .While t8;14), the cytogenetic hallmark of Burkitt’s lymphoma, is a primary genetic event found in about 70-80% of cases, it is usually a rare secondary anomaly in other B-cell disorders including CLL (about 0.2% to <1%) [, joining (J), diversity  regions, but other heavy-chain regions are occasionally involved [MYC and IGH@ (IG heavy chain) [IGK) at 2p12, or lambda light chain (IGL) at 22q11.2 [MYC is also involved with IGH in DLBCL [TCR alpha/delta in T-acute lymphoblastic leukemia/lymphoma, and IG kappa and lambda chains in plasma cell myeloma [In a typical t translocation, the ve 14q32 -15. The  in size , localizlocation . The IGHotential . Whereasinvolved . While ay chain) , others  22q11.2 -18. MYC  myeloma .MYC is a proto-oncogene that encodes for a transcription factor that regulates cell cycle progression, growth, differentiation, apoptosis, survival and biosynthesis [MYC drive cells into lymphomagenesis often through its deregulation and overexpression [MYC rearrangements is implicated not only in the initiation of lymphomagenesis but also in its transformation and progression of low-grade lymphomas into a more advanced disease and an unfavorable outcome [MYC expression of different stages of aberrant cellular maturation and differentiation may influence the neoplastic phenotype [ynthesis ,6,20. Itpression ,12,21,22 outcome ,18,21-23henotype .MYC: regulatory region within MYC, from exon 1 to intron 1, (Class I and most common); transcription factor binding-site at or adjacent to 5’-MYC (Class II); and long-range regions up to 100-300 Kb or more upstream from an intact 5’-MYC-3’ (Class III) [MYC expression is influenced by breakpoint location, mutation within the translocated region, deletion of regulatory elements, or transcription at cryptic sites other than the usual P1 or P2 initiation start site (promoter shift) [MYC expression through chromatin looping bringing the enhancers in close proximity to MYC[MYC, are associated with increased susceptibility to prostate, colorectal, bladder, breast cancer, or chronic lymphocytic leukemia [MYC transcription, the clinical significance of MYC rearrangements upstream of MYC remain unclear and a subject of burgeoning field of investigation [At 8q24.1 locus, translocation breakpoints are located within or surrounding the ass III) ,16,20. Ir shift) ,20,24. Ir shift) . Long-raty to MYC,26, or tty to MYC. Multiplleukemia . AlthougMYC[MYC (+MYC) [To date, there are only very few reported cases of CLL with apparently balanced t8;14) and atypical rearrangements ,8, none ;14 and aMYC,8, gain C (+MYC) ,29, or dC (+MYC) ,30,31. TIGH@ and at least 400 Kb upstream of 5’-MYC, unreported complex atypical rearrangements of IGH@ and MYC loci that did not result in IGH-MYC fusion and no subsequent MYC expression.Here we report a CLL case with complex cytogenetic findings of deletions of 11q and 13q, in addition to the apparently balanced t. We also present an undocumented atypical complex rearrangements involving 3’-3 per μL. Peripheral blood smear showed marked lymphocytosis with numerous atypical lymphoid cells including prolymphocytes, smudge cells, normocytic normochromic anemia and thrombocytopenia. The lymphoid-gated population constituted 87% of total cells, and consisted of 2% T cells, 70% B cells, and <2% NK cells  was elevated and measured at 23.15 × 10A complete chromosome analysis was not possible due to low mitotic index. G-banded karyotype analysis of available metaphase cells revealed an abnormal male karyotype with an apparently balanced tq24.1;q32) seen in 50% (6/12) of total cells examined   nor trisomy 12 was detected (data not shown). Sequential GPG-metaphase FISH studies were performed on the same chromosome metaphase spread to determine the clonality of the structural abnormalities seen in our patient. Results showed that two different clones exist in the peripheral blood of our patient: one with deletion 13q14.3 (seen only in interphase nuclei in our study), and another with concurrent deletion 11q  of nuclei examined (data not shown). In addition, deletions of the 13q14.3 (D13S319) ; and, on derivative 14q32: one yellow (relocation of 5’-MYC-3’ and its flanking regions adjacent to 3’-IGH@). The estimated location of the translocation breakpoint upstream of 5’-MYC was determined by in silico mapping . We based our calculations on the Spectrum Orange of the MYC break apart probe, the farthest probe from 5’ of MYC (as compared to the MYC probe in the IGH-MYC fusion probe). The estimated distance of the translocation breakpoint (STS marker WI-1302) from 5’of MYC is at least 400 Kb . This interval includes two RefSeq genes: POU5F1B (POU class 5 homeobox 1B), an intronless gene that encodes for a transcription factor  and a gene with no known function, LOC727677 . It also includes the SNPs implicated in several cancer types, rs1447295 (Region 1), rs16901979 (Region 2) and rs6983267 (Region 3) [Further sequential FISH studies on 10 metaphase nuclei using the IGH@ break apart probe showed splitting or rearrangement (1Y1G1R), with the 5’-IGH@ (green) translocated on chromosome 8q24.1 and the 3’-IGH@ (red) retained on 14q , a gene that encodes for a cell cycle checkpoint phosphorylating kinase that functions for regulating proteins for tumor suppression, checkpoint, DNA repair and maintenance of genome stability [MYC locus despite its removal from der 8q24.1 locus. This suggests that there was no net gain or loss despite the unbalanced rearrangements detected by FISH. In a lesser extent, a 61 Kb gain on 8q24.12 was detected, but found to be unreportable with further in silico investigations. There were no clinically relevant gains or losses detected on chromosomes 12, 13 and 17.SNP CMA refined the 11q22.3 deletion breakpoints detected by FISH. Results showed a 7.1 Mb heterozygous copy number loss at chromosome 11q22.3-q23.3, arr 11q22.3q23.3x1 (data not shown). It deleted 62 RefSeq genes including tability . In additability . CMA didAccording to the ISCN , the ove(ATM-),13q14.3q14.3(D13S319x2).nuc ish, detected by interphase and sequential metaphase FISH studies, are found as two different abnormal clones, indicative of mosaicism. CMA failed to detect gains or losses on 13q, since it only accounts for 8% of the total cell population, a number way below the detection limit 10-30%) of either SNP or BAC microarrays [% of eithMYC antibodies did not detect any staining in our patient’s sample, suggestive of absence of MYC activation  usually confers poor prognosis in CLL, a consistent genotype and phenotype correlation remains an unresolved issue. Our patient’s case exhibits an unreported rearrangement involving MYC[MYC (+MYC) [MYC-specific signal on der 8q24.1 locus was also observed in our patient using IGH-MYC fusion probe (1Y2G1R), it is not identical to the reported deletion by Reddy et al. in 2006 [MYC locus. Instead, it showed two unsplit MYC probes (yellow) on the normal chromosome 8 and on der 14q32. We interpreted these findings as an atypical rearrangement never reported elsewhere, with the 5’-MYC-3’ removed from the 8q24.1 locus at a breakpoint at least 400 Kb upstream of its 5’ region. We also showed that this deleted region is relocated to the 14q32 locus and apposed to the 3’-IGH@ locus. Neither gain of MYC nor deletion of the 5’-IGH@ locus was observed by FISH or CMA in our case. We have exhaustively searched the available literature and did not find any cases similar to the signal patterns reported on here.In our patient, the FISH signal patterns detected are unique from those previously reported in CLL cases with atypical rearrangements and an apparently balanced t8;14). These include a cryptic deletion on 8q24.1 including MYC,8, gain ;14. ThesC (+MYC) ,29, or dC (+MYC) ,30,31. AMYC and its correlation to disease progression have not been established in CLL with t, with or without MYC translocations [MYC expression is generally at low levels in CLL [MYC rearrangement has also been described in CLL with malignant Richter transformation and other higher risk cases for CLL progression [MYC are expressed as a result of the t and its variant translocations in Burkitt’s lymphoma and in some other B-cell malignancies including DLBCL and plasma cell myeloma, these translocations may not necessarily lead to increased expression of MYC in CLL [MYC expression are most likely dependent on specific disruptions of regulatory regions, or characteristic genomic translocation breakpoints either at the MYC or IGH@ locus.To the best of our knowledge, expression levels of with t8;1, with orocations ,7. MYC es in CLL , and sims in CLL . Increasgression . Althougwith t8;1, with orC in CLL ,7. TheseMYC-IGH fusion at der 14q32 expresses the MYC -deregulatting product, while the reciprocal IGH-MYC fusion at 8q24.1 locus is transcriptionally silent [MYC expression in some CLL cases remains within the normal range [MYC expression, with highest level when involving Class I breakpoints [MYC-IGH fusion on der 14q32, with a Class III breakpoint (at least 400 Kb upstream of MYC) [The typical y silent . Despiteal range , or overal range . It has akpoints ,24. The  of MYC) .MYC extends up to about 629 Kb [POU5F1B and several genetic variants or SNPs  . It also highlights the Class III translocation breakpoint upstream of MYC, including the cancer and CLL-associated SNPs within the interval. This report also provides important and promising findings for further studies correlating Myc expression with a specific type of genomic translocation breakpoint or copy number variants in CLL and in other B-cell disorders. Lastly, overall findings in our report highlight the utility of karyotype analysis, interphase and sequential metaphase FISH studies, CMA, and other molecular tools in approaching the diagnosis and prognosis of CLL in a more comprehensive manner.This paper presents an unreported atypical rearrangement involving the Conventional GPG-banded chromosomal analysis was performed on peripheral blood lymphocytes that were cultured for 48 and 72 hours with and without pokeweed mitogen stimulation, following standard cytogenetics protocols. The karyotypes were described according to the ISCN 2009 nomenclature .ATM) and 17p13.1 (TP53). Sequential GPG-metaphase FISH studies were performed using IGH-MYC fusion (with centromere 8-specific probe), and break apart probes for the IGH@ and MYC loci.Initial FISH studies were performed on interphase cells using CLL panel probes  specific for centromere 12, IGH@ break apart or IGH@-CCND1 fusion, and chromosome loci 13q14.3 (D13S319)/13q34, 11q22.3 .Chromosome microarray analysis was performed on DNA sample from 48-hr culture of peripheral blood lymphocytes. DNA was extracted from Carnoy’s fixed pellet cells Qiagen DNA extraction kit . DNA concentration and quality was checked using Nanodrop  and gel electrophoresis, respectively. Whole genome chromosome SNP microarray was performed to assess for imbalances (i.e. gain or losses) in the genomic DNA sample tested. The assay compared the patient’s DNA to a reference set from 380 normal controls (284 HapMap and 96 Affymetrix reference), using the Genome-Wide SNP Array CytoScan HD. This array platform contains 2.6 million markers for Copy Number Variant detection , which 750,000 are genotype SNPs and 1.9 million are non–polymorphic probes, for the whole genome coverage. The analysis was performed using the chromosome analysis suite (ChAS), version CytoB-N1.2.2.271(r4615). Oligonucleotide probe information is based on the 37 build of the Human Genome  were stained for Myc using rabbit monoclonal human anti-Myc antibody . Heat-induced epitope retrieval was accomplished by using ER1 for 20 min. Endogenous peroxidase was blocked using hydrogen peroxide. The slide was incubated in the primary antibody Myc for 30 min, followed by incubation in a post-primary 3,3-diaminobenzidine for 10 min, polymer 3,3-diaminobenzidine for 10 min, and chromogen 3,3-diaminobenzidine for 10 min. Subsequently, slide was incubated with post-primary alkaline phosphatase for 20 min, polymer alkaline phosphatase for 30 min, and fast red for 20 min. The nuclei were counterstained with hematoxylin and the slide was then dehydrated, cleared in xylene, and coverslipped. Appropriate positive controls were used.The authors declare they have no competing interests.IA performed metaphase studies; gathered data for karyotype, interphase and metaphase FISH studies; analyzed, interpreted and wrote the cytogenetic report; analyzed and interpreted chromosome microarray data; and did necessary revisions in the manuscript as requested by the reviewers. PHB analyzed the molecular data, correlated clinical findings, and wrote the initial draft of the paper. BS provided the flow cytometry and immunohistochemistry data. SK reviewed the microarray data and paper draft. CT analyzed and reviewed all the data and drafted the paper. All authors read and approved the final manuscript.


Genome-wide studies of bacterial gene expression are shifting from microarray technology to second generation sequencing platforms. RNA-seq has a number of advantages over hybridization-based techniques, such as annotation-independent detection of transcription, improved sensitivity and increased dynamic range. Early studies have uncovered a wealth of novel coding sequences and non-coding RNA, and are revealing a transcriptional landscape that increasingly mirrors that of eukaryotes. Already basic RNA-seq protocols have been improved and adapted to looking at particular aspects of RNA biology, often with an emphasis on non-coding RNAs, and further refinements to current techniques will improve our understanding of gene expression, and genome content, in the future. The advent of second generation sequencing technologies has created many opportunities to improve functional genomics experiments, including quantitative gene expression studies. Most previous transcriptional analysis methods have relied on hybridization of targeted oligonucleotides to particular loci for their sequence specificity: either primers binding to target cDNA in quantitative reverse transcription polymerase chain reaction (qRT-PCR), labeled probes binding to RNA in Northern blotting or hybridization of cDNA to probes on microarray chips. RNA-seq is different in principle in that data are matched to genes by sequence alignment instead.This has intrinsic advantages: first, because no probe sequences are specified, all transcription is studied in an unbiased manner, and experimental design does not need to be altered in accordance with differences in genome sequence. This promises to be a particular advantage in the study of bacteria with large amounts of genetic variation between strains . It alsoSecond, mapping of sequence data is more precise than hybridization between oligonucleotides. This allows transcription to be studied at a much higher resolution by sequencing, thereby also permitting the study of more repetitive regions of the genome. Additionally, it means quantification of gene expression by RNA-seq does not suffer from the issues of interference between genes due to non-specific hybridization of cDNA to probes .Third, whereas hybridization-based methods measure gene expression levels through detection of fluorescence or radioactivity, RNA-seq uses the amount of data matching a given coding sequence (CDS), typically quantified as reads per kilobase CDS length per million reads analyzed (RPKM) . This meRNA is typically extracted using organic solvents or commercially available kits; however, care should be taken to ensure the method does not bias the sampling of the transcriptome  and is cMycoplasma pneumoniae and Pseudomonas syringae transcriptomes [Helicobacter pylori and Salmonella enterica Typhimurium [N-glycosylase [In the original RNA-seq protocols, following extensive DNase treatment, RNA was typically converted into cDNA through random hexamer-primed reverse transcription followed by second DNA strand synthesis •. Howeveriptomes ••,17• anhimurium ••,18• (Fhimurium , bisulfihimurium , additiohimurium  and incocosylase . The impS. Typhimurium, coimmunoprecipitation of RNA molecules with Hfq, a chaperone that facilitates hybridization between ncRNA and mRNA, was used to enrich a sample for transcripts participating in such interactions [Vibrio cholerae, a very stringent depletion and size-selection process was used to specifically sequence small ncRNA [As well as surveying the entire transcriptomes of bacterial strains, RNA-seq can be adapted to other experiments as well. For instance, techniques have been developed to specifically sequence the 5′ region of RNA molecules, allowing the identification of putative transcriptional start sites and helping to define operons and ncRNA ••,13• F. In S. Tractions •, while ll ncRNA . RNA-seqll ncRNA  and soilll ncRNA .Illumina, 454 and SOLiD sequencing platforms have been used in bacterial RNA-seq studies . Each ofHowever, above a certain threshold, obtaining longer reads results in a relatively small increase in the amount of the genome that can be studied, hence read depth will be the more important consideration in almost all cases.After sequencing, reads can be assembled using software either based on overlap graphs, such as EDENA , or de BRNA-seq, as with comparable methods, requires biological replicates for robust quantification of differential expression. However, the greater cost of sequencing relative to microarray hybridization makes such repetition expensive, so statistical methods have been developed to overcome this by modeling the expected distributions of sequence reads mapping to a locus in different samples. DEGseq  uses a PThe results of bacterial RNA-seq studies have done much to refine our understanding of bacterial gene expression. One initial insight was that genome-wide CDS expression levels appear to be continuously distributed, with no obvious division between actively expressed genes and a ‘background’ transcription level . By contM. pneumoniae, which has a genome just 816 kb in size [Annotation of CDSs has been significantly improved using RNA-seq data. Novel CDSs have been identified in most studies •,13•,17•M. pneumoniae and H. pylori, annotation of transcriptional units was complicated by an unexpectedly high level of flexibility in the structure of operons [M. pneumoniae was estimated to be similar to that in some eukaryotes [However, in both  operons ••,16••. Sulfolobus solfataricus, mapping these ends was also used to detect putative transcript degradation products. Enrichment of such sites was found to inversely correlate with the half life of the RNA molecule, suggesting an endoribonucleolytic cleavage mechanism may be important in gene regulation [All these amendments to genome annotation are aided by having information on the 5′ ends of transcripts; in gulation •.H. pylori, both in silico analysis and mutational inactivation suggested that one novel ncRNA uncovered by RNA-seq regulated a chemotaxis receptor as an antisense RNA [V. cholerae, which was found to down regulate mannitol metabolism [Bacterial whole transcriptome studies have thus far had a very high success rate of ncRNA discovery. Such transcripts have even been identified and mapped to genomes from marine metatranscriptome data, where certain putative ncRNA showed distinct spatial distributions throughout the water column . Validatense RNA ••, and atabolism .cis antisense ncRNA: regions of CDSs that are bidirectionally transcribed, and suggested to act to block expression of the encoded protein [Directional RNA-seq data are particularly helpful in annotating ncRNA, as it allows reads to be assigned to a particular strand. Furthermore, it has allowed the detection of large amounts of  protein , but thecis or trans, may prove to be much more important than previously appreciated.Overall, bacterial transcription is starting to appear to more closely mirror that of eukaryotes. Rather than operons being fixed polycistronic transcriptional units, they may represent one way, of several, of transcribing a particular gene, with CDSs having a greater than expected level of independence from their neighbors. Additionally, antisense RNAs, acting either in RNA-seq datasets have proved to be highly consistent, when comparing either technical or biological replicates, making them appropriate for expression studies . HoweverTo circumvent such issues, techniques such as direct RNA sequencing  and FRT-RNA-seq promises to gradually replace microarrays in most, if not all, genome-wide gene expression studies. Both technologies have their own limitations, but the opportunity to quantitatively study transcription to single nucleotide resolution makes RNA-seq increasingly attractive as sequencing become cheaper and easier. The use of protocols that sequence RNA in a strand-specific manner, and identify transcriptional start sites, will prove especially useful in the identification of ncRNA and defining the operons to which genes belong. Hence there is the potential for this technique to greatly refine our understanding of bacterial gene regulation.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis.Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR).  Steroid hormones are essential for maintaining normal homeostasis and reproductive capability. Biosynthesis of all steroid hormones starts in the mitochondrion with conversion of cholesterol into pregnenolone by the cholesterol side-chain cleavage enzyme cytochrome P450  Star promoter and mediate transcription of this gene Star transcription involves acetylation and methylation of histones bound to the Star promoter In the adrenal and gonads, regulation of steroidogenesis is mediated partially by mechanisms that enhance the transcription, translation, and/or activity of StAR Star mRNA. Rodent steroidogenic cells express two main transcripts (1.6- and 3.5-kb), as well as a minor 2.8-kb form Star possess similar polyadenylation sites that yield equivalent alternative transcripts Star transcript is preferentially synthesized relative to the 1.6-kb mRNA after cAMP stimulation and then preferentially degraded after removal of the stimulus Star. Most genes that are controlled at the level of mRNA stability are involved in acute cellular responses to stimuli, such as early response genes, cytokines, and inflammatory mediators Star form contains a region containing AU-rich elements (AUREs) and a sequence called the basal instability region (BIR) Star mRNA. A cAMP-stimulated AURE-independent mechanism that targets selective turnover of the 3.5-kb Star mRNA has also been suggested Star gene also reveals putative cytoplasmic polyadenylation elements (CPEs) flanking one of the distal poly(A) signals Star mRNA In addition, post-transcriptional mechanisms, such as polyadenylation, also regulate Recently, the discovery of natural antisense transcripts (NATs) has added an additional level of regulation to gene expression. NATs, also named endogenous antisense transcripts, are single-stranded RNAs that are complementary to mRNA sequences  Star mRNA. We also demonstrated its expression in MA-10 Leydig cells and steroidogenic murine tissues. Finally, we established that human chorionic gonadotropin (hCG) increases Star NAT expression via cAMP in MA-10 Leydig cells. Taken together, these data confirm the involvement of NATs in hormonal regulation.As mentioned above, regulation of StAR protein is a complex process. Given the functional diversity of NATs and growing evidence of their regulatory impact on protein expression, we investigated whether antisense transcription plays a role in modulating StAR protein expression and thus steroidogenesis. We identified a novel NAT that is perfectly complementary to Star mRNA, we performed a computational analysis by aligning the murine Star gene (Gene ID: 20845) with mouse expressed sequence tags (ESTs) using BLAST and the UCSC Genome Browser Star mRNA (NCBI Reference Sequence: NM_011485) and properties such as sequence orientation and localization of poly(A) signals and tails were analyzed. According to these criteria, several EST clones demonstrated transcription from the opposite direction  and the 3′-UTR of the longest Star sense mRNA -treated RNA control was analyzed to identify any RNA contamination in the reaction. In addition, RT-PCR of a DNase I-treated RNA sample without reverse transcriptase was performed. Two controls were conducted in order to evaluate the strand specificity of the reverse transcription: first, an RT reaction conducted in the absence of primer and a second RT reaction done in the presence of a non-specific primer. PCR product was not detected from these negative control reactions. Moreover, RT reactions were carried out at high temperature  in order to avoid primer-independent cDNA synthesis +] and non-polyadenylated [poly(A)-] RNA molecules. To determine the class in which Star NAT should be categorized, MA-10 cell total RNA was divided into two fractions and analyzed in parallel by sequence-specific RT, PCR, and nested PCR following DNase I treatment. Star NAT was amplified from poly(A)+ but not poly(A)− RNA, indicating that this transcript belongs to the class of polyadenylated RNAs. However, neither a classical polyadenylation consensus region nor a poly(A) tail was detected in the Star NAT sequence.Since total RNA contains both polyadenylated -labeled riboprobes were synthesized by in vitro transcription. The Star NAT RNA probe  was complementary to 719 bp of Star NAT sequence. The template for its synthesis was a pGEM®-T Easy vector containing a Star sense fragment amplified by semi-quantitative RT-PCR using the Sense 2 primer pair and cloned in the sense position relative to the T7 RNA polymerase promoter. The Star sense RNA probe  was complementary to 566 bp of the coding region of sense transcripts. The template for its synthesis was a pGEM®-T Easy vector containing a Star sense fragment amplified by semi-quantitative RT-PCR using the Sense 3 primer pair and cloned in the antisense position relative to the T7 RNA polymerase promoter. Both plasmids were linearized with Sal I and gel-purified. Another Star NAT probe  was synthesized to be complementary to 590 bp of the Star NAT sequence in a region different from the previous NAT probe used. The template for its synthesis was the Star cDNA sequence cloned into a pGEM®-T Easy vector in the sense position relative to the T7 RNA polymerase promoter as previously generated in our laboratory Xho I and gel-purified. The choice of probes size  was conducted according to the recommendations for RPA methodology 32P] UTP ; 5 mM dithiothreitol; 20 U RNAsin® inhibitor; 20 U T7 RNA polymerase; and 1X transcription buffer. After 2 h at 37°C, DNA templates were digested with RNase-free DNase RQ1. Transcription products were resolved on 5% denaturing polyacrylamide gels (8 M urea). Gel slices containing full-length transcripts were excised and incubated overnight at 37°C in gel elution buffer . The concentration of RNA probes was 3–6×104 cpm/µl, with a theoretical specific activity of 3×108 cpm/µg.Single-chain 4 cpm each) and DNase I-treated total MA-10 cells RNA (80 µg) were co-precipitated and hybridized overnight at 42°C in hybridization buffer . Then, RNase A (40 µg/ml) digestion was performed for 1 h at 32°C. The reaction was stopped by adding 0.5% SDS, followed by digestion with 12.5 mg/ml proteinase K at 37°C for 15 min. Next, a phenol/chloroform extraction was performed by adding 10 µg yeast tRNA to each sample as carrier. After ethanol precipitation, protected double-chain RNA fragments were subjected to a 5% denaturing polyacrylamide gel (8 M urea) chromatography and then visualized by autoradiography. The negative control (tRNA(+)RNase) consisted of yeast tRNA instead of MA-10 RNA. No bands are expected in this sample following digestion. The tRNA(−)RNase control consisted of yeast tRNA without ribonuclease treatment and was used to visualize the full-length probes.Probes , was subcloned from a pGEM®-T Easy vector into the eukaryotic expression vector pcDNA3.1(+) using Not I restriction sites.Star NAT or an empty vector in Opti-MEM medium and LipofectamineTM 2000 reagent according to the manufacturer's instructions. Transfection efficiency was estimated at approximately 30% by counting fluorescent cells transfected with the pRc/CMVi plasmid expressing enhanced green fluorescent protein (EGFP) For transient transfection, MA-10 cells were seeded the day before and grown up to 80% confluence. Transfection was performed with a pcDNA3.1(+) plasmid expressing Star sense RNA probe was synthesized by in vitro transcription as we detailed previously here. After prehybridization for 6 h at 56°C, blots were hybridized at 56°C overnight with [32P]-labeled riboprobe. The hybridization solution contained 6X SSPE, 5X Denhardt's solution, 0.5% formamide, 10 ng/ml yeast tRNA, and 100 µg/ml denatured salmon sperm DNA. Blots were subsequently washed twice with 2X SSPE /0.5% SDS at room temperature; twice with 1X SSPE/0.1% SDS at 65°C; and twice with 0.1X SSPE/0.1% SDS at 65°C. StAR hybridization signals were detected using a Storm PhosphorImager  and normalized to 18S RNA levels.Samples of total RNA (24 µg) were resolved on 1.2% agarose/2.2 M formaldehyde gels and transferred onto Hybond-N+ nylon membranes . A single-strand Mitochondria were isolated as described previously Progesterone production in cell culture media was measured by RIA as described previously Figure S1Genomic view of Star gene related RNA and EST sequences. The potential antisense transcripts (NATs predicted in silico) are highlighted with a pink circle in front of the sequence name.(PPT)Click here for additional data file.


Saccharomyces cerevisiae several nutrient transporters have been identified that possess an additional function as nutrient receptor. These transporters are induced when yeast cells are starved for their substrate, which triggers entry into stationary phase and acquirement of a low protein kinase A (PKA) phenotype. Re-addition of the lacking nutrient triggers exit from stationary phase and sudden activation of the PKA pathway, the latter being mediated by the nutrient transceptors. At the same time, the transceptors are ubiquitinated, endocytosed and sorted to the vacuole for breakdown. Investigation of the signaling function of the transceptors has provided a new read-out and new tools for gaining insight into the functionality of transporters. Identification of amino acid residues that bind co-transported ions in symporters has been challenging because the inactivation of transport by site-directed mutagenesis is not conclusive with respect to the cause of the inactivation. The discovery of nontransported agonists of the signaling function in transceptors has shown that transport is not required for signaling. Inactivation of transport with maintenance of signaling in transceptors supports that a true proton-binding residue was mutagenised. Determining the relationship between transport and induction of endocytosis has also been challenging, since inactivation of transport by mutagenesis easily causes loss of all affinity for the substrate. The use of analogues with different combinations of transport and signaling capacities has revealed that transport, ubiquitination and endocytosis can be uncoupled in several unexpected ways. The results obtained are consistent with transporters undergoing multiple substrate-induced conformational changes, which allow interaction with different accessory proteins to trigger specific downstream events.In the yeast  Plasma membrane transporters are the entry gate for most of the nutrients used by cells to generate energy and building blocks for cellular maintenance, growth and development. In this sense, they may exert important control over all cellular activities, either as simple providers of the nutrient molecules into the cellular interior or because they may signal the presence of the extracellular nutrients to the cellular machinery. A striking example of this sensing role of transporters has been discovered in yeast where multiple nutrient transporters appear to control activation of the protein kinase A pathway at the onset of fermentable growth induction. When yeast cells grow on a fermentable sugar, like glucose, they display high activity of the protein kinase A (PKA) pathway, causing low levels of storage carbohydrates, low stress tolerance, and high growth and fermentation rates. When such fermenting cells are starved for a single essential nutrient, like nitrogen, phosphate or sulfate, they will arrest growth, reduce their fermentation rate, accumulate high levels of storage carbohydrates and develop high stress tolerance, indicating that the activity of the PKA pathway is being downregulated  coupling between substrate binding/dissociation and conformational changes in the transporter; (2) coupling between the substrate binding/dissociation and protonation/deprotonation of residues; and (3) coupling between protonation/deprotonation and conformational changes in the transporter.Escherichiacoli, LacY, serves as a paradigm when assessing proton-driven transport  the substrate is initially recognized by Trp151, Arg144 and Glu126. This will lead to a disruption of the salt bridge between Arg144 and Glu126, bringing His322 in contact with Glu325. This may induce proton transfer from His322 to Glu325, leading to a rapid conformational change and to the cytoplasm facing conformation, Ci; (3) the substrate is released into the cytoplasm; (4) the salt bridge between Arg144 and Glu126 is re-established. H+ is released from Glu325. It has been suggested that Arg302 could interact with Glu325 to drive proton release from Glu325 because mutants in either residue exhibit the same specific defect in proton-coupled lactose translocation reactions, with no effect on sugar binding, exchange or counterflow    . For the human POT members PepT1 and PepT2, His57 and His87, respectively, have been suggested as primary protonation sites based on results of site-directed mutagenesis  family members. The crystal structures of oligopeptide transporters from the bacteria + transport is also believed to involve residues that undergo a protonation/deprotonation cycle. Along the transport pathway residues Asp46 and Glu135  are thought to be involved in this protonation/deprotonation cycle. Transport studies, involving site-directed mutagenesis, have confirmed that these residues play an important role. Asp46 is believed to be essential for proton-dependent active transport, whereas Glu135 might be involved in substrate recognition  proton translocation from Asp46 to Glu135 will result in abolishment of the hydrogen bond between Glu135 and Tyr365 enabling binding of the substrate with the protonated Glu135; (3) the protonation and binding of the substrate to Glu135 will trigger rigid body rotation of the N- and C-domains, resulting in the Ci conformation of the transporter. The transport cycle is completed with the deprotonation of Glu135 and the release of the substrate into the cytoplasm.The proposed mechanism for fucose:HP. indica phosphate transporter, (PiPT), the crystal structure was resolved with inorganic phosphate bound  family , T254 (TM6), N27 (TM1), N286 (TM7) and T254 (TM6). The coordination of the Na+ in Na2 is executed by G20 (TM1), V23 (TM1), A351 (TM8), T354 (TM8) and S355 (TM8).Several transporters, which were originally not assigned to sequence-related families, have in common the LeuT-like fold. To date, from the eight transporters having the LeuT-like fold . These molecules provided first of all a major new argument for the receptor function of the transporters. With these nontransported signaling agonists, the transceptors function as pure receptors. In addition, the compounds turned out to be interesting new tools for studying the signaling function of the transceptors independently of their transport function, but have also turned out to be powerful tools to investigate other outstanding questions with respect to transporter functionality and regulation.A major breakthrough in transceptor analysis was the identification of nontransported substrate analogues that were able to trigger the signaling function of the transceptor: The molecular mechanisms underlying substrate-induced endocytosis of nutrient transporters have been studied in great detail in yeast, with Gap1 serving as the main model system   Fig. b. This ion) Fig.  and tranon) Fig. . Also inon) Fig. .l-Leu-Gly is a competitive inhibitor of Gap1 transport. The same is true for l-Asp-γ-Phe, but this dipeptide is unable to trigger signaling. It was also unable to trigger endocytosis, but unexpectedly induced oligo-ubiquitination   Fig. c. This pl-lysine, l-histidine and l-tryptophan, are very well transported by Gap1 but they are not able to trigger signaling   Fig. e. These on) Fig. . Hence, These new results on substrate-induced signaling, ubiquitination and endocytosis of transceptors may also indicate that different substrates follow a somewhat different passageway when carried through the transporter, and/or that they trigger different conformational changes during their passage. Alternatively, the different substrates may trigger the same series of conformational changes but with different kinetics. To trigger a downstream event, a specific conformation would have to persist long enough to allow proper interaction with a signal transmission protein or a protein that in some way supports initiation of ubiquitination or endocytosis.Several examples have been reported where transporters appeared to carry out an additional regulatory function triggered by sensing of the nutrient (Gojon et al. AnUapA, in A. nidulans (Gournas et al. Substrate-induced internalization of nutrient transporters has also been documented in other organisms. As mentioned previously, detailed studies have been made on the uric acid/xanthine transporter, The discovery of a nutrient receptor function in a set of yeast plasma membrane transporters that are induced by starvation for their substrate is providing unexpected insight and new approaches and tools to study the mechanisms involved in transport and regulation of the transporter protein level in the membrane. It can be expected that further elucidation of the mechanisms involved in signaling by these transceptors and their connection with transceptor downregulation will provide further unanticipated findings that would have been overlooked if the proteins would just have been studied as transporters.


Arabidopsis thaliana coilin (Atcoilin) suggests that the protein is composed of three main domains. Analysis of the physical properties of deletion mutants indicates that Atcoilin might consist of an N-terminal globular domain, a central highly disordered domain and a C-terminal domain containing a presumable Tudor-like structure adjacent to a disordered C terminus. Despite the low homology in amino acid sequences, a similar type of domain organization is likely shared by human and animal coilin proteins and coilin-like proteins of various plant species. Atcoilin is able to bind RNA effectively and in a non-specific manner. This activity is provided by three RNA-binding sites: two sets of basic amino acids in the N-terminal domain and one set in the central domain. Interaction with RNA induces the multimerization of the Atcoilin molecule, a consequence of the structural alterations in the N-terminal domain. The interaction with RNA and subsequent multimerization may facilitate coilin’s function as a scaffolding protein. A model of the N-terminal domain is also proposed.Cajal bodies (CBs) are dynamic subnuclear compartments involved in the biogenesis of ribonucleoproteins. Coilin is a major structural scaffolding protein necessary for CB formation, composition and activity. The predicted secondary structure of  In addition, CBs associate with snRNA genes, histone gene clusters, Gems, and PML bodies, and also participates in the biogenesis and delivery of telomerase to telomeres. CBs are most frequently detected in cells with high transcriptional demands, such as neuronal and cancer cells or in cell lines infected by viruses , subnuclear compartments physically and functionally associated with nucleoli, participate in the maturation of splicing small nuclear ribonucleoproteins (snRNPs) and facilitates their modification and assembly XenopusArabidopsisDrosophilaXenopus coilin is only 42%, with human coilin having 576 amino acid residues compared with the 508 residues found in Xenopus coilin Caenorhabditis and SaccharomycesCoilin, a major structural scaffolding protein necessary for CB formation, composition and activity, is predominantly found in CBs, but is also distributed throughout the nucleoplasm Arabidopsis thaliana mutants that have lesions in the coilin gene are completely viable even though they cannot form CBs Drosophila, where the absence of CBs has no effect on their survivability Recent knockout and knockdown studies have demonstrated that coilin is necessary for proper CB formation, composition and activity Two important regions with sequence conservation have been identified in coilin proteins from different species Arabidopsis thaliana coilin (Atcoilin) does not contain an RG box, and it is currently not known whether any of the arginine residues in Atcoilin are dimethylated. Similarly, no homologue of SMN has currently been identified in the Arabidopsis genome Xenopus coilins was shown to bind ssDNA and poly r(G) in vitroin vitro and might take part in snRNA processing Coilin interacts directly with the survivor motor neuron (SMN), U snRNPs and Sm proteins Arabidopsis thaliana coilin molecule. The isolated domains were expressed as recombinant proteins and the physical and RNA-binding characteristics were elucidated, allowing identification of the sites responsible for the RNA-binding activity.In this work we demonstrate that coilin proteins from different origins share a similar structural organization, which enabled us to reveal three structural domains within the Protein samples at a concentration of 100 µg/ml in 1 mM phosphate buffer pH 7.5 were loaded into 1–2 mm cells, and CD spectra were recorded from 185 to 250 nm at 25°C in a “Chiroscan” CD spectrometer . The CD spectra were recorded at a speed of 0.5–1.0 nm/s with base-line subtraction. The measured spectra were smoothed using the instrument software. [θ] value calculations were based on the mean amino acid residue molecular weight of 110.Coilin proteins (0.03 mg/ml) or protein-RNA complexes in 1 mM phosphate buffer pH 7.5, were loaded into 1 cm cells of a FluoroMax  spectrofluorimeter. Samples were excited at 280-nm and emission spectra were recorded in the 300- to 400-nm range. Readings took place at 25°C.Protein samples at 0.05 mg/ml in 1 mM phosphate buffer pH 7.5 were loaded into 1 cm cells of the Zetasizer Nano ZS  dynamic light scattering device, and measurements were obtained using the He-Ne laser (633 nm). Curves were fitted using Dispersion Technology Software (DTS) version 5.10.Arabidopsis thaliana sequence . The deletion mutants were produced via PCR amplification of the desired sequence and cloned into the PQE30 expression vector using SphI and SalI restriction sites. The resulting proteins contained a 6×histidine sequence on the N-terminus. The substitution mutations (replacement of R or K with Alanines) were created using overlap PCR.The mutant variants of coilin protein were constructed using the E. coli expression strain JM109 was transformed with the plasmids, and the proteins of interest were expressed by adding 1 mM IPTG to a 1/20 diluted overnight culture, which was further shaken at 37°C for 2 h. Proteins were purified under denaturing conditions using Ni-NTA Qiagen agarose , according to the manufacturer’s protocol.The For structural studies and RNA-binding assays the recombinant proteins were renatured and the urea was removed by dialyzing the 0.1 ml sample in MilliQ water for 1.5 h at room temperature with 5 changes of water. Samples were spun to clear by centrifugation for 10 min at 10 000 rpm.2; 50 mM NaCl), and after incubation on ice for 15 min samples were loaded onto Tris-acetate agarose gels stained with ethidium bromide or Sybr Gold Nucleic Acid Gel Stain (Invitrogen). Gels were photographed and the amount of free non-retarded RNA was quantified using Gel-Pro Analyzer . U1 snRNA, U2 snRNA and pGEM7 RNA substrates were prepared by T7 transcription of PCR products containing a T7 promoter adjacent to the corresponding DNA sequence.RNA-binding activity of the proteins was tested using a gel-shift approach. RNA and protein were mixed at various ratios in RNA binding buffer , and the surface was then air dried. This sample preparation method was used to eliminate any remaining salts and minimize artifactual aggregation during drying. Atomic force microscopy (AFM) analysis was performed on these samples using a Multimode AFM with a Nanoscope IIIA Controller  in tapping mode with a typical scan rate of 1 Hz. The measurements were performed in air in tapping mode using sharp silicon cantilevers  with a guaranteed tip radius of 10 nm.Arabidopsis thaliana , Atcoilin, a 608-amino acid protein The schematic representation of coilin from Mr and the predicted MW do not correlate Xenopus coilin proteins. This is also true of Atcoilin which has a Mr near 90 kDa, although it has a predicted MW of 68.7 kDa. Secondly, there have been several unsuccessful attempts to produce stable full-length human coilin crystals There are several indications which may point to the presence of intrinsically disordered regions within coilin molecules. Firstly, it has been reported that coilin molecules from different organisms have unexpected electrophoretic mobilities, whereby the Using bioinformatic predictions of coilin structure, we suggest that Atcoilin contains two structural domains in the N-terminal part including an ordered domain on the N terminus (NOD) and a central internal disordered domain (IDD) and at least one domain in the C-terminal part  with a presumable Tudor-like structure abutting an intrinsically disordered C-terminus .Danio rerio and Xenopus laevis share a structured N-terminal region (about 100 aa), a long central disordered region and a C-terminal region which contains ordered and disordered parts  Atcoilin and its presumable NOD (1–117 aa residues), IDD (117–350 aa residues) and CTD (350–608 aa residues) domains  were expE. coli . Their sE. coli .The CD spectrum of wt coilin has two negative maxima at 208 nm and 217 nm and also has a weak signal intensity; a curve trend typical of proteins that contain a considerable amount of β-structural elements A tryptophan fluorescence approach was used to determine the packing density of the Atcoilin molecule . This meIn addition to the wavelengths at which maxima occur, it is necessary to take into account the comparative intensity of the fluorescence signal. NOD and CTD provide the peaks of pretty close intensity, despite the fact that the NOD has only one tryptophan residue compared to 6 tryptophans found in CTD. With CTD, the high signal despite the hydrophilic position of the maximum may be explained by the non-involvement of tryptophan residues in the formation of its tertiary structure. All these residues are localized in unfolded regions of the CTD: one is within a loop between the Tudor fold β-sheets, two are more upstream, and the remainder is in the disordered extreme C-terminus. The intensity of the fluorescence signal of wt coilin is much lower, whereas that of central IDD is the lowest. These data might indicate that NOD has the most stable structure, while IDD and CTD domains are rather labile with significant amounts of disorder. The intensity and maximum of the wt coilin fluorescence differs from that of the isolated domains. The fluorescence peak position of the wt coilin is nearly intermediate between the peak maximums of NOD and CTD . SimilarThus, the tryptophan fluorescence and CD data verifies the various degrees of disorder in the different coilin domains and supports the suggestion that coilin is a multidomain protein with a high degree of intrinsic disorder.Coilin is a nucleic acid-binding protein, as shown by its interaction with ds/ss DNA, poly r(U) and poly r(G) Kd values are 0.18–0.2 µM, with 50% of the RNA being incorporated into the complex at the protein:RNA molar ratio of 2∶1, and 100% of the RNA was bound at the molar ratio of 6∶1 . In the electrophoretic mobility shift assay (EMSA) the recombinant Atcoilin is able to bind all these substrates with similar characteristics: apparent o of 6∶1 , 4A. TheKd value is about 0.76 µM and full RNA binding is achieved at the high protein:RNA molar ratio of 36∶1. The higher Kd value of NOD could be due to stereochemical obstacles; in the high-order oligomer that this domain forms some of the RNA-binding sites may be deeply embedded in the complex and consequently they may not be able to interact with RNA. In contrast, the IDD complexes with RNA enter into the agarose gel. In this case no free RNA remains in the gel at the protein:RNA molar ratio 2∶1. Further addition of the protein gradually increases retardation of the complex. The apparent Kd (0.04 µM) is lower than that of the wt protein  sequence (aa 1–13) including arginine residues R7 and R9 (Kd was about 2.25 µM –3 times more than that of NOD) . Since the RNA-binding of NOD is provided by its N-terminal part, we removed some basic amino acids within it. The deletion of the N-terminal highly conservative (between humans and 7 and R9  has no e of NOD) , 4B. The of NOD) .Alignment of Atcoilin to the human coilin reveals a common pattern they share: 34RCR36 in human coilin, which corresponds to the 36RKCHR40 sequence in Atcoilin . ReplaceKd value of 1.42 uM; Kd of 0.06 uM  produces no change in the RNA binding activity, since the  protein . When th 0.04 µM . These dThus, RNA-binding sites have been identified within the Atcoilin molecule; two positive charged sets in the N-terminal half of the NOD and at least one positively charged cluster (the presumable NLS2) in the IDD. These sites are able to function independently and provide effective RNA binding in a non-cooperative or low cooperative manner.We have assessed the effect of RNA binding on Atcoilin structure and multimerization. Using tryptophan fluorescence we have demonstrated that the addition of U1 snRNA to wt Atcoilin alters its protein structure, such that the emission peak of tryptophan fluorescence is shifted from 347nm to the more hydrophobic 340 nm after complexing RNA . A similTo determine the size of the protein complexes we have used dynamic laser light scattering (DLS), an approach which allows the evaluation of the hydrodynamic radii of particles In addition, complexes of Atcoilin with U1 snRNA were visualized by AFM . In the The above data suggests that interaction with RNA not only changes the structure of Atcoilin but also results in its multimerization due to the structural alterations in the NOD.Of the two structured regions within the coilin molecule there is no structural data for NOD. We have used two bioinformatic web-services: QUARK To test the relevance of the model, we have constructed mutant Atcoilin, where one of the basic components of the fold - the long α-helix – was deleted. The secondary structure of the NOD-dα mutant  was analAddition of U1 snRNA to the NOD with the deleted α-helix (NOD-dα) results in the formation of complexes much smaller than that of the intact NOD combined with U1 snRNA . The decBioinformatic analysis has shown that despite the low homology in amino acid sequences, the coilins of different organisms demonstrate a high level of structural and organizational similarity, such that their consistent functionalities may be localized into three main domains. Our data demonstrates that coilin proteins might structurally consist of an N-terminal globular domain (NOD), a central intrinsically disordered domain (IDD) and a C-terminal domain (CTD) containing a presumable Tudor-like structure abutting a disordered C-terminus . The bioIn previous works the coilin N-terminus as well as a region in the C-terminal part was defined as being rather conserved across species http://myhits.isb-sib.ch/cgi-bin/motif_scan) to be E-rich and K-rich   . These past.cgi) . The C-tast.cgi) , S6. MorAtcoilin demonstrates effective and non-specific RNA-binding activity. This activity is provided by three sites of positively charged amino acids - two in the NOD and one in the central IDD (the predicted NLS2). All these sites are localized in disordered regions on the surface of the molecule. Both NOD RNA-binding sites, according to the structural models, are located in the loops (The RNA-binding and multimerization of coilin seem to be connected. The RNA-binding pattern of various Atcoilin mutants points to the fact that the oligomerization region lies within NOD (14–78 aa), a region which is key for RNA binding. It has been demonstrated that interaction of NOD and full-length coilin with RNA leads to multimerization/aggregation of these proteins, a process induced by the structural remodeling of NOD. The interaction of NOD with RNA may be one of the factors that induce coilin to switch from a soluble nucleoplasmic state into an aggregated complex that likely accumulates to form Cajal bodies. It is suggested that CBs facilitate snRNP biogenesis by providing a locally high concentration of the active molecules Figure S1Comparison of the FoldIndex predicted folded/unfolded regions of coilin proteins from different species.(TIF)Click here for additional data file.Figure S2E. coli (Coomassie blue staining).SDS-PAGE of Atcoilin and its isolated domains  expressed and purified from (TIF)Click here for additional data file.Figure S3Arabidopsis thaliana  and Homo sapiens . (A) Alignment of predicted NODs from these two proteins, which have a similarity score of 30%. The positive amino acids whose replacement with Alanines affect RNA-binding, are shown in bold. (B) Alignment between the Tudor-like fold of human coilin and the homologous sequence from Arabidopsis thaliana coilin. The similarity score between the compared sequences is 45%. The alignment was performed by CLUSTAW2 multiple sequence alignment . Asterisks mark the identical residues in all sequences, colons denote conserved substitutions, and dots highlight the semi-conserved substitutions.Sequence alignment of the most conserved coilin protein sequences from (TIF)Click here for additional data file.Figure S4Influence of salt concentration on the pattern of the oligomerization of coilin and its isolated domains. DLS measurement of the hydrodynamic radius was used to determine the level aggregation of Atcoilin (A), NOD (B), IDD (C) and CTD (D) after exposure to different NaCl concentrations. Atcoilin and NOD have similar levels of resistance to salt induced aggregation, whereas IDD and CTD were much more susceptible to aggregation upon salt exposure (precipitation occurred at 50 mM NaCl).(TIF)Click here for additional data file.Figure S5The putative structural organisation of the Atcoilin molecule. Domains and functionally important regions are indicated. NLS1, NLS2 and 7K (cryptic NoLS) are indicated as black boxes. The alternate charged regions within the central part of the protein are indicated by “+” or “−”, according to their charge. Folded regions are shown as spheres.(PDF)Click here for additional data file.Figure S6Arabidopsis thaliana coilin and several coilin-like proteins from various plant species. The protein sequences were obtained by using Arabidopsis coilin as a query in a Basic protein BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Plant proteins with the highest homology ratios (above 39) were selected for the alignment. We refer to them as coilin-like proteins: Brassica , Ricinus , Populus , Vitis , Medicago , Brachypodium . The alignment was performed using the CLUSTAW2 multiple sequence alignment tool. Asterisks mark identical residues in all sequences, colons indicate conserved substitutions, dots denote the semi-conserved substitutions. Extensive charged regions are marked in blue (negatively charged), red (positively charged) and yellow . The positive amino acids whose replacement with Alanines affect RNA binding of different domains in the Arabidopsis thaliana coilin (and the homologous aa residues in other proteins) are shown as white letters on black and the aa residues homologous to them are highlighted in gray. Conserved octamer KKKGQKWG is shown in a box.Sequence alignment of (TIF)Click here for additional data file.