Neat1. While interaction of protein components of paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with paraspeckles. Here, by varying paraspeckle number and size, we investigate how paraspeckles influence the nuclear organization of their non-structural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of paraspeckles, transcriptional reactivation resulted in increased number of paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-paraspeckle localization. Collectively, these results show that while paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA  The eukaryotic nucleus is the site where crucial cellular events such as DNA replication, RNA synthesis and processing take place1234314NEAT1  is a 3.17-kb long poly(A) RNA, while Men β (Neat1_v2) is a 20.7-kb long transcript that harbors a triple helical structure in its 3′end121416Neat1, paraspeckles contain several paraspeckle-localized RNA-binding proteins, collectively termed as PSPs (paraspeckle-associated proteins). In addition to the three core PSPs that are members of the Drosophila Behavior Human Splicing (DBHS) family - NonO, SFPQ and PSP1, paraspeckles also contain an additional ~40 or more PSPs78Paraspeckles are sub-nuclear bodies that are detected as variable number of discrete dots, and are preferentially located adjacent to nuclear speckles7Neat1 as the organizational component of paraspeckles49101213NEAT1_v2 forms the paraspeckle core, whereas NEAT1_v1 is recruited as a subsidiary factor12Neat1 has been shown to disrupt paraspeckle structure91012Neat1 and paraspeckle proteins demonstrate that active transcription of Neat1 regulates paraspeckle maintenancePrevious studies demonstrated Neat1 plays an important role in basic physiological functions and diseases20NEAT1 facilitates the relocation of splicing factor proline/glutamine-rich (SFPQ), a NEAT1-associated PSP, from the IL8 (Interleukine 8) promoter to paraspeckles, leading to transcriptional activation of IL8NEAT1 is also known to repress transcription of several genes, including ADARB2 (Adenosine deaminase that Acts on RNA 3), by sequestering the transcription repressor SFPQ from the promoters of these protein-coding genesNEAT1-enriched paraspeckles have been suggested to be involved in the nuclear retention of A-to-I edited transcriptsNeat1-knockout mice are viable under laboratory growth conditions, and thus, paraspeckles are considered to be nonessential nuclear bodies that are formed upon certain environmental triggers such as viral infection, proteasome inhibition and differentiation142223Neat1 and influence the spatial arrangement of Neat1 within paraspeckles. For example, NonO and SFPQ selectively associate with, and stabilize NEAT1_v2, thus contributing to the organization of the paraspeckle structureNeat1 has been studied in detail, information regarding the organization and behavior of non-structural RNA components of paraspeckles is scant25Ctn RNA is an 8 kb long, mouse-specific, nuclear-retained RNA that is induced as part of the antiviral responseCtn RNA regulates the expression of its protein-coding partner, Cat2 (mouse cationic amino acid transporter 2)Ctn RNA and mCat2 mRNA are encoded by the Slc7a2 gene, however, due to alternative poly(A) site selection, Ctn RNA contains a longer unique 3′UTRCtn RNA contains several inverted repeats of SINE origin, and several of the adenosines within these repeats undergo Adenosine-to-Inosine (A-to-I) editing by ADAR family of cellular enzymesCtn RNA is cleaved at the 3′UTR and is exported into the cytoplasm where it is translated to form mCAT2 proteinCtn RNA does not affect paraspeckle integrity, suggesting that it is a non-structural RNA component of paraspeckles10Ctn RNA has been shown to interact with PSPs – NonO and PSP1Ctn RNA with paraspeckles. In this study, we investigated how alteration in paraspeckle number and size affects the association of Ctn RNA with paraspeckles. In addition, by utilizing Ctn RNA as a model system, we determined the potential involvement of A-to-I editing in the nuclear retention and paraspeckle association of RNA.Members of the paraspeckle-resident PSPs interact with Ctn RNA is a paraspeckle-associated transcript that is A-to-I edited within its long 3′UTRCtn RNA, we determined the cellular localization of Ctn RNA in WT-MEFs (Mouse embryonic fibroblasts) and Neat1-KO (knockout) MEFs by RNA-FISH (RNA-Fluorescent in situ hybridization) analysisNeat1 lncRNA has been shown to nucleate paraspeckles and thus, in the absence of Neat1, paraspeckle structure is disrupted9101214Ctn RNA co-localized with Neat1 with in the intact paraspeckles. In addition, Ctn RNA also displayed homogenous nuclear distribution  analysis. In order to measure Ctn RNA levels specifically, and not mCat2, we used a primer pair that is unique to the long 3′UTR of Ctn RNA   . In the ar space . On the Ctn RNA with other paraspeckle components. Ctn RNA has been shown to interact with paraspeckle proteins such as NonO and PSP1Ctn RNA continues to associate with paraspeckle proteins in the absence of Neat1. To this end, we determined the localization of Ctn RNA and paraspeckle protein NonO by performing RNA-FISH followed by immunostaining for NonO in WT and Neat1-KO MEFs. We observed that as shown in previous studies, Ctn RNA and NonO co-localized in WT-MEFs  studies using the NonO antibody followed by RT-qPCR. We observed that NonO displayed increased interaction with Ctn RNA in Neat1-KO cells but not with Malat1, another highly abundant nuclear-retained long noncoding RNA  of SINE origin that are inverted with respect to the forward repeat (FwR)  . Furthermore, Ctn RNA showed similar paraspeckle localization both control and Adar1-depleted (using siRNAs) transformed WT-MEFs  26. DoublNA-FISH) 3536. In th Neat1 . Ctn RNA-KO MEFs , even th WT-MEFs . TogetheNeat1 transcription12Ctn RNA foci were also disrupted upon RNA pol II transcription inhibition, including treatment of cells with DRBCtn RNA foci upon transcription reactivation. To this end, we treated transformed WT-MEFs with DRB for three hours. Next, we removed DRB by washing cells with medium, cultured the cells in fresh media for another 3 hours, performed co-RNA-FISH and counted the number of Ctn RNA foci in both untreated and DRB recovered cells (Neat1 staining (~4/cell in control to ~8/cell in treated) (Ctn RNA decorating Neat1 positive paraspeckles (Ctn RNA foci in control cells whereas ~70% of transcription-reactivated cells showed Ctn RNA positive paraspeckles). Furthermore, Ctn RNA staining within the paraspeckles appeared more prominent in transcription-reactivated cells, indicating a possible increase in the number of Ctn RNA molecules per paraspeckle during DRB-recovery.The adenosine analogue 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) inhibits RNA polymerase II-mediated transcriptional elongation by inactivating the activity of pTEFb kinase. Previous studies have shown that paraspeckle protein PSP1 re-localized to perinucleolar caps after 5 min of DRB treatment and remarkably, paraspeckles structure was disrupted ~40 min after DRB treatment12ed cells . In agretreated) 19. In adspeckles  , Ctn RNA constitutes a stable pool of RNA 26Ctn RNA association with paraspeckles during transcription reactivation was due to changes in the total levels of Neat1 and Ctn RNA. To this end, we measured the total levels of Neat1 and Ctn RNA in control and DRB-recovered transformed WT-MEFs by RT-qPCR  cells  cells . We obsereatment  and S4.Ctn RNA locus. To test this aspect, we measured Ctn RNA levels in DMSO and MG132-treated transformed WT-MEFs by RT-qPCR analysis . Collectively, these results showed that upon proteasome inhibition, Ctn RNA formed enlarged foci. In addition, the Ctn RNA levels were only moderately increased in comparison to the marked increase in Neat1 levels.We wondered whether proteasome-inhibition also altered transcription from analysis . Consisted cells 23. On thCtn RNA and Neat1 localization studies, especially in the proteasome-inhibited cells, using conventional fluorescent microscopy indicated that only a fraction of the paraspeckle-associated Neat1 and Ctn RNA displayed complete co-localization  random self-organization or (2) ordered assembly random s438Ctn RNA forms discrete foci that localize to paraspecklesCtn RNA continues to form residual foci, a subset of Ctn RNA localizes to the perinucleolar space. This suggests that paraspeckles mediate the efficient compartmentalization of Ctn RNA, and in their absence Ctn RNA appears to acquire a more stochastic distribution in the nucleoplasm. Furthermore, in the absence of Neat1, Ctn RNA continues to associate with other PSPs, as indicated by RNA-FISH and RNA immunoprecipitation studies. This is in contrast to a previous study where the authors showed that structures induced by tethering of individual PSPs (instead of Neat1) to a LacI reporter construct did not retain Ctn RNACtn RNA, which might otherwise occur under physiological conditions.In ~30% of MEFs, a significant fraction of Neat1 and the consequent disruption of paraspeckle structure in HeLa cells resulted in the nucleocytoplasmic export of inverted Alu (IRAlu) containing mRNA40Neat1) or A-to-I editing (due to deletion of both ADAR1 & 2), Ctn RNA continued to localize in the nucleoplasm. Thus, in the case of Ctn RNA, Neat1 or intact paraspeckles do not influence its nuclear retention in MEFs. We observed Ctn RNA positive residual nuclear foci in Neat1 KO cells, some of which co-localized with other PSPs. At present, we cannot exclude the potential involvement of these residual nuclear foci in the nuclear retention of Ctn RNA. It is possible that association of Ctn RNA with other PSPs (such as SFPQ) in Neat1 or NonO-depleted cells could facilitate the nuclear retention of Ctn RNA. Our results also demonstrate that A-to-I editing of Ctn RNA is not essential for its association with paraspeckles since Adar1/Adar2 double KO MEFs showed paraspeckle localization of Ctn RNA.Previous studies have suggested that paraspeckles are potentially involved in the nuclear retention of A-to-I edited transcripts in human cellsNeat1 transcription12Ctn RNA showed increased localization to paraspeckles upon transcriptional reactivation without a concomitant increase in Ctn RNA or Neat1 levels. Earlier studies have demonstrated that paraspeckles tend to assemble in close proximity to the site of transcription of Neat1410Ctn RNA gene loci are located further away from paraspeckles as compared to Neat1 transcription siteCtn RNA localizes to paraspeckles possibly for its further processing or A-to-I editing. Future studies will investigate the potential involvement of paraspeckle in the processing of Ctn RNA.Previous studies have shown that transcriptional reactivation results in the reassembly of paraspeckles due to initiation of Neat1. The positive effect of proteasome inhibition on transcription has also been shown to occur in case of cyclooxygenase-2 (Cox-2) where increased level of this protein in response to proteasome inhibition has been attributed to its increased transcriptionCtn RNA, while we see only a marginal increase in total Ctn RNA levels upon proteasome treatment, we observe a significant increase in the size of Ctn RNA foci. At the ultrastructural level, as observed by SR-SIM, Ctn RNA adopts a more heterogeneous intraparaspeckle distribution in MG132-treated cells – with altered Neat1: Ctn RNA ratios and foci overlap. Therefore, Ctn RNA shows altered intra-paraspeckle organization upon proteasome inhibitionIn addition to being responsive to transcriptional reactivation, paraspeckles also show enlargement upon proteasome inhibitionCtn RNA forms residual foci in the absence of Neat1, though at a significantly lower level. Therefore, while Ctn RNA by itself forms ‘paraspeckle-like’ foci, Neat1 improves the efficiency of foci formation. Second, Ctn RNA can associate with other paraspeckle components (PSPs) in the absence of Neat1. The efficiency of such interactions remains largely unaffected even in the presence or absence of intact paraspeckles. From this, we infer that the non-structural RNA components do not require an intact paraspeckle structure to associate with other PSPs. Lastly, RNA components of paraspeckles – both Neat1 and Ctn RNA are responsive to environmental triggers, strengthening the view that paraspeckles function in response to certain stimuli.In summary, the results from this study further our knowledge about the organization and behavior of RNA components within the paraspeckle at several levels. First, non-structural RNA components of paraspeckles, namely, Adar1/Adar2-KO MEFs35Neat1-KO MEFsAdar1/Adar2-KO MEFs were grown in DMEM containing high glucose, supplemented with penicillin-streptomycin and 10% foetal bovine serum (FBS) . For the 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) recovery experiments, cells were treated with Ethanol or 25 μg/ml DRB . For proteasome inhibition experiments, cells were treated with DMSO or 5 μM MG132 .Total cellular RNA was isolated using Trizol  according to the manufacturer’s instructions and reverse transcribed into cDNA using Superscript III First-Strand Synthesis System for RT-PCR . qPCRs were performed using the Applied Biosystems StepOne Plus Real-Time PCR Systems . Transcript levels were quantitated against a standard curve by Real-Time RT-PCR using the SYBR Green fluorogenic dye and data analysed using the Applied Biosystems StepOne Plus Real-Time PCR Systems . Primer sets showing comparably high efficiencies were used for the analyses. The qPCR results were analysed using the comparative CtmethodNonO   and Adar1 siRNA   were used to deplete NonO and Adar1, respectively. The siRNAs were transfected to cells using Lipofectamine RNAiMAX reagent as per the manufacturer’s instructions  and incubated for 48 hrs. Knockdown was confirmed using NonO antibody Neat1-KO cells (1 × 107) were used for RNA immunoprecipitation utilizing reversible chemical crosslinking of RNA-protein interactions by formaldehyde followed by immunoprecipitation using Anti-NonO antibody . Following IP, extracts were reverse cross-linked and total RNA was extracted using Trizol LS . Extracted RNA was treated with RNase-free DNase I , and RT was conducted using random-hexamer primers as per the manufacturer’s instructions . qPCR was performed using gene-specific primers.RIP was performed using an established protocolNeat1-KO MEFs, control and NonO siRNA treated transformed WT-MEFs (1 × 106) cells were used for fractionation. Cells were washed with PBS and re-suspended in RSB buffer  and lysed in RSB buffer containing Digitonin (8 μg/ml)  for 10 min on ice. Cells were centrifuged  and the supernatant (cytoplasmic fraction) collected. The pellet (nuclear fraction) was washed with RSB and digitonin by the procedure described above. Trizol LS  was added to the cytoplasmic fraction while Trizol was added to the nuclear fraction. Ct values of nuclear or cytoplasmic fractions were normalized to total RNA.Transformed WT-MEFs, Ctn RNA and Neat1, RNA-FISH analysis was performed as previously describedCtn RNA localization to paraspeckles was increased during transcriptional reactivation. Therefore, for Ctn RNA FISH, cells were treated with the transcriptional inhibitor DRB followed by reactivation of transcription by removal of inhibitor with medium for 3 hrs. After RNA-FISH, immunofluorescence staining of NonO was performed using NonO antibody  as previously describedCtn RNA/Neat1/NonO foci are very distinct and prominent. Unless indicated, hundred cells were counted in each experiment and the experiments were performed in biological replicate.To detect Images were acquired by SR-SIM ELYRA system with Axio Observer Z1 microscope from ZEISS. 3-color imaging was performed using 488 nm, 561 nm and 632 nm lasers. Exposure time was 100 ms or less for all three channels. Exposure time and excitation power was adjusted to maximize the signal without saturating the camera. SIM images were reconstructed using commercial softwares (ZEN 2012 and ZEN 2011 from Zeiss). Profile along the drawn line was created applying default setting in ZEN 2012.All methods were carried out in accordance with relevant guidelines and regulations. The University of Illinois biosafety committee approved all the experimental protocols.How to cite this article: Anantharaman, A. et al. Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA.Sci. Rep.6, 34043; doi: 10.1038/srep34043 (2016).


Salvia miltiorrhiza is one of the most widely-used medicinal plants. Here, we systematically analyzed the RNA editing events in its mitochondria. We developed a pipeline using REDItools to predict RNA editing events from stand-specific RNA-Seq data. The predictions were validated using reverse transcription, RT-PCR amplification and Sanger sequencing experiments. Putative sequences motifs were characterized. Comparative analyses were carried out between S. miltiorrhiza, Arabidopsis thaliana and Oryza sativa. We discovered 1123 editing sites, including 225 “C to U” sites in the protein-coding regions. Fourteen of sixteen (87.5%) sites were validated. Three putative DNA motifs were identified around the predicted sites. The nucleotides on both strands at 115 of the 225 sites had undergone RNA editing, which we called symmetrical RNA editing (SRE). Four of six these SRE sites (66.7%) were experimentally confirmed. Re-examination of strand-specific RNA-Seq data from A. thaliana and O. sativa identified 327 and 369 SRE sites respectively. 78, 20 and 13 SRE sites were found to be conserved among A. thaliana, O. sativa and S. miltiorrhiza respectively. This study provides a comprehensive picture of RNA editing events in the mitochondrial genome of S. miltiorrhiza. We identified SREs for the first time, which may represent a universal phenomenon. RNA editing is an important mechanism to increase the diversities of transcriptomes and proteomes in eukaryotic organisms through post-transcriptionally modifications of mRNA sequences1145671111Arabidopsis thaliana)Oryza sativa)Brassica napus)Beta vulgaris)Vitis vinifera)Nicotiana tabacum)atp6 RNA editing in sorghum causes male sterility, which is also known as the CMS phenotyperpl16 and affected seed development in maizeOGR1 also encodes a PPR protein that is essential for RNA editing in the mitochondria and required for normal growth and developmentMitochondria are membrane-bound organelles found in most eukaryotic cells and play important roles in energy conversion, the storage of calcium ions, and other metabolic tasks, such as regulation of the membrane potential, steroid synthesis and etcSalvia miltiorrhiza (Danshen) is one of the most economically important medicinal plants, and its products have been widely used for centuries to treat various human diseases, such as cardiovascular disease, dysmenorrhea, and amenorrheaS. miltiorrhiza have been reported21S. miltiorrhiza transcriptome have been carried out2324252627S. miltiorrhiza have also been studied in details. These include genes involved in the biosynthesis of terpenoid and phenolic acid31S. miltiorrhiza’s biology. Nevertheless, as an important functional diversification mechanism, RNA editing has not been investigated in S. miltiorrhiza so far.S. miltiorrhiza based on strand-specific RNA-Seq data previously generated by our groupS. mitiorrhiza’s biology. Most interestingly, we identified 115 sites, at which the nucleotides on both strands were edited. This so-called symmetrical RNA editing (SRE) phenomenon was further identified in the A. thaliana and O. sativa mitochondria, suggesting the universal presence of SRE in plant mitochondria.With the rapid development of DNA sequencing technologies, RNA sequencing (RNA-Seq) has become an effective method for the discovery of RNA editing sites because of its massive throughput, low cost, and superior sensitivityS. miltiorrhiza has not been published by the time this study was initiated, we examined the quality of the genome assembly using three methods. Firstly, we mapped short reads generated from the same species and sequenced using the illumina platform to the reference genome. The entire assembly was covered with short reads at an average coverage of 1390×  sequence of of 1390× . Secondlof 1390× . Last, wof 1390× . The aliS. miltiorrhiza. Briefly, clean reads were obtained from the leaf , flower , and root  samples, these reads were mapped to the mitogenome with mismatch = 7. The results showed that 569,489 reads from the leaf, 221,727 reads from the flower, and 52,485 reads from the root were successfully mapped. These mapped reads were grouped based on the strands to which they were mapped by samtools. Consequently, 285,146 reads from the leaf, 111,155 reads from the flower, and 26,387 reads from the root were mapped to the forward strand. By contrast, 284,343 leaf, 110,572 flower, and 26,098 root reads were mapped to the reverse strand.We developed a computational pipeline  to identWe first carried out the analysis using data combined from the three tissue types. RNA editing sites from each strand were identified by REDItools. A total of 1123 sites were found to have undergone RNA editing, these sites will be called RNA editing site for simplicity. Among them, 575 sites were found on the forward strand and 548 were found on the reverse strand .As some of the observed nucleotide changes in mRNAs might result from the polymorphism at these sites in the genome DNA; thus, we need to identify possible SNP sites in the genome and exclude them from the steps for the prediction of RNA editing sites. We conducted a low-coverage sequencing (~40×) with whole genomic DNA and identified putative SNP sites in the mitogenome. A total of 63 SNPs and 32 indels were identified ; 24 of tS. miltiorrhiza mitogenome as constructed with OrganellarGenomeDRAWThe schematic representation of all predicted RNA editing sites is shown in We then predicted RNA editing sites using data of each tissue. All RNA editing sites found in each tissue were shown in A RNA editing site that were found missing in a particular type of tissue might be due to two reasons. Firstly, no reads were found in the region containing the RNA editing sites. Alternatively, there were reads found in the regions containing the RNA editing sites. However, the coverage and/or the editing frequency did not pass our threshold. To show how many RNA editing sites were not predicted due to each of the two reasons, we summarized the tissue distribution patterns for all RNA editing sites in In our analysis, we filtered out the sites that have undergone more than one type of RNA editing. As a result, the sites reported here are associated with only one type of RNA editing. We found 12 types of RNA editing events ; these eIn the CDS regions, the “C to U” transition is also the most abundant type, which represents 64.17% and 14.29% of all events on the forward and reverse strands, respectively . SimilarWe also examined the distribution of RNA editing sites across the CDS, intron, rRNA, tRNA, and intergenic regions . The sitA. thalianaThe 225 “C to U” RNA editing sites are unevenly distributed among the 26 protein-coding genes . OverallWe randomly selected sixteen predicted RNA editing sites for experimental validation. The results for the sixteen RNA editing sites: cox2-544, cox2-557, cox2-632, cox3-419, cox3-422, nad1-889, nad1-919, nad5-835, nad6-463, nad6-569, rps3-1471, rps-1525, rpl12-196, rpl16-146, orf214-305 and orf456-493 are shown in S. miltiorrhiza mitogenome. A total of 2899 and 2784 putative RNA editing sites were predicted on the positive and negative strands of NUMT sequences (data not shown). Comparing the set of RNA editing sites identified on NUMTs with those on the mitogenome revealed eleven possible identical RNA editing sites  was divided into two smaller loops , which contain repeats with a ~35-amino acid sequence motiftrans-factors involved in RNA editing48cis-NATs) might also be involved in the process to generate SRE. They can perfectly pair with the edited sense coding transcripts and may form double-stranded RNA in vivo in some casesPrevious research showed that several RNA modification reactions required a Taken together, we propose two possible mechanisms for the generation of SRE. First, the sequence around the RNA editing sites on the sense and antisense strands contain motifs that are recognized by the proteins involved in RNA editing. Consequently, the complementing sites on both strands were edited. Second, the double-strand RNA structure might be involved in the RNA editing process. The two sites on both strands were not distinguishable in certain contexts, thereby leading to the editing of the sites on both strands. In the future, data from other organisms need to be examined to further examine the universality and prevalence of the SRE events. Additional functional studies are needed to illustrate the mechanism for the genesis of SRE.S. miltiorrhiza strain 99-3 was grown in the experimental field of the Institute of Medicinal Plant Development . The genomic DNA was extracted by the CTAB methodS. miltiorrhiza plants with the RNAprep Pure Plant Kit  according to the manufacturers’ recommendations. The total RNA samples were treated with RNase-free DNase I (Promega) to remove genomic DNA contamination.The S. miltiorrhiza was downloaded from REFSeq (NC_023209). The RNA-Seq data of the flower, leaf, and root samples  were generated in our previous studyThe flowchart for the identification of RNA editing sites is shown in FSeq NC_0209. The A. thaliana, 455 RNA editing sites are distributed in the CDS regions of 34 genes. An average of 3.4 RNA editing sites can be found on every 100 bp of sequence. The ccb206 gene has 40 RNA editing sites; the average distance between two RNA editing sites is only 14.5 bp. Consequently, the default parameters, such as mismatch number of 2, will exclude the mapping of edited reads to these regions. On the other hand, a large mismatch number for the reads mapping might lead to a high false-positive rate (results not shown).Given this mapping-based method, a critical parameter is the mismatch number used to map the reads to the reference genome. Our preliminary analysis showed that the use of a small mismatch number might lead to the significant underestimation of the RNA editing sites because several genes have RNA editing sites that are very closely located. For example, in the mitogenome of To determine the “true” number of RNA editing sites, we then developed an “assembly-based” method to predict the RNA editing sites. In this method, the clean reads obtained for the three tissue types were separately assembled by trinityTo find the optimal mismatch numbers, the mapping-based method (method 1) was used to identify RNA editing sites with the mismatch numbers ranging from 2 to 10. The identified RNA editing sites were compared with those identified by the assembly-based method (method 2). The sensitivity and specificity were calculated for the results obtained with different mismatch numbers by the following formulas: Sensitivity = (RNA editing sites discovered by both methods 1 and 2)/(RNA editing sites discovered by method 2) and Specificity = (RNA editing sites discovered by both methods 1 and method 2)/(RNA editing sites discovered by method 1). Overall, the best combination of sensitivity and specificity was observed with mismatch number = 7 . This nuThe whole genomic DNA samples were subjected to next-generation sequencing (NGS) with Hiseq2000 (Illumina) sequencing platform. A total of 93,695,535 high-quality reads with 100 bp were obtained. These high-quality reads were mapped to the mitogenome by BWA (version 0.7.12)Total genomic DNA and RNA were extracted and processed as described above. Two types of primers  were useThe sequence context of the RNA editing sites was analyzed to discover potential motifs that may determine its interaction with the RNA editing enzyme complexes. The regions covering the upstream 25 bp around the RNA editing siteshttp://raptorx.uchicago.edu/) with the default parameters.The secondary structures of RNA were predicted by RNAfold (version 2.1.6) with the default parametersA. thaliana (NC_001284.2) and O. sativa (NC_011033) mitochondria were downloaded from GenBank. The strand-specific RNA-Seq data of A. thaliana (SRR1004790) and O. sativa (SRR1618549) were downloaded from the GenBank SRA database. The SRE sites on the mitochondrial genomes of A. thaliana and O. sativa were identified by the mapping-based method with the same parameters as those described for S. miltiorrhiza.The genome sequences of To identify the conserved SRE sites, we first extracted all CDS sequences from each of the three genomes. Only CDS having homologs across the three species were subjected to the following analysis. Secondly, for each cluster of homologous CDS, multiple sequence alignment was conducted. Thirdly, the consensus sequence for each cluster was obtained. The conserved RNA editing sites were those that were aligned to the same consensus position and had undergone the same type of editing.How to cite this article: Wu, B. et al. Identification of Symmetrical RNA Editing Events in the Mitochondria of Salvia miltiorrhiza by Strand-specific RNA Sequencing. Sci. Rep.7, 42250; doi: 10.1038/srep42250 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


At low εPR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate εPR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At εPR > εPP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.Recently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA and other macromolecular regulators, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with regulatory components. Protein-like particles have four patches, with attraction strength  Upon phase separation, these organelles emerge as droplets containing condensed component proteins, from the cytoplasm or nucleoplasm where the component proteins are dilute. Membraneless organelles have been associated with many cellular functions. In particular, the condensation of component proteins may facilitate the assembly of complexes for biochemical processes, as exemplified by the nucleolus, an organelle with a primary function in ribosome pre-assembly. Component proteins usually contain intrinsically disordered regions (IDRs) and RNA-binding domains. The effects of RNA on LLPS have been investigated in many experimental studies, but conflicting results have been reported15. The aim of this study was to use computation to elucidate potential roles of RNA and other regulatory components in LLPS and the underlying physical mechanisms.There is intense current interest in membraneless organelles, thought to be formed by liquid-liquid phase separation (LLPS) of protein-RNA mixtures16. The low free energy is achieved by distinct means in the two phases: e.g., high entropy (due to low concentration) in the dispersed phase but low enthalpy (from intermolecular bonding) in the droplet phase. Raising temperature or salt concentration leads to reduced stabilization of the droplet phase by intermolecular bonding. Beyond a critical point, the distinction between the two phases vanishes, and hence droplets dissolve. The thermodynamic equilibrium between the two phases is characterized by a phase diagram, or binodal, which is specified by the concentrations in the two coexisting phases for conditions below the critical point. In general, the stronger the intermolecular bonding, the higher the critical point.A hallmark of LLPS  is thermodynamic reversibility. That is, droplets can easily dissolve upon raising temperature or salt concentration and can reform when conditions are reverted. The reversibility comes because the two phases, droplet and dispersed, are in thermodynamic equilibrium. The two phases separate when the molecules can achieve the same low free energy by adopting two distinct types of configurations, with disparate concentrations and extents of intermolecular bonding13. Reinforcing the promotional effects, both Saha et al.12 and Smith et al.13 found that molecules that competed for RNA binding with the droplet-forming proteins also suppressed phase separation. Intriguingly, Burke et al.6, Zhang et al.9, and Banerjee et al.14 observed that RNA promoted LLPS up to a point in the RNA to protein molar ratio; further increase in RNA led to LLPS suppression. The mechanism of such a dual effect remains unresolved; Zhang et al. suggested screening of interprotein electrostatic interactions by RNA  at higher concentrations, whereas Banerjee et al. speculated that increased RNA led to a charge inversion of the protein component. In yet another variation, Wei et al.15 recently found that RNA had little effects on the threshold protein concentration and critical salt concentration, but significantly reduced the protein concentration in the droplet phase. The binodals in the absence and presence of RNA were fitted to a polymer theory, with RNA treated only implicitly. Based on the fitting parameter values, the authors attributed the leftward shift of the high-concentration arm of the binodal in the presence of RNA to the latter’s weakening of interprotein attraction.Among the experimental studies on LLPS of protein-RNA mixtures, most reported that the addition of RNA promoted LLPS, indicated by either reduced threshold protein concentrations for droplet formation or increased critical salt concentrations10 and in another case, instead of RNA, the regulatory component was arginine-rich peptides 11. LLPS also occurs for, and in fact, was first observed on, globular proteins18. The phase separation of droplet-forming proteins was suppressed, with decreased critical temperatures, when another protein was added20. For both disordered and globular proteins, LLPS was promoted by crowding agents such as polyethylene glycol (PEG) or Ficoll22. Interestingly, in the latter study, bovine serum albumin (BSA) was found to suppress the phase separation of hnRNPA1 or its IDR fragment, but promoted, as if as a crowding agent, the phase separation of a protein-RNA mixture. Moreover, lysozyme exhibited a concentration-dependent dual effect on the LLPS of the IDR fragment of FUS (FUSIDR). In short, RNA and other regulatory components can exert similarly diverse effects on the LLPS of disordered and globular proteins. This is not surprising, since the LLPS of disordered and globular proteins have a common physical basis, though their phase behaviors show characteristic differences23.It should be recognized that the effects of RNA on protein LLPS are a reflection of intermolecular interactions rather than anything unique about RNA. Indeed, similar promotional effects were observed in one case when RNA was replaced by heparin 25, coarse-grained simulations26, and hybrids thereof27 have been used to determine phase diagrams of chain molecules, in particular as models of intrinsically disordered proteins (IDPs), and to address several important questions. These include how phase boundaries are affected by charge patterns along the sequence, chain length, charge mutations, and salt. The study that relates most closely to the present one is by Lin et al.25, who treated the phase separation of a mixture of two IDP sequences within a polymer theory. All these approaches have limitations and hence their predictions are necessarily qualitative. In particular, polymer theories often provide only a very crude account of excluded-volume effects23.Experimental studies have presented many observations on LLPS that beg for mechanistic answers. In principle, theoretical calculations and molecular simulations can provide these answers but still face significant technical challenges. Polymer theories29, when RNA or another regulatory component is attractive toward the droplet-forming component and hence is recruited into the droplet phase, the physical rules governing the foregoing disparate effects on LLPS are yet to be established. Toward that aim, we chose a relatively simple model for protein-regulator mixtures, to calculate phase diagrams over a wide range of parameter values. To our knowledge, this is the first computational study devoted to uncovering the physical determinants underlying the roles of RNA and other regulatory components in LLPS. Our results qualitatively recapitulate the behaviors observed in the experimental studies, and provide a physical basis, in terms of the relative strengths of protein-protein and protein-regulator interactions, to reconcile the apparently conflicting effects of RNA on droplet formation.Here we used Gibbs-ensemble simulations to investigate how regulatory components affect protein droplet formation, a pressing question that has received only scant attention in previous theoretical and simulation studies. While the promotion of LLPS by crowding agents can be understood from the perspective of excluded-volume effects23. As alluded to above, we reason that the disparate effects on LLPS are generic instead of unique to RNA, and hence can be captured by models that possess common features of mixtures of proteins with RNA or other regulatory components, in particular, polyvalent interactions. Here we used patchy particles to model protein and regulatory molecules , whereas inter-regulator interactions are weakly attractive or even purely repulsive. The latter scenario is certainly true for RNA, due to the uniform anionic charges of the phosphates. Meanwhile protein-regulator interactions are expected to be highly dependent on the particular molecules involved. Lastly, all particles occupy volume so they experience steric repulsion, which, just like the attractive interactions between patches, should play a role in determining phase diagrams, as demonstrated by effects of crowding agents.mS, where S = P or R. The patches together cover the same fraction, χ, of the surface areas of the hard cores, which have the same diameter σ for both types of particles. Each patch is a spherical disk, with the spanning polar angle θS given byIn our models, protein-like (“P”) and regulatory (“R”) particles have 4 and 2 patches, respectively, to be denoted as n with appropriate indices. The centers of the patches are positioned as a tetrahedron on a P particle but at the poles on an R particle. The greater spreading of the patches on a P particle gives it an advantage in forming bonds with multiple partner particles. A bond is formed when a patch on one particle is in contact with a patch on another particle.Each patch is identified by the unit vector from the particle center to the patch center, denoted by i and j, has a directional square-well form34,rij is the distance between the particle centers, Ωi and Ωj denote the orientations of the particles. The square-well part has the usual form,εij and λ are the strength and width, respectively, of the interparticle attraction. The interparticle attraction operates only when the patches of the two particles form a bond. The latter condition is specified by α or β refers to any of the patches on particle i or j, and θiα or θjβ is the corresponding spanning polar angle of the patch. HenceThe interaction energy between any two particles, Note that each patch can simultaneously form bonds with multiple partner particles. The total energy of the two-component patchy particle system is the sum of all pairwise interactions between particles.χ was fixed at 0.7 and λ was fixed at 0.5 σ. We set εRR = 0 (so the R particles experienced only steric repulsion between each other), and varied the strength of attraction between P and R particles, εPR, over a wide range, from 0 to 1.5εPP.In the present work, εPR, we used Gibbs-ensemble Monte Carlo simulations35 to determine phase diagrams for the protein-regulator model system over a wide range of molar ratios between P and R particles. In these simulations, fixed numbers of P and R particles were placed in a box with a fixed volume and maintained at a constant temperature. The box was divided into two regions, and the volume and the particle numbers in each region were changed to achieve equality in pressure and chemical potentials. After reaching equilibrium, the two regions represent different phases, with distinct concentrations (denoted by ρ with appropriate subscript and superscript) for the two types of particles . Likewise the temperature, T, is in units of εPP/kB, where kB is the Boltzmann constant. We denote the ratio of total R and P particle numbers as X.For each les Fig. . We refeεPR  for the pure P system  is 0.75 (in agreement with a previous study34). With increasing X, Tc increases . Here the effects of the R particles depend on the R to P molar ratio. With increasing X, Tc initially increases, reaching a maximum at X = 0.35  persists over a wider range of X .The critical temperature for LLPS is determined by the strength of the bonding networks in the droplet phase. We measured the latter by the average number of bonds, h X Fig. . At εPR nb. At low εPR, as typified by the situation at εPR = 0, the R particles preferentially partition into the dispersed phase, thereby displacing more and more of the P particles into the droplet phase. As nb at T = 0.7 increases from 6.6 at X = 0 to 7.5 at X = 1.5 . Indeed, when surrounded by pure P particles, a P on average forms 7 bonds [X = 0 in Fig. T = 0.68), while an R forms only 4.5 bonds (extrapolation of X = 0). As X increases, more and more R particles are recruited into the droplet phase, and hence they weight down the overall nb. In addition, at the highest X value  studied, In Fig. nb at εPR = 1.0 is similar to that at εPR = 0.5, but shows subtle differences  enables an R to form more bonds than a P when each is surrounded by pure P particles  by R particles  can have divergent effects (active promoter vs. active suppressor) on the phase separation of a given protein (FUSIDR), a given regulatory protein can also have distinct effects on the phase separation of two different proteins. The latter was observed with BSA acting as an active suppressor for the LLPS of hnRNPA1 but as a volume-exclusion crowder for the LLPS of a protein-RNA mixture22. Again, the distinct effects can be attributed to the differential strengths of attraction of the regulatory protein toward the droplet-forming proteins. We note that the strengths of intermolecular interactions can be directly measured by the second virial coefficient or the binding affinity, determined at concentrations below the threshold for LLPS15.As support for our contention that the findings in the present study are applicable to both rigid and flexible molecules, a concentration-dependent dual effect was also found for lysozyme, a globular protein, on the LLPS of FUS15, polyadenylate RNA had little effects on the threshold LAF-1 concentration for LLPS and critical salt concentration, but significantly reduced the protein concentration in the droplet phase. These effects are similar to our calculation results at εPR = 1.0, where the dominant effect of the R particles is to reduce et al.15 related the reduction of In another intriguing reportPII Fig. . This corks Fig. . In contet al.6 actually tested such a construct, FUSIDR, for RNA effects but the results were null. It is possible that the FUSIDR concentration in that test was too low, and that a higher FUSIDR concentration may exist where LLPS can be induced by adding RNA. We hope that these and other predicted trends in Fig. Our study has identified the relative strengths of protein-protein and protein-regulator interactions as an important physical determinant for the effects of regulatory components on protein droplet formation. In particular, active promotion of LLPS occurs only when RNA or other regulators have stronger attraction toward proteins than the latter toward themselves. Conversely, RNA is expected to behave like volume-exclusion crowders when droplet-forming proteins do not have much affinity for RNA. This can happen when droplet-forming proteins are deleted of their RNA-binding regions. Burke Different membraneless organelles may use RNA for different roles, some as clients whereas others for active promotion of LLPS. Our study demonstrates that these different roles can be fulfilled by modulating the relative strengths of protein-protein and protein-RNA interactions. Furthermore, if RNA does promote LLPS, it can do so only within a certain concentration range. This turnover behavior affords cells the ability to use RNA expression level for regulating the formation of membraneless organelles.εPR (strength of P-R attraction) between 0 and 1.5 and X (R to P molar ratio) between 0 and 1.5 were determined from Gibbs-ensemble Monte Carlo simulations35. In most simulations, the total number of particles was 512 and the total concentration, ρ0, was 0.3, a setup where the binodal for the pure P system has been obtained previously34; our simulations at X = 0 reproduced this result. Some simulations were also carried out with a total of 1000 particles (while preserving ρ0); the resulting coexistence concentrations were unchanged. In other simulations ρ0 was changed to 0.1 and the results are qualitatively consistent with those at ρ0 = 0.3. Each binodal was obtained from simulations at a series of temperature ranging from 0.65 to 0.80. At each temperature, the first 1 to 1.5 million cycles were discarded to ensure equilibration, and the subsequent 2 to 3 million cycles were used for data collection. Each cycle consisted of 500 attempts of particle displacement, 500 attempts of particle exchange, and 5 attempts of volume exchange.The phase diagrams of the protein-regulator model system for 38A and B are other fitting parameters. Each fit was performed first by including data at all available temperatures and then gradually removing data that were closer to Tc, until the fitted values of Tc were stable.The critical point for each binodal was determined by fitting the temperature dependence of the coexisting P concentrations, The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information


Vice versa, ectopic expression of GABARAPL1 decreases the invasiveness of CWR22Rv1 cells. Our previous in vitro shRNA screening identified FOXO4, a PI3K/Akt-inactivating downstream target, as a potential CaP metastasis suppressor. We show here that silencing FOXOs leads to reduced GABARAPL1 expression and enhanced invasion in LNCaP cells. Transfection of constitutively-activated Akt (myr-Akt) increased the invasion of LNCaP cells, which is associated with the inactivation of FOXOs and decreased GABARAPL1 expression. Indeed, forced expression of GABARAPL1 reversed the increased invasiveness of LNCaP/myr-Akt cells. Finally, immunohistochemistry analysis shows that Akt phosphorylation is negatively correlated with GABARAPL1 expression in human CaP tissues. Taken together, our data indicate that the suppression of FOXOs-GABARAPL1 signaling by Akt is an important mechanism for CaP progression and metastasis.Metastasis remains the primary cause of prostate cancer (CaP)-related death. Using a genome wide shRNA screen, we identified GABARAPL1 as a potential CaP metastasis suppressor. GABARAPL1 mRNA levels inversely correlate with the invasive potential of a panel of human CaP cell lines. Lower mRNA levels correlate with higher Gleason scores in clinical CaP tumor samples. Moreover, Kaplan-Meier curves analysis showed that GABARAPL1 down-regulation in cancer tissues is associated with decreased disease-free survival in CaP patients. Knockdown of GABARAPL1 in human LNCaP cells results in increased invasion  Metastasis remains the primary cause of prostate cancer (CaP)-related death, which is the second leading cause of cancer deaths in men in the U.S. . UnfortuPI3K/Akt pathway is a major contributor to CaP progression , 5. In 4in vivo. Using DNA sequence analysis and the BLAST databank search, we identified gamma-aminobutyric acid (GABA) A receptor-associated protein-like 1 (GABARAPL1) as a metastasis suppressor candidate. GABARAPL1, also known as autophagy-related 8 (ATG8) or Glandular epithelial cell protein 1 (GEC1), belongs to the GABARAP family [In order to understand the genetic components necessary for CaP metastasis, we used a high throughput shRNA screening approach to identify potential metastasis suppressors in the LNCaP orthotopic CaP model P family . GABARAPP family .in vitro and identified FOXO4, a PI3K/Akt-inactivating downstream target, as a potential CaP metastasis suppressor [Previously, we used this functional shRNA screening approach in an invasion assay ppressor , 11. Intppressor –14, indippressor . Therefoin vivo shRNA library screening of potential metastasis suppressor genes is presented in Figure in vitro. Single colonies were established and PCR sequencing analysis with the bar codes and shRNA sequences followed by the BLAST databank searching (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed to identify the genes that were silenced in these metastatic LNCaP cells by the shRNAs. These genes include GABA(A) receptor-assocated protein like 1 (GABARAPL1), Leucine proline-enriched proteoglycan (leprecan)1(LEPRE1), Myosin IIIB (MYO3B), zinc finger protein 763 (ZNF763), tRNA aspartic acid methyltransferase 1 (TRDMT1), Opoid growth factor receptor-like 1 (OGFRL1), and Dyslexia susceptibility 1 candidate 1 (DYX1C1) . Further, data mining in the Oncomine database (http://www.oncomine.org) including 4 individual studies revealed that GABARAPL1 is significantly down-regulated in metastatic CaP compared to primary tumor samples, and normal prostate tissue samples have the highest GABARAPL1 expression  , revealed that 32 cases (18%) that displayed GABARAPL1 downregulation in CaP tumor tissues had shorter disease free time compared with the 142 cases (82%) whose GABARAPL1 expression levels were not altered in CaP tumor tissues  patients by qRT-PCR. Overall, GABARAPL1 mRNA expression levels in CaP tumor tissues were significantly lower than those in the benign prostate tissues  Figure . Among t) Figure . Moreoves Figure . These dP < 0.05) increased invasion in LNCaP cells compared to control shRNA infection   or scrambled control siRNA, and subjected to Matrigel-based invasion assays. Three FOXO proteins were successfully knocked down as validated by Western blot analysis Figure . The kno) Figure . These dPI3K/Akt pathway is a major contributor to CaP progression , 5. WhenSer473 was observed in CaP tumor tissues, while no positive staining was observed in adjacent non-tumor tissues  increased the invasion of LNCaP cells associated with inactivation of FOXOs and decreased GABARAPL1. Indeed, forced expression of GABARAPL1 partially reversed the increased invasiveness of LNCaP/myr-Akt cells.Recently, a growing body of work –35 has rin vitro and in vivo RNAi screening results strongly suggest that CaP metastasis is promoted by Akt-mediated inhibition of FOXOs activation and the expression of its downstream targets such as GABARAPL1. Hence, we hypothesize that the inhibition of FOXOs-GABARAPL1 signaling by Akt is an important mechanism for CaP progression and metastasis  were used in this study: rabbit polyclonal Abs specific for pan-Akt (#ab8805), p-Akt (Ser473) (#ab81283), anti-GABARAPL1 (#ab86503), and SP rabbit HRP kit were purchased from Abcam . Anti-FOXO1 (#2880), FOXO3 (#12829), FOXO4 (#9472), p-FOXO1 (Ser256) (#9461), and p-FOXO3a (Ser253) (#9466) were from Cell Signaling Technology . Mouse monoclonal anti-GAPDH and β-actin was from Santa Cruz Biotechnology .Human CaP LNCaP cells infected with the DECODE (OpenBiosystems) pooled pGIPZ lentivirus library encoding human shRNAs were established as previously described . LNCaP, A total of 80 CaP tissue samples and 59 control prostate tissue samples from BPH patients were obtained by needle biopsy in the Department of Urology in Huashan Hospital and the Department of Urology in Cancer Hospital affiliated with Fudan University  between March and September 2013. Ten pairs of primary tumor and matched adjacent non-tumor tissue samples were obtained from the Affiliated Hospital of Xuzhou Medical College . Written consents were obtained from all subjects prior to the recruitment. The study protocol was approved by the Ethics Committee of Huashan Hospital, Cancer Hospital of Fudan University, and the Affiliated Hospital of Xuzhou Medical College. All tumor samples were confirmed to contain more than 80% tumor cells by histological examination of sequential sections by pathologists. Staging was assessed after pathological examination of formalin fixed specimens according to the 1997 TNM classification. The clinical and pathologic characteristics of the subjects were reported previously .pLKO.1-shRNA-GABARAPL1 plasmid , constit6 of LNCaP cells infected with GFP-labeled lentivirus library encoding human genomic shRNAs  were orthotopically injected into the dorsal lobes of the prostate of these male nude mice as previously described [Testosterone pellets were embedded in the flanks of 6–8 weeks old male nude mice (five mice per group) as previously described . 106 of 4 per well) overnight. Cells were transfected with 50 nM of NS-siRNA or three FOXO-specific siRNAs for 24 h using DharmaFECT1 following the manufacturer's protocol.Synthetic ON-TARGETplus SMARTpool siRNA specific for FOXO1, FOXO3 or FOXO4, siCONTROL non-specific siRNA (NS-siRNA), and DharmaFECT-1 transfection reagent were purchased from Dharmacon . LNCaP cells were plated in 6-well plates . Protein samples (40 μg) were separated by SDS-PAGE, blotted onto polyvinylidene fluoride membranes which were blocked for 30 min with 5% bovine serum albumin (Sigma) in 1 × TBS/T  and then probed with primary antibodies as indicated. Digital imaging and signal quantification were performed on a Chemi-Genius2 Bio-Imager  using GeneTools software.Modified Matrigel-based Boyden chamber assays were performed as previously described . BrieflyIHC assays were performed as previous described . Briefly−ΔΔCt method [Total RNA was isolated using TRIZOL Reagent (Sigma) and was reverse transcripted into cDNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific). Real-time PCR was performed using SYBR premix EX Taq (TaKaRa) and analyzed with CFX96 Real-Time System (BIO-RAD). PCR primer sequences are as follows: GABARAPL1: forward, 5′-AGGAGGACCATCCCTTTGAGT-3′; and reverse, 5′-TGGCCAACAGTAAGGTCAGA-3′. β-actin: forward, 5′-GACCTGACTGACTACCTCATGAAGAT-3′; and reverse, 5′-GTCACACTTCATGATGGAGTTGAAG G-3′. β-actin was used as a housekeeping gene for the qRT-PCR reactions. Each test was done in triple replication and the 2t method  was usedt-test. The differences in gene expression levels between CaP tumor tissues and BPH tissue specimens were tested by chi-square test. All statistical analyses were performed by using SPSS 16.0 software program. P < 0.05 was considered statistically significant.Statistical significances between groups were determined by two-tailed student's


Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual components contribute to these processes. In this review we discuss the roles of cellular RNA regulatory mechanisms and RNA granules in plant virus infection in the light of current knowledge and compare the findings to those made in animal virus studies. Endogenous mRNAs produced in the nucleus of eukaryotic cells are subsequently exported to the cytoplasm for protein synthesis. There they are exposed to a complex RNA regulatory network. This network is able to adjust the amount of individual mRNAs within the total RNA population and to keep translationally active and inactive mRNAs in a delicate balance. Regulation of RNA allocation and turnover concerns not only mRNAs but also other RNA types in the cells including intruding viral RNAs (vRNAs). vRNAs possess features of aberrant RNAs which are often recognized as targets of the RNA decay machinery. Typically virus infection is initiated by the production of viral replication proteins and establishment of the replication complex in which progeny genomes and subgenomic RNAs (sgRNAs) are transcribed. Newly synthesized vRNAs and sgRNAs are translated into various viral proteins which carry out all viral functions together with compatible host factors. They also fight against the host's defense pathways. Therefore, to achieve a robust and productive infection, the vRNA molecules need to be able to cope with the cellular degradation and translation machineries. The eukaryotic RNA regulatory network including antiviral defense involves several complex pathways that have been shown to interact, compete and overlap on many levels Figure  and altoArabidopsis XRN exoribonucleases participate not only in mRNA decay but also in the degradation and processing pathways of rRNAs, miRNAs and other small RNAs  RNA in yeast and cucumber necrosis virus (genus Tombusvirus) RNA in Nicotiana benthamiana  infection in N. benthamiana  tail are in general stable and translatable. They all have their individual turnover rates, some being long-lived and some taken to degradation within minutes. A constant exchange of protein factors within the messenger ribonucleoprotein (mRNP)-complexes assembling around the transcripts ensures the correct fate for each mRNA molecule during their dynamic life span  and turnip crinkle virus (genus Carmovirus) infection was detected in N. benthamiana plants expressing transiently a dominant negative mutant UPF1 protein , indicated that depletion of NMD components UPF1, Smg5 and Smg7 increase the level of replication and viral protein production in human cells  of the tobacco etch virus (genus Potyvirus) inhibited translation . SGs are sites where the already assembled ribosomal 43S and 48S pre-initiation complexes are relocated for storage  have been shown to suppress RDR6-dependent post-transcriptional gene silencing (PTGS) , RNA silencing-associated proteins and proteins regulating translational repression  nuclear shuttle protein BV1 which also causes AS2 to exit the nucleus. In the cytoplasm AS2 activates decapping by DCP2, reduces siRNA production, weakens RNAi and promotes virus infection  is a PB component and an activator of DCP2-mediated decapping. It also inhibits siRNA production and functions as an endogenous PTGS suppressor  -induced RNA granules contain both OLIGOURIDYLATE BINDING-PROTEIN 1 (UBP1) of plant SGs and VCS and AGO1 of PBs , Ras-GAP SH3 domain-binding protein (G3BP), and tristetraprolin (TTP) are among the RNA-binding proteins promoting SG formation in animal cells , DCP5, VCS, and possibly DEA(D/H)-box RNA helicase DHH1/AtRH12  form a heptameric structure that requires a protein-associated with topoisomerases (PAT1) to form a bridge between deadenylated mRNA and the decapping complex Tharun, . An ArabArabidopsis. AtG3BP was identified through its interaction with a viral protein, the nuclear shuttle protein 3 (NSP3) of the abutilon mosaic virus (genus Begomovirus), in SGs  in HeLa cells  and Poliovirus  have evolved a different strategy: viral proteins sequester TIA1 so that it cannot form functional SGs. The Dengue virus interferes with SG formation by using nonstructural protein NS1 to sequester TIA1 to the perinuclear space -type zinc finger proteins and tandem zinc finger proteins (TZF) comprise a large protein family well conserved across eukaryotes. The PB and SG component Tristetraprolin (TTP) belongs to this protein family. It has a critical role in mRNA metabolism in recruiting enzymes involved in deadenylation, decapping, and exonucleolytic activities to specific mRNAs related to innate immunity and inflammatory responses in animals proposing a role in virus infection  translation and replication . Cucumber mosaic virus 2b co-localizes with AGO1 to PBs and directly blocks AGO1's slicer activity both in vitro and in vivo  P1 protein which blocks target RNA binding to preassembled AGO1  P0  coat protein  infection proposing an important role for PBs in the elimination of vRNAs , a fish DNA virus,  V2 protein interacts directly with SGS3. Mutational analysis showed that this interaction is required for V2 silencing suppression function  TGBp1 (P25) co-localizes to siRNA bodies and inhibits dsRNA synthesis by the SGS3/RDR6 complex  interacts with SGS3 suggesting that it could act through inactivation of the RDR6/SGS3 pathway  up-regulates the expression of N. benthamiana calmodulin-like protein REGULATOR OF RNA SILENCING (Nbrgs-CaM) which is an endogenous silencing suppressor. In turn, up-regulation of Nbrgs-CaM leads to the suppression of RDR6 gene expression resulting in both PTGS suppression and symptom induction  interacts with rice and Arabidopsis RDR6 in vivo and affects the production of secondary siRNAs by RDR6  modifications of vRNA. Human immunodeficiency virus (genus Lentivirus) and hepatitis C virus (genus Hepacivirus) use this mechanism to regulate viral gene expression and infection  genomic RNA is regulated by Arabidopsis AtALKBH9B, which carries m6A demethylase activity , NMD and mechanisms targeting RNAs displaying specific secondary structures. An interesting hypothesis of the exaptive origins of regulated mRNA decay in eukaryotes has been set forward by Hamid and Makeyev . They prWhile many investigations have paid attention to the amplification of vRNA in replication complexes and post-replication fate of vRNA in relation to gene silencing, the virus-host interactions regulating the replicated vRNA through the rest of the cellular RNA regulatory network is a research area that only recently gained attention in plant biology. Viruses have evolved to regulate host RNA metabolism and SGs, PBs, siRNA bodies and other subcellular structures associated with it in order to maintain the cellular environment optimal for robust viral gene expression and avoid the elimination of vRNA. Apart from the core proteins, the protein composition of RNA granules formed by different plant viruses likely varies. Methods based on the enrichment of the RNA granules via centrifugation and subsequent affinity purification via antibodies or specific affinity tags have been developed for plant (Hafrén et al., Even with the best studied animal viruses there are still substantial knowledge gaps in understanding the viral interactions with RNA granule components as discussed by Lloyd . For exaKM: Writing the text; AL: Writing the text, drawing the figures; MP: Writing the text, Compiling the tables.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thalassoma, with abundances up to 15 times higher than any other labrid. A key locomotor modification—a winged pectoral fin that facilitates efficient underwater flight in high‐flow environments—is likely to have underpinned this global success, as numerical dominance by Thalassoma was contingent upon the presence of high‐intensity wave energy. The ecological success of the most abundant species also varied with species richness and the presence of congeneric competitors. While several fish taxa have independently evolved winged pectoral fins, Thalassoma appears to have combined efficient high‐speed swimming (to relax abiotic selection) with trophic versatility (to maximize exploitation of rich resources) to exploit and dominate extreme coral reef habitats around the world.Phenotypic adaptations can allow organisms to relax abiotic selection and facilitate their ecological success in challenging habitats, yet we have relatively little data for the prevalence of this phenomenon at macroecological scales. Using data on the relative abundance of coral reef wrasses and parrotfishes (f. Labridae) spread across three ocean basins and the Red Sea, we reveal the consistent global dominance of extreme wave‐swept habitats by fishes in the genus  In identifying the dominant taxa among locations and wave exposures, we explored the macroecological consequences arising from an ecologically relevant adaptation  that has independently evolved among several lineages of ray‐finned fishes Fulton, . We focu2−2), and the total number of reef crest labrid species (richness) tallied for each location. To explore the relationship between species richness and relative abundance, we divided the density of the most abundant species by the density of the second most abundant species as a conservative measure of numerical dominance. We then explored the extent to which numerical dominance of the most abundant species varied across a sixfold gradient in labrid species richness, which can represent a gradient of competitive pressure from confamiliar fishes that tend to have very similar resource requirements  wave‐swept crest habitats were investigated for eleven coral reef locations across three ocean basins and the Red Sea Table . We usedThalassoma (the most abundant labrid genus) and all other genera, we quantified the abundance of all labrid fishes within shallow reef habitats (3 m depth) that are either sheltered (back reef) or directly exposed (crest) to incident trade wind‐driven waves across a 40‐km cline on the northern Great Barrier Reef (GBR), Australia ‐transformed data to explore differences in the relative density of Thalassoma versus other genera with the fixed factors of continental shelf position and habitat zone (crest/back), and random reef replicates nested within shelf position. A minimum of 9,500 permutations of residuals under a reduced model were performed using type III sums of squares within the PERMANOVA+ (v1.0.6) module of Primer (v6.1.16). We then fitted least‐squares linear and nonlinear (quadratic) regressions between fish density and mean habitat flow velocities to explore trends in the abundance of the two fish groups. All regression were fitted using Sigmaplot (v9.01). To examine links with fin morphology, we plotted mean density against the pectoral fin aspect ratio for each species present within the most exposed reef crests of the outer Great Barrier Reef, and documented the pectoral fin aspect ratio for the top three species present in each of our global locations. Pectoral fin aspect ratios were mostly taken from published sources  were calculated from calibrated images taken by the first author or by J. E. Randall  of all labrid individuals within exposed coral reef crests  had a density that was 5.3 ± 1.1 times higher than the next most abundant labrid species. The extent of numerical dominance by the top species increased along a gradient in species richness, with the greatest dominance evident in low‐diversity systems  was seven to eight times more abundant than their confamiliars, versus two to three times more abundant in Indonesia and Papua New Guinea , and over ten times more abundant than species of all the other labrid genera  pectoral fins have emerged as a significant adaptation in the biology and ecology of several independent reef fish lineages , possibly because the next most abundant species was often another Thalassoma. Presumably, this pattern is influenced by the tendency for close relatives to be most ecologically similar and compete most strongly for the same resources , Thalassoma fishes are known for their flexible trophic morphology and broad dietary preferences relative to their confamiliars, and generalized foraging among a wide spectrum of coral reef microhabitats  with broad trophic preferences, but rounded pectoral fins, is one of the most common labrids within wave‐sheltered and deeper reef habitats across tropical and temperate latitudes (Fulton & Bellwood Thalassoma exhibit a range of traits that predispose them to success within a range of habitat types to which they gain access according to their locomotor capabilities.Trophic versatility may be one such trait that helps explain the global success of Thalassoma may have been a key component of this invasion, which coincided with a wave of phenotypic innovations among the Labridae, and speciation of Thalassoma (Bernardi et al., Thalassoma to gain a competitive edge in exploiting challenging high‐flow environments around the world. For Thalassoma, it appears the energy savings afforded by a highly tapered pectoral fin have allowed the relaxation of environmental selection and an ecological release in shallow wave‐swept habitats, where the ecological success of Thalassoma has manifested in the global domination of these extreme coral reef habitats.Colonization of wave‐swept habitats by fishes appears to have been a relatively recent phenomenon in the evolution of coral reef ecosystems, with an initial invasion in the early Miocene about 23–20 Ma (Bellwood, Goatley, & Bellwood, We have no conflict of interests to declare.10.5061/dryad.24q8p.Supporting data is available in Table


Amphistegina lobifera collected along a cross-shelf gradient of temperature and nutrients fluctuations. We analysed survivorship, bleaching frequency, chlorophyll a content and fecundity. Elevated temperature and nitrate significantly reduced survivorship and fecundity of A. lobifera across populations studied. This pattern was exacerbated when combined with below optimum light levels. Inshore populations showed a consistent resistance to increased temperature and nitrate levels, but all populations studied were significantly affected by light reduction. These findings demonstrated the capacity of some populations of LBF to acclimate to local conditions; nonetheless improvements in local water quality can ultimately ameliorate effects of climate change in local LBF populations.Large benthic foraminifera (LBF) are crucial marine calcifiers in coral reefs, and sensitive to environmental changes. Yet, many species successfully colonise a wide range of habitats including highly fluctuating environments. We tested the combined effects of ocean warming, local impacts and different light levels on populations of the common LBF  Marine environments are projected to change at unprecedented rates due to global warming and local disturbances136Phenotypic plasticity is one mechanism that allows organisms to adjust their physiology and morphology according to local conditions3) test, producing mainly calcitic tests3 cycling in the ocean14Large benthic foraminifera (LBF) are a group of single-celled protists that build a calcium carbonate A. gibbosaA. radiataA. lobifera living in different reef locations respond differently to elevated temperatures26Amphistegina are well adapted to live under low-nutrient conditions due to their symbiotic relationship with algal endosymbiontsA. radiataA. lobifera to increases in nitrate levels (up to 4.5 μM) can also be dependent on their local habitatA. lobifera as a model organism to study the influence of local habitat in the responses of holobiont systems to the combined effects of ocean warming and local impacts. We compared populations that have previously showed differential sensitivity to the independent effects of elevated temperature or nitratea (Chl a) content and fecundity of A. lobifera individuals collected from different reef sites. Specimens were exposed to the combined effects of different conditions of temperature and nitrate in two independent full orthogonal design experiments using two different light levels. This approach allows us to significantly improve our understanding of how predicted changes in the local environment, caused by the combinations of local and global impacts, are likely to affect the life history and biology of Amphistegina populations and possibly other holobiont systems.Here, we use the LBF morpho-species A. lobifera. However, the combined effect of reef site and temperature had a significant influence on survivorship  to an average of ~5% when exposed to high temperature and nitrate levels .Bleaching frequency was variable across treatments, and the single effect of temperature or nitrate was significant, as well as the interaction between these factors . In conta content of foraminifera showed no significant differences across reef sites and treatments  coupled with intense visible light can also cause loss of symbionts. This might be the case for inner- and mid-shelf individuals, which showed a reduction in chl a content but consistent reproduction and low mortality. However, outer-shelf populations not only showed signs of symbiont loss, but also significant declines in survivorship and fecundity when exposed to the combined effects of heat and nitrate stress.The number of bleached individuals was not different between light treatments, and most populations across different reef sites showed relatively low bleaching frequencies of ~25–30%, even when exposed to high temperature and nitrate levels. In general, individuals exposed to reduced light levels across reef sites showed a consistently high concentration of chl Survivorship was also divergent across reef sites. In the outer-shelf population, the interaction of parameters tested likely caused initial bleaching (within a few days), which quickly progressed to mortality, as evidenced by the reduction in survivorship after 30 days under control light levels. The decline in survivorship was even more severe under reduced light levels. The addition of nitrate has been demonstrated to lower the threshold of tolerance of holobiont organisms to the effects of heat stress333729A. lobifera tends to attain large sizes, compared to other species within the genus, thereby increasing fecundityA. lobiferaA. lobifera populations is essential for survival in turbulent, shallow, subtidal environments40Reduced light levels completely suppressed asexual reproduction, indicating the important role of light in the reproduction of this species. A. lobifera was greatly reduced across all reef sites studied under reduced light levels. Thus, even though inner-shelf reef populations were more resistant than mid- and outer-shelf populations to elevated temperature and nitrate, they failed to consistently reproduce asexually under reduced light levels. Decreases of LBF abundance and diversity on inshore reefs of the GBR are commonly correlated to terrestrial runoff and decreases in water quality1846A. lobifera populations can survive and reproduce under heat and nutrient stress, the reduction in light levels below their optimum is likely to be the key factor affecting their abundance and occurrence on inshore reefs of the GBR and elsewhere.The interaction between light, temperature and nitrate can be complex. On the Great Barrier Reef, flood events typically result in a significant transitory reduction in light availability and peaks in nutrients on inshore reefs, which can occasionally reach offshore reefs43A. lobifera populations collected across a water quality and temperature gradient. Whether this phenomenon is driven by adaptation of endosymbionts or the host itself remains to be investigated.Our results demonstrate that, similar to other holobiont organisms, such as reef building coralsAlong with reef-building corals, reef-dwelling LBFs play a fundamental role in the formation and maintenance of reef ecosystems, as they constitute a significant proportion of the reef sediment and carbonate budget1519Amphistegina, have been present on reefs worldwide for over 50 million yearsCoupled with results published previously26A. lobifera were collected from inner-, mid- and outer-shelf reefs located on the northern Great Barrier Reef in August and September 2014. Samples were collected by SCUBA divers from the back slope of reefs with similar habitat located on: (1) inner-shelf – Martin reef ; (2) mid-shelf – Lizard Island ; and (3) outer-shelf – Yonge reef  at depths of 6 to 8 m 26et al.et al.−1). Amphistegina individuals were extracted and separated for the experiments. We selected adults (>0.5 mm in diameter) of uniform brown colour that displayed reticulopodial activity , and specimens were acclimatised for ten days prior to commencing the experiments.Pieces of dead coral rubble containing A. lobifera individuals to different light levels (control and reduced levels). We used a flow-through system (~750 ml min−1) supplied with raw seawater over a period of 30 days in outdoor aquaria at the LIRS. For each combined-effect experiment, we tested three different conditions of temperature and nutrients with three replicate glass tanks per treatment (n = 3), for total of 27 tanks per experiment. 100 specimens from each site were randomly assigned and placed into two separate Petri dishes in each tank (50 specimens per dish), and used to assess two important biological parametersa content. A total of three replicate Petri dishes per biological parameter analysed was used. These biological processes have been shown to be negatively affected by increases in temperature and nitrate levels in both laboratory and field settings2526A. lobifera were kept in Petri dishes containing small pieces of reef rubble (<1 cm2).We tested the combined-effect of different levels of temperature and nutrient conditions on the foraminifera in two independent experiments exposing 3−) to the seawater. Similar to the temperature, water was either fed straight into the tanks (ambient control: 0.09 ± 0.01 μM NO3−), or stored in sumps. Two concentrated solutions of NaNO3− (0.1 mM and 0.4 mM) were delivered into 5 l sumps at a constant rate (1 ml min−1) using peristaltic pumps. Water from the sumps was distributed into each of the respective treatment tanks at 150 ml min−, and NO3− concentrations in the tanks were kept at either 1.1 ± 0.3 or 3.2 ± 0.9 μM NO3−. Tanks were kept in the outdoor aquaria under shade cloth reducing natural sunlight light levels down to 98.7 ± 8.2 μmol photon m−2 s−1 at midday.Incoming natural seawater was either fed straight into the tanks  or stored in two different sumps, heated to either 27 ± 0.7 °C or 30 ± 0.8 °C using feedback controlled heaters, and then gravity fed into each relevant treatment tank. Nutrient levels were manipulated by adding nitrate , and temperature and nutrient manipulations were regulated in the overhead sumps and then fed into the relevant treatment tanks. Temperature was kept constant at 24 ± 0.3 °C (ambient controls), or heated up to 27 ± 0.8 °C or 30 ± 0.2 °C. Nitrate levels in ambient control tanks were 0.07 ± 0.01 μM NO® data loggers (Model UA-002-64) were placed in each sump, and in one tank per treatment to record temperature and light intensity every 15 min, for the duration of the experiments. Additionally, two replicate water samples were collected from each tank every week, filtered (0.45 μm) and frozen for further laboratory analysis. Dissolved nitrate concentrations were determined using a Lachat flow injection analyser in the Advanced Water Management Centre at the University of Queensland .For both experiment 1 and 2, HOBOA total number of 50 individuals from experiments 1 and 2 were assessed daily regarding the number of dead individuals and reproduction. Using a stereomicroscope, dead individuals were counted and percentage calculated. Dead individuals were adult-sized (from 0.5 mm to 1.2 mm of diameter), had no obvious reticulopodial activity, were usually epiphytised and contained no trace of internal debris.Amphistegina sp. present a trimorphic life cycle, which consists of alternations between gamont, agamont and schizont forms58Fecundity was calculated according to Hallocka (Chl a) content of live individuals of A. lobifera holobionts was also measured. Each specimen was individually placed into 1.5 ml vials filled with 320 μl of 90% ethanol to extract pigment25® Plus 384, Molecular Devices, Australia). After extraction, specimens were dried at room temperature for 24 h, and weighed using a very fine scale . Chl a concentration was calculated according to Hosono et al.For both experiments 1 and 2, we determined the frequency of bleaching by counting the number of individuals showing any sign of symbiont loss  and the percentage of bleached individuals was calculated. Bleached individuals were easily identified as they usually contain some remanent of cytoplasm, resulting from the digestion of diatomslme4 in Ra (Chl a) content was analysed with a Partly Nested ANOVA using the function aov in R. Because the Chl a content at control light levels violated the homogeneity of variance assumption, data were square root transformed. In all cases, reef site, temperature and nitrate concentration were considered fixed factors, while tank was the random effect. Tank was considered the blocking factor, and temperature and nitrate between-block effects. Reef site was used as a within-block effect. Significance of single and interaction effects of fixed factors was quantified using Type III Sum of Squares. Tukey’s post-hoc test was conducted using the package multcomp in R. Fecundity was analysed using an unbalanced ANOVA to test for differences in number of new individuals produced by adults exposed to different treatments across reef sites. A linear regression using the total number of new individuals produced and size of adults that reproduced in each reef site population at any given treatment was plotted. Due to the small sample size of overall outer-shelf individuals that reproduced, assumptions of normality and homogeneity of variance were not met. Therefore, we performed a non-parametric Kruskal-Wallis test to detect differences in the number of offspring between treatments, instead of an unbalanced ANOVA. A non-parametric  robust regression using the package mblm in R was performed to assess the correlation between test size and number of individuals produced by outer-shelf A. lobifera exposed to different treatments. Regression slope coefficients of the linearised function for each reef site population, which defines the relationship between number of offspring and size of adult individuals from each reef site population, were compared using a Student’s t-test. In all cases, assessment of normality and homogeneity of variance were carried out using Shapiro-Wilk’s and Levene’s tests, respectively.Each combined-effect experiment was analysed separately, as they represent two independent experiments. Survivorship and bleaching frequency were analysed with a Generalised Linear Mixed Model (GLMM) using the package How to cite this article: Prazeres, M. et al. Variation in sensitivity of large benthic Foraminifera to the combined effects of ocean warming and local impacts. Sci. Rep.7, 45227; doi: 10.1038/srep45227 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.