Drosophila RNA-binding protein Shep associates with the gypsy insulator complex and inhibits insulator activities. By mutating key residues in the RRM domains, we generated a Shep mutant protein incapable of RNA-binding, and this mutant lost the ability to inhibit barrier activity. In addition, we found that one of many wildtype Shep isoforms but not RRM mutant Shep was sufficient to repress enhancer blocking activities. Finally, wildtype Shep rescued synthetic lethality of shep, mod(mdg4) double-mutants and developmental defects of shep mutant neurons, whereas mutant Shep failed to do so. These results indicate that the RNA-binding ability of Shep is essential for its ability to antagonize insulator activities and promote neuronal maturation. Our findings suggest that regulation of insulator function by RNA-binding proteins relies on RNA-mediated interactions.Chromatin insulators are DNA-protein complexes that regulate chromatin structure and gene expression in a wide range of organisms. These complexes also harbor enhancer blocking and barrier activities. Increasing evidence suggests that RNA molecules are integral components of insulator complexes. However, how these RNA molecules are involved in insulator function remains unclear. The  Drosophila and mammals  and binds thousands of transcripts (gypsy insulator protein components Su(Hw), Mod(mdg4)67.2, and CP190  participate in insulator antagonism. Previous work showed that ectopic expression of the Shep E isoform in a wildtype background can antagonize barrier activity  double mutants and developmental defects of shep mutant neurons. Our results suggest that the RNA-binding capability of Shep is necessary to antagonize gypsy insulator activities and promote neuronal maturation.In this study, we mutated key residues in the RRMs of Shep isoform E and verified loss of RNA-binding capability. We ectopically expressed wildtype and a Shep Eshep E and pUASt-attB shep E RRM mutant constructs were transformed using PhiC31 integrase into the attP40 docking site on chromosome 2L  and verified by genomic PCR and Western Blotting upon driving with Gal4. The 6ct phenotype was scored in flies on the first day after eclosion. Other fly strains used in this study include luciferase transgenes (insulated and uninsulated UAS-luciferase) , Ser::Gal4 (BSC 6791), Mef2::Gal4 (BSC 27390), arm::Gal4 (BSC 1560), UAS-Su(Hw)-RNAi (10724 GD), BG00836shep , and 6ct; u1mod(mdg4) (BSC 59960).Stocks were raised at 25° on standard cornmeal medium. pUASt-attB Drosophila Elav and Sxl RNA-binding proteins  were designed based on structural and RNA-binding studies of the E. coli BL21(DE3)  cells and purified with Ni-NTA-agarose (Qiagen) using manufacturer protocols.Both wildtype and RRM mutant His-tagged Shep E were produced in in vitro transcribed using 32P-UTP incorporation with T7 polymerase  followed by removal of unincorporated nucleotides . 10 pmol of labeled probe was mixed with 10 μg tRNA in binding buffer  and heated to 95° for 2 min, then rapidly cooled on ice. 47 pmol protein was added and incubated on ice for 30 min. Samples were run at 4° initially at 1000 V for 1.5 min then at 250 V for approximately 3 h on a pre-run 6% GTG acrylamide gel .Single stranded RNA probes were P14 pupae were dissected and fixed with 4% paraformaldehyde for 1 h and incubated with anti-BURS . We generated single stranded 32P labeled RNA probes corresponding to fragments of two transcripts, brahma (455 nt) and CG10555 (470 nt), which are full length transcripts shown to stably associate with Shep in nuclear extracts of CNS-derived BG3 cells  null mutant at the 6ct locus  null mutant is low, but additional mutation of d05714shep results in improvement of enhancer blocking activity. We found that expression of wildtype UAS-shep E in the d05714shep, u1mod(mdg4) mutant background using the Ser::Gal4 driver substantially decreased enhancer blocking activity, represented as reduced wing notching u1shep double mutant  double mutants or developmental defects of shep mutant neurons as did wildtype Shep E. Our results suggest that Shep antagonism of the gypsy insulator requires its RNA-binding capacity.In this manuscript, we tested whether the RNA-binding capacity of Shep is essential for antagonism of gypsy enhancer blocking activities and the viability of the shep, mod(mdg4) double mutant. The observation further extends previous findings that Shep E is sufficient to inhibit the gypsy barrier activities (shep mutants (shep has been shown to be regulated in a tissue and stage-specific manner (We showed that expression of Shep E was sufficient to restore the inhibition of in cis (shep mutants are highly enriched as genomic and transcript binding targets of Shep, implying co-transcriptional regulation of gene expression by Shep (gypsy insulator and regulate neuronal maturation relies on its RNA-binding capacity, raising the possibility that RNA-mediated interactions underlie these functions. These RNA molecules may be co-transcriptionally captured as insulator components that nucleate complexes or further recruit other regulatory factors. Future studies will address these intriguing possibilities.Our findings are consistent with a recently proposed model that Shep is co-transcriptionally recruited to regulate insulator activities. Previous studies observed that stably associated RNA immunoprecipitation targets of Shep are often derived from genes to which Shep binds in chromatin, suggesting Shep recruitment


HERV-K (HML-2) is the most recent retroviral candidate to be proposed following the observation of elevated HERV-K expression in cortical and spinal neurons of ALS patients and the demonstration of HERV-K envelope protein neurotoxicity in vitro and in transgenic mice. This retroviral hypothesis is an attractive one, not least because it raises the possibility that ALS might become treatable using antiretroviral drugs. In the present study we have attempted independent confirmation of the observation that HERV-K RNA levels are elevated in ALS brain. Total RNA was extracted from the postmortem premotor cortex of 34 patients with ALS and 23 controls. Quantitative real-time reverse transcription PCR (RT-qPCR) was performed according to the MIQE guidelines using HERV-K gag, pol and env primer sets. Data was analysed by the 2-∆∆Ct method with normalisation against two reference genes, GAPDH and XPNPEP1. Geometric mean HERV-K RNA expression levels in the premotor cortex of ALS patients were not found to be different from the expression levels in non-ALS controls. Our findings do not confirm the recently reported association between elevated cortical HERV-K RNA levels and ALS, and thus raise doubts about the role of this endogenous retrovirus in ALS pathogenesis. The results of this study may have implications for ongoing clinical trials aiming to suppress HERV-K activity with antiretroviral drugs.Over the past two decades a number of studies have demonstrated activity of the retroviral enzyme reverse transcriptase in the serum of patients with sporadic amyotrophic lateral sclerosis (ALS). Known human The online version of this article (10.1186/s40478-019-0698-2) contains supplementary material, which is available to authorized users. Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease, is a fatal neurodegenerative disease characterised by loss of motor neurons from the brain and spinal cord. In Europe and the USA the incidence is about 2 cases per 100,000 person-years and survival post diagnosis is typically 3 to 5 years. In about 5–10% a family history of ALS may be obtained in a first or second degree relative. Variants causative of or associated with ALS have been identified in at least 25 different genes, and in people with or without a family history, but the aetiology of most apparently sporadic cases remains uncertain .Numerous non-genetic environmental risk factors for apparently sporadic ALS have been proposed including electromagnetic fields, heavy metals, pesticides, smoking, dietary factors, physical trauma and viral infection . EnterovIn a series of studies looking directly for evidence of retroviral involvement in ALS we have been able to exclude known exogenous retroviruses whilst repeatedly demonstrating an increased prevalence and raised levels of reverse transcriptase activity  in the serum of patients with ALS , 25, 29.Human endogenous retroviruses (HERVs), which constitute around 8% of the human genome, are thought to be the relics of retroviral germline infections that occurred millions of years ago . Most HEGAPDH -normalised RT-qPCR methods for quantification of HERV-K gag, pol and env transcripts as those described previously [XPNPEP1 (X-prolyl aminopeptidase 1), as recommended by the MIQE guidelines [env RNA expression using a similar RT-qPCR method, to exclude it as a possible cause of the previously observed raised levels of serum reverse transcriptase in ALS.In the present study we have attempted independent confirmation of elevated HERV-K transcripts in the cortex of ALS patients by using exactly the same eviously . We haveidelines . Additioidelines , in the Frozen postmortem brain material was obtained from the Medical Research Council (MRC) Neurodegenerative Disease Brain Bank Network, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, UK. Premotor cortex  from 34 patients with sporadic ALS and 23 non-ALS controls was analysed. Details including age, gender, diagnosis, postmortem delay and RNA integrity number (RIN) are presented in Additional file 60 mg pieces of frozen (− 80 °C) premotor cortex were homogenised  on dry ice in QIAzol lysis buffer (Qiagen) and extracted using the RNeasy Lipid Tissue kit (Qiagen) with on-column DNase treatment  according to the manufacturer’s instructions. RNA concentration was measured by Qubit™ RNA BR assay  and RNA quality established by Agilent RNA 6000 Nano assay .Reverse transcription of 1 μg of total RNA in a 20 μl reaction was performed using the Invitrogen SuperScript III First-Strand Synthesis Supermix for qRT-PCR (Thermo Fisher) according to manufacturer’s instructions. RNase H digestion was carried out at 37 °C for 20 mins and cDNA stored at − 20 °C. Negative control reverse transcription reactions without adding RNA were included in each batch of cDNA syntheses.XPNPEP1. Primer sequences for amplification of HERV-K gag, pol and env, and GAPDH were as described previously [-∆∆Ct method [GAPDH and XPNPEP1 reference transcripts. RT-qPCR procedures were performed in accordance with the MIQE guidelines [Real-time PCR was performed in an Applied Biosystems QuantStudio™ 5 thermocycler (Thermo Fisher), 96 well format, using Fast SYBR Green Master Mix (Thermo Fisher) in a 20 μl reaction with 2 μL cDNA and final concentrations of 0.25 μM forward primer and 0.25 μM reverse primer or 1X primer pool for eviously  and detaidelines  , which utilises the geNorm selection algorithm [A panel of 9 candidate reference gene primer sets were evaluated in order to establish the one with the most stable level of expression in ALS and non-ALS control premotor cortex samples. Briefly, RT-qPCR was used to quantify the RNA expression levels of lgorithm , along wlgorithm  which exlgorithm , geNorm lgorithm  to comprgag, HERV-K pol, HERV-K env, HERV-W env, GAPDH and XPNPEP1 PCRs were subjected to Sanger dideoxy sequencing  and sequences analysed by BLAST  in order to check the specificity of each assay. In each case amplicon size was confirmed by agarose gel electrophoresis.Amplicons generated by the HERV-K p-values of < 0.05 were considered significant. Linear regression p-values were calculated using Microsoft Excel Add-In Daniel’s XL toolbox v6.22.The statistical significance of differences between groups was assessed by 2-tailed Mann-Whitney U test (GraphPad Prism 7 software). p = 0.03). There was also a difference in gender distribution between the ALS and control groups. The ALS group was 29% female whereas the control group was 48% female. There was no significant difference in mean postmortem delay between the patients and controls; for the ALS group it was 45.1 h and for the control group 41.7 h (p = 0.64). Finally, there was a small difference between the mean RNA integrity value (RIN) of the ALS samples, 6.53, and that of the controls, 6.05 (p = 0.01). The potential effect of such imperfectly matched parameters on HERV-K expression comparisons between ALS patients and controls is considered below.Due to limited availability of suitable postmortem material it was not always possible to obtain a perfect match of all clinical parameters between the ALS patient group and the non-ALS control group. The mean age at death was 66.9 years for the ALS patients and 73.5 years for the controls  were single bands of the expected size. Melt curve analysis also revealed single dominant peaks for all assays. Amplification specificity was further confirmed by Sanger sequencing of the amplicons which demonstrated that the nucleotide sequence was as expected for all six RT-qPCR assays . PCR efficiencies for each assay were estimated from slopes of serial dilution standard curves using the formula E = 10^(− 1/slope) – 1 and all fell within the range 99% ± 6%. For each assay the R2 value of the standard curve was > 0.99. The no template controls were negative in all experimental runs and the no reverse transcriptase controls revealed that residual genomic DNA in RNA extracts typically contributed less than 1%, but always less than 3%, of the total signal in all samples.Agarose gel electrophoresis confirmed that the amplicons generated by each of the six different RT-qPCR assays . RIN values were not correlated with HERV-K gag, pol or env RNA levels when GAPDH normalisation was used but there was a slight negative correlation between high RIN values and XPNPEP1-normalised relative HERV-K gag and env RNA levels . However, when XPNPEP1 was used for normalisation the geometric mean HERV-W env RNA level in ALS was lower at 0.75 than in controls at 1.00 (p = 0.04).Figure env RNA levels showed no correlation with age or gender whether normalised by GAPDH or XPNPEP1   but there was no correlation when GAPDH normalisation was used , the same 2-∆∆Ct data analysis method and identical primer sets to those used previously [gag, pol and env, suggesting that the entire viral genome was expressed. However, in contrast to the findings of Li et al. [gag, pol or env RNA levels between ALS patients and controls, whether the data were normalised by GAPDH or XPNPEP1 reference genes.Overexpression of HERV-K has recently been proposed as a possible causative factor in patients with ALS . Althougeviously . Our obseviously  regardini et al. , we wereGAPDH-normalised HERV-K RNA expression and so their imperfect matching does not appear to explain why our results differ from those published previously [GAPDH-normalised HERV-K RNA expression and RIN we did observe a trend towards lower GAPDH-normalised HERV-K RNA levels (pol and env only) with increasing postmortem delay. Nevertheless, we consider it unlikely that this difference in mean postmortem delay explains our failure to confirm the previously published findings [n = 11) may have resulted in less statistically robust findings than those generated by our larger analysis of 34 cases.The reasons for this conflict are uncertain but several possibilities exist. Firstly, our ALS patients and non-ALS controls were not perfectly matched for age, gender, postmortem delay or RIN values and the differences between the groups were statistically significant for age, gender and RIN. However, none of these three parameters was found to be correlated with eviously . Secondleviously  and our eviously . Althougfindings  because findings  we foundfindings  it couldGAPDH which had been used previously [XPNPEP1 and GAPDH had the most stable expression in ALS and non-ALS control material, and XPNPEP1 was therefore chosen as the additional reference gene. It is noteworthy that Durrenberger et al. [XPNPEP1 as the most stable and suitable candidate. GAPDH-normalised relative expression levels and XPNPEP1-normalised relative expression levels were generally well correlated. Our conclusion that HERV-K RNA levels are not elevated in the premotor cortex of ALS patients with respect to non-ALS controls was the same whichever reference gene was used for data normalisation.For ensuring accurate quantification, the importance of selecting appropriate reference genes for normalisation in RNA expression studies has been stressed by many authors, as has the advantage of using more than one reference gene , 15. In eviously . This evr et al.  in an evGAPDH-normalised, a small negative correlation (p = 0.04) between HERV-W RNA expression and ALS was observed with XPNPEP1 normalisation due in part to four control samples showing relatively high HERV-W env expression when normalised to this reference gene. We speculate however that this small negative correlation may be unreliable due to our ALS samples having a slightly higher mean RIN value than controls, in conjunction with high RIN values being significantly correlated with lower XPNPEP1-normalised relative HERV-W expression.In the study by Li et al. , cerebraGAPDH-normalised HERV-K gag RNA in frozen postmortem neural tissues from a total of 108 ALS and control samples obtained from brain banks in the USA. As in our study, Mayer and colleagues were unable to confirm the previous findings [Another study investigating the potential role of HERV-K in ALS has just been published by Mayer et al. . Using afindings  and concfindings  who obsefindings  that expIn conclusion, our observations and those recently published by Mayer and colleagues  fail to Additional file 1:Figures S1.–Figure S11. and Tables S1–Table S3. (PDF 874 kb)


Eukaryotic genomes are pervasively transcribed. Besides protein-coding RNAs, there are different types of non-coding RNAs that modulate complex molecular and cellular processes. RNA sequencing technologies and bioinformatics methods greatly promoted the study of ncRNAs, which revealed ncRNAs’ essential roles in diverse aspects of biological functions. As important key players in gene regulatory networks, ncRNAs work with other biomolecules, including coding and non-coding RNAs, DNAs and proteins. In this review, we discuss the distinct types of ncRNAs, including housekeeping ncRNAs and regulatory ncRNAs, their versatile functions and interactions, transcription, translation, and modification. Moreover, we summarize the integrated networks of ncRNA interactions, providing a comprehensive landscape of ncRNAs regulatory roles. Th. Thst ceof human . The larof human , NONCODEof human , miRbaseof human , and cirof human .In general, ncRNAs are found to participate in multiple biological processes, regulate physiological and developmental processes or even disease. They have been identified as tumor suppressors and oncogenic drivers in various cancer types . In plan2Eukaryotic transcription from different genomic regions and RNA processing produce various ncRNA species. Several parts of DNAs could be transcribed into ncRNAs, such as protein-coding genes, enhancer regions or transposon elements . Conside2.1As early discovered ncRNA species, housekeeping ncRNAs have been intensively studied in the past decades, including rRNAs, tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs) and telomerase RNAs , 21]. T. T21]. T2.2Based on their average size, regulatory ncRNAs could be further classified as small non-coding RNAs (sncRNAs) with comprising transcripts fewer than 200 nt, and lncRNAs with greater than 200 nt. The main classes of small ncRNAs are microRNA (miRNA), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs). However, some ncRNAs with variable length might belong to two classifications at the same time, such as promoter-associated transcripts (PATs), enhancer RNAs (eRNAs), and circular RNAs (circRNAs).miRNAs are the most abundant class of small ncRNAs that are generated from transcribed hairpin loop structures , 30], , . They resiRNAs are a class of double-stranded RNA molecules and play essential roles in RNA interference (RNAi) pathway. Based on their origins, siRNAs can be divided into two subcategories, exogenous siRNA (exo-siRNA), products of exogenous nucleic acid resulting from artificial insertion or virus infections, and endogenous siRNAs (endo-siRNA) that are arising from endogenous genomic locus and mostly transcribed from transposon elements (TEs) , 34]. I. I34]. INamed after PIWI proteins, piRNAs are a class of animal-specific small ncRNAs. Mature piRNAs are derived from two major pathways, precursors are transcribed from piRNA clusters and cleaved by PIWI proteins in the primary pathway, and then modified through “ping-pong” cycle in the second , 39]. D. D39]. DLncRNAs are defined as transcripts that longer than 200 nt and lack of protein-coding ability. Based on their location with respect to protein-coding genes, lncRNA can be divided into five subcategories . Long ineRNAs are emerging as a group of ncRNAs transcribed from transcriptional enhancers. Besides similar length and protein-coding potentiality, eRNAs share some transcriptional features with lncRNAs, like having both sense and antisense transcripts by bidirectional transcription , 49], , . BidirecCircRNAs are a unique class of endogenous ncRNAs that form covalently closed loop structures. Although discovered for decades, advanced deep sequencing and bioinformatics analysis made circRNA gain more attention recently . CircRNA33.1in vivo [To define biological functions for ncRNAs, some experimental approaches are employed for the investigation of ncRNA-based targeting, like PAR-CLIP , HITS-CLin vivo , which ein vivo .Furthermore, miRNA-mRNA interactions would result in the silencing of mRNA expression. In neoplasms, upregulated miRNAs could act as oncogenes through silencing the mRNAs encoding tumor suppressor proteins . ncRNAs 3.2ncRNA-ncRNA crosstalk indicates the intersecting non-coding world, and these interactions could impact numerous biological processes, such as epigenetic modifications, transcription, and translation; hence gain a new layer of genomic regulation. Those miRNA response elements (MREs) such as circRNAs, lncRNAs, and eRNA act as competitive endogenous RNAs (ceRNAs), which has abundant implications for gene regulation during multiple physiological and pathophysiological processes at the post-transcriptional level. For example, functional experiments suggested that the circular RNA hsa_circ_0001368 decelerates the growth of gastric cancer by regulating miR-6506-5p/FOXO3 axis by serving as a ceRNAs to sponge miR-6506-5p. Thus hsa_circ_0001368 plays a tumor-suppression role in gastric cancer and may be adapted to the gastric cancer therapy .In addition to the interaction between different ncRNAs, such as ceRNAs mentioned above, there are interactions between the same kind of ncRNAs, such as miRNA-miRNA and lncRNA-lncRNA. The interactions among multiple miRNAs have enriched the complex mechanisms of post-transcriptional regulations . In mamm3.3Many ncRNAs serve as direct regulators on chromatin, as exemplified by lncRNAs and tiRNAs. Recent progress in high-throughput RNA sequencing facilitates the detection of promoter-proximal small RNAs, including tiRNAs, promoter-associated small RNAs (PASRs) , and traAn RNA sequencing technology called global RNA interactions with DNA by deep sequencing (GRID-seq) has been reported to detect chromatin-RNA interactions, comprehensively . Two hig3.4ncRNA-protein interactions have critical roles in all aspects of gene expression, therefore, plenty of approaches have been created for comprehensively analysis. Development of deep-sequencing methods together with immunoprecipitation of RNA binding proteins (RBPs) has manifested a great diversity of ncRNA-associated proteins .Housekeeping ncRNAs interacting with proteins can shape into varied ribonucleoprotein (RNP) complexes that perform various functions. For instance, snRNAs together with multiple proteins form spliceosomes (snRNPs) that participate in canonical splicing and alternative splicing. Many nucleotides in the pre-rRNA, pre-snRNA, and pre-tRNA undergo post-transcriptional modifications via small nucleolar RNP particles . What’s 4More and more evidence proves that ncRNAs provide diverse regulatory functions in the cell. Those RNA mediated interactions are often interconnected, thus, constructing complex regulatory RNA networks. Most ncRNAs, especially housekeeping ncRNAs, have been characterized through their functions in the nucleus and their interactions with various types of nuclear machinery may contribute to their nuclear retention . However4.1Based on the ceRNA hypothesis, the cross-talk between RNAs through MREs could form a large-scale regulatory network across the transcriptome, including both coding and non-coding RNAs . Like otAccording to ceRNA theory, the existence of this network has three important conditions . First, In ceRNA networks, the majority of validated ceRNAs are mRNAs. They compete for miRNA binding to sequester miRNAs from potential alternative targets, this biological role depends on their ability to compete for miRNA binding and are independent of protein-coding function . Zinc fi4.2Besides ncRNA-ncRNA interactions, ncRNAs could regulate biological processes through diverse mechanisms. ncRNAs and their mediated networks are an integral part of genomics and proteomics .ncRNAs could contribute to local and long-range genomic interactions . FunctioAt the transcriptional and the post-transcriptional levels, regulated gene expression determines the cell type-specific proteome, and ncRNAs are widely involved in protein-mediated interaction networks . There a5Benefited from the rapid advance in sequencing technologies and bioinformatics approaches, different classes of ncRNAs have been detected and discovered in the past decades, leading to a complex ncRNA world. Sufficient evidence shows that ncRNA plays significant biological roles in regulation of cellular mechanisms, and is associated to higher eukaryotes complexity. ncRNAs can interact with different species of molecules, and regulate them through various molecular mechanisms generating a complex network. ncRNA-mediated networks not only contained interplay among RNAs, but also involved in genomics and proteomics, in both nucleus and cytoplasm.The ceRNA hypothesis attributes potentially predictable function to the coding and noncoding transcriptome, which helps to systemically realize the ncRNA network. Although ceRNA networks are one of the most extensively studied ncRNA networks, novel ncRNA species will increase the complexity of these networks. Multiple types of molecules and biology pathways make it more difficult to comprehensively understand ncRNA functions in multi-level networks. Even though the accumulation of information about ncRNA interactions and their mediated networks is relatively slow, the development of tools in silico predictors and network analysis module may accelerate this process. ncRNA-mediated interactions could be captured by experiment detections, and those associations have been systematically analyzed and extended to more connections in various computation method. Based on those intricate interactions, network model could be constructed and predict potential ncRNA related associations.


Targeted needle biopsies are increasingly performed for the genetic characterization of cancer. While the nucleic acid content of core needle biopsies after standard pathology processing ) has been previously reported, little is known about the potential yield for molecular analysis at the time of biopsy sample acquisition.  Our objective was to improve the understanding of DNA and RNA yields from commonly used core needle biopsy techniques prior to sample processing.  We performed 552 ex vivo 18 and 20G core biopsies in the lungs, liver, and kidneys. DNA and RNA were extracted from fresh-frozen core samples and quantified for statistical comparisons based on needle gauge, biopsy site, and tissue type. p < 0.001).  Median tumor DNA yields from all 18G and 20G samples were 5880 ng and 2710 ng, respectively. Median tumor RNA yields from all 18G and 20G samples were 1100 ng and 230 ng, respectively. A wide range of DNA and RNA quantities  were acquired. Median DNA and RNA yields from 18G needles were significantly greater than those from 20G needles across all organs ( Core needle biopsy techniques for cancer diagnostics yield a broad range of DNA and RNA for molecular pathology, though quantities are greater than what has been reported for FFPE processed material. Since non-formalin-fixed DNA is advantageous for molecular studies, workflows that optimize core needle biopsy yield for molecular characterization should be explored. Image-guided solid tumor needle biopsies are frequently the starting point for modern cancer care. The ability to genomically characterize tumors has amplified the importance of tissue biopsies for cancer treatment selection, determining eligibility for clinical trials and understanding disease progression. In recent years, the brisk pace of discoveries revealing the genetic basis for malignant transformation has empowered oncologists, enabling therapies targeting specific molecular aberrations –3. NeedlA high-quality, high-value biopsy is now defined by sufficient cancer cellularity for diagnosis and genomic analysis . DiagnosDeoxyribonucleic acid (DNA) and ribonucleic acid (RNA) quantities required for a combination of routine clinical care, clinical trials, and research protocols often vary by individual institution and clinical team. Quantities sufficient for analysis will also vary in relation to the increasing number and range of molecular tests and technical advances in tissue analytics. Furthermore, several analyses of preanalytic factors related to tumor sequencing have raised concerns about low DNA and RNA yields from percutaneous tumor biopsies , 9.Importantly, standard core biopsy processing in pathology laboratories includes formalin fixation and paraffin embedding (FFPE). All downstream diagnostic and molecular assays are generally performed on thin sections prepared by microtomy from FFPE tissue blocks. In most studies to date, DNA and RNA content in core biopsies has been analyzed from FFPE material, whereas quantities of nucleic acid in unprocessed core biopsies are not well established. The goal of this study was to assess DNA and RNA quantities obtained using widely used core biopsy techniques from different cancer types in order to facilitate planning and decision-making with regard to molecular oncology testing. Knowledge of needle biopsy sampling capabilities can be essential for patient management in the setting of either known or suspected cancer. For both patient and healthcare provider, the anticipated value of quantitative data to plan needle biopsies is a better understanding of the potential risk versus clinical benefit –12.We performed an Institutional Review Board-approved prospective study of surgically resected specimens at a comprehensive cancer center with a waiver of informed consent. Biopsies were performed in a tissue procurement service facility under direct visualization within 2 hours of surgical excision using 18-gauge (18G) and 20-gauge (20G) core biopsy needles . Each surgical specimen was first dissected to allow direct visualization of the tumor and surrounding normal tissues. Biopsies were acquired from a variety of locations in normal parenchyma and tumor, avoiding areas of visible necrosis, and each 2 cm long core needle sampling tray was visually inspected. Core specimens that did not fill at least 85% of the sampling tray were discarded. Biopsies were performed in triplicate using 18G and 20G needles for both DNA and RNA processing. Biopsy sample sizes were estimated based on the number of samples required to achieve statistical significance from a preliminary kidney biopsy cohort. Each specimen was immediately placed in a 1.7 ml Eppendorf tube and snap frozen in liquid nitrogen. Samples were then stored in a −80°C freezer until molecular extractions were performed.μl RNase A added immediately after incubation. 50 μl of 10 nM Tris-Cl and 0.5 mM EDTA buffer  were used for the elution step.DNA was extracted using a standard protocol  with 4 μl of lysis buffer  with the addition of on-column DNase digestion before RNA purification. 30 μl of RNase-free water was used to elute all samples.RNA was extracted in an RNase-free environment according to the standard product protocol . All RNA samples were kept on dry ice during extraction. Tissues were lysed using 1.4 mm ceramic spheres  in a tissue homogenizer  and 650 DNA and RNA quantity  was calculated from concentration multiplied by volume. Concentration was measured using a spectrophotometer . If the measured ratio of absorbance at 260 : 280 was less than 1.6 for DNA or 1.8 for RNA, the samples were run for an additional time on the chromatography columns in the extraction protocol until the purity threshold was reached.p value < 0.05 was considered statistically significant. Analyses were conducted using R software version 3.1.0 .The three repeated observations for each tumor sample were averaged into a single observation for analysis after examining the variation of repeated observations using descriptive statistics and graphical displays. Box plots of averaged data were generated for each tumor separately for RNA and DNA and by needle gauge (18G versus 20G) and tissue type . For comparisons between tissue type and needle gauge, the Wilcoxon signed-rank test for paired data was used. For comparisons across organ sites (lung versus liver versus kidney), the Kruskal-Wallis rank sum test was applied. A n = 15), liver (n = 15), and kidney (n = 16) specimens were performed. A total of 552 ex vivo biopsies from 46 surgically resected lung (p < 0.001) than that from 20G needles in both tumor and normal tissue samples. Median DNA quantities were greater for lung tumor samples compared to normal lung tissue . There was no statistical difference in median DNA obtained from normal versus tumor tissues in the liver or kidney. For all cancer types sampled, the median DNA quantity acquired from single-needle pass 18G and 20G core biopsies was 5880 ng (range 1060–13390 ng) and 2710 ng (range 370–6280 ng), respectively. Box plots in For all pooled organ sites, the median DNA yield from the larger 18G biopsy needles was significantly greater (p < 0.001) than that from 20G biopsies when tumor and normal samples were pooled for all organs. Median RNA quantities were greater for lung tumor tissue compared to nonmalignant tissues for 18G and 20G from the lungs (18G biopsies (p = 0.001) and 20G biopsies (p < 0.001), respectively) and liver , but not from the kidney. The median RNA quantity from 18G and 20G cancer biopsies was 1100 ng (range 110–17210 ng) and 230 ng (range 60–5210 ng), respectively. Box plots in The median RNA yield from 18G needles was also significantly greater  have evolved, offering insights beyond traditional histopathologic or radiographic diagnoses . IncreasWhile tumor heterogeneity, cellularity, and size as well as other preanalytic parameters and factors can impact downstream analytic success, important information can be gained from studies examining DNA and RNA yield using standardized ex vivo conditions , 17. NotOnly in the lung, and not in the liver or kidney, did we observe a statistically significant difference in DNA quantities obtained from normal parenchyma versus tumor tissues. In this case, increased cell density, particularly relative to normally air-filled lung tissues, and higher nuclear to cytoplasmic ratios may account for higher quantities of genetic material in lung tumor samples versus normal aerated lung , 19. RNASimilar to other studies, we found that larger 18G needles acquired twofold more DNA and fivefold more RNA on average compared to 20G needles, suggesting that additional needle passes may be necessary to obtain sufficient genetic material when using smaller-gauge needles. The clinical implications of substantial yield variance should not be minimized however, as linear models have not been validated in clinical practice, and smaller 20G needles can effectively reveal clinically meaningful mutations in lung tumors , 21. BasThe practical implications of this study are most apparent in relation to contemporary genetic testing requirements and sources of preanalytic biopsy sample variation. Minimum DNA for NGS can vary depending upon the clinical laboratory technology platform, as well as the target enrichment strategy and number of genes tested in a panel. For example, for the NGS platform currently used at our institution—hybridization capture MiSeq Illumina-based MSK-IMPACT assay —200–250 Biopsy sample RNA profiling is highly dependent upon quality, as fragmentation or degradation by RNases can hamper mutational analysis. Microarray technologies can be used to analyze 500 ng of RNA, while NGS and amplification techniques can substantially lower thresholds for clinically meaningful sequencing, even to the level of single-cell genetic material , 25.Assuming no degradation, fragmentation, or other preanalytic disruption of biopsy samples, based on the median DNA content revealed in this study, a single 2 cm long 18G or 20G biopsy should be sufficient for most contemporary NGS assays. The same holds true for RNA; however, at the lower end of the RNA range, as low as 60 ng for 20G core needle biopsies, a single biopsy sample may be more susceptible to analytic failure. In practice, sample degradation and disruption do occur during acquisition and processing. The routine fixation methods fail to conserve the structure of nucleic acids and proteins in tissues. Even short-term treatment of sections with formalin has been shown to significantly reduce the DNA solubility. Similarly, the extraction of useful RNA from FFPE tissue is often compromised because of incomplete lysis leading to poor extraction efficiency. In a recent study, RNA extracted from FFPE samples was severely degraded compared to fresh-frozen samples . In our Although the biopsy yields reported here can serve as a reference for physicians planning or performing molecular studies, the following limitations must be considered in regard to the generalizability of our data. Many factors may reduce the quantity of RNA and DNA suitable for analysis within a small solid tumor biopsy sample, including prior chemotherapy  or tumorIn summary, we report a wide range of nucleic acid quantities obtained from core needle biopsies in organs commonly afflicted with primary or metastatic cancer. Overall, unprocessed sample nucleic acid quantities are increased relative to FFPE processed tissues; therefore, workflows that bypass fixation and paraffin-based processing may improve yield and utility of core needle sampling for molecular diagnostics.


Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5’ end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5’ genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5’ end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5’ end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood. With the advent of potent direct-acting antivirals (DAA), hepatitis C virus (HCV) can now be eliminated from the majority of patients, using multidrug therapy with DAAs. However, such DAAs are not available for the treatment of most RNA virus infections. The main problem is the high error rate by which RNA-dependent RNA polymerases copy viral RNA genomes, allowing the selection of mutations that are resistant to DAAs. Thus, targeting host-encoded functions that are essential for growth of the virus but not for the host cell offer promising, novel approaches. HCV needs host-encoded microRNA miR-122 for its viral RNA replication in the liver, and depletion of miR-122 in HCV patients results in loss of viral RNA. This study shows that a single-nucleotide mutation in HCV allows viral RNA amplification when miR-122 abundances are low, concomitant with changes in its tropism. Many cell- and virus-encoded microRNAs (miRNAs) regulate the expression of mRNAs by binding to the 3’ noncoding regions of target mRNAs. The binding is facilitated by an RNA-induced silencing complex (RISC) that mediates base-pair interactions between nucleotides two through seven in the microRNA (seed sequences) and their complementary sites in the target mRNA (seed-match sequences). This targeting event inhibits the translation of the mRNA. In addition, deadenylation at the 3’ of the mRNA, followed by decapping and 5’ to 3’ degradation of the mRNA greatly increases its turnover , 2.The growth of hepatitis C virus (HCV), a member of the flaviviridae, is dependent on the most abundant miRNA in the liver, miR-122 . In the Clinical applications of miR-122 anti-miRs first showed that sequestration of miR-122 in mice , 5 and iSera from five out of six HCV patients whose viral titers rebounded following miR-122 anti-miR treatment contained HCV genomes with a C3U mutation. Two patients contained a C3U/C27U or C3U/G28A double mutation, respectively . These fGaussia luciferase-expressing H77S.3/GLuc viral RNAs , an analog of uridine. Total RNA was isolated and conjugated to biotin in a copper-catalyzed reaction. Newly synthesized RNA was subsequently captured using magnetic streptavidin beads and quantitated by qRT-PCR using HCV-specific primers. While the abundance of wild-type RNA synthesis was reduced by ~80% in miravirsen-treated samples, only a 30% reduction was observed in C3U variant samples following miR-122 depletion compared to control miR-106b-LNA treatment .To determine whether the binding of miR-122 molecules to the C3U HCV genome plays any role at all in its replication, infectious RNAs were transfected into miR-122 knock-out Huh7.5 cells  that werTo test this hypothesis, C3U-complementary duplex miR-122 molecules (referred to as “mut-miR-122”) were employed to monitor C3U RNA replication phenotypes in cells that expressed no wild-type version of miR-122. Again, pre-treatment with miR-106b RNA mimetics did not rescue replication of wild-type or C3U viral RNAs . As was To substantiate this finding further, miR-122 knock-out cells were pre-transfected with wild-type and mut-miR-122 duplexes alone or in combination and abundances of transfected infectious RNAs were monitored over time. Co-transfection of both wild-type and mut-miR-122 had minimal additive effect on the accumulation of wild-type HCV RNA compared to transfection of wild-type miR-122 alone . In shar3 FFU/ml, virus yield was ten-fold lower in cells infected with C3U HCV virus  wild-type and mutant HCV RNA synthesized with and without a 5’ non-methylated guanosine cap analog after transfection into Huh7.5 cells. Viral RNAs containing a cap structure or a 5’ terminal ppp-N moiety in C3U HCV displayed similar stabilities to that of wild-type RNA across multiple infection time points . Data frThe interaction between the 5’ noncoding region of HCV and miR-122 at sites 1 and 2  extends Adopting Mortimer’s and Doudna’s electrophoretic mobility shift assays (EMSA) approach , we invein silico prediction and mutational analyses indicated that the C3U in the viral positive or the complementary negative strand RNA is not part of a secondary structure . As. As33]. Traditional antivirals have overwhelmingly focused on targeting virus-encoded proteins that are essential components of the viral life cycle using small molecule inhibitors. Due to the high mutation rates of RNA-dependent viral RNA polymerases, non-lethal mutations in the viral genome can result in drug-resistance phenotypes to these direct-acting antivirals. On the other hand, targeting the expression or function of host cellular factors that are vital for viral growth is predicted to result in a higher genetic barrier to resistance than direct-acting antivirals. The liver-expressed microRNA miR-122 was recently targeted in a miRNA-based anti-HCV therapeutic strategy. Pharmacological inhibition of miR-122 using modified anti-sense oligonucleotides (anti-miRs) significantly lowered the viral load in the serum of HCV-infected patients , 21.A C3U resistance-associated mutation in the miR-122 binding site 1 of the HCV 5’ noncoding region was observed in sera from patients that experienced virologic rebound several weeks following treatment with miR-122 anti-miRs. However, late rebounders did not display the C3U mutation. In follow-up studies with 18 patients, 6 of them displayed the C3U mutation at the time of rebound, and the mutation disappeared in 3 of the 6 patients before they underwent standard-of-care treatment . This leWhat is the mechanism by which the C3U variant can replicate in the presence of low amounts of miR-122? Using genetic and biochemical approaches, we discovered that miR-122 binds only at site 2 in the C3U RNA. Using EMSA, Mortimer and Doudna showed that miR-122 binds at miRNA binding site 2 with an affinity 50-fold greater than at site 1 , explainHuh7.5 Sec14L2 and Huh7.5 ΔmiR-122 cells  were maintained in DMEM supplemented with 10% FBS, 1% non-essential amino acids, and 2 mM L-glutamine (Gibco).Plasmids H77S.3 and H77S.3/GLuc genotype 1a  (generou4) were seeded in a 48-well plate and incubated overnight. Serial dilutions of virus stock were added to cells and incubated for six hours at 37°C. The diluted virus supernatant was removed and replaced with fresh medium. Media in each plate were exchanged daily. At day three post-infection, cells were washed once with PBS and fixed with cold methanol/acetone (1:1). HCV infection was analyzed by using a mouse monoclonal antibody directed against HCV core (Abcam) at 1:300 dilution in 1% fish gelatin/PBS at room temperature for two hours and an AlexFluor488-conjugated goat anti-mouse antibody (Invitrogen) at 1:200 dilution at room temperature for 1 hour. The fluorescent focus forming units were counted using a fluorescence microscope.Infectious titers were determined by a fluorescent focus forming units (FFU) assay. Huh7.5 cells (3X10Antisense miR-122 locked nucleic acid (LNA) and RG101 have been previously described , 21.3,000 Huh7.5 cells were seeded in quadruplicates in a 96-well plate one day prior to infection. The following day, cells were infected with wild-type and C3U virus at an MOI of 0.005. Six hours post infection, media was aspirated and replaced with fresh media. Media was replaced with fresh media every day. At the indicated time post-infection, cells were lysed using the Power SYBR Green Cell-to-Ct kit (Ambion), and RNAs were quantified on a Bio-rad CFX Connect quantitative-PCR (qPCR) machine and Ct values were normalized to internal control 18S ribosomal RNA expression values. The primer sequences for the HCV H77.S3 genotype were: forward 5’- CCAACTGATCAACACCAACG -3’ and reverse 5’- AGCTGGTCAACCTCTCAGGA -3’. The primer sequences for human 18S rRNA gene were: forward 5’- AGAAACGGCTACCACATCCA -3’ and reverse 5’-CACCAGACTTGCCCTCCA -3’.AGUGUUUGU-3’; hsa-miR-106b, 5’-UAAAGUGCUGACAGUGCAGAU-3’.Huh7.5 ΔmiR-122 cells were plated in 12-well dishes and transfected with annealed native miR-122 and mut-miR-122 duplexes (50nM) alone or in combination using Dharmafect I (Dharmacon) following the manufacturer’s instruction. The following day, cells were transfected with 1 μg H77 GLuc IVT RNA as stated above. Twenty-four hours post H77 RNA transfection, cells were re-supplemented with 50nM of native or mut-miR-122. Supernatants from transfected cells were collected at the indicated time points. The following oligonucleotides were used in this study: native miR-122, 5’- UGGAGUGUGACAAUGGUGUUUGU-3’; mut-miR-122, 5’-UGGAGUGUGACAAUHuh7.5 cells, plated in 60mm dishes, were transfected with 2 μg of H77 GLuc IVT RNA as stated above. Three days post-transfection, media was removed and replaced with media containing MK-0608 at a final concentration of 25 μM. RNA was collected at the indicated time points and HCV RNA was analyzed by Northern blot analysis. RNA half-lives were calculated from three independent experiments using GraphPad Prism.Fifty micrograms of IVT H77 WT GLuc-AAG and H77 C3U GLuc-AAG RNAs were capped using the ScriptGap m7G Capping System (Cellscript C-SCC30610) according to the manufacturer’s protocol. RNA was extracted using the RNeasy Mini kit (QIAGEN) following the manufacturer’s instructions. Purified RNA was transfected into Huh7.5 cells and samples were collected at the indicated time points.Following RNA transfection, secreted GLuc activity was measured in 20 μl aliquots from supernatants using the Luciferase Assay System (Promega), according to the manufacturer’s instructions. The luminescent readings were taken using Glomax 20/20 luminometer using a 10 second integration time.Virus production was done as previously described Nucleotide substitutions to pH77S.3 and pH77S.3/GLuc were completed using the QuickChange Site Directed Mutagenesis Kit (Agilent), according to the manufacturer’s protocol.For the C3U mutation, the following primers were utilized: 5’-ACGACTCACTATAGCTAGCCCCCTGATGGG-3’ and 5’- CCCATCAGGGGGCTAGCTATAGTGAGTCGT-3’. To introduce the lethal mutation to the NS5B polymerase (GDD>AAG), the following primer were used: 5’-CCATGCTCGTGTGTGCCGCCGGCTTAGTCGTTATCTG-3’ and 5’-CAGATAACGACTAAGCCGGCGGCACACACGAGCATGG-3’.For XRN1-mediated depletion, the following RNAs were utilized: sense 5’-GAGGUGUUGUUUCGCAUUAUUdTdT-3’ and antisense 5’-AATAATGCGAAACAACACCTCdTdT-3’. Sense and antisense strands were combined in 1X siRNA Buffer (Dharmacon) at a final concentration of 20 μM, denatured for 2 minutes at 98°C, and annealed for 1 hour at 37°C. As a negative control siRNA, the following oligonucleotides were used: sense 5′- GAUCAUACGUGCGAUCAGAdTdT-3’ and antisense 5’-UCUGAUCGCACGUAUGAUCdTdT-3’.Huh7.5 cells were seeded overnight in 12 well plates. The following day, 50 nM of siRNA duplexes were transfected using Dharmafect I (Dharmacon). Following overnight incubation at 37°C, cells were transfected with H77S.3/GLuc, supernatants were collected at the indicated time points and total RNA was extracted 3 days post-transfection. Depletion of XRN1 was assessed by western blot analysis.Cells transfected for 3 days were washed with PBS once and lysed in RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) in the presence of cOmplete™, EDTA-free protease inhibitor cocktail (Roche) for 30 min on ice. Lysates were clarified to remove the non-soluble fraction by centrifugation at 14,000 rpm for 10 min at 4°C. Protein concentrations were measured by Bradford Assay and 50 μg of total protein lysate was mixed with 5X protein sample buffer containing reducing agent. Samples were separated on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane (Millipore), and blocked with 5% non-fat milk in PBS-T. The following primary antibodies were used to probe the membranes: anti-Core (C7-50) , anti-Actin (Sigma), and anti-XRN1 (Bethyl Lab A300-443A.) Immunoblots were developed using Pierce ECL Western Blot Substrate (Thermo Fisher) following the manufacturer’s suggested instructions.32P dATP-RadPrime DNA labeled probes.Total RNA was extracted using the RNeasy Mini kit (QIAGEN). For Northern blot analysis of HCV and actin RNA, 10 μg of total RNA in RNA loading buffer , and 4.4% formaldehyde) was denatured for 10 minutes at 65°C, separated in a 1% agarose gel containing 1X MESA and 3.7% formaldehyde, transferred and UV-crosslinked to a Zeta-probe membrane (Bio-Rad) overnight. The membrane was blocked and hybridized using ExpressHyb hybridization buffer (Clontech) and α-Nascent HCV RNA transcripts were quantified using the Click-iT Nascent RNA Capture Kit (Thermo Fisher) following the manufacturer’s instructions. Complementary DNA was synthesized using Superscript III reverse transcriptase (Thermo Fisher) following the manufacturer’s protocol. Newly synthesized HCV transcripts were quantified using Power SYBR Green PCR Master Mix (Thermo Fisher). HCV transcript abundances were determined by comparisons to standard curves obtained from in vitro transcribed H77.S3 RNA. The primer sequences for the HCV H77.S3 genotype were: forward 5’- CGTGTGCTGCTCAATGTCTT -3’ and reverse 5’- AATGGCTGTCCAGAACTTGC -3’. Newly C3U HCV synthesized RNA abundances were represented as fold change relative to EU labeling of wild-type HCV RNA.2 in a 5 μl reaction. Reactions were heated to 98°C for 3 min, cooled to 35°C for 5 min, and incubated with miR-122 oligos in molar ratios of 0.5, 1, 2, 3, and 4 at 37°C for 30 min. For anti-miR experiments, 1 μl of miR-122 antisense oligonucleotide was added to each reaction and incubated for an additional 10 minutes. Five μl of RNA loading dye  was added and samples were separated in a non-denaturing gel  at 4°C for 2.5 hours at 20 Amps. Gel was stained with SYBR Gold (Invitrogen) to visualize the HCV RNA-miR-122 interactions.Wild-type and mutant HCV RNA oligonucleotides corresponding to H77S.3 domain I (nucleotides 1–47) were synthesized (Stanford PAN facility). HCV domain I RNA (5 μ) was mixed with 100 mM HEPES (pH 7.5), 100 mM KCl, and 5mM MgClWildtype and C3U H77.S3 IRES stem-loop I was mutated to the BoxB RNA motif sequence 5’-GCCCTGAAAAAGGGC-3’  using thStatistical analyses were performed with Prism 5 (GraphPad). A two-tailed paired Student’s t-test was employed to assess significant differences between two groups. Error bars represent standard error of the mean.S1 FigShown are the non-normalized data from (TIF)Click here for additional data file.S2 FigHuh7.5 cells were transfected with wild-type or C3U J6/JFH1 RLuc genomes and RNA replication was determined at the indicated time points. P values are < 0.0001.(TIF)Click here for additional data file.S3 FigHuh7.5 cells were transfected with chimeric J6/JFH1 RLuc genomes and RNA replication was determined at the indicated time points. P values are < 0.0001.(TIF)Click here for additional data file.S4 Fig(A) HCV RNA distribution across a sucrose gradient was determined three days following transfection of Huh7.5 cells with wild-type and C3U H77.S3/GLuc RNAs. Polysomal profile trace of lysates separated in 10–60% sucrose gradients. Individual subunits, monosomal, and polysomal peaks are indicated (top). Detection of HCV and actin RNA in sucrose fractions 2 through 13 by Northern blot analysis (middle). Small (S6 rp) and large (L13a rp) ribosomal protein abundances detected by Western blot of total protein isolated from input (6%) and from fractions 4 through 13 (bottom). (B) Percent of HCV RNA distributed across the polysomal gradients of three independent experiments. Error bars display +/- SD.(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.


Hepatitis delta virus (HDV) is unique among animal viruses. HDV is a satellite virus of the hepatitis B virus (HBV), however it shares no sequence similarity with its helper virus and replicates independently in infected cells. HDV is the smallest human pathogenic RNA virus and shares numerous characteristics with viroids. Like viroids, HDV has a circular RNA genome which adopts a rod-like secondary structure, possesses ribozyme domains, replicates in the nucleus of infected cells by redirecting host DNA-dependent RNA polymerases (RNAP), and relies heavily on host proteins for its replication due to its small size and limited protein coding capacity. These similarities suggest an evolutionary relationship between HDV and viroids, and information on HDV could allow a better understanding of viroids and might globally help understanding the pathogenesis and molecular biology of these subviral RNAs. In this review, we discuss the host involvement in HDV replication and its implication for HDV pathogenesis. Hepatitis delta virus (HDV) was discovered in 1977 following the identification of a new antigen present exclusively in hepatocytes from a patient having the hepatitis B virus (HBV) surface antigen (HBsAg) . HDV cauHDV is a defective and satellite virus of HBV, requiring HBV envelope proteins (HBsAg) for its propagation (reviewed in Reference ). HoweveHDV is the smallest known human RNA virus. Its genome consists of a small (~1700 nucleotides) single-stranded, circular RNA molecule and folds into an unbranched, rod-like structure due to 74% self-complementarity  5]. It . It 5]. Even though it requires the HBV envelope proteins for virion production, HDV replicates independently of HBV and relies entirely on its host for replication. HDV replicates via a symmetrical, rolling circle mechanism . ReplicaHDV does not encode its own replicase and there are no known HDV DNA intermediates. Accordingly, at least one host RNA polymerase  is involved in the replication and transcription of HDV. HDV replication and transcription takes place in the nucleus. HDAg mRNA is post-transcriptionally processed with a 5′-cap and a 3′-poly(A) tail, which are typical features of transcripts generated by RNAP II ,17. HDV Several host proteins involved in post-translational modifications, sub-cellular localization, RNA synthesis, and gene regulation, interact with HDAgs (reviewed by ). Among HDAg-S was reported to co-immunoprecipitate with more than 100 host proteins and more than 25% of them were demonstrated to affect HDV RNA accumulation . These pAmong the proteins reported to associate with HDV RNA genome, ADAR-1 interacts with HDV antigenome to edit the position corresponding to the stop codon of HDAg-S allowing the production of HDAg-L . In addiBecause HDV interacts with numerous host proteins, several studies were initiated to understand the consequence of these interactions on gene and protein expression. The effect of the accumulation of HDAg and/or HDV RNA has been investigated using proteomic analyses in several experimental systems . Using hAnother proteomic analysis reported a total of 89 proteins affected using a HEK-293 cell system mimicking HDV replication. This cellular system is comprised of three cell lines: 293 cells, 293 cells expressing HDAg-S under the control of a TET-ON promoter (293-Ag), and 293-Ag cells transfected with an HDV RNA genome unable to produce HDAg-S (293-HDV) . In thesActive HDV replication induces the production of interferon beta (IFN-β) and interferon lambda (ifn-λ1/2/3) . The patRecently, the involvement of paraspeckle proteins in HDV replication was demonstrated . ParaspeAlso consistent with the cellular stress response induced by HDV, HDAg-L can activate the nuclear factor kappa B (NF-κB), as well as the signal transducer and activator of transcription-3 (STAT-3) ,67. ReacOver the years, our knowledge of HDV, its replication, propagation and effects on the host has expanded. However, a lot remains unknown and more research is required to improve our understanding of HDV pathogenesis. Predominantly, most of what was reported on the contribution and disruption of host processes during HDV replication was obtained in vitro, or in conditions where HDAgs accumulation or HDV replication was triggered artificially in non-hepatocytes cells and in the absence of HBV. Although the restricted tissue tropism of HDV could be due to HBV, it is likely that the levels of HDAgs and HDV RNA are different during an infection, and together with the presence of HBV, might affect both interactions and host gene disruption. Because HDV is the only mammalian virus of its kind, but shares a lot of similarity with plant viroids, this virus might constitute a good model to study other subviral RNA pathogens, such as viroids ,71. Both


Rpb5 is one of the five common subunits to all eukaryotic RNA polymerases, which is conserved in archaea, but not in bacteria. Among these common subunits, it is the only one that is not interchangeable between yeasts and humans, and accounts for the functional incompatibility of yeast and human subunits. Rpb5 has been proposed to contribute to the gene-specific activation of RNA pol II, notably during the infectious cycle of the hepatitis B virus, and also to participate in general transcription mediated by all eukaryotic RNA pol. The structural analysis of Rpb5 and its interaction with different transcription factors, regulators and DNA, accounts for Rpb5 being necessary to maintain the correct conformation of the shelf module of RNA pol II, which favors the proper organization of the transcription bubble and the clamp closure of the enzyme.In this work we provide details about subunit Rpb5's structure, conservation and the role it plays in transcription regulation by analyzing the different interactions with several factors, as well as its participation in the assembly of the three RNA pols, in cooperation with prefoldin-like Bud27/URI. While the genome in bacteria and archaea is entirely transcribed by one single RNA pol, most eukaryotic cells use three distinct enzymes , which carry out type-specific transcription programs in conjunction with different accessory factors. RNA pol I transcribes the precursor of the three largest rRNAs (35S rRNA), RNA pol II produces all mRNAs and many non coding RNAs, while RNA pol III synthesizes a set of small non translated RNAs, including all tRNAs, 5S rRNA and other short RNAs Eukaryotic RNA pols are characterized by their complex heteromultimeric composition. In yeast and other eukaryotes, RNA pol I, II, III are composed of 14, 12, and 17 subunits, respectively One of these RNA pol common subunits, Rpb5, is an evolutionarily highly conserved RNA pol component that is described as a key structural and functional component of all eukaryotic RNA pols, which suggests a more general role in transcription In this report we provide details about subunit Rpb5 and its relationship with prefoldin-like Bud27 and their roles in transcription regulation.2.S. cerevisiae, Rpb5, also called ABC27, is located in the periphery on all three RNA pols and occupies the “lower” far-end of the DNA cleft RPB5 null allele in S. cerevisiaeIn jaw domain), which corresponds to positions 1–142 in S. cerevisiae, and a C-terminal globe (assembly domain), which is largely conserved in all non bacterial enzymes in vivo, and are functionally exchangeable with their human homologs, except for a small central segment located between positions 121–146 in S. cerevisiae, which marks the end of the eukaryotic domain and extends to the first four amino acids of the C-terminal domain lower” far end of the DNA cleft. Two prolines (P86 and P118 in S. cerevisiae) have been proposed to contact DNA. Moreover, the C-terminal module is highly conserved (positions 143–215 in S. cerevisiae) and binds a conserved fold on the largest subunits of RNA pols I, II and III , which correspond to a bundle of four helices (Rpb1-α44/47) S. cerevisiae Rpb5 and Sulfolobus solfataricus RpoH archaeal subunits Rpb5 shows a bipartite organization  by combi3.S. cerevisiae RNA pol II jaw domain of Rpb5 through a conserved “TPSA domain”, apparently an interaction that has already been established during transcription initiation, and is maintained during the transition from initiation to elongation cleft by translocation along Rpb5 upon TFIIH action and DNA opening The N-terminal domain of Rpb5 accounts for the Rpb5/DNA contacts ahead of the transcription fork (15–20 nucleotides) in S. cerevisiae, Rpb5 interacts with Rsc4, a subunit of the RSC complex (chromatin remodeler complex) that regulates transcription negatively or positively through its ability to mobilize nucleosomes The peripheral localization of Rpb5 4.Many studies point out a role of Rpb5 in transcription regulation, but the specific role of Rpb5 in transcription remains unclear.in vitro and in vivo analyses done with an Rpb5 and RNA pol II CTD mutants showing that Rpb5 and CTD had overlapping functions in the activation, account for this function Its peripheral localization also enables interactions with general transcription factors or specific gene regulators. Accordingly a role for yeast Rpb5 in transcription activation has been previously proposed. The in vitro and in vivo by negatively modulating transcription, and by probably interfering with the trimeric interaction among Rpb5, TFIIB and HBx As shown in in vitro with TIP120 (TATA-binding protein-interacting protein 120) which stimulates the enzymatic activity of both RNA Pol I and II Human Rpb5 also interacts with the Human TATA-binding protein (TBP)-associated factor 68 (TAFII68), an RNA/ssDNA binding protein that was originally identified for its homology to the proto-oncogenes EWS (Ewing's Sarcoma) and TLS  In yeast a major role has been described for Rpb5 in maintaining the proper organization of the RNA pol II structure around the transcription bubble In line with a role for Rpb5 that favors transcription elongation, loss of the Rpb5-Rsc4 interaction alters the chromatin structure in the promoter region of several RSC-regulated genes, and consequently impairs transcription 5.Bud27 and its ortholog URI , are members of the prefoldin (PFD) family of ATP-independent molecular chaperones in higher eukaryotes S. cerevisiaeS. cerevisiae and humans by participating in the gene expression controlled by TOR kinase In mammals, URI forms a complex with PFDN2, PFDN6, UXT, WDR92/Monad, PDRG1 and RPB5 S. cerevisiae Paf1, a complex involved in cell-cycle control, RNA pol II phosphorylation, and histone modification during transcription elongation C. elegansDrosophilade novo NAD + synthesis in mouse Other functions for URI have been described. In human HEK-293 cell extracts, URI interacts with the human homolog of S. cerevisiae Bud27 has been described to coordinate translation initiation and cotranslational quality control Finally, 6.The importance of the Rpb5-Bud27 (RPB5-URI) interaction has been described in transcription regulation. In fact it was originally described how these two human proteins, in association with transcriptional activator HBx (Hepatitis B virus X protein), modulate its action The role of URI controlling androgen receptor (AR) transcription supports the idea of an RPB5-URI interaction, where Rpb5 could participate in regulating the androgen receptor in human cells S. cerevisiae has recently been described as the first protein to mediate the cytoplasmic assembly of the three RNA polymerases prior to their entry to the nucleus, a function that depends on Rpb5 Bud27 in S. cerevisiae, probably through the correct association with Sth1 (a RSC complex's subunit). We speculate that this action may involve a tripartite association among Bud27, Rpb5 and Sth1 Notably, Bud27 participates in transcription elongation in 7.Rpb5, as the only one of the five RNA polymerase common subunits to contact DNA and chromatin remodelers, may play a more relevant role than a general element in transcription. In fact its recently described participation in the transition from transcription initiation to elongation by modulating Spt5 elongation factor association and backtracking demonstrates a specific role of this subunit in RNA pol II activity. The association between Rpb5 and Bud27, a prefoldin-like protein that participates in RNA pols assembly, transcription, translation and the TOR pathway, among other important cellular processes, prompted us to speculate that this connection could regulates transcription, ribosomal biogenesis and growth. Notably URI, the human ortholog of Bud27, is altered in cancer processes.The importance of these two proteins and their association in regulating important cellular processes is currently being studied.


Shark recovery in reef habitat was accompanied by a two and a half‐fold decline in the abundance of small mesopredatory fishes (≤50 cm TL) (14.00 hr−1 ± 3.79 to 5.6 hr−1 ± 1.20) and a concomitant increase in large mesopredatory fishes (≥100 cm TL) from 1.82 hr−1 ± 0.48 to 4.27 hr−1 ± 0.93. In contrast, near‐reef habitats showed an increase in abundance of large mesopredatory fishes between years (2.00 hr−1 ± 0.65 to 4.56 hr−1 ± 1.11), although only smaller increases in sharks (0.67 hr−1 ± 0.25 to 1.22 hr−1 ± 0.34) and smaller mesopredatory fishes. Although the abundance of most mesopredatory groups increased with recovery from fishing, we suggest that the large decline of small mesopredatory fish in reef habitat was mostly due to higher predation pressure following the increase in sharks and large mesopredatory fishes. At the regional scale, the structure of fished communities at Ashmore Reef in 2004 resembled those of present day Scott Reefs, where fishing still continues today. In 2016, Ashmore fish communities resembled those of the Rowley Shoals, which have been protected from fishing for decades.The recovery of communities of predatory fishes within a no‐take marine reserve after the eradication of illegal fishing provides an opportunity to examine the role of sharks and other large‐bodied mesopredatory fishes in structuring reef fish communities. We used baited remote underwater video stations to investigate whether an increase in sharks was associated with a change in structure of the mesopredatory fish community at Ashmore Reef, Western Australia. We found an almost fourfold increase in shark abundance in reef habitat from 0.64 hr We observed abundance and community composition of shark and mesopredatory reef fish at Ashmore Reef in Western Australia, before and after recovery from illegal fishing pressure. After eight years of recovery, there was a large decrease in small (≤50 cm TL) mesopredatory reef fish abundance and a concomitant increase in shark and large (>100 cm TL) mesopredatory fish abundance in reef habitat. Our findings provide support for fish community restructuring with reef shark recovery, which has implications for management time frames of marine protected areas. Ashmore2.2The Scott Reefs consist of three large atolls on the edge of the continental shelf: Seringapatam Reef (8 × 9.4 km); Scott Reef North (16.3 × 14.4 km); and Scott Reef South (27.4 × 17 km), which are ~260 km from the mainland of Australia  Figure . The ScoThe Rowley Shoals are composed of three large reefs: Imperieuse (17.8 × 9.5 km); Clerke (15.8 × 7.6 km); and Mermaid (14.5 × 7.6 km) Berry, , which aA very similar suite of species is present on the offshore reefs of the entire North West Shelf, including Rowley Shoals, Scott Reefs, Ashmore Reef, and Cartier and Hibernia Reefs, and at the level of genus, these communities are similar to other reefs in the tropical Indo‐West Pacific  and 2016 (n = 29), and the Scott Reefs (n = 20) and the Rowley Shoals (n = 20) in 2016. All deployments were within 1.5 km from the reef edge to ensure reef‐associated species were the primary focus. The 2004 survey at Ashmore Reef was part of a larger program to assess shark stocks in the MOU Box  were used in housings in 2004, and GoPro Hero4 Silver Edition was used in 2016.Data were collected using baited remote underwater video stations (BRUVS) in shallow water (10–30 m) around Ashmore Reef in both 2004 (Sardinops spp.) was suspended at the end of a 1.5 m pole in front of the camera. BRUVS were deployed to provide a minimum of 60 min of video recorded at the seabed. Successive deployments within a set were spaced between 400 and 1,000 m apart in shallow depths (10–30 m) around the reef during daylight hours.A bait bag containing 1 kg of crushed pilchards , a purpose‐built event logger that allows an operator to record the number of fish observed and their species identification. To quantify the abundance of sharks and mesopredatory reef fishes, we recorded the maximum number of individuals of each species occurring in a single video frame (MaxN) during the entire video , Lethrinus olivaceus and Lethrinusmicrodon (Lethrinus oli_micro), and Plectropomus laevis and Plectropomus maculatus (Plectropomus spp). All mesopredatory fishes were grouped into three size classes based on maximum obtainable lengths (TL) from regional estimates at Fishes of Australia http://fishesofaustralia.net.au/ , bull (Carcharhinus leucas), and greater hammerhead (Sphyrna mokarran) sharks, although these larger species are not necessarily limited by gape width due to a variety of prey manipulation strategies  at Ashmore Reef. Generalized linear models (GLMs) with negative binomial error structures were used to model abundance with factors that included year and a size class grouping , and their interaction. The variables' complexity and coral were found to be collinear in near‐reef data (Pearson Correlation = 0.6) and therefore not included in the same models. Changes in the mesopredatory fish communities were also assessed for Ashmore Reef between habitats and years using a principal component analysis (PCA) on scaled MaxN data for species that occurred on ≥10% of deployments. A permutational multivariate analysis of variance (PERMANOVA) test was then used to examine whether there were significant differences in composition of fish communities between years (2004 and 2016), habitats (reef and near‐reef), or between year and habitat combinations (interaction) at Ashmore Reef. Values of MaxN for each species were square root transformed prior to analyses to reduce the influence of dominant species, while still retaining the major differences in community structure according to abundances.The second set of analyses investigated differences in the communities of mesopredatory fishes and reef sharks in reef habitats among reefs across the northwest region, and tested for an effect of protection status (fished vs. no‐take). Negative binomial GLMs were used to test for an effect of depth (10–30 m), coral cover (0%–100%), habitat complexity , and year (2004 & 2016), on the combined abundance of sharks and mesopredatory fish of all size classes  across the northwest region of Western Australia. As data exploration within the region‐wide analyses revealed significant correlations between year with site and complexity with site , these factors were not included within the same models for this analysis. A PERMANOVA test was used to examine whether there were significant differences in composition of fish communities between fished (Ashmore 2004 and Scott Reef 2016) and protected reefs  across the northwest region. Values of MaxN for each species were square root transformed prior to analyses.c) and AICc weights (wAICc) . Near‐reef fish communities had fewer noticeable changes in mean abundance of size classes, with only large mesopredatory fishes showing a clear increase between years from 2.00 hr−1 ± 0.65 SE in 2004 to 4.56 hr−1 ± 1.11 SE in 2016.The top‐ranked negative binomial GLMs used to model the difference in total combined abundance of predatory fishes and shark communities between years at Ashmore Reef included year, size class, and depth (reef habitat model only) Appendix . Both toLutjanus gibbus and Lethrinus rubrioperculatus), whereas the increase in abundance of the large size class was primarily driven by representatives of the Carangidae   and years  at Ashmore Reef . Separation was driven by higher abundances of sharks and large mesopredatory fishes at protected reefs, whereas smaller mesopredatory fishes were more abundant at fished reefs . The abundance of communities of predatory fishes (sharks and mesopredatory fishes) was strongly associated with the variable site, as indicated by the top‐ranked negative binomial GLM , although depth and coral cover were also important explanatory variables with similar ranked models falling within two AIC6. The abc Table . Partialc Table . PrincipLutjanus gibbus was one of the small species of mesopredatory fishes that was more common at fished reefs (Ashmore Reef in 2004 and Scott Reef in 2016) and typically occurred in schools , although this effect appears to be species‐dependent , which are known to have a largely piscivorous diet (Dale, Meyer, & Clark, Increases in large mesopredators were mostly driven by carangids, including species such as the bluefin trevally (The similarities between abundances of reef sharks and the structure of mesopredatory reef fish communities now present at Ashmore Reef and the protected Rowley Shoals implies that both the reef shark and mesopredatory fish communities have largely recovered from illegal fishing in the eight years since enforcement of the no‐take status of the reef. However, it must be acknowledged that our study represents two snapshots in time and therefore requires further sampling to confirm that long‐term patterns in recovery continue. Nevertheless, the rapid rate of recovery in shark and mesopredatory fish numbers at Ashmore Reef will be encouraging for managers seeking time frames in which to predict for stakeholders when impacts from conservation actions might occur.None declared.MGM, CWS, and MR conceived and designed the project. MGM, CWS, and KC collected the data. BV, KC, MR, and CWS performed BRUVS footage processing. CWS statistically analyzed the data. MGM, CWS, MR, KC, and BV wrote and edited the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.