Hevea brasiliensis). Numerous studies have been conducted to define its origin and nature, but the molecular nature and mechanism of TPD occurrence remains unknown. This study is committed to de novo sequencing and comparative analysis of the transcriptomes of healthy (H) and TPD-affected (T) rubber trees to identify the genes and pathways related to the TPD.Tapping panel dryness (TPD) involves in the partial or complete cessation of latex flow thus seriously affect latex production in rubber tree  contains supplementary material, which is available to authorized users. Hevea brasiliensis Muell.) is a perennial plant of the Euphorbiaceae family and is commercially cultivated in the tropical areas worldwide (particularly in the Southeast Asia) for the production of natural rubber  which is an strategic raw material for over 40,000 products [Rubber tree , implying that it may play a role in apoptosis. Venkatachalam et al. [HbTOM20 gene , suggesting that it may involve in alteration of mitochondrial metabolism. Venkatachalam et al. [HbTK) related to TPD by random amplified polymorphic DNA screening. Moreover, Venkatachalam et al. [Several protein markers linked to TPD have been identified –30, but m et al.  using DDm et al.  identifim et al.  and Li em et al.  studied m et al.  identifiRecently RNA sequencing (RNA-Seq), referring to the whole-transcriptome shotgun sequencing of fragmented mRNA or cDNA, has emerged as a powerful tool for profiling expressed genes in plants and other organisms –41. RNA-In the rubber tree, several reports about the transcriptional profile of different organs and tissues were published –52, but To identify the genes associated with TPD, two sequencing cDNA libraries were constructed: H library from bark tissue in healthy rubber trees with normal latex flow Figure A and T lFor function annotation analysis, 68621, 63025, 44183, 41042, 28238 and 53709 unigenes were annotated to the NR, NT, Swiss-Prot, KEGG, COG and GO database, respectively, with the total annotation unigenes of 73597. The unigenes were assigned to three main categories  of the GO classification. COG function classification for H and T is shown in Figure Unigene expression difference analysis was conducted with the total number of unigenes of 107020 (73597 unigenes with annotations). 22577 annotated DEGs and 29702 DEGs with no function and no hit were obtained between the healthy and TPD affected trees using FDR ≤ 0.001 and |log2Ratio| ≥ 1 as a threshold to determine the statistical significance of gene expreesion  of −11.347), chain A of anomalous substructure of hydroxynitrile lyase (with a log2 ratio (T/H) of −11.0766), uncharacterized protein LOC101298879 (with a log2 ratio (T/H) of −10.7788) and osmotin precursor (with a log2 ratio (T/H) of −10.6413). The genes coding arginine N-methyltransferase (with a log2 ratio (T/H) of 9.655192) and Dip1-associated protein C (with a log2 ratio (T/H) of 9.438686) were the top up-regulated DEGs in T compared to H.Of the 2660 DEGs with differences greater than 15-fold, 1206 unigenes were present at higher levels in the healthy trees than the TPD affected trees, and 1454 in the TPD affected trees than the healthy trees. The DEGs exhibiting the highest down-regulated expression in T compared to H were the genes coding 6-phosphogluconolactonase  synthesis is involved in isoprenoid metabolism, and TPD syndrome is often an abrupt and complete shut down of the latex biosynthesis machinery, we focused on pathway analysis of IPP metabolism, volatile mevalonic pathway and jasmonate synthesis.Based on the KEGG pathway enrichment analysis, four and six DEGs that encode exzymes involved in mevalonic acid (MVA) pathway and methylerythritol phosphate (MEP) pathway, respectively  synthase (GPPS) and farnesyl diphosphate (FPP) synthase (FPPS) which catalyze the formation of GPP and FPP respectively was also dramatically down-regulated , allene oxide cyclase (AOC) and 12-oxophytodienoic acid (OPDA) reductase (OPR) were found to be down-regulated  of −8.371071 and PWWP domain-containing protein with a log2 ratio (T/H) of −6.1634774, and the top up-regulated TFs in T compared to H were LOL1 (LSD ONE LIKE 1) with a log2 ratio (T/H) of 6.102857 and zinc finger (FYVE type) family protein with a log2 ratio (T/H) of 7.7258673. But their exact functions in occurrence and maintenance of TPD remained to be elucidated.TPD of rubber tree is generally considered as a complex physiological disorder, and genetic, developmental, histological, cytological and biochemical processes would be involved in. Transcription factors (TFs) understandably control and coordinate those diverse pathways and processes as master regulators. In our study, 415 proteins were identified as putative TFs categorized into different familes  selected at random was verified by qRT-PCR. These eight genes were involved in isoprenoid biosynthesis and jasmonate synthesis, and their expressions were remarkably down-regulated in T library. The results obtained by qRT-PCR fitted in well with the expression pattern by transcriptome analysis Figure .Figure 8In this study, we have first profiled the gene expression between the healthy rubber trees and the TPD-affected trees using Illumina sequencing technology. This transcriptome dataset will serve as an important public information platform for the discovery of TPD-related genes, the functional genomic investigation of TPD and the elucidation of the TPD molecular mechanism.de novo sequencing and transcriptome profiling. The reason for this is not clear, but it is possibly that there were fewer laticifer differentiation/regeneration and more active biological processes related to the tree growth occurred in TPD-affected bark showing complete dryness, although the biosynthesis of latex was turned off. In fact, a more vigorous growth and more trunk girth increment are often observed in rubber trees after tapping cut becomes entirely dry.Previously, Venkatachalam et al.  and Li eH. brasiliensis [H. brasiliensis) [Biosynthesis of natural rubber, poly, is from IPP units, and in higher plants the monomeric subunit of natural rubber IPP is formed by the MVA pathway and the MEP pathway ,54. Moreiliensis . The proiliensis . The genliensis) ,58. HMGRliensis) ,60.Hevea trees to synthesize rubber (cis-poly isoprene) and oxidative damage of laticiferous vessels may be the cause for the complete shut down of the rubber biosynthetic machinery. Chrestin et al. [Hevea, and in particular overstimulation with Ethrel can lead to TPD and an imbalance between the toxic peroxidative activities and the scavenging activities within the latex vessels should be responsible for the disorganization of the organelle membrane structure. Siswanto [In TPD affected rubber trees, latex production is severely decreased and can be completely shut down in the ultimate stage . Krishnan et al.  and Chren et al.  suggesteSiswanto  concludeSiswanto  and a TOSiswanto  implicatSiswanto . We firsThe expressions of the majority of genes involved in JA biosynthesis including 13-LOX, AOC and OPR were found to be down-regulated. It is noteworthy that the suppression of OPR was the greatest (with a log2 Ratio (T/H) of −12.915768) and it suggested that the OPR might be a key enzyme for JA biosynthesis pathway. As we know, the first half of the JA biosynthesis from α-LeA to OPDA occurs in a plastid, and after the transportation of OPDA into a peroxisome, the second half taking place in the peroxisome is begun with the conversion of OPDA to cyclopentanone catalyzed by OPR .HMGR1 in the MVA pathway was concurrently suppressed. The mevalonate is generated from HMG-CoA by HMGR and is further converted to isoprenoid compounds as well as natural rubber. Although there are genes  encoding HMGR in H. brasiliensis, but only HMGR1 is likely to be involved in rubber biosynthesis [HMGR1 in the MVA pathway was regulated by methyl jasmonate [H. brasiliensis [Interestingly, ynthesis . The facasmonate –70 suggeiliensis ,72. In siliensis ,74. TherMany study showed that over-stimulation with ethphon and employing intensive tapping systems increase the incidence of dryness –16. But In future, we need to research the influence of those factors on JA biosynthesis and latex biosynthesis. Especially, in the case of TPD resulted from intensive exploitation and over-stimulation with ethephon, the study should be conducted to show that JA biosynthesis is suppressed and further resulted in the decline of the latex biosynthesis after intensive exploitation and over-stimulation with ethphon. As for the phenomena that the incidence of TPD varies with varieties and the occurrence of TPD in trees which were not opened for tapping , it seemOur findings shed a new light on the nature of, and even the cause of TPD. In completely dried panel trees, the expression of genes associated with the latex biosynthesis was significantly inhibited and it is possibly resulted from the reduction of the signaling molecule JA biosynthesis. In addition, the transcriptomes in this study provide valuable information for further identification of genes related with TPD.The high yielding clone PR107 planted at the experimental farm of Chinese Academy of Tropical Agricultural Sciences in 2000 was selected for the present experiment. Since 2008, the rubber trees have been regularly tapped with a tapping system  plus application of 1.5% ethephon two days before tapping (once every three tappings). The bark tissues were respectively collected from nine healthy rubber trees and nine TPD-affected ones showing complete dryness. The samples were washed with diethyl pyrocarbonate treated water to remove the latex and immediately frozen in liquid nitrogen and shipped on dry ice to BGI Life Tech Co., Ltd  for Illumina sequencing.Bark RNA was extracted using the TRIzol® Reagent (Invitrogen) according to the manufacturer's instructions. RNA samples were detected for quantification and quality by a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) and a 2100 Bioanalyzer (Agilent Technologies). Two cDNA libraries, H  and T (TPD-affected trees), were prepared using the mRNA-Seq 8 sample prep Kit (Illumina) according to the manufacturer's instructions. Poly(A) mRNA was first isolated from 20 μg of total RNA using magnetic beads containing poly-T molecules. Then, the purified samples were fragmented into small pieces using divalent cations at 94 °C for 5 minutes and reverse transcribed into cDNAs with the SuperScript double-stranded cDNA synthesis kit . Then, end repair and adenylation of 3' ends were conducted and purified using the QIAquick PCR Purification Kit (QIAGEN). After that, Illumina paired end adapters were ligated to the resulting cDNA fragments. Finally, each cDNA library with an insert size of 200 bp was generated. After quality checking by an Agilent Technologies 2100 Bioanalyzer, the libraries were deep sequenced using Illumina HiSeqTM 2000 .de novo assembly was then carried out with short reads assembling program – Trinity (release 20130225 ) (http://trinityrnaseq.github.io/) [Raw reads produced from sequencing machines were filtered by the Illumina pipeline and the dirty raw reads with adaptors and unknown nucleotides larger than 5%, and low quality reads  were removed. Transcriptome hub.io/) ,77. FirsWith the NR annotation, the Blast2GO program  was usedFPKM method (Fragments Per kb per Million reads) was applied to calculate the expression of the Unigenes . Therefo−△△Ct method. The forward and reverse primers used in this study are listed in Table Seven cDNAs (lowly expressed in the T library) encoding DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase), HMGR (3-hydroxy-3-methyl-glutaryl-CoA reductase), MVK , GPPS (geranyl diphosphate synthase), 13-LOX (13-lipoxygenase), AOC (Allene oxide cyclase) and OPR (12-oxophytodienoic acid reductase) with potential roles in rubber biosynthesis and its regulation were chosen for validation using qRT-PCR. Total RNA was respectively extracted from the equal amount of bark tissues of three H healthy rubber trees and three TPD-affected ones showing complete dryness as described by Venkatachalam et al.  for qRT-


Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both  RNA plays a central role in many cellular processes, acting as a major player in gene expression control46A modification of the mRNA, the RNA looping, and the direct entry driven by Internal Ribosome Entry Site (IRES) elements10111415In eukaryotic mRNAs, a variety of mechanisms have been proposed to allow the ribosomal machinery to recognize the translation start codon. These are the scanning of the 5′UTR following cap-dependent recruitment of the translation machinery, the cap-independent mechanisms guided by the mThe RNA sequences and secondary structures of picornaviruses show higher heterogeneity than in hepatitis C virus (HCV). Accordingly, the picornavirus IRES elements are grouped into five different types ; each type harbors conserved sequence motifs and a common RNA structure core maintained by evolutionarily conserved substitutions18Picornavirus IRES elements are organized in modular domains. In type I and type II IRES, each domain consists of one or more stem-loops that provide the binding site for RBPs and various eIFs, with the exception of eIF4E20212224Experimental evidence supports the involvement of RNA structural motifs for IRES activity262724303133RNA viruses in general, and FMDV in particular, are characterized by a high genetic variability3822in silico design can be successful in constructing complex functional RNA elements.In spite of the mutational analysis carried out in the picornavirus IRES Py tract, it remains elusive whether having the Py tract in a unique structural conformation is an absolute requirement for IRES activity. To answer this question we have designed candidate RNA sequences adopting different conformations of domain 5, but harboring a pyrimidine tract of the same length as that of wild type IRES. Hence, the pyrimidine tract could be either unpaired or base-paired in stem-loops with different stability. For this, we have made use of the FMDV IRES, a type II IRES element whose secondary structure is well characterized25To design RNA structural motifs with unique folding patterns, potentially affecting initiation of protein synthesis, we focused our attention on domain 5 of the FMDV IRES, located immediately upstream of the functional translation initiation codon . ModificThe experimentally determined RNA structure of domain 5 consists of three elements , of whic22in silico design and posterior filtering process produced sequences with high probability of having the Py base-paired. Among these sequences (which were arbitrarily named), sequence I-20 was selected because it had the highest probability for the Py tract to be base-paired. Moreover, four additional sequences  with moderate and low probability among the filtered sequences were selected in order to later establish correlations between measures and IRES activity. Measures shown to have high correlation with IRES activity can then be used as optimization criteria in subsequent design rounds.The target structure used as input to RNAiFoldAs devised, the selected candidates (RNA family I) had a pyrimidine tract of the same length as the wild type RNA , and U/Cin vitro synthesized RNA. As shown in 35S-labeled luciferase (LUC) polypeptide to chloramphenicol acetyl transferase (CAT) polypeptide in rabbit reticulocyte lysates (RRL) was reduced in all selected candidates relative to the wild type RNA. Note that the activity of RNAs I-2, I-3, I-4, and I-7 was very similar. However, the activity of RNA I-20 was reduced to a higher extent than all other candidates  activity expressed from bicistronic RNAs in transfected BHK-21 cells . The samild type . Again, p values and absolute SHAPE differences in To gain information about the RNA structure of the selected candidates, we chose a representative member of the family, RNA I-3, to analyze its local RNA flexibility in solution using SHAPE methodology . SHAPE rUsing RNAstructure software to predict the secondary structure including SHAPE reactivity data for wild type RNA and candidate I-3 , we showTo further prove the effect of structural accessibility of the pyrimidine tract on IRES activity, we plotted the parameters used to select final candidates of family I against the efficiency of protein synthesis measured in cell-free systems . This daSince the reduced activity of RNA I-20 could be related to the higher stability of the hairpin sequestering the Py tract, we attempted to generate a second round of candidates, selected on the basis of adopting a stable hairpin that sequesters the Py tract within a long stem-loop. In order to generate sequences for this family, we again used RNAiFold with the target structure shown in in vitro assay indicated a strong decrease of luciferase synthesis , confirming that the selected candidate harbored a modified RNA structure. As observed before for candidate I-3, the RNA structure model of candidate II-B obtained by imposing SHAPE reactivity on RNAstructure  separated by 84 nts21Concerning the differences among type I and type II picornavirus IRES elements, experimental evidence points to a differential role of the spacer structure on protein synthesis. For instance, in the case of FMDV RNA, the presence of a highly stable hairpin (six repetitions of the Xma linker) within the spacer  but not the presence of unstructured sequences up to 99 nts corresponding to the polylinker sequence of pGem3, hinders IRES activityThe lack of conserved pathways to recognize start codons among different IRES elements2332515354To determine potential similarities in Py accessibility of different IRES elements, we have analyzed the Rfam families of viral IRES for structural analogy. We used INFERNAL 1.1It is worth noting that expression vectors used to express more than one protein in eukaryotic cells usually contain type II picornavirus IRES elements, due to their high efficiency and their proven resistance to inhibitory conditions for cap-dependent translation. Given that spacers of variable sequences and length, often inherited from previous constructs, are found in expression vectors harboring IRES elements, our rational design provides useful data for the definition of RNA structural features of this spacer region affecting internal initiation of translation. In summary, we show here that a critical step for FMDV IRES function is the presence of an unpaired Py tract, along with the immediately upstream conserved stem-loop. All the selected candidates that form a stable hairpin sequestering the Py tract within the stem of a downstream hairpin show a significant decrease in protein synthesis despite conservation of the Py residues. To our knowledge, this is a novel finding since previous studies investigated the effect of modified Py sequences but did not investigate structural accessibility of the Py tract. Thus, future studies aimed to develop improved expression vectors should take into account the accessibility of this conserved structural motif.H. sapiens. A high-throughput version of the hybrid computational/experimental method described in this paper could generate sufficient data to train a regression support vector machine to predict translation efficiency of IRES elements. Such a tool could possibly provide insights concerning the ratio of protein products from genes that contain an IRES element.Several machine learning algorithms have been developed57RNAiFold computes all sequences that fold into a given characteristic secondary structureFamily I was designed to fold into the target structure shown in Family II was designed to fold into the target structure shown in We computationally estimated the accessibility of the polypyrimidine tract at 30 °C for each candidate using three different methods  and 2: F2 10 mM, 15 min at 37 °C and then inserted into the HindIII and XhoI restriction sites of pBIC, previously linearized with the same enzymes. Colonies that carried the correct insert were selected for further studies. Prior to expression analysis, the nucleotide sequence of the entire length of each region under study was determined (Macrogen).Insertion of the candidate sequences into the bicistronic plasmid pBICIn vitro transcription was performed for 1 h at 37 °C using T7 RNA polymerase, as describedin vitro were translated in 70% rabbit reticulocyte lysate (RRL) (Promega) supplemented with 35S-methionine (10 μCi)The relative IRES activity obtained in assays conducted with monocistronic RNAs (100 ng of the IRES-firefly luciferase RNA and 25 ng of the renilla luciferase RNA) was monitored in a luminometer using the dual system (Promega).5 cells were prepared 20 h after transfection in 50 μl of 50 mM Tris-HCl, pH 7.8, 120 mM NaCl, 0.5% NP40. Luciferase and chloramphenicol acetyl transferase (CAT) activities were measured as describedPlasmids carrying the candidate sequences upstream of the luciferase reporter gene were assayed in BHK-21 cells. Transfection of 90% confluent monolayers was carried out using cationic liposomes 1 h after infection with the Vaccinia virus VT7F-3 expressing T7 RNA polymerase. This assay excludes the presence of cryptic promoters since the transfected plasmid is transcribed in the cell cytoplasm by the T7 RNA polymerase. Extracts from 10in vitro. RNAs (2 pmol) were treated with N-methylisatoic anhydride (NMIA)in vitro synthesized RNA was renatured by heating at 95 °C for 2 min, snap cooling on ice for 2 min, and subsequently incubated in a final volume of 18 μl of folding mix  for 20 min at 37 °C. Then, RNA were incubated with dimethyl sulfoxide (DMSO) (untreated RNA) or 6.5 mM NMIA for 45 min at 37 °C, precipitated and resuspended in 10 μl of 0.5X TEt-test.Monocistronic constructs were linearized with SphI prior to synthesis of RNA transcripts http://www.clustal.org). The RNA sequences were obtained from GenBank; only domain 5 unique sequences were included in the alignment. The phylogenetic tree was inferred by the neighbour-joining method from the multiple-sequence alignment using MEGA4 program (www.megasoftware.net). Robustness of evolutionary relationships was assessed by 1000 bootstrap replicates. Bootstrap values are shown as percentages, only nodes with percentage values higher than 50% are indicated.The alignment of forty-six sequences of FMDV IRES field isolates was performed using CLUSTALX software with default parameters (http://weblogo.berkeley.edu) was generated from the multiple-sequence alignment of the IRES sequences. The overall height of each stack indicates the sequence conservation at that position (measured in bits), and the height of symbols within the stack reflects the relative frequency of the corresponding nucleic acid at that position.The sequence logo (http://varna.lri.fr/).Secondary RNA structure prediction accuracy can be improved incorporating SHAPE reactivity values as constraints in RNAstructure softwareHow to cite this article: Fernandez-Chamorro, J. et al. Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements. Sci. Rep.6, 24243; doi: 10.1038/srep24243 (2016).


We examined the performance of three RNA-Sequencing library preparation protocols as a function of RNA integrity, comparing gene expressions between heat-degraded samples to their high-quality counterparts. This work is invaluable given the difficulty of obtaining high-quality RNA from tissues, particularly those from individuals with disease phenotypes.With the integrity of total RNA being a critical parameter for RNA-Sequencing analysis, degraded RNA can heavily influence the results of gene expression profiles. We discovered that gene expression read results are influenced by RNA quality when a common library construction protocol is used. These results are based on one technical experiment from a pool of 4 neural progenitor cell lines.The use of alternative protocols can allow samples with a wider range of RNA qualities to be used, facilitating the investigation of disease tissues. Adiconis et al.[s et al. examinedSpecifically, 20 ug of high-quality total RNA  was constructed by pooling RNA extracted using a Direct-zol RNA MiniPrep kit  from neural progenitor cell lines made from 4 individuals . RNA-SeqEach DNA library was sequenced with 4.5–60 million 100 bp single-end reads on an Illumina HiSeq2000. The reads were uniquely mapped with three or fewer mismatches using PerM [For confirmation of mapper accuracy, we mapped all of the samples using TopHat v1.4.0  to GENCOIt is likely that the poor performance of protocol #1 at lower RINs can be explained by the poly-A selection step. As RNA integrity decreases, less full length poly-A RNA is recovered, leading to a cDNA library that is increasingly 3′ biased. This is supported by analysis of the 5′ to 3′ read distribution of each library. Those from protocols #2 and #3 are essentially unchanged at decreasing RIN, while the distribution for samples from protocol #1 is severely 3′ biased by RIN 4.5 (data not shown).We recognize that our results are based on a single experiment using an RNA pool from 4 neural progenitor cell lines and are not broadly applicable. Hence, other investigators may want to use this method to determine the effect of RNA integrity on RNA-Seq from their tissue source of interest.In summary, our data show that the results of RNA-Seq are influenced by RNA quality with a widely-used cDNA/sequencing library construction protocol. However, this problem can be avoided with alternative protocols, allowing samples with a wider range of RNA qualities to be used, facilitating the investigation of disease tissues.


Different cell types and multiple cellular connections characterize the human brain. Gene expression analysis using a specific population of cells is more accurate than conducting analysis of the whole tissue homogenate, particularly in the context of neurodegenerative diseases, where a specific subset of cells is affected by the different pathology. Due to the difficulty of obtaining homogenous cell populations, gene expression in specific cell-types  has been understudied. To leverage the use of archive resources of frozen human brains in studies of neurodegenerative diseases, we developed and calibrated a method to quantify cell-type specific—neuronal, astrocytes—expression profiles of genes implicated in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining using cell-specific antibodies. The immunoreactive-cells were isolated by Laser Capture Microdissection (LCM). The enrichment for a particular cell-type of interest was validated in post-analysis stage by the expression of cell-specific markers. We optimized the technique to preserve the RNA integrity, so that the RNA was suitable for downstream expression analyses. Following RNA extraction, the expression levels were determined digitally using nCounter Single Cell Gene Expression assay (NanoString Technologies®). The results demonstrated that using our optimized technique we successfully isolated single neurons and astrocytes from human frozen brain tissues and obtained RNA of a good quality that was suitable for mRNA expression analysis. We present here new advancements compared to previous reported methods, which improve the method's feasibility and its applicability for a variety of downstream molecular analyses. Our new developed method can be implemented in genetic and functional genomic research of neurodegenerative diseases and has the potential to significantly advance the field. The human brain is characterized by an immense number of cells and cellular connections . Twenty-five subjects were selected with post-mortem intervals (PMI) of <13 h. The project was approved by the Duke Institutional Review Board (IRB). RNA Integrity Number (RIN) evaluation was done using RNA extracts from brain tissue. Brain samples that showed RIN values >7 were selected for conducting the following steps of the method introduced in this paper.3) using a clean mortar and pestle chilled in dry ice. Next, the brain pieces were embedded over dry ice into disposable vinyl specimen cryomolds using Optimal Cutting Temperature Compound  and held at −80°C. After 24 h, tissues from the molded blocks were sliced onto the pre-cleaned microscope slides. Samples were sliced into 8 μm sections using the Microm HM 505 N Cryostat at −20°C utilizing High Profile Microtome Blades . Slides with brain tissue slices were stored at −80°C. All methods were performed under RNase-free conditions.Microscope slides  were cleaned with RNase zap  and 100% ethanol , decontaminated under UV light for 30 min, and then chilled at −20°C until usage. Brain tissue samples were broken into smaller sections  and gently scraped from the slide surface. RNA was extracted directly from the slide following the manufacturer's protocol along with the addition of DNase I treatment . The quantity of RNA was analyzed using a NanoDrop 8000 UV-Vis spectrophotometer . The quality of the RNA was assessed on a 2100 Bioanalyzer  using the RNA 6000 Pico Chip . Samples above the upper limit of the qualitative range for the Agilent RNA 6000 Pico Chip (50–5000 pg/μl), as determined by the NanoDrop results, were diluted 20-fold with elution buffer before analysis on the Bioanalyzer. All samples with RIN values of <4.6, post-mounting and slicing, fail at this step and were excluded from the next steps of the LCM isolation for RNA analysis.Slides were held in place using a pre-chilled slide holder block. The holder block was submerged in ice throughout the staining process to maintain the cool temperature of the slide with the tissue section. Staining was performed rapidly using the Vectastain ABC Kit peroxidase enzyme system . Prior to conducting the staining protocol we prepared a 10X working solution by applying 50 μL of avidin/biotinalyted enzyme complex (ABC) into 500 μL of cold 1X Tris Buffered Saline  and incubating for ~30 min. The staining procedure was then performed. First, the tissue was hydrated onto the slide using a series of cold (−20°C) fresh ethanol dilutions: 100% (1 min) to 95% (15 s) to 75% (15 s). Next, the tissue was blocked using a 2% normal goat serum in 1X TBS for 3 min. The primary and secondary antibodies were diluted in the blocking solution. The primary antibody  was attached onto the collector stage. The corresponding PALM Robomover Z software was used to position the cap over the desired tissue section on the slide. Cells were visualized on the computer interface and manually selected using the CenterRoboLPC function. Subsequently, the microscope laser cut the tissue and catapulted the cells into the hovering adhesive cap. From each used slide 60–200 cells were collected. After microdissection, the adhesive caps were sterilely removed from the plastic and placed into 100 μl aliquots of lysis buffer . The cap and lysis buffer were incubated at 42°C for 30 min and then stored at −80°C until RNA extraction.For each tissue sample collected cells were combined in aliquots of a total of 200 cells. RNA was extracted from each aliquot of 200 cells using the Ambion RNAqueous-Micro Total RNA Isolation kit following the manufacturer's instructions. Samples were incubated with DNase I  for 15 min at room temperature to control for DNA contamination. RNA was eluted in 8 μl of Elution Solution and stored at −80°C.Six microliters of RNA were used for reverse-transcription (RT) into complementary DNA (cDNA) using 2 μL of SuperScript VILO Master Mix , under the following conditions: 10 min at 25°C, 120 min at 42°C, and 5 min at 85°C. Next, Multiple Target Enrichment (MTE) was performed using specific primers (Table http://www.nanostring.com/products/single_cell). Each sample was hybridized in a strip tube by adding buffer and a CodeSet of target-specific reporter and capture probes  human brain tissues were used to evaluate the RNA quality. RNA was extracted from three frozen brains and matched FFPE tissues (same ID#), and RIN was evaluated Table . All theThe PMI, age, and RIN for each of the initial sample set chosen for quality control (QC) are summarized in Table Figure We identified neurons and astrocytes in normal and diseased samples by utilizing rapid immunohistochemistry Figures , 3. We cOverall, we collected neurons from eight samples and astrocytes from three samples. Neurons were identified by Neurofilament immunoreactivity, i.e., positive cells presented with a golden-brown color differentiating them from the underlying background tissue Figure . AstrocyUsing the data generated by nCounter Single Cell Gene Expression Assay (Nanostring Technologies®), we evaluated two parameters relating directly to the immuno-LCM procedure that potentially may affect the successful accomplishment of the method: (1) duration of LCM Figure , and (2)Different neuronal genes  have been used to evaluate the neuronal enrichment. The expression of seven housekeeping genes  and GFAP as an astrocyte marker has been also analyzed. Raw data are reported in Supplementary Table Using the data generated by nCounter Single Cell Gene Expression Assay (Nanostring Technologies®), we found that RNA obtained from neurons displayed higher levels of specific neuronal marker transcripts relative to a calibrator. The calibrator sample consisted of RNA extracted from a whole section of normal brain tissue (heterogeneous cell populations). We evaluated the enrichment of neurons using three neuronal specific probes: ENO2 and two different probes to identify SYP, here labeled as SYP 1 and SYP 2. Counts for these neuronal markers were obtained for all samples and were normalized relative to the geometric mean of the housekeeping gene counts for each sample, respectively. The housekeeping genes used in this study were B2M, LDHA, and SDHA. The selection of these housekeeping genes was based on the counts obtained: specifically, we selected genes with high, medium and low counts (B2M>LDHA>SDHA). Our findings demonstrated neuronal enrichment in the collected neuron aliquots. Comparison of the computed relative expression level of each neuronal marker gene to that of the calibrator is shown in Supplementary Table Similarly, for the validation of the astrocyte enrichment we collected 200 astrocytes, GFAP-immunoreactive cells, from brain slides of three samples. We compared the levels of GFAP expression relative to the calibrator Table . The astThe total amount of RNA per cell is estimated to be 10 pg, however the expected yield of the procedure due to manipulations steps is lower than 100%. The initial amount of RNA we collected from 200 cells was measured in the range of <1 ng. The nCounter protocol required an input of a total of >50 ng RNA for collection of readable expression data. Therefore, a step of RNA amplification was necessary. We evaluated the number of cycles needed to detect mRNA expression in the LCM-RNA samples and yet remain within the quantitative range. We perform RNA amplification using three different cycle numbers: 12, 18, and 22. A comparison analysis of the nCounter Single Cell Gene Expression Assay (Nanostring Technologies®) data using RNA generated different number of cycles showed that we were able to detect mRNA using as little as 12 RNA amplification cycles and the obtained data using the 12 cycles was consistent and in the quantitative range.Archived brains from patients and control donors represent a great resource for studying neurodegenerative diseases. Changes in gene expression in brain tissues from neurodegenerative disease patients compared to healthy controls have been reported  prior to the manipulation steps of the method. Furthermore, to preserve the RNA quality, we optimized the rapid immunostaining on ice under RNAse-free conditions. Other factors that affect the RNA stability were examined as well. We observed that the factors such as the duration of LCM and days elapsed between LCM, and RNA extraction determined the successful performance of the technique. We found that the probability of success decreased with increasing duration of these parameters that are likely to affect the RNA stability.in situ hybridization, single cell quantitative real-time PCR, and microarrays. The in situ hybridization technique is prone to false positive results due to non-specific binding of the probes. Moreover, only fairly abundant mRNA can be detected using this technique. PCR-based techniques require substantial amounts of amplification cycles to allow the detection from RNA input extracted from single cells. Microarrays enable measurements of thousands of genes at once from single cells. However, small RNA input (<200 ng) requires amplification step, which might introduce bias and non-specific products  requires a MTE amplification step . We considered samples that showed an increase in SYP2/GFAP ratio >100-fold compared to the calibrator, to be enriched for neurons. We also collected homogenous aliquots of astrocytes and examined their enrichment by evaluating the expression of GFAP. GFAP is a widely accepted marker for astrocytes  developed here, will advance the understanding of the neuronal vs. astroglia-specific processes involved in the development of neurodegenerative diseases. The method can be implemented in a broader range of molecular analyses in addition to gene expression and is potentially applicable for studies of other diseases in which specific cell-type is affected.OC Conceived goals, guided method development, performed analysis, wrote the paper. LT, Guided method development, performed experiments and analysis, wrote the paper. KB, OG Performed experiments and analysis, wrote the paper.This work was funded in part by the National Institutes of Health/National Institute of Neurological Disorders and Stroke .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.


TP53) target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R) injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3) construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I) are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA) inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases.DNA damage-regulated autophagy modulator protein 1 (DRAM1), a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 ( Cerebral homeostasis and function rely on an adequate supply of oxygenated blood. Stroke, often caused by hypoxic ischemic encephalopathy and acute cerebrovascular incidents, interrupts cerebral blood supply and results in high mortality and long-term disability worldwide . The goaAutophagy is a basic catabolic mechanism involving intracellular components and damaged organelle degradation that maintains normal organelle function and nutrient restoration , and it TP53) target gene involved in autophagy and apoptosis [in vitro. We found that OGD/R induced DRAM1-dependent stimulation of autophagy in Neuro-2a cells. Knockdown of DRAM1 inhibited autophagy flux by decreasing autophagosome-lysosome fusion.DNA damage-regulated autophagy modulator protein 1 (DRAM1) is a multi-pass membrane lysosomal protein that has shown a high degree of conservation throughout evolution and has been reported as a tumor protein p53  to suppress autophagy activation. We found that the increases in DRAM1 expression and the LC3II/I ratio in the Neuro-2a cells treated with OGD/R were significantly inhibited after co-treatment with 3-MA, and the p62 decline was also attenuated B,C. In ain vitro model. DRAM1 encodes a 238-amino acid protein and is highly conserved in a number of species, including humans, mice, zebra fish, Drosophila, and Caenorhabditis elegans (C. elegans) [et al. also documented that DRAM1 knockdown inhibits p62 targeting to autophagosomes and reduces its degradation [DRAM1 expression was significantly up-regulated in OGD/R-induced Neuro-2a cells, peaking at 12 h of reperfusion along with the LC3II/I ratio. Meanwhile, 3-MA treatment reduced DRAM1 expression and LC3II/I ratio. These data suggest that the DRAM1 expression pattern and autophagy were regulated in the in the elegans) ,28. A prelegans) . Althougradation . Howeverradation , and knoradation . We hypoin vivo. After autophagosome formation, autophagosome turnover is mostly determined by the process of autophagosome-lysosome fusion and the degradation of autophagy contents by lysosomal enzymes [et al. reported that DRAM1 regulates autophagy in a 3-Nitropropionic Acid (3-NP) treatment A549 cell model through promoting lysosomal acidification and activating lysosomal enzymes [To investigate the mechanism by which DRAM1 regulates autophagy, we investigated the effects of DRAM1 on autophagosome-lysosome fusion because of its subcellular location. Lysosomes are rich in hydrolytic enzymes and are responsible for the degradation of intracellular materials captured by autophagy. LysoTracker Red was used to stain lysosomes. OGD/R treatment increased the number of LysoTracker-labeled lysosomes A. Neuro- enzymes . Due to  enzymes . Pretrea enzymes . Lysosom enzymes . Zhang e enzymes . Lysosom enzymes . Autopha enzymes . DRAM1-m2.The mouse neuroblastoma cell line Neuro-2a was purchased from the American Type Culture Collection  and cultured in Dulbecco’s Modified Eagle’s Medium  with 10% fetal bovine serum , 100 μg/mL streptomycin  and 100 U/mL penicillin . The cells were maintained at 37 °C in a saturated-humidity atmosphere containing 95% air/5% CONeuro-2a cells were transfected with the GFP–RFP–LC3 expression vector or DRAM1 siRNA using Lipofectamine 2000  according to the manufacturer’s instructions. Empty vector controls or negative siRNA controls were used for each transfection. The GFP–RFP–LC3 reporter plasmid  has previously been described . Small i2 and 5% CO2 at 37 °C. After 4 h, the cells were maintained in DMEM with 10% FBS and returned to normal conditions for 0, 6, 12 or 24 h.The OGD and reperfusion model was established as described previously . DMEM meThe cell viability assay was performed using the LIVE/DEAD viability/cytotoxicity kit for mammalian cells . Neuro-2a cells were cultured in 96-well plates for 24–48 h until acceptable cell density levels were reached. The cells were rinsed twice with D-PBS  to remove serum esterases and then incubated for 30 min at room temperature in a mixture of 2 μM calcein AM  and 4 μM ethidium homodimer-1 . The live  and dead  cells were visualized on a fluorescence microscope  and automatically counted using ImageJ software .The LysoTracker Red and Hoechst stains were diluted in DMEM. The cells were cultured in 37 °C medium containing 50 nM LysoTracker Red  and 10 μg/mL Hoechst  for 30 min. The cells were observed and analyzed using a fluorescence microscope. After GFP–RFP–LC3 reporter plasmid transfection and different treatment, the cells were observed and imaged with a confocal microscope , more than 30 cells were calculated.Western blotting was performed as previously described . The celp < 0.05 was considered significant.All results were expressed as means ± SDs. The differences between multiple groups were computed using a one-way analysis of variance (ANOVA) followed by Tukey’s test. A value of In conclusion, we demonstrated that DRAM1 regulated autophagy in an OGD/R-induced mouse Neuro-2a cell injury model. DRAM1 was initially described as regulating autophagy in cancer; here, we extended its role in the context of cerebral I/R. Our study suggested that DRAM1 expression patterns and autophagic pathways were regulated in OGD/R-induced Neuro-2a cell injury. Inhibition of autophagy exacerbated OGD/R-induced Neuro-2a cell injury, which involved the effects of DRAM1 and autophagosome-lysosome infusion. These findings may provide experimental and therapeutic options for the treatment of a broad range of pathologic disorders associated with cerebral ischemia reperfusion injury.


C9orf72 in amyotrophic lateral sclerosis/frontotemporal dementia and with polyglutamine diseases, respectively, localize to neuritic granules that undergo active transport into distal neuritic segments. In cultured mammalian spinal cord neurons, the presence of neuritic GGGGCC repeat RNA correlates with neuronal branching defects, and the repeat RNA localizes to granules that label with fragile X mental retardation protein (FMRP), a transport granule component. Using a Drosophila GGGGCC expansion disease model, we characterize dendritic branching defects that are modulated by FMRP and Orb2. The human orthologs of these modifiers are misregulated in induced pluripotent stem cell-differentiated neurons (iPSNs) from GGGGCC expansion carriers. These data suggest that expanded repeat RNAs interact with the messenger RNA transport and translation machinery, causing transport granule dysfunction. This could be a novel mechanism contributing to the neuronal defects associated with C9orf72 and other microsatellite expansion diseases.Microsatellite expansions are the leading cause of numerous neurodegenerative disorders. Here we demonstrate that GGGGCC and CAG microsatellite repeat RNAs associated with DOI:http://dx.doi.org/10.7554/eLife.08881.001 Genes contain instructions to build proteins, but these instructions are often interrupted by stretches of DNA that do not code for protein. Typically the entire length of a gene is copied to produce an intermediate molecule of RNA, which is processed to remove the non-coding regions before being translated to make a protein. The genes associated with various neurodegenerative diseases, including Huntington's disease and myotonic muscular dystrophies, often also carry short stretches of DNA sequence that are repeated one after the other.An increase in the number of the repeats within one of these genes can lead to a neurodegenerative disease. These disorders often have similar features, but are associated with different repeat sequences that can occur either in regions of the gene that code for protein or regions that do not. When the repeats lie in a non-coding region, it is thought that the RNA itself and not the protein causes the damage to nerve cells. While it is not known how this happens, it could be related to the shape of the RNA molecules, which in turn controls where the RNA molecules will go within a cell.Inside nerve cells, some RNAs  are directed to particles called 'transport granules'. These particles carry specific RNAs into the tips of the nerve fibers where they are then translated into proteins. Burguete et al. wanted to test whether disease-associated RNAs that contain repeats might interfere with this process in nerve cells. Microscopy showed that many RNAs with expanded stretches of repeats ended up in the transport granules by mistake, and were carried toward the tips of the nerve branches.When the repeat-containing RNAs localized to the transport granules, the nerve endings tended to form fewer branches. By analyzing the components of the granules, Burguete et al. could show that the incorrect localization of RNA molecules in the granules appeared to interfere with the production of proteins in the nerve branches. This disruption could contribute to the nerve cell defects seen in the many neurodegenerative diseases associated with these types of repeat expansions. These data suggest that preventing the disruption of transport granules’ activity could represent a potential therapeutic avenue against these diseases.DOI:http://dx.doi.org/10.7554/eLife.08881.002 C9orf72 gene has been shown to be associated with amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) . Most rALS/FTD) . How micn, occur in non-coding sequence, suggesting that the RNA product may be toxic (repeat associated non-ATG (RAN) translated peptides . These line #11 . We confdization . The GGGdization . In addidization . We did 100, in primary rat stage E14 mixed spinal cord neurons  neurons  and probtructure , conferstructure , and hastructure . As withtructure . We alsotructure . These dcoat-binding protein, which is fused to a nuclear localization signal and to green fluorescent protein (NLS-CP-GFP). When expressed alone, the NLS-CP-GFP signal was predominantly nuclear (not shown). Co-expression of NLS-CP-GFP with either of two control RNAs, the MS2 100 by in situ hybridization 10neurites . RNA par-MS2 RNA . In cont compare , with 94 compare .48-MS2 RNA and found it neuritically localized in 21.1 ± 3.7% SD  of all neuron types in the cultures (100-MS2 and (CCUG)100-MS2 RNA particles were also present in neurites in over 75% of the transfected neurons (100-MS2), multiple microsatellite RNA repeats  with high structural context became localized to RNA particles in neurites, by independent detection methods and in a variety of neural systems.Next, we examined (GGGGCC)cultures , and in cultures . The RNAcultures . Indeed, neurons . Thus, i3-MS2 RNA particles (48-MS2 particles (0.24 ± 0.04 SD) , than for (GGGGCC)48-MS2 . These data indicate that three GGGGCC units, at the low end of non-expanded C9orf72 alleles (Disease severity and age of onset in patients with trinucleotide repeat expansion disorders (e.g. CAG and CUG) correlates with increasing repeat number . Therefoarticles  was high0.04 SD) , and the alleles , are suf48-MS2 and (CAG)100-MS2 particles were undergoing anterograde, retrograde, and bidirectional movement, both proximally as well as distally along neurites 100-MS2, and the average max velocity was 1.40 μm/s for (GGGGCC)48-MS2, and 1.85 μm/s for (CAG)100-MS2 neurites –3. Similneurites , the mea)100-MS2 . By cont)100-MS2 . We coulC9orf72 hexanucleotide expansion carrier-derived neurons and in transfected rat spinal cord neurons raised the possibility that such expanded repeat RNA may confer local toxicity. We therefore analyzed neuritic arborization patterns in rat mixed spinal cord neurons with (GGGGCC)48-MS2 RNA localized to the nucleus, the soma, or the neurites. While 37.5 ± 3.7% SD  of neurons in the total population contained RNA in the soma  than neurons with nuclear (GGGGCC)48-MS2 RNA foci , or than neurons with somatic but without neuritic (GGGGCC)48-MS2 RNA  , which lacked neuritic RNA particles 48-MS2 RNA . Similarly, the expression level of the RNA in the soma did not affect primary branch number 48-MS2 RNA association into neuritic particles or to induce branching defects. These data argue that the presence of the (GGGGCC)48-MS2 RNA in neuritic particles is associated with deleterious effects on neuronal branching.The presence of the GGGGCC repeat RNA in distal neuritic particles in the soma , about hRNA foci . Neurons compare . They al compare . In cont-MS2 RNA  and E. W3-MS2 RNA did not confer dramatic primary branch loss.Expanded repeat RNAs have also been reported to undergo translation into peptide repeat proteins. We looked for RAN translated peptide repeat proteins derived from the repeat RNAs  by immunDrosophila. To visualize this, we used fly lines expressing (GGGGCC)48 repeat RNA or DsRed control RNA (UAS-(GGGGCC)48 resulted in dramatic dendritic branching defects compared with the UAS-DsRed control at late third larval instar (compare UAS-(GGGGCC)48 RNA appeared nearly normal (compare UAS-DsRed control neurons (compare yellow bars in UAS-(GGGGCC)48 RNA had failed to extend far from the cell body (compare pink bars in boxed areas in UAS-(GGGGCC)48 RNA, coinciding with a 53% loss of higher order branches (orders 13–24) (UAS-(GGGGCC)48 RNA were capable of establishing a complex dendritic arbor; however, they subsequently failed to extend and underwent late-stage degeneration of pre-established branches.To further address the functional impact of the expanded microsatellite repeat on neuron morphology, we analyzed repeat RNA-induced dendritic degeneration in trol RNA  in the htrol RNA , 2003. Ttrol RNA . Express compare . To dete compare ; however compare . At earl compare , with a s 13–24) . These dC9orf72 patients 48-induced dendritic branching defects (compare dFMR1 in the context of UAS-(GGGGCC)48 expression potentiated the branching defects, reducing intersections by 70% 48-induced branching defects: distal intersections were doubled upon downregulation (103% increase 140–380 μm from cell body), while upregulation resulted in a 34% overall loss (dFMR1 RNAi), and to 94% (orb2 RNAi) (compare UAS-(GGGGCC)48, up- or downregulation of dFMR1 and orb2 resulted in minimal changes in the dendrite intersection distribution. Upregulation of dFMR1 or orb2 alone did not reduce the number of intersections , respectively , a component of mRNA transport granules and a local translational regulator , and ass compare  with a n compare . In contall loss  with 4B. compare . Our consections . Knockdoectively . The eff neurons . Taken tdFMR1 raised the possibility of a functional association between the repeat RNA in the RNA-granules and FMRP protein. In rat spinal cord neurons, both endogenous and exogenous FMRP colocalized in neuritic granules with (GGGGCC)48-MS2 and (CAG)100-MS2 repeat RNAs in neuronal processes  can be recognized by the mRNA localization machinery, can become incorporated into neuritic RNA transport granules, and, at least for expanded GGGGCC hexanucleotide repeat RNA, may disrupt RNA granule function. The RNAs expressed are directed to the cytoplasm with a poly(A) tail, as are repeats that occur within the mRNA of the respective disease genes. In the case of the hexanucleotide expansion in C9orf72, although the repeat is defined as intronic, we saw neuritic GGGGCC RNA granule localization in iPSNs, indicating the repeat can localize to the cytoplasm in disease. Notably among the large portion of mRNAs that are localized, RNAs with stem-loop structure commonly function as cis-acting localization signals . Indeed,3 repeat—branching in neurons with neuritic non-expanded repeat is not significantly different from branching in neurons with somatic or nuclear expanded repeat. These data indicate that the incorporation of expanded microsatellite repeat RNAs into granules within neurites induces dysfunction. The finding of branching defects in rat primary spinal cord neurons in culture was also extended to da neurons in Drosophila. In vivo, the dendritic arbors of da neurons are normal early, but later show a different pattern with fewer intersections and smaller field. This effect is distinct from defects in endosomal transport, as described for dynein loss-of-function mutations   . Howeverin vivo in a Drosophila model. In culture, we have shown many different expanded microsatellite repeat RNAs are incorporated into neuritic granules, and at least several are actively transported. For the GGGGCC repeat, a number of proteins that bind the repeat (hnRNP A3) or are modifiers of GGGGCC repeat toxicity  are implicated in transport granule function 70, (CAG)100, (CUG)100, (CCUG)100, (GGGGCC)48, and (GAA)100. The backbone, as well as the repeat sequences, were synthesized and ligated into pUC57 . The repeat sequences contained 5’ EcoRI and 3’ BamHI sites, and the first base of the first tandem repeat was omitted if it started with cytosine. The backbone contained the following in 5’ to 3’ order: a 6Stop sequence (carrying six 5’ stop codons (underlined) in the leader sequence, two in each reading frame) containing a 3’ EcoRI site (TAGCTAGGTAACTAAGTAACTAGAATTC (20-MS2 and (GGGGCC)3-MS2 were made by polymerase chain reaction (PCR) using complimentary oligos and ligated into pGW containing the backbone, as described above. LacZ was amplified by PCR and ligated into pGW-MS2 to generate LacZ-MS2. (CAG)100, shown in 100-FLAG-HA-Myc and cloned into pcDNA, and lacked an MS2 tag. CP-(GFP)2 , and FMRP-RFP was a kind gift from Dr. Ian Macara . A backbone was designed to receive the repeat sequences (CAG)TAGAATTC ), followTAGAATTC  (of sequP-(GFP)2  with a 5Embryonic Sprague Dawley rat spinal cord neurons from embryonic day 14 were grown on previously established cortical postnatal d1-3 astrocyte monolayers . Neurons8 and (CAG)8 sense and antisense oligonucleotide probes were generated , in situ hybridization was performed (4 oligonucleotide probe was used for in situ hybridization of iPSNs as described (DIG labeled (CUG)erformed , and theescribed . A LeicaEarly and late third instar larvae were filleted in ice cold phosphate-buffered saline (PBS), fixed in PBS/4% paraformaldehyde, and stained with chicken α-GFP as described above. Post-fix and post-stain washes included three rinses and 3 × 15 min in PBS/0.3% Triton X-100. Secondary antibodies were conjugated to Alexa 488 .Drosophila lines to knock down orb2 , and dFMR1  were from the Bloomington stock center. The UAS-(GGGGCC)48 repeat sequence 48 and the construct was injected to generate transgenic strains . UAS-dFMR1 was from Dr. Thomas Jongens . The UAS-DsRed strain was used as a control for UAS-(GGGGCC)48. UAS-DsRed  software . Sequential acquisition was applied when capturing an image in multiple channels. Similar voltage settings were applied when capturing images of rat neurons transfected with different constructs, and a saturation threshold was applied. For 2. Individual frames were generated at 1 s intervals for single channel imaging. The percentage of neurons with distal (GGGGCC)48-MS2, and (CAG)100-MS2 RNA particles detected by live imaging (six sessions for (GGGGCC)48-MS2, and two sessions for (CAG)100-MS2 was similar to that seen in fixed neurons from multiple biological replicates. Two sessions for (GGGGCC)48-MS2 were excluded due to low transfection efficiency.Images of spinal cord neurons at 17–24 hr post transfection, were collected with a Deltavision Core Deconvolution Microscope , equipped with an Olympus IX70 microscope and a Photometrics CoolSNAP HQ camera, a 60X, 1.42 NA oil immersion PlanApo lens , and softWoRx  acquisition software. Environmental control was provided by a home-built plexiglass cage surrounding the entire microscope, kept at 37ºC and 5% CODrosophila da neurons were traced, and the tracings were analyzed with Neurolucida and Neuroexplorer software, respectively . For the rat dendritic arbor analysis, only neurons that had a cell body diameter of >20 μm, and had more than two primary arbors were included. A pre-established standard cell sample size  was used for this type of analysis, except for samples that had nuclear or neuritic (GGGGCC)48-MS2 RNA (n=12), due to the limiting inclusion criteria used. For quantitation of PSD-95 and CPEB3 particles in iPSNs, z-stacks taken with a 63× objective acquired on a Leica confocal microscope equipped with a HyD detector were projected, and the cell body was outlined. The particle number and size were analyzed using the 'analyze particle' function of Image J (NIH), using the Yen or Max Entropy auto-thresholding methods. Our analysis of PSD-95 in the entire cell body differs from previous quantitation solely in dendrites . The colocalization coefficients (M1 and M2) were computed  for regiendrites . Total pAll samples, including all animal experiments, were randomly assigned to processing order, and for cell transfections, the positions in the wells were random. Data was also collected randomly.C9orf72 patient samples were compared with several gene lists as indicated in the text: RNA binding proteins and mRBP classifications were from . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , and two reviewers (Roy Parker and Harry Orr).Thank you for submitting your work entitled "GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function" for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript addresses the possible mechanisms by which microsatellite expansions in mRNAs may lead to neurodegenerative disease. The main contribution is to show that GGGGCC and CAG repeat RNAs can localize to granules within neurites, which is clearly documented. Additional work leads the authors to argue that repeat RNAs in neurites leads to loss of dendritic branching and toxicity, which may be related to changes in the levels of key components of RNP granules.Essential revisions:Roy Parker raises some important points that would need to be addressed, primarily stronger data showing that the repeats are causative for both localization and toxicity (see complete comments below). If you think that you can substantially address these comments within the next few months, we would be willing to consider a revised manuscript.Additional points:Additional comments from Harry Orr: “I think reviewer 1 (Roy Parker) raised some important points (1-4). Demonstrating neurite pathology in disease, I assume in humans, is a tall order particularly since there is not a mouse model of at least C9ORF except for the viral mediated disease recently published in Science”.C9orf72 papers show RNA foci only in cell body or nucleus). The protein partner of GGGGCC RNA foci they found was FMRP, which unfortunately for these authors, is now published by another group . Although the binding between GGGGCC and FMRP is not novel anymore, they showed nice rescue with FMRP knockdown in flies, suggesting functional importance of this interaction”.The Reviewing editor also consulted another expert, who provided the following comments: “The only novelty in this paper is the existence of GGGGCC RNA foci in dendrites  clear documentation that the neurite localization is specific to the repeat sequences, 2) that the toxicity is due to neurite localization of RNAs, and 3) some limitations in specific experiments. I would think the manuscript should be improved technically before further consideration. A downstream issue will be whether this type of toxicity, without demonstration in disease, or an understanding of mechanism will be sufficient for publication in Specific Comments:1) It is clear that the repeat RNAs can be seen in neurites. However, the basis for this specificity is not clear. Is the control the exact same RNA without the repeats inserted? From my reading of Methods it seems not. Would any highly expressed RNA end up in neurites? Evidence that the repeats are what allows accumulation should be either provided, or if already present, clarified by more explicit description of the experiments and control RNAs examined. A simple figure showing the compared RNAs would be helpful.I think it would also improve the clarity and strength of this experiment to describe the cells more completely, e.g. what percentage of cells has nuclear foci? Somatic foci? Neurite foci? Does neurite foci correlate with expression level in the soma? If not, showing so would improve the rigor of the experiment.2) A second key contribution is the conclusion that neurite RNA limits arborization. To make this rigorous it would be important to again show that this only correlates with neurite RNA and not soma foci, nuclear foci, or expression level. Since one is showing correlations, being sure to examine if there are other possible correlations is important.3) It was unclear how the authors are concluding there are changes in the levels of mRNP granule components. As I read the work, it seems they are showing a change in average intensity in granules when the repeat RNA is expressed. Is this due to less protein in the cells? To less granule formation? This should be clarified.4) The presentation of the effects on neurite arborization is difficult to follow. I request the following alterations:A) For the experiments in rat neurons it would be appropriate to examine each neuron examined for the overall level of the repeat RNA, the presence of soma foci and nuclear foci. This is an attempt to show that the correlation with neurite foci is at least as strong a correlation as one can assess.B) For the experiments in flies I request the following:48 RNA. Are they the same mRNAs just with an insertion of repeats? Or are they actually different? (dsRed vs. just a repeat RNA?) From my reading of Methods it seems they are different RNAs.i) A clear description of the differences between the "control" RNA and the (GGGGCC)48 expansion, the key comparison is the control to the (GGGGCC)48 expression lines at early and late time. The figures would be easier to interpret if 48 and 48 RNA.ii) For comparison of the effects of the (GGGGCC)iii) Earlier work  had shown that loss of function mutations in FMR result in higher-order dendritic branches, while the overexpression of FMR dramatically decreases dendritic branching. Given this it is surprising that no effects of FMR OE or RNAi are seen in the wild-type control. It might be worth commenting on this and clarifying that they see a different result. I raise this issue since OE of many FMR interacting proteins decreased dendritic branching in similar experiments . Perhaps a different driver is being used that is not as strong, which should just be clarified?48 lines since this is critical to interpretation of the experiment.I would suggest that these key wild-type controls be included in the main text and presented in the same manner with the same type of images as the (GGGGCC)Reviewer #2:Since discovery of the C9ORF hexanucleotide repeat as a major cause of inherited ALS/FTD much effort has gone into understanding its pathogenic basis. This study presents data in support of the concept that the repeat, based on its structural features, directs repeat-containing RNA to transport granules in neurons. Interestingly, they show that the ability of the RNA to form a structure is critical for its targeting to RNA granules – for example the Frataxin repeat that does not for a structure did not localize to granules. They go onto demonstrate that association of repeat-RNA to granules is linked to neurite degeneration and alterations in proteins, whose translation is regulated by FMRP and Orb2. Overall this a strong study that uses results from several approaches and model systems to support their novel conclusion that repeat containing RNA disrupts RNA transport granule function that may be another pathogenic mechanism. A minor point that I think should be addressed is:In the subsection “Neuritic subcellular localization is a common property of highly structured microsatellite repeat RNAs”, the authors describe that the number of granules with wt G4C2 RNA is much higher than those form in the presence of a G4C repeat with 48 repeats. Does this suggest that while targeting information is retained by expanded repeat the ability to form granules is compromised? Essential revisions: Roy Parker raises some important points that would need to be addressed, primarily stronger data showing that the repeats are causative for both localization and toxicity (see complete comments below). If you think that you can substantially address these comments within the next few months, we would be willing to consider a revised manuscript. Additional points: Additional comments from Harry Orr: “I think reviewer 1 (Roy Parker) raised some important points (1-4). Demonstrating neurite pathology in disease, I assume in humans, is a tall order particularly since there is not a mouse model of at least C9ORF except for the viral mediated disease recently published in Science”. The Reviewing editor also consulted another expert, who provided the following comments: “The only novelty in this paper is the existence of GGGGCC RNA foci in dendrites (whereas other C9orf72 papers show RNA foci only in cell body or nucleus). The protein partner of GGGGCC RNA foci they found was FMRP, which unfortunately for these authors, is now published by another group . Although the binding between GGGGCC and FMRP is not novel anymore, they showed nice rescue with FMRP knockdown in flies, suggesting functional importance of this interaction”.We thank the external consultant for appreciating the rescue of GGGGCC-induced defects by dFMR1 knockdown, which we agree suggests that the RNA-dFMR1 interaction is functionally important. We likewise agree that a dFMR1 interaction with GGGGCC RNA has been previously described, by in vitropull down and mass spectrometry .31 RNA induced formation of Purα and FMRP foci in NSC34 and in HeLa cells, but the (GGGGCC)31 RNA and FMRP were not found to colocalize, see However neither of these studies shows colocalization between GGGGCC repeat RNA and FMRP. In the Rossi et al. paper, overexpression of (GGGGCC)Reviewer #1:The strength of the manuscript is to show repeat RNAs in neurites, and to suggest possible phenotypic consequences of that localization. The limitations of the work are: 1) clear documentation that the neurite localization is specific to the repeat sequences, 2) that the toxicity is due to neurite localization of RNAs, and 3) some limitations in specific experiments. I would think the manuscript should be improved technically before further consideration. A downstream issue will be whether this type of toxicity, without demonstration in disease, or an understanding of mechanism will be sufficient for publication in eLife. Specific Comments: 1) It is clear that the repeat RNAs can be seen in neurites. However, the basis for this specificity is not clear. Is the control the exact same RNA without the repeats inserted? From my reading of Methods it seems not.We thank Dr. Parker for noting that one of our major findings, that expanded structured repeat RNAs are localized to neurites, is clear. Below we address his concerns regarding the specificity of this result.To verify the specificity of the localization of the experimental repeat RNAs, several control RNAs were examined. As suggested, we now include a figure depicting all of these constructs (100-MS2 RNA. These control RNAs contain exactly the same sequences as the (CAG)100-MS2, (CAG)70-MS2, (CAG)40-MS2, (CAG)20-MS2, (CUG)100-MS2, (CCUG)100-MS2, (GGGGCC)48-MS2, and (GGGGCC)3-MS2 RNAs, except that they either have no microsatellite repeats (MS2 RNA), or contain 100 GAA repeats ((GAA)100-MS2), not predicted to have stem-loop structure). All other parts of the sequence are identical. As described in the first paragraph of the Methods section, all of these experimental and control RNAs have a 5’ leader sequence that includes 6 stop codons (two in each reading frames), an EcoRI site, a BamHI site, as well as a 3’ sequence that includes FLAG-, HA-, and Myc-tags, and 12 MS2 stem loops.The control constructs arguing for specificity include MS2 RNA and (GAA)100-MS2 control also indicates that a non-structured expanded repeat sequence present in the context of the leader and 3’ sequences is not sufficient to confer neuritic localization.The MS2 RNA and (GAA)100-MS2 RNA controls demonstrate that the leader sequence and 3’ sequence are not sufficient to confer neuritic localization. The (GAA)In addition we used a LacZ-MS2 RNA control. This control RNA did not include the leader sequence or the FLAG-, HA-, and Myc- tags, but did include the 12 MS2 stem loop tag, thus demonstrating that the 12 MS2 stem loops are not sufficient to confer neuritic localization to an overexpressed mRNA. SeeFigure 1—figure supplement 1 and accompanying legend.We have added Would any highly expressed RNA end up in neurites? Evidence that the repeats are what allows accumulation should be either provided, or if already present, clarified by more explicit description of the experiments and control RNAs examined.In 100 RNAs. As described above, MS2 and (GAA)100-MS2 have the exact same flanking sequences as the experimental RNAs, but these RNAs are not localized in neurites. Likewise LacZ-MS2, expressed from the same promoter, is not localized in neurites. These findings show that high expression of any RNA is not sufficient for localization to neurites, and that there is selectivity for the repeat RNAs in question – the structured repeat RNAs tested do localize, but the unstructured control (GAA)100-MS2, and the MS2 controls (MS2 and LacZ-MS2) do not. This is shown in For the experiments in rat primary culture where we employed exogenous expression, as noted above, we provide several controls including MS2 only, LacZ-MS2, and MS2 tagged (GAA)We also show in 48-MS2 vs. (GGGGCC)3-MS2 below, under Dr. Orr’s minor point.Also see Discussion for (GGGGCC)A simple figure showing the compared RNAs would be helpful.We thank Dr. Parker for the suggestion, and to clarify the nature of the constructs, we have added I think it would also improve the clarity and strength of this experiment to describe the cells more completely, e.g. what percentage of cells has nuclear foci? Somatic foci? Neurite foci? Does neurite foci correlate with expression level in the soma? If not, showing so would improve the rigor of the experiment.We have added the requested data in the text, figures, and figure legends. The percentage of neurons with nuclear foci in the total mixed rat spinal cord neuron population is 10.0 ± 4.0% s.d. (n=108 neurons). These data are in the Results, subsection “Expanded GGGGCC microsatellite repeat RNA causes neuritic branching defects”, first paragraph, and we also show an example of a neuron with nuclear foci in a new The percentage of neurons with somatic RNA in the transfected population is 37.5 ± 3.7% s.d. , see the aforementioned paragraph in the Results section. In original We note that nuclear foci cannot be scored simultaneously with somatic or neuritic foci due to the limitations of the MS2 system: nuclear foci are visualized by expressing NES-CP-GFP with the MS2-tagged repeat RNA whereas somatic and neuritic foci are visualized by expressing NLS-CP-GFP with the MS2-tagged repeat RNA.Does neurite foci correlate with expression level in the soma? If not, showing so would improve the rigor of the experiment.We show in 48-MS2 RNA particles and expression level in the soma. Indeed, neurons with neuritic (GGGGCC)48-MS2 RNA did not have significantly higher expression level in the soma than neurons without neuritic (and only somatic) (GGGGCC)48-MS2 RNA . This is now indicated in the Results (subsection “Expanded GGGGCC microsatellite repeat RNA causes neuritic branching defects”), shown in a new To address Dr. Parker’s concern in greater detail, we have also now tested whether expression levels of the exogenous RNA correlated with neuritic localization in primary rat neurons. Expression level of neurons was quantified using ImageJ on digital immunofluorescent images, as now detailed in the Methods, subsections “Data analysis” and “Neuron culture and immunostain”. We do not find a significant correlation between neurons with neuritic (GGGGCC)2) A second key contribution is the conclusion that neurite RNA limits arborization. To make this rigorous it would be important to again show that this only correlates with neurite RNA and not soma foci, nuclear foci, or expression level. Since one is showing correlations, being sure to examine if there are other possible correlations is important.We appreciate Dr. Parker’s concern and want to clarify that, in the original As Dr. Parker suggests, we now add new data examining the relationships between nuclear foci and branching. We examined neurons co-expressing NES-CP-GFP and (GGGGCC)48-MS2: these data show that neurons with nuclear foci have significantly more primary branches  than neurons that contain neuritic RNA , see new 48-MS2 – we do not find a correlation between expression level and primary branch number, and in Further, we add new data examining the relationship between expression level and branching, determined by scoring the fluorescence intensity of outlined neuronal cell bodies using imageJ, and scoring primary branches in neurons with a minimum of 2 primary branches, and a cell body diameter that was larger than 20 μm. New 3) It was unclear how the authors are concluding there are changes in the levels of mRNP granule components. As I read the work, it seems they are showing a change in average intensity in granules when the repeat RNA is expressed. Is this due to less protein in the cells? To less granule formation? This should be clarified.The changes in mRNP granule components were scored in iPSNs derived from human carriers of GGGGCC expansions, as compared to iPSNs derived from non-carrier controls; there was no transfection in this experiment .For PSD-95  and CPEB3 (third paragraph): we examined the number of foci and the total protein levels using imaging analysis. The number of foci was scored by automatic thresholding (to avoid user bias) and particle analysis using ImageJ as described in Materials and methods, subsection “Data analysis”. Total levels of protein were scored as total pixel intensity per neuron using ImageJ; the non-carrier control pixel intensity was normalized to one.For all of these analyses, carrier and control iPSNs were stained in the same experiment and captured with the same confocal/laser settings. For FMRP , total protein levels were assessed as for PSD-95 and CPEB3. Based on these data we conclude that there are more PSD-95 and CPEB3 granules and more CPEB3, FMRP and PSD-95 protein in carrier-derived iPSNs.To make this clearer in 4) The presentation of the effects on neurite arborization is difficult to follow. I request the following alterations:A) For the experiments in rat neurons it would be appropriate to examine each neuron examined for the overall level of the repeat RNA, the presence of soma foci and nuclear foci. This is an attempt to show that the correlation with neurite foci is at least as strong a correlation as one can assess.Please see Specific Comment 2, which addresses all of these points.B) For the experiments in flies I request the following:48 RNA. Are they the same mRNAs just with an insertion of repeats? Or are they actually different? (dsRed vs. just a repeat RNA?) From my reading of Methods it seems they are different RNAs.i) A clear description of the differences between the "control" RNA and the (GGGGCC)48 repeat expressed in da neurons is the UAS-DsRed transgene, previously described as a control for expanded CAG repeat constructs . This transgene encodes DsRed expressed from the same UAS vector as UAS-(GGGGCC)48.The control used for the (GGGGCC)Drosophila strains”), and in a new A clear description of the constructs is now provided, in the main text , in the Methods section 48 expression lines at early and late time. The figures would be easier to interpret if 48 and 48 RNA.ii) For comparison of the effects of the (GGGGCC)48, and between late control to late (GGGGCC)48 are key for showing the effects of GGGGCC expansion.We appreciate Dr. Parker’s point that the comparison between early control to early (GGGGCC)A comparison between beige bars in The original 48, and of late control to late (GGGGCC)48 in new graphs requires showing exactly the same data that is displayed in Because a direct comparison of early control to early (GGGGCC)iii) Earlier work  had shown that loss of function mutations in FMR result in higher-order dendritic branches, while the overexpression of FMR dramatically decreases dendritic branching. Given this it is surprising that no effects of FMR OE or RNAi are seen in the wild-type control. It might be worth commenting on this and clarifying that they see a different result. I raise this issue since OE of many FMR interacting proteins decreased dendritic branching in similar experiments . Perhaps a different driver is being used that is not as strong, which should just be clarified?48 lines since this is critical to interpretation of the experiment.I would suggest that these key wild-type controls be included in the main text and presented in the same manner with the same type of images as the (GGGGCC) As Dr. Parker points out, Lee et al. (2003) described da branching defects in dFMR1 loss of function and overexpression studies.dFMR1 null animals and described a mild excess branching in ventral da neurons in segments 5 and 6 (Thoracic segments T2 and T3); they note that these changes are subtle and the da neuron branching characteristics are overlapping with the normal variation in wild type, but with a shift in distribution. They do not mention an effect on dorsal da neurons for dFMR1 loss of function.In the case of loss of function, they generated dFMR1 using a UAS-dFMR1 RNAi line reveals little, if any, effect on dorsal da neuron branching in a wild type background (see 48 induced branching defects in a (GGGGCC)48 background . We used a different Gal4 driver line  to drive expression of dFMR1, and scored dorsal da neurons in Abdominal segments A3 and A4; we found no adverse effect on dorsal da neurons in a wild type background (dFMR1 expression in a (GGGGCC)48 background dramatically enhanced the expanded repeat-induced phenotype . These differences are now noted in the subsection “Transport granule components modulate GGGGCC-induced branching defects in Drosophila da neurons”.In their overexpression studies, Lee et al. used Gal4 line 10980 to drickground , whereashenotype . Hence, orb2 down- and upregulation to orb2 RNAi and overexpression on da neuron morphology have not been previously described.We have moved these critical controls to 477 driven upregulation of dFMR1 .


Candida albicans is an important polymorphic human pathogen. It can switch from a unicellular yeast form to germinating hypha, which may play a role in making it the successful pathogen it is. This hyphal transformation can be triggered by various extracellular stimuli, the most potent one being serum from any source. We have previously reported that Candida albicans transiently polyadenylates portions of both the large and small subunits of ribosomal RNA, shortly after serum exposure. Northern blots at the same time suggested that serum might induce a decrease in total ribosomal RNA. We have carried out a number of experiments to carefully assess this possibility and now report that serum significantly reduces ribosomal RNA in Candida albicans. Fluorometric measurements, Northern blotting and quantitative RT-PCR, have all confirmed this decrease. Timed experiments show that serum induces this decrease rapidly, as it was seen in as early as five minutes. Cell mass is not decreased as total cellular protein content remains the same and metabolic activity does not appear to slow, as assessed by XTT assay, and by the observation that cells form hyphal structures robustly. Another hyphal inducer, N-acetylglucosamine, also caused RNA decrease, but to a lesser extent. We also observed it in non-germinating yeast, such as Candida glabrata. The reason for this decrease is unknown and overall our data suggests that decrease in rRNA does not play a causal role in hyphal transformation. Rapid and significant decrease in a molecule so central to the yeast’s biology is of some importance, and further studies, such as its effect on protein metabolism, will be required to better understand its purpose. Candida species are important pathogens especially for immunocompromised patients. They are the fungal species most commonly recovered from normally sterile sites in these patients [Candida species that cause human disease, variable phenotypic behaviors are observed. These range from polymorphic changes exhibited to external signals by Candida albicans, to Candida glabrata which remains in a yeast form exclusively [Candida albicans and a number of these are now well established, including some with negative regulatory activity [patients . Among tlusively . Yet, clactivity , 4. It wactivity , 6, 7. Tactivity , 9. At tCandida albicans SC5314 (purchased from ATCC MYA 2876) was maintained in 50% glycerol in YPD broth  at -80°C. Cells were activated in YPD broth at 30°C and maintained on Sabouraud dextrose agar at 4°C, passaged every 4–6 weeks up to 4–5 times. Additional yeasts were received from the Greater Los Angeles VA System microbiology laboratory. These included Candida krusei ATCC 14243, Candida tropicalis ATCC 750, Candida glabrata MYA 2950 and a Candida dubliniensis WLA 7.15 a clinical isolate identified by Vitek 2 (Biomerieux) and ChromAgar Candida (Hardy Diagnostics). Yeast were lifted from agar surface and grown in YPD broth for 16 hours at 30°C. Yeast cell concentrations were established using a hemocytometer. 5 X 107 cells were collected by centrifugation, washed with sterile phosphate buffered saline (PBS) and were put on ice to await total RNA extraction (referred to as T0 min). For germination assays again 5 X 107 cells were suspended in 50 ml of pre-heated (37°C) 10% v/v heat inactivated fetal bovine serum (FBS) in sterile double distilled water for a final concentration of 1 X 106 cells ml-1. Additional assays with other media included water alone, undiluted FBS, fresh YPD broth alone, YPD broth with 10% (v\v) FBS and 2% (w\v) N-acetyl-D-glucosamine (GlcNAc). All of these assays had yeast concentration of 1 X 106 cells ml-1 and the same 50 ml volumes at 37°C. The length of assays was as reported in the result sections. It has also been reported for C. albicans, that change in temperature alone from 37°C to 39°C in YPD induces hyphal transformation [7 ml-1. At the same time a second group of cells same in number were put on ice (T0). After 1 hour total RNA was extracted from both groups and measured as above. Germ tube formations were followed microscopically for all assays.After incubation for germination, cells were collected with centrifugation at 5°C, washed with cold PBS and had RNA extracted simultaneously with T0 min cells. Total RNA isolation was done by using the components and the protocol of the RiboPure Yeast RNA Purification Kit (Life technologies). This included cell disruption with RNAse-free zirconium beads in the presence of RNAse inhibiting lysis solution and column purification. The isolated RNA was treated with RNAse-free DNAse I. To insure that another mode of isolation gave us similar results as the kit, RNA was extracted simultaneously once, using the hot acid phenol isolation protocol . RNA qua-1 was made in pre-heated (37°C) PBS. 370 μl of this solution was combined with 30 μl of 1 mM menadione (Sigma-Aldrich) previously prepared in acetone. Yeast cells were collected by centrifugation, washed with PBS and suspended in the 400 μl of working solution in 96-well plates. Plates were incubated at 37°C with moderate shaking. To measure formazan derivative formation, spectrophotometric readings at 490 nm were obtained at appropriate times, using the Epoch Micro-plate Spectrophotometer (BioTek).To insure that cell viability is not a confounding factor, XTT viability assays were performed simultaneous to RNA extraction . Yeast cTo exclude significant loss in cell mass, protein was extracted using Y-PER Yeast Protein Extraction Reagent (Thermo Scientific). Yeast numbers, germination conditions and timing were the same as those for the RNA extraction and XTT experiments. After the incubation periods, cells were pelleted at 3000 x g for 5 minutes at 4°C. The pellets were suspended in 250 μL Y-PER reagent and vortexed gently at room temperature for 20 minutes. The mixture was centrifuged at 14,000 x g for 10 minutes saving the supernatant containing the soluble protein. The protein concentration was measured with the Qubit 2.0 fluorometer using the Qubit Protein Assay Kit (Life Technologies).Candida albicans SC5314 were synthesized (Blue Heron Gene Synthesis Co.) . Inserts were released with BamH I/EcoR I for 25S, 5S, and BamH I/Nco I for 18S, and were purified with a QIAquick Gel Extraction Kit (Qiagen). 50–100 ng of purified inserts were biotinylated using the BrightStar Psoralen-Biotin Nonisotopic Labeling Kit (Life Technologies). Membranes were pre-hybridized at 55°C in North2South hybridization buffer (Pierce) for 30 min followed by overnight hybridization with denatured biotinylated probes at 5ng ml-1 in the same buffer at 55°C. Membranes were washed with low and high stringency buffers at room temperature and RNA was detected using Chemiluminescent Nucleic Acid Detection Kit (Pierce) according to the manufacturer’s protocol. Film was developed with the SRX-101A Konica film processor. Northern blot and Sybr Gold images were densometrically analyzed by ImageJ software from NIH.RNA was separated on pre-fabricated formaldehyde agarose gels (Lonza) and stained with SYBR Gold Nucleic Acid Gel Stain (Life Technologies) for 30 minutes. Gel images were captured with a digital camera (Canon Vixia HFS30). RNA was transferred by electro-blotting (Thermo Scientific Owl Hep-1) to a positively charged nylon membrane (Life Technologies) in 0.5 x TBE (standard Tris/Borate/EDTA buffer). The RNA was cross-linked to the membrane using UV (Stratagene UV Crosslinker). Probes for the 25S, 18S, and 5S components of the ribosomal RNA gene of sis Co.)  and clonRT-PCR. Quantitative Real Time PCR was performed in the CFX Connect Detection System (Bio-Rad) using the QuantiTect SYBR Green (Qiagen) kit. Total RNA was isolated from the same number of yeast cells as described above. The same volume of total RNA for each time point was diluted one hundred fold and one μL of diluted RNA was used for the cDNA synthesis and amplification for each time point and subunit. Primers  were desStandard deviations and P-values using 1-sample T test were calculated on Excel.Candida albicans to shift from yeast to hypha. By 30 minutes, after transfer into 10% serum at 37°C, hypha can be easily seen (Ten percent serum in water remains among the most potent stimuli for ily seen . As seenily seen . Resultsily seen . Water aily seen  and showily seen . Using 1ily seen  in additily seen , yet forily seen . A numbeily seen . Extensiily seen  and formily seen  mirroredily seen . Loss inily seen . Poly-A While new classes of RNAs have been recently recognized, ribosomal RNA remains the predominant form of this molecule in eukaryotes; therefore any decrease in total RNA will likely include rRNA. Using Northern blot analysis for rRNA quantitation presents a special challenge, since the large and small subunits are usually used as controls for loading. However rRNA analysis does have one advantage that subunits can clearly be seen on stained gels and quantitative estimates can be obtained without the loss that can occur on blotting. To minimize any artifacts related to loading, assays were run in same volumes, total RNA always suspended in final volume of 50μL and the same volumes of T0 and assay pairs were carefully loaded unto gels. We are confident with our results as more than a dozen stained gels and Northern Blots gave similar results. One representative blot showing results after 30 minutes of serum exposure is shown in C. albicans is already significantly decreased as early as five minutes. To get to that level, one needs to assume that whatever mechanism underlies this decrease, it was activated almost immediately after 10% serum exposure.We took several approaches to see if we could get any indication whether RNA levels have any role in hyphal transition. For RNA decrease to play such a role it would have to precede the appearance of hypha. Therefore we looked at total RNA levels as early as five minutes after start of serum exposure. As can be seen in Candida species with variable capacity to form hypha in 10% serum. As can be seen C. tropicalis (C. dubliniensis (C. krusei (C. glabrata (C. krusei does not form hypha in 10% serum, it does form them on agar surface after a few days, whereas C. glabrata does not form them even on agar surface. Their data which includes both RNA content and early timing is also shown in C. albicans except C. krusei which shows an initial decrease with full recovery by 30 minutes.Another approach was to compare opicalis  and C. diniensis  form ger. krusei  and C. gglabrata  do not. 2O there was a decrease in RNA content (We also looked at GlcNac, a well-studied hyphal inducer that acts via Ngt1 directly activating the transcription factor Efg1, unlike serum that utilizes the MAPK and cAMP pathways . The res content  without  content .We also did assays where nutrition for the cells was maintained. When 10% serum was added to YPD at 37°C, hyphal formation could be seen by 30 minutes. As can be seen in 2O, after initial decline of RNA by 30 minutes there is no additional decline at 60 minutes (Candida species show similar decline in RNA on 10% serum exposure, indicating this to be an independent event. Thus overall it appears that this decrease in rRNA does not play a direct causal role in hyphal formation. The possibility exists that rRNA decline potentiates hyphal transformation in a multi-step process in those that do form hypha, but since we did not do any quantitation of newly formed hypha among the various conditions, we cannot draw such a conclusion from our data.These data confirm our previous observation that rRNA is decreased on serum exposure. Additionally, GlcNac another potent hypha inducer also mirrors 10% serum both in RNA reduction and formazan formation. The obvious question this raises is whether this change in RNA plays any role in hyphal transformation? That two germination inducers also decrease rRNA and the rapidity of rRNA decrease , occurri minutes , yet rob minutes . Similar minutes . When 10 minutes , both RN minutes , again i2O seems to affect rRNA content specifically. Indeed with water alone, there was a significant decrease of total RNA and metabolic activity was also somewhat decreased by 30 minutes (Our data however clearly indicate that 10% serum in H minutes . With 10 minutes , yet tot minutes . At 60 m minutes  metaboli minutes , abundan minutes . We also minutes , metabol minutes , yet tot minutes  though t2O significantly and rapidly decreases rRNA in C. albicans. Microarray studies by Nantel et al [C. albicans is well established [Saccharomyces cerevisae have been described [C. albicans. It is interesting that when we previously reported polyadenylation of both the large and small subunits of rRNA in C. albicans, we found that this polyadenylation occurred rapidly, as early as five minutes after 10% serum exposure and it came down to baseline by fifteen minutes [Our data clearly shows that 10% serum in Hel et al  profilinablished . Tor 1 sablished . The rapescribed . One of escribed . It is v minutes ,9. This  minutes . It is n