Pre-mRNA processing is mechanistically linked to transcription with RNA pol II serving as a platform to recruit RNA processing factors to nascent transcripts. The TREX complex member, REF/Aly, has been suggested to play roles in transcription and nuclear RNA stability in addition to its more broadly characterized role in mRNA export. We employed RNA-seq to identify a subset of transcripts with decreased expression in both nuclear and cytoplasmic fractions upon REF/Aly knockdown, which implies that REF/Aly affects their expression upstream of its role in mRNA export. Transcription inhibition experiments and metabolic labeling assays argue that REF/Aly does not affect stability of selected candidate transcripts. Instead, ChIP assays and nuclear run-on analysis reveal that REF/Aly depletion diminishes the transcription of these candidate genes. Furthermore, we determined that REF/Aly binds directly to candidate transcripts, supporting a direct effect of REF/Aly on candidate gene transcription. Taken together, our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription. Our data are consistent with the model that REF/Aly is involved in linking splicing with transcription efficiency. In the eukaryotic cell, a pre-mRNA must undergo multiple processing events to generate a mature mRNA. Many of these nuclear pre-mRNA processing steps including capping, splicing and 3′-end formation occur co-transcriptionally –4. In faMuch work has been done demonstrating that cells link transcription with downstream events in RNA processing, but recent investigations suggest that RNA processing can, in turn, modulate transcription rates. For example, several aspects of pre-mRNA splicing have been associated with transcription. Splicing efficiency and splice site mutations have been shown to impair transcription activity by decreasing assembly of the pre-initiation complex (PIC)  and repoPre-mRNA splicing changes ribonucleoprotein (RNP) composition to facilitate downstream events in gene expression. Subsequent to intron removal by the spliceosome, the exon junction complex (EJC), is deposited ∼20 nucleotides (nt) upstream of the exon–exon junction . The EJCCaenorhabditis elegans and Drosophila melanogaster  enhancer . REF/Alyormation . This isormation . Thus, RREF/Aly is also known to stabilize at least one RNA, PAN RNA, a Kaposi's sarcoma-associated herpesvirus nuclear non-coding RNA . ArtificHere, we aimed to identify endogenous RNAs affected by REF/Aly early in their biogenesis. In particular, we looked for transcripts that were destabilized or had decreased transcription in the absence of REF/Aly. Using an RNA-Seq approach, we identified transcripts whose levels were altered upon REF/Aly depletion and focused on several candidates that were down-regulated in the absence of REF/Aly in both the nuclear and cytoplasmic fractions, suggesting that the changes in expression were not driven solely by mRNA export defects. Half-life determinations, metabolic labeling assays, pol II chromatin immunoprecipitation (ChIP) and nuclear run-on (NRO) assays showed that REF/Aly enhances the transcription of specific target genes. Additionally, we show that REF/Aly binds directly to candidate transcripts, consistent with a direct effect of REF/Aly on the candidates. Our data suggest that the importance of REF/Aly is not limited to RNA export, but that REF/Aly is also critical for gene expression at the level of transcription and may be involved in linking transcription efficiency with splicing.293A-TOA cells were grown in Dulbecco's modified Eagle's medium (Sigma) containing 10% tetracycline-free fetal bovine serum (Clontech), 1× penicillin-streptomycin (Sigma), 2 mM L-glutamate and 100 μg/ml G418 (Fisher BioReagents).Silencer Select siRNAs targeting REF (Ambion). Control samples were transfected with Silencer Select Negative Control #2 siRNA. Cells were transfected with siRNAs for 24 h before replating on larger plates. For example, for RNA-Seq and ChIP sample preparation, 293A-TOA cells were initially plated on 6-well plates for siRNA transfection. Twenty-four hours after transfection, cells were trypsinized, re-plated on 10-cm plates and allowed to grow for 72 more hours, 96 h total. We found that efficient knockdown required cell division, not just additional time, presumably because the REF/Aly protein is stable. Ultraviolet (UV) crosslinking experiments were performed in 10-cm plates with 10 μg Flag-REF2–I plasmid DNA  according to manufacturer instructions. For siREF samples, cells were transfected with a pool of two (8 nM each) smid DNA  using TrImage Studio software (LI-COR Biosciences).All antibodies were purchased commercially from the following sources: REF , Pol II , H3K36me3 , hnRNP C1/C2 (clone 4F4), TAP , Hpr1 , UIF , UAP56/URH49 . Protein was harvested following REF/Aly knockdown with siRNAs at various time points, separated on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, and quantitative western blots were performed using infrared detection with an Odyssey Fc machine. Quantification of western blots was performed using 2, 2 mM MgCl2, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris pH 8.0) which was immediately added to 5 ml Tri-Reagent (Molecular Research Center); this is the ‘total’ fraction. Cells were then harvested and combined from the other two 10-cm plates and resuspended in 1 ml Buffer I with fresh 1 mM DTT, 0.04 U/μl RNaseIN and 0.5% TritonX-100, and incubated on ice for 5 min. Samples were then centrifuged at 500 × g for 5 min at 4°C. To obtain the ‘cytoplasmic’ fraction, the supernatant was removed and added to 10 ml Tri-Reagent. The pellet was then resuspended in 1 ml Buffer I and added to 10 ml Tri-Reagent; this is the ‘nuclear’ fraction.For RNA-Seq sample preparation, 293A-TOA cells were transfected with 16 nM of siREF (pooled) or siControl and grown on three 10-cm plates per siRNA for 96 h. After knockdown, cells were harvested from one 10-cm plate, washed with ice-cold phosphate-buffered saline (PBS), and resuspended in 500 μl Buffer I  Purist Kit (Ambion) to enrich for polyadenylated RNA following manufacturer specifications. Analysis using an Agilent Bioanalyzer indicated that rRNA contamination in samples was minimal. Two biological replicates were prepared and sequenced using the SOLiD 5500 sequencing platform. Reads were mapped back to the UCSC hg19 reference genome using LifeScope. Mapped reads were then post-processed for transcript assembly and these reads were assembled into their respective transcripts using Cufflinks version 1.2.0 . Transcr20 (fractionation experiments) as per the manufacturer's protocol. cDNAs were then treated with RNase A and RNase H for 30 min at 37°C and purified with phenol:chloroform:isoamyl alcohol (PCA). cDNAs were resuspended in 20 μl H2O and diluted in an assay-dependent manner. Primer sequences and efficiencies are described in Supplementary Table S3. Relative quantities (RQ) were determined based on amplification efficiency and Ct value. To determine the relative expression of candidate transcripts after fractionation, single knockdown or knockdown over time, RQ values were normalized to RQ values of 7SK RNA.All RNA analyzed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was treated with RQ1 DNase (Promega) before the reverse transcription (RT) reaction. RT reactions were performed using SuperScript™ II RT (Invitrogen) or M-MuLV-RT (New England Biolabs) and random hexamers (Sigma) or dTTo assess relative stability of candidate transcripts, cells transfected with REF or Control siRNAs were treated with 1 ug/ml Actinomycin D to inhibit transcription. Cells were harvested at various time points from 0 to 4 h by addition of Tri-Reagent. RNA was extracted and analyzed by RT-qPCR to determine RNA abundance after transcription shut-off. Transcription shut-off RT-qPCR was performed using a one-step reaction with MultiScribe Reverse Transcriptase (Invitrogen).3, 1% SDS) and incubation at 65°C for 15 min. To reverse protein crosslinks and degrade protein, chromatin was incubated at 65°C overnight in elution buffer containing 20 mg/ml Proteinase K and 40 μl Tris pH 6.8. Chromatin was purified using PCA (25:24:1) and ethanol precipitation and DNA was analyzed by qPCR. ChIP amplicon primer sequences and efficiencies can be found in Supplementary Table S3.293A-TOA cells were grown on 6-well plates and transfected with 16 nM siControl or pooled siREF. Twenty-four hours after transfection, cells were replated on 10-cm plates and allowed to grow until REF/Aly was depleted for 96 h. To crosslink cells, 0.75% formaldehyde was added to each plate and plates were rocked for 10 min at room temperature. Formaldehyde was quenched by addition of 125 mM glycine. Cells were harvested from plates, washed three times with PBS and resuspended in 500 μl RIPA buffer  containing protease inhibitors. Cells were sonicated for 45 cycles (30 s on and 30 s off per cycle) at 4°C using a Digenode Bioruptor on the high setting to shear DNA to ∼200–300 base pairs. Sheared chromatin was centrifuged at max speed for 10 min at 4°C and then DNA was measured using a NanoDrop Spectrophotometer. Samples were normalized and ∼200 μg DNA was added to protein A beads conjugated with either 8WG16 (4 μl) or H3K36me3 (3 μg) antibodies. DNA was immunoprecipitated for at least 2 h at 4°C, and then washed with 1 ml of the following washes: (i) RIPA, (ii) low salt wash , 150 mM NaCl), (iii) high salt wash , 500 mM NaCl), (iv) LiCl wash , (v) T.E. buffer , (vi) T.E. buffer. Chromatin was eluted by addition of 200 μl elution buffer  MPG-I, (ii) 65°C MPG , (iii–vi) MPG-I, (vii) MPG without NaCl, (viii) MPG-1:10 (MPG-I diluted 1:10). Selected RNAs were eluted by first adding 200 μl elution buffer (MPG 1:10 + 5% β-mercaptoethanol) and incubated at room temperature for 5 min. Eluted RNA was moved to a new tube and beads were resuspended in another 200 μl elution buffer and incubated at 65°C for 10 min. Eluted fractions were combined and RNA was PCA extracted and ethanol precipitated. Relative expression of RNAs was measured by RT-qPCR.For 4-thiouridine (4SU) labeling experiments ,56, 293A40 probe.Analysis of bulk poly(A) RNA with ethynyl uridine (EU) was performed as previously described  using 1.7 cells (siControl or siREF as indicated) were used per sample. Cells were harvested by trypsin digestion, trypsin was quenched in ice-cold media and cells were washed in ice-cold 1× PBS. Cell pellets were then incubated for 5 min on ice in 1 ml of HLB (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2) supplemented with 1 mM DTT and 0.5% IGEPAL CA-630. The solution was then underlaid with 1 ml of HLB supplemented with 25% sucrose (w/v), 1 mM DTT and 0.5% IGEPAL CA-630 to create a step gradient and then centrifuged at 600 × g for 5 min at 4°C. After discarding the supernatant, the pellets were resuspended in 60 μl of 2× TXN buffer (20 mM TrisHCl (pH8.0), 180 mM KCl, 10 mM MgCl2, 50% Glycerol, 5 mM DTT, 40 U RNasin). Nts were then added to final concentrations of 250 μM ATP, GTP and CTP and 1 μM 4SUTP. The ‘-4SU’ samples substituted UTP for 4SUTP. Sarkosyl was added to 0.5% and transcription was allowed to proceed for 5 min at 30°C prior to stopping the reaction by addition of 4 ml Tri-Reagent. RNA was extracted and treated with DNase. Next, 40 μg of RNA was subjected to limited hydrolysis on ice in a volume of 50 μl of 200 mM NaOH for 4 min. To neutralize the solution, a mix of 50 μl 1M TrisHCl (pH 6.8), 10 μl 3M NaOAc (pH 5.2) and 1 μl glycoblue was added and samples were ethanol precipitated.We developed a NRO assay using 4-thiouridine 5′-triphosphate  based on two established protocols ,59. Appr2O, heated to 65°C for 3 min, cooled on ice for 1 min and 50 μl of a 4× biotinylation buffer was added . Samples were warmed to 25°C prior to adding 100 μl of 0.4 mg/ml biotin-HPDP (in dimethylformamide) for a final concentration of 0.2 mg/ml biotin-HPDP. After a 3-h incubation at 25°C, samples were chloroform extracted once, then ammonium acetate was added to 1 M, 1 μl of glycoblue was added and the aqueous volume brought to 200 μl with H2O. After two more chloroform extractions, the samples were ethanol precipitated. For SA selection, precipitated RNA was washed with 70% ethanol, resuspended in 60 μl H2O and heated at 65°C for 5 min. For each sample, 20 μl of Dynal MyOne Streptavidin T1 bead slurry was washed three times in MPG-1:10 and pre-blocked for 1 h in 1 ml of MPG-1:10 supplemented with 0.1% SDS, 0.1 mg/ml sheared salmon sperm DNA, 0.1 mg/ml poly(A) RNA (Sigma) and 0.1 mg/ml Torula yeast RNA (Sigma). This pre-blocked solution (950 μl) was then added to the heated RNA samples and binding proceeded for 1 h at room temperature. Beads were washed 11 times using the following washes: (i) MPG-1:10, (ii and iii) 55°C MPG-1:10 (without IGEPAL), (iv) MPG-1:10, (v–vii) MPG-I, (viii) MPG-1:10, (ix and x) MPG-I, no NaCl, (xi) MPG-1:10. Selected RNAs were eluted by two successive steps of 200 μl elution buffer (MPG 1:10 + 5% β-mercaptoethanol) incubated at room temperature for 5 min. Eluted fractions were combined and RNA was PCA extracted, chloroform extracted and ethanol precipitated. Relative expression of RNAs was measured by RT-qPCR.Due to the lower signal-to-noise for NROs, modifications to the 4SU-biotinylation and SA selection protocols were necessary. To biotinlylate RNAs, the hydrolyzed RNA samples were resuspended in 50 μl HOur goal was to find specific mRNAs that were affected by REF/Aly early in their biogenesis, particularly at the levels of nuclear RNA stability or transcription. Therefore, we wanted to exclude RNAs that were affected by REF/Aly primarily at the level of mRNA export. To identify candidate transcripts, we employed RNA-Seq to analyze RNAs in total, nuclear or cytoplasmic fractions upon siRNA-mediated REF/Aly knockdown. In the simplest scenario, RNAs that are exported in a REF/Aly-dependent fashion will decrease in the cytoplasm and increase in the nucleus upon REF/Aly depletion, but be relatively unchanged in the total. However, the factors involved in nuclear and cytoplasmic RNA decay differ, and this simple model does not account for inherent differences in RNA decay rates between the nuclear and cytoplasmic compartments. In principle, if REF/Aly is necessary for the export of a transcript, its total levels may decrease upon REF/Aly knockdown if the transcript is more rapidly degraded when sequestered in the nucleus. However, if REF/Aly acts exclusively at the level of mRNA export, the nuclear levels should not decrease upon REF/Aly knockdown. As such, we reasoned that if a transcript displays decreased levels in both nuclear and cytoplasmic compartments upon REF/Aly depletion, then either the nuclear stability or transcription of that mRNA must be altered. Therefore, in our RNA-Seq experiments we examined the effects of REF/Aly depletion on unfractionated cells, as well as nuclear and cytoplasmic fractions, and focused on transcripts reproducibly down-regulated in all three samples. Following identification of transcripts that are down-regulated in the nucleus, cytoplasm and total samples, we can subsequently determine whether REF/Aly acts on their stability or transcription.Drosophila cells , nuclear or cytoplasmic fractions. The purity of the nuclear and cytoplasmic fractionation was verified by examination of nuclear pre-mRNAs and their cytoplasmic mRNA counterparts . Two biological replicates were sequenced, mapped and DEGs were identified  see Mat. As expeAs described above, our goal was to identify transcripts whose expression is enhanced by REF/Aly upstream of export, so we focused on transcripts that were down-regulated in all samples upon REF/Aly depletion and found 646 DEGs Figure ,62 did nAs expected, visual inspection of the mapped reads showed larger peaks in the siControl sample compared to the siREF/Aly sample, corresponding with the identified DEGs Figure . To confAs described above, we hypothesized that REF/Aly acts early in biogenesis of our selected target mRNAs. However, examination of steady-state RNA levels can be misleading because some transcripts might have been generated prior to functional knockdown of REF/Aly. In order to look at changes in RNA accumulation over a brief window subsequent to REF/Aly knockdown, we metabolically labeled RNAs using the modified uridine analog, 4-thiouridine (4SU), to capture newly made RNAs ,56. UponTo determine if changes in the abundance of candidate transcripts upon REF/Aly depletion is a result of decreased RNA stability, we performed transcription inhibition experiments using the transcription inhibitor Actinomycin D (ActD). Following a 96-h depletion of REF/Aly, cells were treated with 1 μg/ml ActD and RNA was harvested at time points from 0 to 4 h (0–240 min) after transcription shut-off. Comparison of the decay rates of specific RNAs in cells transfected with control siRNA or with REF/Aly siRNAs revealed no differences in RNA stability when REF/Aly was depleted Figure . We therTo more directly assess REF/Aly's role in transcription, we employed ChIP to look at polymerase density on our genes of interest. Following a 96-h knockdown, ChIP was performed with a pol II antibody that preferentially recognizes a hypophosphorylated form of the CTD (8WG16) . Five amTrimethylation of lysine 36 on histone 3 (H3K36me3) is a marker for active chromatin found preferentially at the 3′ ends of genes . H3K36meWhile REF/Aly depletion affects the steady-state levels of specific transcripts, we cannot conclusively state that these are due to direct effects of REF/Aly. For example, changes in RNA levels and transcription of our candidate transcripts could result from cell toxicity or other indirect effects. If so, we would expect direct effects of REF/Aly to be temporally linked to its depletion; that is, direct targets should be diminished relatively quickly upon REF/Aly depletion. To test this, we depleted REF/Aly for 48, 72 or 96 h and measured RNA levels by RT-qPCR. As seen in Figure Because REF/Aly is an RNA-binding protein, we hypothesized that REF/Aly binds directly to our candidate RNAs. To test this, we employed UV irradiation cross-linking followed by RNA immunoprecipitation . 293A-TOREF/Aly has been ascribed both general and gene-specific roles in RNA processing. Our analysis suggests that, while some transcript levels are more robustly influenced by REF/Aly depletion, the effects of REF/Aly may extend beyond the identified DEGs Figures  and 5. TOur ChIP assays demonstrated that REF/Aly depletion correlates with decreased pol II occupancy on candidate genes, but most of the detected pol II signal was at promoters Figures  and 6. MMuch work has been done demonstrating the importance of coupling between nuclear events in gene expression. The data presented here suggest that the RNA export factor, REF/Aly, may be an important component linking transcription, RNA processing and RNA export. While REF/Aly and its homologs function in mRNA export, REF/Aly is also capable of stabilizing a viral nuclear RNA and has been implicated in transcriptional control. Our RNA-Seq analysis suggests that a considerable number of transcripts are affected by REF/Aly upstream of export Figure . We chosOur work shows that depletion of REF/Aly leads to decreased transcription of particular transcripts, but we cannot conclusively state whether that role is direct or indirect. REF/Aly plays important roles in the cell, particularly in facilitating export of mature mRNAs into the cytoplasm. In our studies, we have depleted REF/Aly, potentially disrupting important cellular processes and causing non-specific decreases in gene expression. Thus, we must allow for the possibility that changes in steady-state RNA levels, and particularly changes in transcription, are a result of indirect effects. However, several pieces of data argue against that idea. After a 48-h knockdown of REF/Aly, a significant decrease in candidate steady-state RNA levels was observed Figure , even thet al. has shown that in vitro, Yra1 is capable of binding directly to the hyperphosphorylated CTD  interacts with pol II. In yeast, Yra1 is both directly and indirectly associated with pol II. Specifically, work from Mackellar ated CTD . Additioated CTD ,71. Whilated CTD .et al. established that mutation of the 5′ splice site reduces steady-state RNA levels by impairing transcription (et al. (Furthermore, Damgaard cription , and the (et al.  demonstret al. identified REF/Aly as an E2F2-interacting protein, and a modulator of E2F2-responsive genes (In a non-mutually exclusive model, REF/Aly could affect transcription directly by interacting with transcription factors. Indeed, REF/Aly was first identified by its ability to interact with AML-1 and LEF-1 transcription factors and it has been reported to enhance b-ZIP protein interactions with DNA ,45. Moreve genes . In theiDepending on the species and cell type, REF/Aly has been proposed to act as a general mRNA export factor or as a gene-specific factor. We cannot make definitive conclusions regarding the generality of REF/Aly in transcription from our data. Our RNA-Seq data led to the identification of only a subset of genes, suggesting a specific effect, but it is important to point out that normalization schemes used for RNA-Seq experiments can be biased against factors that have general effects . ConsistREF/Aly has been implicated in promoting mRNA export, transcription and nuclear RNA stability. However, the contributions of REF/Aly to each of these activities to the cell remain unknown. Our studies specifically sought to identify REF/Aly's roles in RNA stability and/or transcription, so we cannot make any judgments regarding whether export is the primary role of REF/Aly. Interestingly, our RNA-Seq data showed that virtually all of the DEGs identified in the cytoplasmic fractions were down-regulated, strongly supporting a role for REF/Aly in nuclear mRNA export Figure . ConsistUntil recently, cross-talk between RNA processing events has been thought to take place in a forward manner. That is, transcription machinery positively influences factors involved in pre-mRNA processing. However, a growing body of evidence suggests that enhancement of gene expression occurs in a bidirectional fashion with RNA processing factors positively regulating transcription efficiency. Our work suggests that REF/Aly influences this bidirectional coupling but additional studies are required to determine the mechanisms of REF/Aly-mediated enhancement of pol II recruitment.Supplementary Data are available at NAR Online.


Caenorhabditis elegans. In addition to ethyl methyl sulfonate-based mutagenesis, we employed RNAi-mediated knockdown of candidate chaperones in unc-45 temperature-sensitive mutants and screened for impaired movement at permissive conditions. This approach confirmed the cooperation between UNC-45 and Hsp90. Moreover, the screens identified three novel co-chaperones, CeHop (STI-1), CeAha1 (C01G10.8) and Cep23 (ZC395.10), required for muscle integrity. The specific identification of Hsp90 and Hsp90 co-chaperones highlights the physiological role of Hsp90 in myosin folding. Our work thus provides a clear example of how a combination of mild perturbations to the proteostasis network can uncover specific quality control modules.Proteome stability is central to cellular function and the lifespan of an organism. This is apparent in muscle cells, where incorrect folding and assembly of the sarcomere contributes to disease and aging. Apart from the myosin-assembly factor UNC-45, the complete network of chaperones involved in assembly and maintenance of muscle tissue is currently unknown. To identify additional factors required for sarcomere quality control, we performed genetic screens based on suppressed or synthetic motility defects in  The term molecular chaperone was first coined to describe the transient involvement of the nuclear protein nucleoplasmin in the assembly of the nucleosome subunits  results in only a minor effect on motility  mutants of unc-45 result in motility and sarcomere organization defects at restrictive conditions but have no apparent phenotype at the permissive growth temperature of 15°C. unc-45-associated phenotypes can be exposed under permissive conditions or masked under restrictive conditions by genetic or chemical remodeling of proteostasis  can be viewed as a sensitized background to screen for molecular chaperones specifically associated with muscle function.To study tissue-specific chaperone networks in a multicellular organism, we focused on the essential role of the myosin chaperone UNC-45, a member of the conserved UCS (UNC-45/CRO1/She4p) family, with the aim of uncovering novel chaperones involved in myosin folding. Temperature-sensitive (unc-45(ts) phenotypes at restrictive conditions and targeted RNAi synthetic interaction screens to identify chaperones that aggravate unc-45(ts) phenotypes under permissive conditions. Using an epistatic analysis approach, we genetically verified the interaction between UNC-45 and Hsp90. Moreover, we identified the Hsp90 co-chaperones CeHop (STI-1), CeAha1 (C01G10.8), and Cep23 (ZC395.10) as novel and specific modulators of UNC-45-Hsp90 myosin-associated functions.Here, we used a non-biased mutagenesis screen to identify chaperones that ameliorate C. elegans strains Bristol N2 (wild-type), DR94 (unc-45(m94)), CB286 (unc-45(e286)), RW1596 (myo-3(st386); stEx30) and RB2525 (C01G10.8(ok3501)) were obtained from the Caenorhabditis Genetics Center. Animals were backcrossed to N2 at least four times. Strain ABZ100  was generated by crossing DR94 (unc-45(m94)) males with RB2525 (C01G10.8 (ok3501)) hermaphrodites. The m94 allele was confirmed by incubating eggs at the restrictive temperature (25°C) and testing for the characteristic movement phenotype. The ok3501 allele was confirmed by genotyping PCR .Escherichia coli OP50-1 strain at 15°C. Synchronized L1 larvae were obtained as follows: Synchronized high density plates containing adult worms and L1 progeny were washed with M9 solution and collected into tubes. Then, L1 larvae were separated from the adult worm population by centrifugation. For all experiments, 5–20 μl of M9 containing 30–130 L1 larvae where transferred onto fresh plates seeded with E. coli HT115 (DE3) containing the indicated RNAi vectors and grown at 15 or 20°C for the duration of the experiment. The animals were then taken for phenotypic analysis, imaging and/or antibody staining. At 15°C, the first day of egg-laying was set at 120 h after L1 transfer, at 65 h for growth at 20°C and at 50 h for growth at 25°C. The first day of egg-laying was defined as the point when animals grown on HT115 (DE3) bacteria containing an empty vector control were able to lay eggs.Nematodes were grown on NGM plates seeded with the unc-45(m94) worms were grown at the permissive temperature (15°C) to young adulthood and mutagenized using 50 mM EMS, as described earlier  containing the empty vector (pL4440) or unc-45 RNAi were included as negative and positive controls in each experiment, respectively  movement was assessed by the thrashing assay, as previously described . RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). mRNA levels were measured by quantitative PCR (qPCR), performed on a C1000 Thermal Cycler (Bio-Rad) with KAPA SYBRFAST qPCR Master Mix (KAPA Biosystems). The results were analyzed using CFX Manager software (Bio-Rad). Relative transcript levels were determined by averaging the CAge-synchronized populations were collected and lyzed in SDS sample buffer (98°C for 10 min). Samples were separated by SDS-SB gel electrophoresis and analyzed by western blot, using anti-tubulin (Sigma), anti-CeHop and anti-CeAha1 antibodies (kindly provided by Dr. Klaus Richter). Peroxidase-conjugated AffiniPure goat anti-rabbit antibodies served as secondary antibodies.An age-synchronized population was collected in M9 buffer, flash-frozen and lyzed using a hand drill. The lysate was maintained at 4°C. Samples were diluted in RIPA buffer (Sigma) supplemented with protease inhibitors (Roche) and centrifuged to remove debris. The supernatant was divided into equal aliquots. Two aliquots were incubated for 30 min at 4°C with either anti-CeHop or anti-CeAha1 antibodies and one aliquot was left untreated. Protein A/G PLUS-Agarose beads (Santa Cruz) were added to the samples, which were incubated for additional 30 min at 4°C. The samples were centrifuged  and supernatants and pellet were collected. Pellets containing the agarose beads were washed three times in RIPA buffer. Pellet and supernatant fractions were boiled (95°C for 10 min) in SDS sample buffer, resolved by SDS-SB gel electrophoresis and analyzed by western blot using anti-CeHop, anti-CeAha1, anti-CeHsp90 (kindly provided by Prof. Richard Morimoto) or anti-UNC-45 antibodies , results in temperature-sensitive motility defects and myosin disorganization phenotypes when animals are grown at restrictive conditions (>22°C). However, animals show no phenotypes during development and early adulthood when grown at the permissive temperature (15°C). To identify chaperones that play a redundant role in myosin assembly and muscle maintenance, we screened for modifiers that can mask unc-45-associated phenotypes under restrictive conditions. Because RNAi knockdown of chaperone expression is expected to aggravate unc-45 phenotypes, we used mutagenesis to screen for compensatory factors. Specifically, EMS was used to induce mutations in the germline of unc-45(m94) hermaphrodite worms. F2 animals were shifted to the restrictive temperature (22°C) and screened for mutants with improved motility. Mutant animals, identified by their ability to reach a distant E. coli food source on the plate, were isolated , all proved to be revertants, presenting the A2342G substitution that restores the m94 allele of the unc-45 gene to the wild-type version  elsewhere in the genome was unable to compensate unc-45(m94)-specific phenotypes under restrictive conditions. This indicates an essential role for UNC-45 that cannot be bypassed by other chaperones.The gene encoding UNC-45, an essential muscle-specific chaperone, was used for our mutagenesis screening approach. A single amino acid substitute in the UCS domain of d Figure . The ided Figure . We firsn Figure . This obunc-45(ts) phenotypes at permissive conditions might result from the redundant but not compensatory functions of other chaperones in the muscle  (Table One possible interpretation of the mutagenesis screen results is that the lack of s) Table  and exams) Table . To drawC. elegans life cycle (L1) and its effects were examined after 5 days at 15°C (or after 3 days at 20°C), until animals grown on control RNAi reached adulthood , αB-crystallin/HSPB5 (hsp-25), and DNAJB6 (dnj-24), were suggested to play a key role in sarcomere assembly and maintenance  mutant strains under permissive conditions. Age-synchronized L1 unc-45(e286) mutant animals kept at permissive conditions (15°C) were treated with RNAi for different molecular chaperones and monitored for motility defects. We first sought chaperones that cause unc-45(e286)-specific phenotypes at permissive conditions when depleted  mutant animals. As noted above, down-regulation of unc-45 or unc-23 also induced a loss of coordination in wild-type animals (Table CeHsp90 (daf-21), CeHop (sti-1) and CeAha1 (C01G10.8)  animals but induced a strong motility defect only in unc-45(e286) animals . CeHop and CeAha1 RNAi only affected unc-45(e286)-expressing animals, where complete paralysis resulted  were tested for their effects on animals carrying the unc-45(m94) allele  mutant worms maintained under permissive conditions were treated with RNAi for CeHsp90, CeHop or CeAha1 and compared to animals that took up the empty vector control. Similar to the e286 allele, unc-45(m94) animals treated by RNAi for CeHsp90, CeHop, or CeAha1 were completely paralyzed on day 2 of adulthood under permissive conditions -expressing animals  and a deletion allele of CeAha1 (RB2525 strain) were motile by the second day of adulthood, in contrast to the respective single mutant strains  and Aha1 (Activator of Hsp90 ATPase 1) are co-chaperones of Hsp90 that facilitate the maturation of Hsp90 substrates by modulating the Hsp90 ATPase cycle and thus driving Hsp90 conformational changes  using immuno-staining assays. Animals expressing unc-45(e286) or unc-45(m94) that were shifted to restrictive conditions after the first larval stage exhibited disruption of myofilament structure associated with mislocalization of MYO-3, while wild-type animals were unaffected , or unc-45(m94) mutant animals under permissive conditions was mostly unaffected by the third day of adulthood, although myosin disorganization was observed at later time points . In contrast, treatment of unc-45(e286) mutant animals with CeHop and CeAha1 RNAi resulted in complete disruption of myofilament organization and MYO-3 mislocalization under permissive conditions , although this mutant showed mild myosin disorganization in control animals. CeHop or CeAha1 RNAi-induced myofilament disruption was similar to the MYO-3 disorganization observed when unc-45 mutant animals were shifted to restrictive conditions  and unc-45(m94) muscle organization.To directly test this prediction, we examined whether d Figure . MYO-3 os Figure . Similars Figure . Taken tunc-45(ts) motility. To re-examine the role of these co-chaperones in muscle function, wild-type and unc-45(e286) animals were treated with RNAi for known Hsp90 co-chaperones  resulted in motility defects of unc-45(e286) mutant animals but not wild-type animals. 39 ± 11.3% of the unc-45(e286) mutant animals examined were motile  motility to a lesser extent than CeHop or CeAha1  motility was highly significant (p < 0.005). Cep23 was considered as false negative of the screen and its role as an UNC-45 modifier was next examined.The specific role of the Hsp90 system in modulating UNC-45-associated phenotypes suggested that other Hsp90 co-chaperones not identified in our screen might be required for  Figures , 4B, CepCep23(RNAi) also affected myofilament organization, wild-type and unc-45(e286) mutant animals were monitored for changes in the subcellular organization of MYO-3 using immuno-staining assays. In agreement with the Cep23-mediated effects on motility, treatment of unc-45(e286) animals with Cep23(RNAi) resulted in severe MYO-3 disruption in affected animals  knockout animals were treated with CeHsp90(RNAi), only 13.9 ± 7% of the animals were motile by the first day of egg-laying, while no effect on motility was observed for CeAha1(ok3501) animals grown on the empty vector control  animals, as most of the wild-type animals treated with CeHsp90 RNAi were motile (76.6±4%)  animals treated with CeHsp90(RNAi) showed a complete larval arrest phenotype  Figure . Moreovee Figure . Thus, uC. elegans body wall muscle model to identify novel chaperones that are required for sarcomere assembly and maintenance. To screen for novel sarcomere-associated chaperones, we specifically challenged the proteostasis network by studying animals with mutations affecting a known muscle chaperone, unc-45. These unbiased screens were able to genetically verify the known UNC-45-Hsp90 interaction -based mutagenesis screen, which only identified unc-45(m94) revertants as modifiers of UNC-45 function under restrictive conditions. In agreement, epistatic analyses of yeast genes required for folding in the endoplasmic reticulum identified specific aggravating and improving interactions between molecular chaperones (Jonikas et al., unc-45 modulators, suggests that the proteostasis buffering capacity depends on modulating the function of chaperones that cooperate in the same process. In agreement, DnaK and trigger factor, chaperones that overlap in binding newly synthesized proteins, are redundant for bacterial growth under non-stress conditions but become essential when both are affected (Deuerling et al., Down-regulation of chaperone expression in an C. elegans body wall muscle cells resulted in differential exposure of metastable mutant phenotypes, suggesting that each SOD1 mutant affects the proteostasis network differently (Gidalevitz et al., This specificity is somewhat surprising given that the chronic expression of misfolded proteins or aging unmask many unrelated metastable proteins (Gidalevitz et al., in vivo that are specialized for a set of substrates to specifically impact their folding. This view of functional modules within the proteostasis network is supported by the roles of co-chaperones in directing chaperone function. For example, Hsp90 has different co-chaperones that perform similar functions for different substrates. Accordingly, kinases and steroid receptors interact with different subsets of Hsp90 co-chaperones (Rohl et al., C. elegans is more sensitive to down-regulation of genes encoding ATPase chaperone machines than those encoding their co-chaperones (compare 13/21 Hsp60/Hsp70/Hsp90 genes to 4/58 Hsp60/Hsp70/Hsp90 co-chaperone genes, Table One possible interpretation of our data is that different co-chaperones form functional modules in vivo.By directing our examination to how chaperones orchestrate sarcomere assembly and maintenance, we were able to identify key players in sarcomere folding that safeguard the functionality of muscle cells and are at risk in disease and aging. Any newly identified chaperone can be used to find additional chaperones and/or co-factors associated with muscle folding and assembly. Focusing on the substrate, rather than the chaperone, helps in the drawing of a genetic interaction map describing chaperone-substrate interactions 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.


Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor.RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed,  In tobacco, a phenylalanine codon is present at the DNA level without any editing. When the spinach psbF was introduced into tobacco plastids, the lack of RNA editing led to a defective phenotype, indicating that RNA editing is site-specific [Atropa belladonna, demonstrates that the belladonna nuclear genome is unable to edit the tobacco plastid ATPase α-subunit transcript, resulting in an albino phenotype of the cytoplasmic hybrid plant [RNA editing in angiosperms mainly defines the process that alters a cytosine (C) to uracil (U) in specific positions of RNA so that the sequence in the mature RNA differs from that of genomic DNA. RNA editing is a mechanism that corrects missense mutations of genes at the RNA level. It thereby restores conserved amino acid residues to maintain essential functions of encoded proteins . For exaspecific . Introduid plant .RNA editing is absent in algae and highly abundant 300–500 sites) in hornworts and ferns, but unusually high in lycophytes, where 3,415 RNA-editing events were found 00 sites . Given tHowever, to validate such a hypothesis would require RNA editing studies for species spread throughout the phylogenetic tree. The genomes of most monocot species that have been studied are from the clade of the Commelinids and therefore do not provide a genome of a more distantly related species of monocot plants.Spirodela polyrhiza), belonging to the subfamily of Lemnoideae in the order of Alismatales [Recently, we studied the nuclear and chloroplast genomes of the aquatic plant Spirodela , submitted by the DOE Joint Genome Institute (JGI) from our 454 sequencing of Spirodela ESTs (SRX148325). For unconfirmed sites, we designed specific primers spanning the candidate regions and performed RT-PCR; the resulting products were directly sequenced using the same PCR primers. These sites were confirmed as RNA editing sites only if there were two overlapping peaks at the same location. To further validate editing efficiency, we cloned the RT-PCR products into pGEM-T easy and selected 96 clones for sequencing to validate the edited sites by comparing them with the genomic DNA sequence of Spirodela.Spirodela polyrhiza; coconut—Cocos nucifera; rice–Oryza sativa; maize—Zea mays; tobacco—Nicotiana tabacum; Arabidopsis—Arabidopsis thaliana; tomato–Solanum lycopersicum.All chloroplast genome sequences used in this study were downloaded from Genbank: Spirodela JN160603; coconut KF285453; rice NC_001320; maize NC_001666; tobacco NC_001879; Arabidopsis NC_000932. The editing sites for coconut , maize  and ArabpsbA, rbcL and psaJ were highly expressed, whereas rpoC2, rpoC1 and rpoB were expressed at low levels , ndhD (6 sites) and ndhA (5 sites). As expected, the RNA editing in the first and second position of codons changes the identity of amino acid, whereas it was silent for the third codon. Of the 58 editing sites in protein coding regions, 6 sites (10.3%) were in first, 49 sites (84.5%) in second, and three sites (5.2%) in third codons. The conversions from ACG to AUG in rpl2 and ndhD genes were found to create an initiation codon in Spirodela. Due to the depth of sequencing, we also detected eight silent RNA editing sites  in non-coding sequences that have been rarely reported for chloroplast genome  and coconut (75) were about twice the editing sites of rice (35) and maize (26) ,24.ndh genes. A total of 15 out of 31 sites were from members of the ndhB genes ..ndh generbcL alignment, Spirodela and coconut were sister groups, whereas rice and maize were sister species  out of 26 of the sites were common between Spirodela and coconut, whereas Spirodela shared only 11 (42%) of the sites with rice and 10 (38%) with maize  and 44% for the rpl2 gene  in fronds  were significantly higher in fronds than in turions, whereas six sites  were lower in fronds than in turions  appear not to be required for transcription or the function of the translated protein as coconut, rice, maize, Arabidopsis, tobacco and tomato develop normally even though they have the nucleotide “C” in this position  offers a new approach to identify all the potential RNA editing sites and quantify RNA editing efficiency, and to detect edited sites at very low efficiency .Whereas the powerful technique of RNA-seq has been greatly utilized to study the nuclear transcriptome, it has not been widely applied to the organellar transcriptome because extracting transcripts from purified mitochondria and chloroplast is very time-consuming. Although one could sequence total RNA of both the nuclear and organellar genomes, the experimental method for preparing total RNA needs to be carefully considered. The reason is that organellar transcripts do not undergo polyadenylation like nuclear transcripts and do not have to be transported from one cellular compartment to another. Furthermore, post-transcriptional polyadenylation of organellar transcripts accelerates their degradation . TherefondhA, ndhB, ndhD and ndhF transcripts were shared between Spirodela and its sister species coconut. In contrast, Spirodela shared only 11 (42%) of its sites with rice and 10 (38%) with maize, two more distantly related species Click here for additional data file.S2 TableThe significant change was considered when |Fold change| >2 and p-value < 0.05. The expression unit is FPKM.(XLSB)Click here for additional data file.S3 TableRNA editing sites were compared in monocots including Spirodela, coconut, rice and maize. “NA” means the item was not studied.(XLSB)Click here for additional data file.S4 Table“Reference coverage” means the number of mapped reads identical to reference (four replicates are combined). “Edited coverage” means the number of mapped reads that have been edited (four replicates are combined). "Edit (%)” gives the percentage of RNA editing using the edited reads divided by total mapped reads.(XLSB)Click here for additional data file.


Here we present the results of a large-scale bioinformatics annotation of non-coding RNA loci in 48 avian genomes. Our approach uses probabilistic models of hand-curated families from the Rfam database to infer conserved RNA families within each avian genome. We supplement these annotations with predictions from the tRNA annotation tool, tRNAscan-SE and microRNAs from miRBase. We identify 34 lncRNA-associated loci that are conserved between birds and mammals and validate 12 of these in chicken. We report several intriguing cases where a reported mammalian lncRNA, but not its function, is conserved. We also demonstrate extensive conservation of classical ncRNAs  and more recently discovered ncRNAs  in birds. Furthermore, we describe numerous “losses” of several RNA families, and attribute these to either genuine loss, divergence or missing data. In particular, we show that many of these losses are due to the challenges associated with assembling avian microchromosomes. These combined results illustrate the utility of applying homology-based methods for annotating novel vertebrate genomes. Non-coding RNAs (ncRNAs) are an important class of genes, responsible for the regulation of many key cellular functions. The major RNA families include the classical, highly conserved RNAs, sometimes called “molecular fossils”, such as the transfer RNAs, ribosomal RNAs, RNA components of RNase P and the signal recognition particle . Other cThe ncRNAs pose serious research challenges, particularly for the field of genomics. For example, they lack the strong statistical signals associated with protein coding genes, e.g. open reading frames, G+C content and codon-usage biases .New sequencing technologies have dramatically expanded the rate at which ncRNAs are discovered and their functions are determined . HoweverIndependent benchmarks of bioinformatic annotation tools have shown that the CM approaches out-perform alternative methods , althougThe publication of 48 avian genomes, including the previously published chicken , zebra fIn the following we explore the conservation patterns of the major classes of avian ncRNA loci in further detail. Using homology search tools and evolutionary constraints, we have produced a set of genome annotations for 48 predominantly non-model bird species for ncRNAs that are conserved across the avian species. This conservative set of annotations is expected to contain the core avian ncRNA loci. We focus our report on the unusual results within the avian lineages. These are either unexpectedly well-conserved ncRNAs or unexpectedly poorly-conserved ncRNAs. The former are ncRNA loci that were not expected to be conserved between the birds and the other vertebrates, particularly those ncRNAs whose function is not conserved in birds. The latter are apparent losses of ncRNA loci expected to be conserved; Here, we consider three categories of such “loss”: First, genuine gene losses in the avian lineage where ncRNAs well conserved in other vertebrates are completely absent in birds. Second, “divergence” where ncRNAs have undergone such significant sequence and structural alternations that homology search tools can no longer detect a relationship between other vertebrate exemplars and avian varieties.n ≈ 80) generally consisting of approximately 5 larger “macrochromosomes” and many smaller “microchromosomes”  and (2) the vast majority of avian microchromosomes remain unsequenced [Seven families of “core” ncRNAs were found in some avian genomes but not others . These fequenced , 31. Furequenced . Therefoblastn with E ≤ 10−30. In some cases the sequences appear degraded at the ends, which is likely due to low sequence quality at the very ends of contigs or scaffolds. A blastn search also readily retrieves additional RNase P and RNAse MRP RNAs in the majority of genomes, albeit only the best conserved regions are captured. In many cases these additional candidates are incomplete or contain undetermined sequence, which explains why they are missed by the CMs [To wit, we performed dedicated searches for a selection of these missing ncRNA families. Here, tRNAscan is tuned for specificity and thus misses several occurrences of tRNA-sec that are easily found in the majority of genomes by  the CMs , 76.Many RNA families constitute the most evolutionarily conserved genes across all life on this planet . ExampleUnsurprisingly, the bulk of these classes of RNAs are well represented across the bird genomes See . HoweverA number of the classic RNAs are incorporated into RNA-protein complexes (RNPs) involved in core cellular processes. An example of this are the spliceosomal RNAs. Based upon the presence/absence patterns of the major spliceosomal RNAs they are all well represented in these genome sequences. The exceptions to this observation are the U4 RNA in cormorant and the U5 RNA in the bee eater which are both missing. These two genomes are low coverage, suggesting these genes weren’t captured in the current assembly. The minor spliceosomal RNAs are more interesting, the U4atac and U11 snRNAs show widespread patterns of loss, even in some of the high coverage genomes. These RNAs are frequently missed in bioinformatic screens. Indicating either frequent loss  or sequeThe telomerase RNA is also largely missing from the avian annotations. This RNA acts as a template for the telomerase enzyme that extends the telomeres found on chromosome ends. It is only found in the chicken, bald eagle, kea, budgerigar, crow and zebrafinch. Homology searches searches with the telomerase reverse transcriptase (TERT) protein show that the protein component of the telomerase RNP is conserved across all the bird genomes (data not shown). This pattern of presumably divergent telomerase RNA and conserved telomerase protein has been noted previously, most notably in the fungi , 78.The RNA components of RNase P and RNase MRP also appear to have undergone dramatic losses within the bird lineage. RNase P is required for the maturation of tRNA, the paralogous enzyme, RNase MRP is required for the maturation of rRNA. Each RNP cleaves smaller RNAs from larger transcripts . It is uThe 5.8S component of the ribosome in the turtle, turkey bustard, hoatzin, flamingo, tropicbird, seriema, owl, cuckoo roller, trogon, bee eater and falcon appears to have been lost See . The rRNSmall nucleolar RNAs (snoRNAs) are important ncRNAs that participate in the maturation of other functional RNAs . The bulH. sapiens and S. cerevisiae [There are 66 ribosomal modification sites, guided by 59 snoRNA families, that are preserved between revisiae . Of thesThe first cluster is found at chr11:62620797–62622484 on the human genome (hg19) and contains SNORD27, SNORD29 and SNORD31 of the human-yeast conserved snoRNAs. These snoRNAs are located in the inside-out gene SNHG1 which hosts a total of eight C/D box snoRNAs: SNORD25, SNORD26, SNORD27, SNORD28, SNORD29, SNORD22, SNORD30 and SNORD31 . Each ofThe second cluster is located at chr19:49993222–49994231 on the human genome (hg19) and contains two copies of SNORD33 and one SNORD34 all within a 1 KB genomic region. The turtle and alligator genomes retain the two copies of SNORD33 yet don’t have an obvious SNORD34 gene on the same scaffold. Within the bird genomes, the crow and rifleman each retain a single SNORD33 and SNORD34 gene on the same scaffold. While the ground-finch and bald eagle retain a single SNORD33 and the zebrafinch and seriema retain a single SNORD34  where we identified 543 sequences homologous to known microRNAs.In addition, we can confidently identify a further 3 microRNA families that are present in mammals, and turtle and/or crocodile, but not in any avian genome . This suggests that these sequences were lost in the last common ancestor of archosaurs or birds. There are also a number of microRNAs that are predicted to be present in turtles and/or crocodiles, and only a small number of bird genomes. Indeed, there are many missing annotations, species-specific and otherwise, that are not consistent with the consensus phylogeny, and could be due to either incomplete genomes or widespread microRNA loss.The turkey genome contains a high number (190) of microRNAs so far found only in chicken, which account for the higher number of annotated sequences in this genome compared with other birds. This is consistent with its phylogenetic position as the closest chicken relative among the examined birds. However, 101 chicken microRNAs have no homolog in the turkey or other bird genomes, suggesting that these genes are chicken-specific. This is consistent with previous reports of large number of species specific microRNAs in all animals, and supports the view of fast microRNA turnover during animal evolution .The cis-regulatory RNAs are a group of RNA structures encoded on mRNAs. Generally they are involved in regulating the expression of the mRNA they are encoded within. Others may recode the translated protein product into an alternate sequence.′ UTR (histone3) [This group includes the iron response element (IRE)  and the istone3) . These aistone3)  and the istone3) .′ UTR protein-binding region (Vimentin3) and a connective tissue growth factor (CTGF) 3′ UTR element (CAESAR) are conserved across a diverse group of vertebrates, including the bird lineages explored here , potassium channel RNA editing signal (K chan RES), Antizyme RNA frameshifting stimulation element (Antizyme FSE), vimentin 3here See .Non-coding RNA derived pseudogenes are a major problem for many ncRNA annotation projects. The human genome, for example, contains > 1 million Alu repeats, which are derived from the SRP RNA . The exiIt is possible that the avian annotations also contains excessive pseudogenes. However, it has previously been noted that avian genomes are significantly smaller than other vertebrate species . We haveP < 1016). This number is an underestimate of the fraction of our annotations that are genuinely expressed, as only a fraction of the developmental stages and tissues of chicken have been characterized with RNA-seq. Furthermore, some ncRNAs are expressed in highly specific conditions [The ncRNAs presented here have been identified using homology models and are evolutionarily conserved in multiple avian species. In order to further validate these predictions we have used strand-specific total RNA-seq and small RNA-seq of multiple chicken tissues. After mapping the RNA-seq data to the chicken genome see . This shnditions , 112.The classes of RNAs where the majority of our annotations were experimentally confirmed includes microRNAs, snoRNAs, cis-regulatory elements, tRNAs, SRP RNA and RNase P/MRP RNA. The RNA-seq data could not provide evidence for a telomerase RNA transcript, which are only generally only expressed in embryonic, stem or cancerous tissues. Only a small fraction of the 7SK RNA, the minor spliceosomal RNAs and the lncRNAs could be confirmed with the 10% FDR threshold. There are a number of possible explanations for this: the multiple copies of the 7SK RNA may be functionally redundant and can therefore compensate for one another; The minor spliceosome is, as the name suggests, a rarely used alternative spliceosome; and the lncRNAs are generally expressed at low levels under specific conditions , 113. Nebona fide gene loss. A large fraction of our annotations have been confirmed using RNA-seq data, which also showed a 7-fold enrichment of expression within our annotations relative to unannotated regions.In this work we have provided a comprehensive annotation of non-coding RNAs in genome sequences using homology-based methods. The homology-based tools have distinct advantages over experimental-based approaches as not all RNAs are expressed in any particular tissue-type or developmental-stage, in fact some RNAs have extremely specific expression profiles, e.g. the lsy-6 microRNA . We haveThe collection of ncRNA sequences is generally biased towards model organisms , 87. HowIn summary, these results indicate we are in the very early phases of determining the functions of many RNA families. This is illustrated by the fact that the reported functions of some ncRNAs are mammal-specific, yet these are also found in bird genomes.S1 ResultsFurther results and discussion of poorly conserved ncRNAs, genomic contamination, additional analyses and the sources of avian sequencing data.(PDF)Click here for additional data file.


A report on the Plant and Animal Genome XXII meeting held in San Diego, California, USA, January 11–15, 2014. The Plant and Animal Genome XXII meeting was held in the warm and sunny coastal city of San Diego, California, on 11–15 January this year, during a time when many other US cities were plagued with snowstorms and extreme cold weather. More than 2,800 attendees from 62 countries joined the meeting. There were eight plenary talks and many workshops and posters covering topics ranging from genomics and functional genomics to epigenomics and systems biology. Tremendous progress in our understanding of the structure, evolution and regulation of plant and animal genomes has been made in recent years. Revolutionary high-throughput sequencing techniques have enabled complete sequencing of many large and complex genomes containing extensive transposable elements (TEs) and repeats, and have also helped in the identification of many hidden regulators, such as small RNAs, especially in non-model species. Here, I highlight some of the exciting presentations on epigenomics and small RNAs.Whole genome sequencing analysis has revealed that TEs constitute more than half of the genomes of many animals and crop plants. For example, more than 80 % of the maize genome and 60 % of our human genome is composed of TEs. Many studies and presentations support the notion that proliferation of TEs is one of the major driving forces for genome expansion. Nathan Springer  described the complex interplay between TEs and genes within the maize genome. He showed that although DNA methylation and a repressive histone methylation marker, H3K9me2, are present in almost all TE regions in maize, DNA methylation patterns and their ability to spread to surrounding regions vary among different transposon families. It was also shown that many genes with TEs in their introns are still actively transcribed, suggesting that maize transcription machinery can tolerate blocks of heterochromatin.de novo DNA methylation and histone modifications. Nathaniel Street  presented the genome assembly and small RNA profiles of Norway spruce (Picea abies), a tree with a similar number of genes as the model plant Arabidopsis, but which has a genome 100 times larger and with many more TEs. The large expansion of the genome is due to the slow and steady accumulation of a diverse set of long terminal repeat (LTR) retrotransposons. The 24-nucleotide class of siRNAs is much less abundant in gymnosperms than in angiosperms, which may partially explain the mass proliferation of TEs in gymnosperms. By comparative analysis with related species, it was found that the distribution of small RNA classes was highly diverse and tissue-specific. In animals, PIWI proteins and PIWI-interacting small RNAs (piRNAs) are key regulators in suppressing TE proliferation in germ cells. Michael Vandewege  discussed the interplay between piRNAs and TEs among Laurasiatherian mammals. Small RNAs from the testes of dog, cat, vesper bat and horse were profiled and it was shown that piRNAs play an essential role in suppressing the expression of active retrotransposons in mature testes in dog, cat and horse, whereas the DNA transposons in the vesper bat, which lacks a RNA intermediate, are not targeted by piRNAs. This demonstrates the complex dynamics between TEs and genomic defenses in mammalian genomes.Although TE transposition and replication are beneficial for adaptive evolution in many eukaryotic organisms, uncontrolled transposition activities can be detrimental and even fatal. Therefore, TEs are usually under tight epigenetic control via DNA methylation or histone modifications. In plants, the 24-nucleotide heterochromatic small interfering RNAs (siRNAs) are mainly responsible for silencing TEs by guiding mPing, in rice. In a few rice landraces, mPing has been amplified to more than 750 copies during recent domestication. She further illustrated that one strategy for successful rapid amplification is that mPing has a preference for inserting into AT-rich regions and thus minimizes exon insertions because rice protein coding regions tend to be GC-rich. This study has provided an excellent example that links TEs with genome restructuring and regulation.Despite robust genome integrity maintenance, certain TEs can still circumvent the epigenetic surveillance and amplify rapidly in the genome without killing its host or being silenced. Sue Wessler  gave a plenary talk on one such successful TE - a miniature inverted repeat transposable element (MITE), FLOWERING LOCUS C (FLC) locus, the antisense transcripts COOLAIR are alternatively spliced and polyadenylated, which recruit distinct chromatin-modifying complexes for different FLC chromatin states. FLC is one of the best-studied gene loci that are under precise control via multiple layers of regulation at the epigenetic level.Caroline Dean  gave a plenary talk on co-transcriptional coupling mechanisms that link chromatin dynamics with antisense transcripts for quantitative gene regulation. At the flowering repressor Arabidopsis, soybean and Medicago truncatula) and monocots , and discovered a novel class of 24-nucleotide phasiRNAs from monocots that are generated by a monocot-specific Dicer-like (DCL) protein. This class of phasiRNAs, emerging with germinal cell maturation and meiosis, shares characteristic features with animal piRNAs. Doris Wagner  introduced the function of ATP-dependent SWI/SNF chromatin remodeling proteins, SPLAYED and BRAHMA, during lateral organ formation in Arabidopsis. Meanwhile, the animal epigenetics workshop covered topics ranging from genomic imprinting and transcriptome analysis to public resources, such as the livestock epigenetics database (epiDB).The plant and animal epigenome workshops were held separately during the conference. Rob Martienssen  and Eric Lyons  introduced the Epigenomics of Plants International Consortium (EPIC), a joint effort to support the community working on plant epigenomics and epigenetics. In plants, epigenetic control is associated with the RNA-directed DNA methylation (RdDM) pathway. Plant-specific RNA polymerase IV (Pol IV) is a central player that initiates siRNA biogenesis in the RdDM pathway. Julie Law  presented work on the characterization of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), a protein that targets Pol IV to chromatin. She found that SHH1 was able to recognize both H3K9 methylation and an unmodified H3K4 residue, which connects DNA methylation and H3K9 methylation within the context of the RdDM pathway. Blake Meyers  discussed phased secondary siRNAs (phasiRNAs) in plants. He compared phasiRNAs and their generation loci between dicots  have organized informative ‘Small RNA’ workshops at this meeting for the last eight years. This year, six speakers from both animal and plant fields presented their findings. In addition, small RNAs have become an indispensable part of genomics studies and appeared in many genomics and epigenomics presentations.Small RNAs are short non-coding regulatory RNAs that direct gene silencing in a sequence-dependent manner. It has been 20 years since the first microRNA (miRNA) was discovered in let-7, in cardiovascular differentiation using whole genome miRNA screening, revealing a remarkable conservation of function from amphibians to mammals. Robert Sullivan  reported findings on extracellular microvesicle-associated miRNAs in the male reproductive tract. Distinct miRNA repertories were found in the epididymal intraluminal compartment that are likely to be involved in intercellular communication through the epididymis via extracellular microvesicles. Beth Thompson  introduced the function of DCL1 in maize development. Pamela Green gave a talk on the genome-wide analysis of Brachypodium miRNAs in the Brachypodium Genomics workshop. Conserved and novel miRNAs were identified and their potential targets were predicted and confirmed by parallel analysis of RNA ends.Several talks addressed the function of small RNAs in various cellular processes. For example, Alexandre Colas  presented findings on the function of several miRNAs, including Botrytis cinerea, can be delivered into host plant cells to silence host immunity genes by hijacking host RNA interference (RNAi) machinery to facilitate its infection. This finding represents a naturally occurring cross-kingdom RNAi, which serves as an advanced virulence mechanism for aggressive eukaryotic pathogens. Barbara Baker  described analyses of several plant endogenous small RNAs that regulate host immunity. The tobacco N gene that confers resistance to tobacco mosaic virus is regulated by both promoter-targeted transposon MITE-associated siRNAs and coding-region-targeted miRNAs. They also identified a new class of miRNAs derived from long inverted repeats, one of which targets Cf9, a tomato gene that confers resistance to fungal pathogen Cladosporium fulvum.In addition to their role in animal and plant development, small RNAs also play essential roles in regulating host-pathogen interactions. I  presented our work on pathogen small RNAs that regulate host immunity in the ‘Small RNAs’ and ‘Host-Microbe Interactions’ workshops. A group of small RNAs from an aggressive fungal pathogen, cis-regulatory feature through small RNA processing machinery in Arabidopsis. By performing RNA-seq on double-stranded RNA and single-stranded RNA pools, a negative correlation between mRNA abundance and structure complexity was identified. Thus, in addition to long double-stranded RNAs or miRNA precursors, highly structured mRNAs can also potentially serve as substrates of DCL proteins and give rise to small RNAs with a wide size range, unveiling a new regulatory function of DCLs on highly structured mRNAs.Brian Gregory  showed that the specific secondary structure of mRNAs can act as a potent As the premier and largest ag-genomics meeting in the world, this year’s Plant and Animal Genome Conference was a great success, and there are many more exciting and stimulating talks that I unfortunately do not have space to discuss here. Even if you were overwhelmed by the wealth of information presented at the meeting, you would have certainly been inspired. It was very satisfying to see that epigenetics and small RNAs have become an important part of many genomics studies, especially in complex non-model systems, which indicates their essential roles in regulating genes and genomes.DCL: Dicer-like; miRNA: microRNA; MITE: Miniature inverted repeat transposable element; phasiRNA: phased secondary siRNA; piRNA: PIWI-interacting small RNA; Pol IV: Polymerase IV; RdDM: RNA-directed DNA methylation; RNAi: RNA interference; SHH1: SAWADEE HOMEODOMAIN HOMOLOG 1; siRNA: small interfering RNA; TE: Transposable element.The author declares that she has no competing interests.


Small non-coding RNAs play essential roles in gene regulation, however, the interplay between RNA groups, their expression levels and deregulations in tumorigenesis requires additional exploration. In particular, a comprehensive analysis of microRNA (miRNA), PIWI-interacting RNAs (piRNAs), and tRNA-derived small RNAs in human testis and testicular germ cell tumor (TGCT) is lacking.in situ, and 12 normal testis samples. miRNA was the most common group among the sequences 18–24 nt in length and showed histology-specific expression. In normal samples, most sequences 25–31 nucleotides in length displayed piRNA characteristics, whereas a large proportion of the sequences 32–36 nt length was derived from tRNAs. Expression analyses of the piRNA population demonstrated global loss in all TGCT subtypes compared to normal testis. In addition, three 5′ small tRNA fragments and 23 miRNAs showed significant (p < 10−6) differential expression in cancer vs normal samples.We performed small RNA sequencing on 22 human TGCT samples from 5 histological subtypes, 3 carcinoma We have documented significant changes in the small RNA populations in normal adult testicular tissue and TGCT samples. Although components of the same pathways might be involved in miRNA, piRNA and tRNA-derived small RNA biogenesis, our results showed that the response to the carcinogenic process differs between these pathways, suggesting independent regulation of their biogenesis. Overall, the small RNA deregulation in TGCT provides new insight into the small RNA interplay.The online version of this article (doi:10.1186/s12943-015-0411-4) contains supplementary material, which is available to authorized users. In recent years, it has become increasingly evident that many non-protein-coding regions of the genome are transcribed , giving Unlike miRNA expression, piRNAs are mainly restricted to the male germline , 15–17, Other classes of sncRNAs have also been identified, including tRNA-derived small RNAs, a class of small RNA generated by specific endonucleases from mature tRNAs in response to certain conditions, such as oxidative stress, heat shock, or nutrient deprivation –33. Previn situ  lesions that may arise in utero from primordial germ cells (PGCs) [Small ncRNA may play an important role in TGCT. TGCT develops from carcinoma s (PGCs)  and is ts (PGCs) , 38. Thes (PGCs) –41. In Cs (PGCs) , indicats (PGCs) . DownregDespite the emerging biological significance of sncRNAs, most studies thus far have been conducted in model organisms. Therefore, the abundance, diversity, origin, and function of human sncRNAs are still relatively unknown. Although several studies have been performed on the role of miRNAs in spermatogenesis and TGCT, little is known about the presence and molecular function of other classes of human testicular sncRNAs. This exploratory study elucidates the presence and expression levels of small RNA populations in normal testicular tissue and TGCT histological subtypes. We analyzed the distribution of sncRNAs across the human genome by small RNA sequencing on RNA isolated from a total of 37 human samples.The resulting small RNA dataset encompasses samples from all major histological TGCT subtypes and relevant cell lines, and yielded almost 379 million sequence reads Table . The lowAfter sequence trimming, 76.5 % of the sequences mapped to the reference genome, of which 63.7 % mapped uniquely. In consistence with the piRNA signature observed in previous studies , 25, a pth position (10A). The proportion of 5′U and 10A show a strong decrease from 30 to 33 nt sequence length. The CIS subtype has a base composition pattern that resembles that of the normal samples, whereas the TGCT samples display a random distribution of the nucleotides in position 1 and 10 (probability for each base close to 25 %)  Fig. . Additio %) Fig. . The can %) Fig. .Fig. 1SmThe sequences were assigned to different classes based on annotations of the mapped region and the sequence lengths corresponding to the three peaks observed in the length distribution plot. The aligned sequences make up between 58.8 – 81.1 % of all sequences Table , whereasThe absolute difference in sequence counts between normal and TGCT samples in this size range are mostly due to low abundance of repeat-associated sequences in the TGCT samples   revealed a genome-wide loss of small RNA contigs in the TGCT samples Fig. . This lop > 10−4; data not shown). Differential expression analyses of the tRNA-associated sequences ~20 nt in length (tRFs) indicated a significant difference in the sequences derived from three tRNA genes, all downregulated in TGCT . The top 10 most differentially expressed miRNAs are listed in Table To assess the miRNA population in our samples, sequences with lengths corresponding to miRNAs (17–23 nt in length) were analyzed. A total of 23 miRNAs show differential expression between normal testis and TGCT samples  and TGCT histological subtypes Fig. . The CISIn contrast to the results on small RNA contig expression, the volcano plot for the miRNA data generates a symmetrical pattern, indicating that some miRNAs are downregulated, whereas others are upregulated in TGCT Fig. . The mosWe selected three miRNAs, piRNAs and tRFs for validation using qPCR. The validation shows corresponding up/downregulation to the small RNA sequencing Fig. , althougin situ, as well as in normal testis samples. Overall, we show that the human testis is highly abundant in miRNAs, piRNAs and tRNA-derived small RNAs. More specifically, the small RNA population in our dataset showed characteristics of canonical piRNAs, with an overall genomic distribution closely resembling that of a previous study [During the last decade, several novel classes of sncRNAs have been identified. These molecules are highly complex, and their biogenesis, molecular function and regulation are still largely unknown . With thus study , as wellus study . In addius study , 48–50. us study , 48, 49.Most piRNAs are globally downregulated or completely lost in all TGCT histological subtypes. This is not surprising, as tumor tissue has fewer differentiated germ cells, the main producer of piRNAs , 15–17 cin utero in PGCs or gonocytes [TGCT develops from CIS lesions that may arise onocytes . Althougonocytes  in a 100onocytes . The samThe high abundance of tRNA-derived RNAs 32–36 nt in length, representing tRNA halves, is in accordance with other studies , 35. DifIn addition to tRNA halves, we identified tRNA-derived sequences ~20 nt in length, corresponding to tRNA Fragments (tRFs) . These tWhereas piRNAs are almost exclusively found in the male gonad , 16, 17,Our results confirm previous findings indicating that miRNAs have a relevant role during testicular carcinogenesis, since overexpression of the miR-371-373, miR-302 and miR-367-3p clusters was noted in malignant TGCT tissue , 57, 58.A limitation of the present study is the possible presence of non-piRNAs among the 25–32 nt long sequences. Although we enrich for phosphorylated small RNAs with a 5′-OH group in the library preparations, degraded RNAs and other non-phosphorylated RNAs may be co-sequenced. Compared to using PIWI protein immunoprecipitation , our metn = 9) and phenol extraction (n = 3) of the normal samples were compared. No significant differential expression was found between samples prepared with the two extraction methods. Regardless, a replication study is needed to confirm potential biomarkers.Another possible bias in the small RNA expression measurements is differences in RNA extraction protocols . Most noThere is a need for more sensitive biomarkers for TGCT detection and surveillance . SeveralWe have documented significant changes in the small RNA populations in normal adult testicular tissue and TGCT samples. Most notable, our results indicate loss of the piRNA biogenesis pathway in TGCT, independent of histological subtype, and presence of large amounts of tRNA-derived small RNAs in both normal testicular tissue and in TGCT samples. Although crosstalk between small RNA pathways has been suggested, our results indicate independent regulation of miRNA, piRNA and tRNA-derived sequences in TGCT. These results may contribute to increased understanding of the functional roles of sncRNAs and their role in human spermatogenesis. In addition, they indicate that sncRNAs play an important role in TGCT development and progression, and that they may be promising candidate diagnostic markers for TGCT.Testicular tissue samples were selected from two sources; i) a series of primary TGCTs previously analyzed by Skotheim and co-workers , and ii)http://oslo.genomics.no),Total RNA extracts from all samples ) were used for library preparation and small RNA sequencing. Small RNA sequencing libraries were generated using the Illumina TruSeq Small RNA Sample Preparation protocol . One microgram total RNA was ligated to 3′- and 5′-RNA adapters, and ligation products were reverse transcribed to generate cDNA libraries for each sample. Products were PCR amplified, where unique index sequences were incorporated, before pooling of the libraries and gel purification. The samples were size selected on a 6 % PAGE gel, cutting from the upper side of the 160 bp marker to the lower side of the 140 bp marker, ensuring inclusion of small RNAs in the size rage 18–36 nucleotides. Small RNA libraries were sequenced single read 50 bp using an Illumina HiSeq2500 sequencer . Library construction and sequencing was performed at the Oslo University Hospital Genomics Core Facility (http://www.vicbioinformatics.com/software.nesoni.shtml). Small RNA length distribution was calculated from the trimmed fastq output files, and the nucleotide distribution in position 1 and 10 were calculated . Unique sequences from each histological subtype were used as input to visualize the probability of each nucleotide in a weblogo (v3.3) for sequences 30 and 33 nt in length [www.novocraft.com).Adaptors, short reads (<17 nt) and low quality bases were removed using Nesoni clip v0.128 (n length . Sequenchttp://genome.ucsc.edu), piRNA clusters in piRNA bank [Sequences overlapping with features such as repeatmasker (includes tRNAs), exon and RefSeq tracks from the UCSC table browser  in the sequencing dataset were selected for validation. Primers for miRNA detection were ordered from the Qiagen pre-designed miScript primer catalogue, whereas primers for piRNA and tRF detection were designed using the Qiagen custom miScript primer service  were selected and cDNA synthesis was performed on 0.2 μg totalRNA using miScript II RT Kit  according to the manufacturer’s instructions. The qPCR reactions were carried out on a Stratagene Mx3000p instrument using 1 ng cDNA, miScript SYBR Green PCR kit  and miScript primer assays . Sequences found to have a differential expression (


Escherichia coli there are three main exoribonucleases  involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome.The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms.In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases  contains supplementary material, which is available to authorized users. Escherichia coli has three 3′-5′exoribonucleases that accomplish most of the RNA degradative activity: RNase II, RNase R and PNPase  large subunit) is down-regulated in ∆rnb with a fold-change of 0.36 while in the ∆rnr mutant nirB is up-regulated with a fold-change of 9.11 . We selected some biofilm and flagellum assembly related transcripts that were differentially expressed in at least one of the exoribonucleases mutant. We then determined the fold change of that transcript by qPCR and compared the values obtained with the RNA-Seq Table . All thernb mutant but up-regulated in the ∆rnr mutant. This might indicate that RNase II and RNase R have very distinct roles in the cell. Surprisingly there is a higher overlap in the transcripts affected by PNPase and RNase II or PNPase and RNase R than the overlap between RNase II and RNase R , are differentially expressed in the exoribonuclease mutants when compared to the wild-type cells , a value that was further validated by qPCR . When required, antibiotics were present at the following concentrations: kanamycin, 50 μg ml−1; tetracycline, 20 μg ml−1; streptomycin/spectinomycin 20 μg ml−1.600 ~ 0.03 and grown to exponential phase (OD600 ~ 0.3). RNA was isolated following cell lysis and phenol:chloroform extraction as previously described [Overnight cultures from isolated colonies were diluted in fresh medium to an initial ODescribed . After pRNA samples (20 μg) were sent to Vertis Biotechnologie AG, Germany, for library preparation and sequencing of libraries using an Illumina HiSeq platform . For the library preparation Vertis Biotechnologie AG depleted the ribosomal RNA molecules from the total RNA preparations using the MICROBExpress Bacterial mRNA Enrichment Kit (Ambion). The rRNA depleted RNAs were then fragmented with RNase III and the 5'PPP structures were removed using RNA 5' Polyphosphatase (Epicentre). Afterwards, the RNA fragments were poly(A)-tailed using poly(A) polymerase and a RNA adapter was ligated to the 5´-phosphate of the RNA fragments. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 30 ng/μl using a high fidelity DNA polymerase. After the sequencing of the libraries Vertis Biotechnologie AG bioinformatics department did a preliminary analysis of the high-throughput sequencing results which included the cleaning of the sequences and the mapping of the reads against E. coli genome (NC_000913 downloaded from NCBI genome database). For the cleaning of the sequences Vertis Biotechnologie AG removed low quality and Poly(A) sequences and the adapters were trimmed. We then used the mapped files to run Cufflinks (estimates the relative abundance of the transcripts) and after Cuffdiff to find significant changes in transcript expression when comparing two samples . The tra−1, NaCl 5 g L−1, thymine 50 μg ml−1 and agar 0.25%) with 5 μL of cells in exponential phase. Plates were incubated at 37°C and pictures were regularly taken with Chemidoc Imaging system (BioRad) so as to monitor the increase of the swimming halo size. We used square plates to better compare the swimming of the wild-type cells with the swimming of the different exoribonucleases mutants. The upper left corner of the plate was inoculated with wild-type cells and the bottom right corner was inoculated with either ∆rnb, ∆rnr or ∆pnp cells. Motility assays were done at least three times.To measure bacterial motility we inoculated swimming media plates  were inoculated into the wells of a fresh microtiter plate and left at 37°C for 24 h. OD600 of the cultures was measured and subsequently used to normalise the data. Planktonic bacteria was removed from each microtiter dish by briskly shaking the dish out followed by at least two washing steps by submerging the plates in water and then vigorously shake out the liquid. 130 μl of 0.1% crystal violet solution was added to each well and left to stain the biofilms for 30 min at room temperature. The crystal violet was shaken out from the plates and again we performed at least two washing steps as previously described. The plates were allowed to air-dry and then the crystal violet was dissolved by adding 200 μl of an 80% ethanol/20% acetone solution to each stained well and left to incubate for 15 min. The dissolved crystal violet was transferred to a cuvette and each well was washed with another 200 μl of 80% ethanol/20% acetone solution. These 200 μl were added to the previous ones and OD550 was measured. The biofilm formation was determined by normalizing the OD550 values with the OD600 values measured after the 24 h inoculation of the plates. Then the wild-type was used as reference and all other values were obtained by the formula: normalised OD (mutant)/normalised OD (wt). Three independent biofilms formation assays were done each with at least four replicas per sample.Biofilm formation assay were carried on according to the protocol described by Merritt et al. . BrieflycysG as the reference gene. qPCR was performed in triplicate with, at least, three templates of RNA extracted from independent cultures.cDNA for quantitative RT-PCR was reverse transcribed from extracted RNA with random hexamers using the QuantiTect® Reverse Transcription Kit (Quiagen). The PCR amplification was performed with a Corbett Rotor Gene RG 3000 real-time PCR system and SensiFAST SYBR No-ROX Kit (Bioline). Oligonucleotides used as primers for qPCR are listed in Table The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are


Gram- negative bacteria utilize a diverse array of multidrug transporters to pump toxic compounds out of the cell. Some transporters, together with periplasmic membrane fusion proteins (MFPs) and outer membrane channels, assemble trans-envelope complexes that expel multiple antibiotics across outer membranes of Gram-negative bacteria and into the external medium. Others further potentiate this efflux by pumping drugs across the inner membrane into the periplasm. Together these transporters create a powerful network of efflux that protects bacteria against a broad range of antimicrobial agents. This review is focused on the mechanism of coupling transport reactions located in two different membranes of Gram-negative bacteria. Using a combination of biochemical, genetic and biophysical approaches we have reconstructed the sequence of events leading to the assembly of trans-envelope drug efflux complexes and characterized the roles of periplasmic and outer membrane proteins in this process. Our recent data suggest a critical step in the activation of intermembrane efflux pumps, which is controlled by MFPs. We propose that the reaction cycles of transporters are tightly coupled to the assembly of the trans-envelope complexes. Transporters and MFPs exist in the inner membrane as dormant complexes. The activation of complexes is triggered by MFP binding to the outer membrane channel, which leads to a conformational change in the membrane proximal domain of MFP needed for stimulation of transporters. The activated MFP-transporter complex engages the outer membrane channel to expel substrates across the outer membrane. The recruitment of the channel is likely triggered by binding of effectors (substrates) to MFP or MFP-transporter complexes. This model together with recent structural and functional advances in the field of drug efflux provides a fairly detailed understanding of the mechanism of drug efflux across the two membranes. Multidrug resistance or polyspecific transporters (MDRs) are present in all living systems. However, they are particularly abundant and diverse in bacteria and comprise 2–7% of the total bacterial protein content  are primary active transporters which couple substrate translocation with binding and hydrolysis of ATP. MDRs in all the other superfamilies are secondary transporters which utilize electro-chemical gradients of ions (most frequently protons but sometimes sodium) to transport their diverse substrates. Both primary and secondary transporters are ubiquitous in bacteria, however their relative presence seems to correlate with energy generation: fermentative bacteria tend to rely more on the primary transporters while genomes of aerobic bacteria contain somewhat more secondary transporters  binding of a substrate on the cytoplasmic or periplasmic side of the membrane, (ii) conformational changes in a transporter leading to re-orientation of the binding site to the other side of the membrane, and (iii) the release of the substrate. The conformational change leading to reorientation of substrate binding sites and relaxation of a transporter are usually energy-dependent steps, which is provided by either ATP hydrolysis (ABC pumps) or by a proton motive force (PMF) or sodium motive force. The basic mechanism of energization of transporters by ATP and PMF are well understood on examples of ABC and MF transporters  and the outer membrane channels (OMFs)  which are largely responsible for the low permeability characteristic , ABC  and MF  superfamilies  showed significantly lower affinity to AcrB  by stimulating the efflux activities of transporters and (ii) by engaging OMFs and opening them for substrates to be expelled from the cell  that interact with transporters within the cytoplasmic membrane , the MFP oligomerization was treated more as an artifact of crystallization, partly due to the fact that hydrodynamic studies and the size exclusion chromatography studies showed only monomers in solution   in the transport could be enhanced by additional subunits? In case of MFPs, this could be the separation of roles in the stimulation of a transporter and the recruitment/opening of OMF. In transporters, this could be an added substrate specificity or a separation of energy transduction and substrate translocation as suggested for MdtBC  to identify MFP-transporter and MFP-OMF molecular interfaces that are critical for activation of efflux and (ii) to characterize conformational transitions in all components of the complexes leading to efflux across the outer membrane. Non-typical transporters could be powerful tools in understanding how the trans-envelope efflux is achieved. There is a significant gap in knowledge about functions of MFPs in the context of Gram-positive cell envelopes.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.