Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that contribute to multiple aspects of nucleic acid metabolism including alternative splicing, mRNA stabilization, and transcriptional and translational regulation. Many hnRNPs share general features, but differ in domain composition and functional properties. This review will discuss the current knowledge about the different hnRNP family members, focusing on their structural and functional divergence. Additionally, we will highlight their involvement in neurodegenerative diseases and cancer, and the potential to develop RNA-based therapies. Many ribonucleoproteins (RNPs) assemble on to newly created transcripts in the nucleus of a eukaryotic cell. Among these RNPs are the heterogeneous nuclear ribonucleoproteins (hnRNPs). They assist in controlling the maturation of newly formed heterogeneous nuclear RNAs (hnRNAs/pre-mRNAs) into messenger RNAs (mRNAs), stabilize mRNA during their cellular transport and control their translation. Considering their functional diversity and complexity, hnRNPs act as key proteins in the cellular nucleic acid metabolism Fig.  Dreyfus.Fig. 1ThThe role of hnRNPs in regulating gene expression has gained an increasing interest in disease research. The expression level of hnRNPs is altered in many types of cancer, suggesting their role in tumorigenesis. In addition to cancer, many hnRNPs were also linked to various neurodegenerative diseases, such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD) and fronto-temporal lobe dementia (FTLD). As neurons are non-dividing cells, therefore needing a tight regulation of mRNA homeostasis, they are highly vulnerable to dysfunction of RNA-binding proteins (RBPs), including the hnRNPs.Because of the growing repertoire of different types of RBPs, it is still unclear how and when they are triggered to interact and functionally complement each other. The hnRNPs were initially categorized according to their RNA-binding domains (RBDs) composition  and are predominantly present in the nucleus during steady state. They are able to translocate in the cytosol upon post-translational stimulation or by the recruitment of other hnRNPs Fig.  Han et . The hnRFour unique RBDs were identified in hnRNP proteins: the RNA recognition motif (RRM), the quasi-RRM, a glycine-rich domain constituting an RGG box and a KH domain Fig. .The most common RBD is the RNA recognition motif (RRM). Eukaryotic RRMs are structurally characterized by four β-sheets and two α-helices (βαββαβ) together with two conserved RNP1 octameric and RNP2 hexameric sequences that are positioned approximately 30 residues apart from each other  as triple repeats  and auxiliary domains Fig.  increasemyelin basic protein (MBP) , activity-regulated cytoskeleton-associated protein (Arc) and neurogranin (NRGN) mRNA  gene, which is required for telomere maintenance    and disabled-2 (Dab2) , which belong to the family of intermediate filaments and are highly expressed in neurons , which are capable of creating G-quadruplexes. The RRMs present in hnRNP F and H are not conserved and are therefore alternatively described as quasi-RRMs (qRRMs)  because it regulates splicing by interacting with polypyrimidine stretches often located at the branch point upstream of exons  has 75 % homology with hnRNP I and has broadly the same effects on the regulation of RNA splicing. However, nPTB and PTB are mostly differentially expressed, leading to distinct outcomes for some neuronal-specific alternative splicing events  mRNA   mRNA, where it regulates various signaling pathways during osteoclast differentiation  (Hovhannisyan and Carstens carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1)    has three isoforms called hnRNP Q1-Q3. They are all created by alternative splicing events. The N-terminus has three RRMs and an auxiliary domain composed of acidic residues. Although the exact molecular function of hnRNP Q is not yet fully understood, many steps of mRNA maturation have been found to be associated , which is mutated in familial and sporadic ALS patients  experiments shed light on the differential splicing regulation  translation. An alternative approach used in RNA-based therapy is to prevent the formation of toxic RNA foci. A critical step is to reduce the expression level of the disease-propagating protein, which can be done by protein translation arrest or mRNA degradation. Antisense oligonucleotide drugs (AODs) can directly bind to mRNA or interfere with the binding of miRNAs to their target. Promising research done on transgenic C9orf72 mice already showed that the level of mutant pathogenic mRNAs could be lowered by the administration of AODs, therefore reducing RNA toxicity and disease-associated symptoms , Harvey rat sarcoma viral oncogene homolog (HRAS) and a splice variant of Recepteur d’Origine Nantais (ΔRON), and can therefore control tumorigenesis and metastasis indirectly  was found to be regulated by hnRNP C. It was suggested that hnRNP C also acts as a key regulator controlling the metastatic potential of glioblastoma cells  , originating from these ALS/FTLD patients, clearly showed the presence of hnRNP A1 and A2/B1 in RNA foci, suggesting RNA toxicity via the sequestration of RBPs as a possible pathomechanism  gene encoding for an RNA-binding protein are causative for SMA. A paralog gene, SMN2, differs from SMN1 by a C/T transition in exon 7, which results in a lower expression of full-length SMN protein that cannot rescue the loss of SMN1 expression. A strategy used to treat SMA patients is to promote exon 7 retention in SMN2  and hnRNP C. Both RBPs act competitively and influence APP translation in an opposing way  characterized by amyloid-b plaques formed by the amyloid-beta protein, which is a cleavage product of APP. The translational regulation of


Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3′ untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs. Nuclear factor 90 (NF90) is a double-stranded RNA (dsRNA) binding protein, conserved in vertebrates, which affects gene expression at transcriptional, post-transcriptional and translational levels –3. Also NF90  consistgluA2 pre-mRNA  are widespread in proteins involved in many aspects of RNA metabolism . They arpre-mRNA ,19. GluApre-mRNA . The ADApre-mRNA . Solutiopre-mRNA .n-A motif in the minor groove. We further show that dsRNA fragments lacking the preferred G-Xn-A motif are poor competitors of dsRNA binding. The dsRBD domains of NF90 only contribute part of the RNA binding activity of this protein, with a higher affinity for dsRNA found in the full-length NF90/NF45 complex. The similarity to ADAR2 suggests that NF90 is likely to recognize partner RNAs in a highly specific manner, consistent with observations that NF90 has important roles in post-transcriptional regulation of gene expression.To better understand the RNA recognition properties of NF90, we solved the crystal structure of the tandem dsRBDs of NF90 with a synthetic dsRNA. Surprisingly, NF90 tandem dsRBDs have high structural similarity to ADAR2 dsRBDs and show similar base-specific interactions with a G-XdsRBDs, residues 380–590) and for crystallization and isothermal titration calorimetry (ITC)   and induced overnight with 1 mM isopropyl-β-D-1 thiogalactopyranoside (IPTG) at 18°C. Cells were lysed using a cell disruptor (Constant Systems) in 40 mM HEPES pH 7.5, 150 mM NaCl, 1 mM dithiothreitol (DTT), supplemented with a protease inhibitor cocktail (Roche) and DNAse I (Sigma Aldrich). Cleared lysates were bound in batch to GSH resin  at 4°C, packed into a chromatography column and eluted in 40 mM HEPES pH7.5, 150 mM NaCl, 1 mM DTT and 20 mM reduced glutathione. The protein was dialysed into 20 mM HEPES pH7.5, 50 mM NaCl overnight with 1 mg Tobacco etch virus (TEV) protease. TEV protease and GST were separated from NF90 using heparin sepharose or mono S cation exchange chromatography and eluted with a salt gradient to 1M NaCl. The protein was further purified by size exclusion chromatography using a Superdex S75 column . NF90long (residues 1–590 of mouse NF90) and NF45 (full length or residues 28–390) were expressed separately in BL21(DE3) cells as N-terminally 6×His tagged proteins, induced at 18°C overnight and co-lysed in 20 mM Tris.HCl pH 8.0, 500 mM NaCl, 10 mM imidazole (pH 8.0) and 1 mM β-mercaptoethanol with protease inhibitor cocktail. Tagged proteins were extracted from clarified lysates by batch binding to Ni2+-NTA resin, packed into a column and eluted with a gradient from 0.01–1M imidazole, pH 8.0. Eluted proteins were dialysed overnight into 20 mM Tris.HCl pH 7.5, 50 mM NaCl, 1 mM DTT with TEV protease to remove the tags. NF90long and NF45 were mixed and the complex was purified by heparin sepharose chromatography and size exclusion chromatography in 20 mM Tris.HCl pH 7.5, 150 mM potassium acetate, 1 mM DTT. NF90long alone was purified using the same conditions as the NF90long/NF45 complex. NF90ΔC380/NF45ΔN28 were purified as described previously (Two constructs of mouse NF90 were used for electrophoretic mobility shift assays (EMSA) (NF90) Figure . These ceviously .2O, mixed at a 1:1.2 molar ratio of labelled to unlabelled strand for a final concentration of 100 μM duplex and annealed overnight as for 18mer duplexes. For competition assays, GluA2(R/G) RNA was synthesized by Biomers GmbH and reconstituted in water to a final concentration of 100 μM (CAUUAAGGUGGGUGGAAUAGUAUAACAAUAUGCUAAAUGUUGUUAUAGUAUCCCACCUACCCUGAUG). GluA2(R/G) RNA was annealed by heating to 95°C for 3 min and snap cooled on ice for 30 min. Duplexes for unlabelled 17mer RNA (UCACUUUCAUAAUGCUG) and GC- and AU-rich duplexes were prepared as for 18mer duplexes.18mer 2′F modified RNA was a kind gift from ISIS pharmaceuticals . DuplexedsRBDs–18mer RNA complexes. Crystals were grown in sitting drops in 50 mM sodium citrate pH 5.0, 1.0M ammonium formate. Crystals grown from NF90dsRBDsΔNLS–18mer RNA complexes showed improved diffraction quality. Crystals were cryoprotected in mother liquor supplemented with 30% glycerol and flash cooled to 100 K. Data were collected at beam line I03 at Diamond Light Source using a PILATUS detector. Data were indexed using XDS and scaled and merged using SCALA  was pre-run at 2W at 4°C for 1 h. Samples were mixed with 2 μl of native gel loading buffer and 4 μl was loaded onto the gel. After 2 h at 2W, the gel was scanned on a LICOR Odyssey fluorescent infrared scanner at 800 nm. Images were converted to greyscale and inverted using ImageJ. Competition assays were carried out in a 10 μl reaction volume. A 1:1 molar ratio complex of 21mer dsRNA with NF90long/NF45 was made by pre-incubation on ice for 1 h. The complex was used at a final concentration of 0.5 μM with buffer and/or increasing concentrations of unlabelled competitor RNA.Proteins were dialysed into 20 mM HEPES pH 7.5 and 150 mM potassium acetate. Binding reactions, containing 1 μM labelled RNA and increasing concentrations of NF90dsRBDsΔNLS and 18mer RNA (either native or 2′F modified) were dialysed overnight in 20 mM HEPES pH 7.5 and 50 mM NaCl. Protein and RNA concentrations were determined using 280 nm absorbance on a Nanodrop spectrophotometer. Measurements were made at 25°C using a Microcal Auto-iTC200 system . A total of 300 μM NF90dsRBDs in the syringe was titrated into 12 μM 18mer dsRNA in the cell with a total of 16 × 2.5 μl injections and 3 min between each injection. To control for dilution, NF90dsRBΔsΔNLS was titrated into buffer alone. Experiments were repeated three times and Kd app values were obtained using the Microcal iTC200 software.NF90dsRBDs (residues 380–592) or mNF90long(residues 1–592)-mNF45ΔN28 for 20 min at 23.5°C. Anisotropy was measured on a Spectramax M5 multi-plate reader (Molecular Devices) using 530 and 562 nm wavelengths for excitation and emission, respectively. Experimentally observed anisotropies as a function of protein concentration were fit to equation 6 . Binding constants were determined from three independent experiments, from which mean Kd and standard deviations were calculated.A total of 250 nM Cy3-labelled 21mer dsRNA  was incubated with increasing concentrations of mNF90 6 (from ) to deteet al.  and longer constructs containing all folded domains of NF90  and NF90long/NF45 complex were able to shift the labelled dsRNA  is unable to interact with dsRNA. NF90long/NF45, NF90long and NF90dsRBDs show the formation of two distinct RNA–protein complexes at 1:1 and 1:2 RNA:protein . Crystals of the NF90–RNA complex showed improved diffraction properties when a shorter construct of NF90dsRBDs lacking the NLS was used  of ∼2.4 mM . This indicates that a difference in affinity is not the reason for the difference in their ability to crystallize. Previous observations show that duplexes containing 2′F modified RNAs have higher thermal stability than native RNA molecules and are more resistant to degradation, which may explain the difference between native and modified dsRNA in crystallization experiments . DsRBD1dsRBDsΔNLS in complex with 18mer RNA the two dsRBDs bind either side of the dsRNA . He. He15). Interestingly, both dsRBDs of NF90 approach similar base-pairs in the two minor groove interfaces spanned by each domain: a C-G base pair is found at the Region 2 interface and an A-U base pair at the Region 1 interface of dsRBD2 Figure  and C. SBase-specific readout of the minor groove has previously been observed for the tandem dsRBDs of ADAR2 in a solution structure with GluA2(R/G) editing site hairpin RNA . We obseThe similarity between NF90 and ADAR2 is also apparent at the structural level where dsRBD1 from ADAR2 and NF90 superpose with an r.m.s.d of 1.4 Å over 57 Cα atoms and the respective dsRBD2 domains superpose with an r.m.s.d of 1.4 Å over 61 Cα atoms. Interestingly, GluA2(R/G) RNA curves around dsRBD1 of ADAR2 with a gentle deviation from the main helical axis, similar to the kink generated by the intermolecular stacking region of the 18mer dsRNA around its interaction site with NF90 dsRBD1 Figure .ADAR2 and Met238ADAR2). Hydrogen bonds are formed between main chain carbonyl oxygens in the β1–β2 loop of both ADAR2 dsRBDs  . In thise Figure . In the A Figure . PreviouA Figure ,42. Our dsRBDsΔNLS–18mer RNA and ADAR2–GluA2(R/G) hairpin complexes, it seems likely that NF90 will recognize ADAR2 substrates. To test this hypothesis we titrated 1:1 complexes of NF90long/NF45 pre-incubated with labelled 21mer dsRNA with increasing concentrations of unlabelled GluA2(R/G) as a competitor  hairpin does indeed bind to NF90long/NF45 complexes  motif is slightly distorted by base mismatches. Differences in the NF90dsRBDsΔNLS–18mer RNA complex generated by guanine bases coming from different strands are accommodated by small shifts in conformation of the β1–β2 loop in Region 2 .The primary differences between the ADAR2-GluA2(R/G) and NF90 NF90 Figures  and 4. M Figures . TherefoSPNR has identical sequences to NF90 within its dsRBD domains Figure , suggestin vitro binding assays that NF90 can dimerize on dsRNA although we only observe a 1:1 complex of dsRNA to NF90 in the crystal structure. It is possible that the formation of a 1:2 complex in this system inhibits crystallization. While crystallization of this complex was dependent on the presence of a modified RNA strand, containing 2′F at each position, this modification does not seem to have altered the binding constant of this interaction or hindered the association of the protein with the modified ribose moieties. This suggests that 2′F modifications might be a useful tool to promote crystallization of other protein–RNA complexes.The structure of the tandem dsRBDs of NF90 with dsRNA reveals a complex with dsRBDs bound either side of dsRNA. One dsRBD bridges two dsRNA 18mers, inducing a bend where the two dsRNA molecules meet Figure . We showdsRBDsΔNLS–18mer RNA gives an insight in to ADAR2 sequence requirements for dsRNA recognition. Comparison with our structure shows how subtle deviations from ideal A-form RNA such as RNA curvature; localized disruptions by a G-G mismatch; and overwinding of the RNA helix inherent in the GluA2(R/G) motif facilitate interactions with ADAR2. As with NF90, several reports have suggested that ADAR2 is active as a dimer, although whether dimerization is dsRNA-dependent is currently a matter of debate  hairpin motif. Serendipitously, the structure of NF90f debate –50.Zinc Finger RNA-binding protein, Zfr. Zn72D contains an NF45-heterodimerization domain like that found in NF90 and SPNR but does not encode dsRBD domains . NF90 and SPNR are vertebrate-specific proteins. The nearest relative in fruit flies is the zinc finger protein, Zn72D, a homologue of the vertebrate protein  domains . It is sThat NF90 and ADAR2 share a similar mode of binding on dsRNA gives an insight into NF90 function. An important aspect of ADAR2 function is the ability to direct A-to-I editing at a specific residue and the dsRBD domains play a critical part in substrate selection. While we do not yet understand the full physiological implications of this similarity, our data suggest that, like ADAR2, the binding of NF90 on endogenous dsRNA targets may be influenced by sequence elements as well as structural elements Figure . This woData have been deposited in the PDB with the following accession code: 5DV7.


We show that in addition to KIN-29, signaling is mediated by the DAF-2 insulin-like receptor, OCR-2 TRPV channel, and NPR-1 neuropeptide receptor. Cell-specific rescue experiments suggest that DAF-2 and OCR-2 act in ADL, while NPR-1 acts in the RMG interneurons. NPR-1-mediated regulation of srh-234 is dependent on gap-junctions, implying that circuit inputs regulate the expression of chemoreceptor genes in sensory neurons. Using physical and genetic manipulation of ADL neurons, we show that sensory inputs from food presence and ADL neural output regulate srh-234 expression. While KIN-29 and DAF-2 act primarily via the MEF-2 (MEF2) and DAF-16 (FOXO) transcription factors to regulate srh-234 expression in ADL neurons, OCR-2 and NPR-1 likely act via a calcium-dependent but MEF-2- and DAF-16-independent pathway. Together, our results suggest that sensory- and circuit-mediated regulation of chemoreceptor genes via multiple pathways may allow animals to precisely regulate and fine-tune their chemosensory responses as a function of internal and external conditions.Feeding state and food availability can dramatically alter an animals' sensory response to chemicals in its environment. Dynamic changes in the expression of chemoreceptor genes may underlie some of these food and state-dependent changes in chemosensory behavior, but the mechanisms underlying these expression changes are unknown. Here, we identified a KIN-29 (SIK)-dependent chemoreceptor,  in vivo reporter assay in C. elegans for monitoring the expression of a candidate chemoreceptor gene in a single sensory neuron type, called ADL, as a function of feeding state. Using this reporter assay, we show that sensory inputs into ADL and neural outputs from ADL, as well as inputs from the RMG interneuron, which is electrically connected to ADL, are required to fine-tune expression of chemoreceptor genes in ADL. Sensory and circuit-mediated regulation of chemoreceptor gene expression is dependent on multiple pathways, including the neuropeptide receptor, NPR-1, and the DAF-2 insulin-like receptor. Our results reveal mechanisms underlying chemoreceptor gene expression, and provide insight into how expression changes in chemoreceptor genes may contribute to changes in chemosensory behavior as a function of feeding state.Animals dramatically modify their chemosensory behaviors to attractive and noxious chemical stimuli when starved. This could allow them to alter and optimize their food-search strategies to increase their survival and reproduction. Changes in the gene expression of chemoreceptors specialized in detecting environmental stimuli is observed in fish, insects and nematodes, and may be a general mechanism underlying the changes in chemosensory behaviors observed in starved animals. To elucidate this mechanism, we have developed an  Although these state-dependent changes in chemosensory behaviors have long been thought to arise from plasticity in central processes, there is now growing evidence that feeding state gates responses in peripheral chemosensory neurons themselves, thereby directly modulating changes in chemosensory behaviors An animals' feeding state  is strongly expressed in ADL in fed animals (srh-234p::gfp was significantly downregulated (srh-234p::gfp (oyIs57)  mutants, which is required for pheromone biosynthesis srh-234 expression. Thus, starvation reduces the expression of the kin-29-dependent chemoreceptor, srh-234, in ADL, and this modulation is reversible upon re-feeding with food.Since SIKs regulate feeding state-dependent gene expression, we investigated whether feeding and starvation regulates the expression of (oyIs57) . The exp animals . The eff 6 hours . Moreoveobserved . As bactgfp driven by the cis-regulatory region of the chemoreceptor srh-34 in ADL was altered in fed and starved animals with an opposite phenotype than observed for srh-234; srh-34 is expressed in starved animals but not in fed animals (versus starved) of C. elegans alters the expression levels of at least two chemoreceptor genes in ADL. We focused our subsequent studies on using the srh-234p::gfp reporter assay to further explore the mechanisms underlying starvation modulation of chemoreceptor gene expression in ADL.We next sought to identify additional chemoreceptor genes in ADL regulated by fed and starved conditions. Of the 11 chemoreceptor genes tested, only  animals . Thus, tsrh-234 expression levels we observed in starved wild-type animals could arise as a consequence of an internal state of starvation triggered by a decrease in the ingestion of food, or alternatively, by an external sensory response as a result of a decrease in the perception of food. To distinguish between these possibilities, we exposed fed and starved wild-type adult animals carrying the srh-234p::gfp reporter to E. coli food treated with the antibiotic aztreonam, which results in bacteria that grow in long chains that C. elegans cannot eat due its large size, but still can smell and touch comparable to regular non-treated bacteria E. coli OP50 (inedible food) for 24 and 48 hours significantly reduces srh-234 expression mimicking the effects of starvation when compared to fed animals placed on non-treated E. coli OP50 (edible food) (lf) mutations in eat-2 that result in animals with a pharyngeal pumping defect compromising their food ingestion, also reduce srh-234 expression on edible food  on aztreonam-treated E. coli OP50 (inedible food), we found that the srh-234 expression phenotype was not significantly different from their non-treated E. coli OP50 (edible food) counterpart as if they sense food presence correctly even when they cannot eat this food . Thus, the perception of inedible food can override the effects of starvation on reducing srh-234 expression levels. In summary, our results suggest that the starvation-induced downregulation of srh-234 expression is likely a consequence of both sensory inputs associated with a decreased food presence, and an internal state of starvation triggered by a decrease in food ingestion.The reduced le food) . Consistble food . Thus, this food . It is pC. elegans are conveyed through an insulin signaling pathway with DAF-2 being the main insulin-like receptor srh-234. Consistent with low daf-2 insulin signaling being associated with a starved state, we found that daf-2(e1307) mutants reduce srh-234 expression in ADL in fed conditions, similar to starved wild-type animals  suppressed the daf-2- and starvation-induced downregulation of srh-234 expression. Indeed, we found that both daf-16(mu86) mutants and daf-2(e1307); daf-16(mu86) double mutants showed a significant increase in srh-234 expression during starvation compared to starved wild-type animals  mutants during feeding to near wild-type levels, whereas daf-2 expression in the nervous system had a partial effect (daf-2 in the intestine did not result in a restoration of the reduced srh-234 expression phenotype of daf-2(e1307) mutants  mutants during starvation back to wild-type levels, but similar to daf-2, expression of daf-16a cDNA in the intestine had no effect  expressed in  feeding . These rC. elegans regulates a range of food-related behaviors. For example, mutants lacking npr-1 move rapidly, avoid high oxygen concentrations and aggregate in groups in a food-dependent manner srh-234 in ADL. Indeed, we found a strong reduction in srh-234 expression in ADL in lf mutants of npr-1 , and in a reduction-of-function npr-1 allele, g320, in fed conditions, with the g320 allele having the weakest effect  mutants back to wild-type levels (npr-1 using the sre-1 promoter did not restore the reduced srh-234 expression phenotype of npr-1(ad609) mutants  npr-1(ad609) mutants completely restored the reduced srh-234 expression phenotype of npr-1 mutants to wild-type levels during feeding, and increased srh-234 expression when starved  and unc-9(e101) fully suppressed the reduced srh-234 expression phenotype of npr-1(ad609) mutants in fed conditions (daf-2(e1307) or kin-29(oy38) mutant background  and unc-9(e101) mutants, as well as in unc-7 npr-1 and unc-9 npr-1 double mutants when compared to starved wild-type animals , in flp-21-expressing cells that include RMG interneurons (flp-21::unc-7), and in all neurons  in unc-7(e139) npr-1(ad609) double mutants, and examined their effect on srh-234 expression. Surprisingly, however, we did not observe a restoration of the reduced srh-234 expression phenotype of unc-7 npr-1 double mutants back to npr-1 levels for either transgene in fed conditions (unc-7 or unc-9 by RNAi did not suppress the reduced srh-234 expression phenotype of npr-1(ad609) mutants in fed conditions  and osm-6(p811) mutants that lack functional sensory cilia srh-234 expression in ADL in fed conditions (osm-6(p811) mutants by expressing osm-6 fused to mCherry under control of the sre-1 promoter was sufficient to restore wild-type srh-234 expression in fed conditions . These results suggest that sensory inputs from food presence specifically through ADL cilia and dendrites are essential to promote srh-234 expression.Although these dendritic cuts indicate a direct role for ADL sensory neurons in regulating nditions . Functionditions . We confsrh-234. We therefore generated transgenic animals that express the pkc-1(gf) and the tetanus toxin (TeTx) cDNA under control of the ADL-specific sre-1 promoter (ADL::pkc-1(gf) and ADL::TeTx). The pkc-1(gf) is a constitutively active protein kinase C that enhances synaptic output by promoting secretion of dense-core vesicles containing neuropeptides TeTx prevents secretion of small neurotransmitter molecules by blocking synaptobrevin-mediated fusion of small, synaptic vesicles ADL::pkc-1(gf) strongly increased srh-234 expression in fed wild-type animals compared to non-transgenic siblings  did not significantly change levels of srh-234 expression (srh-234 expression phenotype of ADL::pkc-1(gf) is completely suppressed by npr-1(ad609) (npr-1 may act downstream of pkc-1(gf)-enhanced neuropeptide secretion from ADL to regulate srh-234 expression. Consistent with these findings, lf mutations in genes that disrupt the release and processing of neuropeptides globally  , suggestn levels . No chane amines . Togethesrh-234 in fed conditions, suggesting that a cilia-localized mechanism for sensing food presence may be important for the regulation of srh-234. The TRPV channels, OCR-2 and OSM-9, localize to the cilia of a subset of sensory neurons, including ADL srh-234 expression. Indeed, we found that lf mutations in ocr-2  strongly reduce srh-234 expression in ADL in fed animals, whereas osm-9(ok1667) did so more weakly (ocr-2 and to a lesser extent osm-9 promote srh-234 expression in fed conditions. It is possible that ocr-2(ak47) and osm-9(ok1667) mutants reduce srh-234 expression when fed as a result of a decrease in food ingestion instead of a decrease in food perception; however, consistent with previous findings ocr-2 and osm-9 mutants ingest food similar to wild-type animals as measured by pumping rates . ADL-specific expression of ocr-2 under control of the sre-1 promoter (ADL::ocr-2) fully restored srh-234 expression in ocr-2(ak47) mutants during feeding, and is slightly upregulated when starved ; npr-1(ad609) showed a reduced srh-234 expression phenotype similar to that of npr-1(ad609) or ocr-2(ak47) mutants alone in both fed and starved conditions (ocr-2(ak47) and osm-6(p811) fully suppressed the unc-7(e139) phenotype of upregulated srh-234 expression in starved conditions  suppressed the starvation-induced downregulation in a wild-type background  can suppress the reduced srh-234 expression phenotype of npr-1(ad609), daf-2(e1307) and osm-9(ok1667) mutants (kin-29(oy38) mutants (egl-19(gf) specifically in ADL neurons (ADL::egl-19(gf)) also suppressed the reduced srh-234 expression phenotype of ocr-2(ak47) and npr-1(ad609) mutants in fed conditions  using the genetically encoded Ca2+ sensor GCaMP3. ADL is known to sense C9 as determined by calcium imaging and behavioral assays ADL::GCaMP3 reporter in the absence of any stimuli in wild-type animals starved for 6, 12 or 24 hours, we also observed no significant differences in ADL::GCaMP3 expression when compared to control fed animals (egl-19(gf) suggest that increased calcium signaling is correlated with increased srh-234 expression.To further test the hypothesis whether calcium levels in ADL may be modulated by feeding and starvation responses, we measured intracellular calcium dynamics specifically in ADL neurons in fed and starved wild-type animals in the presence of the C9-pheromone  failed to suppress the reduced srh-234 expression phenotype of ocr-2(ak47), npr-1(ad609) and daf-2(e1307) mutants in fed and starved conditions (kin-29(oy38) mutants can suppress the increased srh-234 expression phenotype when overexpressing OCR-2 and NPR-1  suppressed the starvation-induced downregulation of srh-234 expression, but had no major effect on srh-234 expression during feeding  did not suppress the srh-234 expression phenotype of kin-29(oy38) mutants as well as of ocr-2(ak47) and npr-1(ok1447) mutants , or when eat-2(lf) mutants were exposed to edible food, the expression of srh-234 decreased in ADL, suggesting that the signal needed to reduce srh-234 expression is probably an internal metabolic signal arising after food ingestion. However, when starved animals were presented with either edible or inedible E. coli food, expression of srh-234 is increased in ADL, suggesting that animals perceive the availability of food, even when they are unable to eat the inedible food. Thus, inedible food likely blocks signals associated with starvation to regulate srh-234 expression. Interestingly, this finding is similar to a previous study, showing that the repressive effects of starvation on C. elegans mating can be partially blocked by placing males on inedible food Expression of srh-234 expression. Moreover, restoring only ADL cilia in osm-6(lf) mutants that have severe truncations of all ciliated sensory neurons srh-234 expression phenotype of osm-6(lf) mutants. These results suggest that sensory inputs through ADL sensory endings are essential to regulate srh-234 expression. Thus, we propose that depending on food availability and feeding state, srh-234 expression in ADL is regulated by two inputs, through external signals from food presence mediated directly by ADL and internal state signals resulting from food ingestion.Sensory inputs are known to alter chemoreceptor gene expression in a subset of sensory neurons. We show that loss of all sensory inputs into ADL, as demonstrated by physically cutting the ADL dendrites, causes fed animals to reduce npr-1 in RMG interneurons, which forms gap-junctions with ADL srh-234 expression in ADL. Second, this npr-1-mediated regulation of srh-234 is dependent on the function of unc-7/9 gap-junctions. Third, we show that enhancing the secretion of dense-core vesicles that contain neuropeptides by expressing pkc-1(gf) in ADL neurons promotes expression of srh-234, and this regulation is dependent on npr-1. Fourth, we show that mutants with defects in neuropeptide processing and secretion alter the expression of srh-234. Lastly, insulin signals from other neurons may act on DAF-2 in ADL to regulate srh-234 as suggested by the srh-234 expression phenotype of daf-28(gf) mutants, and daf-2/daf-16 signaling acting cell-autonomously in ADL.Modulation of chemoreceptor gene expression is thought to be defined primarily by external environmental inputs perceived by sensory neurons and not by circuit inputs from other neurons. Our results described here reveal a novel role for circuit-mediated regulation of chemoreceptor genes in sensory neurons. First, we show that npr-1 in RMG interneurons alters the expression of srh-234 in ADL remains to be determined. Current understanding suggests that NPR-1-mediated regulation of aggregative behavior and pheromone responses requires RMG unc-7 and unc-9 gap-junctions are essential for npr-1-mediated regulation of srh-234. Surprisingly, however, cell-specific rescue and RNAi knock down experiments of unc-7/9 in either ADL or RMG neurons suggest that they may not be directly involved in forming gap-junctions between RMG and ADL neurons. One possibility is that unc-7/9 are required in other neurons besides RMG and ADL for regulating srh-234 expression, which is consistent with the partial rescue of the srh-234 expression phenotype of unc-7 npr-1 double mutants by driving unc-7 cDNA in the nervous system  mutants in a daf-16-dependent fashion. Rescue experiments with various promoters suggest that both DAF-2 and DAF-16 act cell-autonomously in ADL to regulate srh-234 expression. A gf mutation in the daf-28 insulin-like peptide (ILP), which has been proposed to block other agonistic ILPs from binding to DAF-2 srh-234 expression in fed conditions. These results indicate that ILPs from other yet unknown neurons may target DAF-2 in ADL, which in turn regulates srh-234 expression. C. elegans contains 40 ILPs, and for some specific functions in particular sensory neurons have been reported. For example, in salt-chemotaxis learning, DAF-2 acts in ASE sensory neurons, and is targeted by the INS-1 ILP released from AIA interneurons ins-1 mutants did not change srh-234 expression in fed or starved conditions, suggesting there may be other ILPs involved in the regulation of srh-234 expression.Insulin signals reflect the feeding state of an animal, which has been suggested to directly affect chemosensory sensitivity to chemical cues. Our results suggest a new role for insulin-mediated regulation of chemoreceptor genes. Consistent with low C. elegans have been implicated in regulating the expression of sensory neuronal genes. For example, loss of these TRPV channels reduces expression of the odr-10 chemoreceptor gene in AWA neurons tph-1, in AFD neurons ocr-2 in ADL neurons. TRPV channels in mammals are permeable to calcium C. elegans, the reduced tph-1 expression phenotype of ocr-2(lf) mutants can be suppressed by activating downstream calcium-dependent pathways egl-19(gf) can bypass the requirement of OCR-2 in regulating the expression of srh-234. Such activity-dependent transcriptional regulation of chemoreceptors may allow TRPV channels to regulate the sensitivity of sensory neurons to particular chemical stimuli as a function of feeding state. MEF2 plays a critical role in activity-dependent transcription in the nervous system mef-2 does not suppress the srh-234 reduced expression phenotype of ocr-2 mutants. Thus, TRPV-mediated transcriptional regulation of srh-234 is independent of MEF-2 function, suggesting other possible downstream effectors of OCR-2 in regulating srh-234 expression. A possible target is the CREB-regulated transcriptional coactivator CRTC1, which recently was shown to regulate TRPV-mediated longevity The OCR-2 and OSM-9 TRPV channels in mef-2(lf) and daf-16(lf) suppress the starvation-induced downregulation of srh-234 expression, but the expression of srh-234 is not altered during feeding. On the basis of these genetic experiments, we suggest that under starved conditions, MEF-2 and DAF-16 are required to repress but not activate srh-234 expression, suggesting that other yet unknown transcription factors may be required to drive srh-234 expression in ADL. Our previous work showed that MEF-2 is able to directly bind a MEF-2 sequence motif upstream of the str-1 chemoreceptor srh-234 revealed a similar MEF2 binding motif as well as an E-box sequence motif  appears to be present in the 165 bp promoter sequence necessary for feeding-state regulation of srh-234 (pers. comm. M.G and A.M.V). In Drosophila, SIK3 activity can be activated by insulin, which antagonizes FOXO-activated gene expression srh-234, suggesting that DAF-16 mainly acts downstream of insulin signaling.The specific transcriptional mechanisms by which feeding and starvation regulate chemoreceptor gene expression remain to be deciphered; however, our genetic experiments implicate a key role for the MEF-2 and DAF-16 transcription factors acting downstream of the KIN-29 and DAF-2 pathways, respectively. We show that ce motif . E-box mC. elegans expresses multiple chemoreceptors in each chemosensory neuron. We and others have proposed that to selectively modify a response to a single chemical may rely on changing the expression of individual or subsets of chemoreceptor genes, rather than altering the response of the entire neuron, which would inadvertently result in changing the response to all stimuli sensed by that neuron. Selectively modulating distinct populations of chemoreceptors in this manner may allow C. elegans to modulate ‘long-term’ changes in chemosensory responses in changing environmental conditions. This expression strategy may be particularly important in ADL, which mediates avoidance responses to a wide variety of chemical signals in its environment, such as odors srh-234 and srh-34, are regulated by fed and starved conditions, but it is unknown what chemical or subset of chemicals are detected by these chemoreceptors. Only a few chemoreceptors with known chemical ligands have been found in C. elegans, such as the chemoreceptor gene, odr-10, expressed in the AWA olfactory neuron type for the attractive chemical diacetyl odr-10 gene was introduced into the repulsive AWB neuron, C. elegans changes its normally attractive response to diacetyl to trigger avoidance of diacetyl srh-234 expression in ADL, allows fed animals to be less tolerant of aversive stimuli detected by srh-234, whereas decreased srh-234 expression in ADL following starvation may allow starved animals to be more tolerant to these aversive stimuli. This dynamic modulation in chemoreceptor gene expression could allow starved C. elegans animals to sample different environments, perhaps increasing their chances of finding food under stress-full conditions.Our results show that the expression of least two candidate chemoreceptor genes in ADL, Danio rerio is induced in temporal waves, which may reflect a mechanism for odorant sensitivity during development Drosophila rapidly modify their chemosensory behaviors to lower their attraction to males such that they can focus on reproduction, and these behavioral changes are accompanied by modulation of different chemoreceptor genes C. elegans, ablation of ASE and ASH neurons of the parasitic nematode S. stercoralis showed that these sensory neuron types mediate attraction and repulsion to soluble chemicals, respectively C. elegans has high anatomical and functional similarity to certain parasitic nematodes, it would be important to determine whether similar mechanisms of feeding state-regulated chemoreceptor gene expression presented here operate in parasitic nematodes. Moreover, identification of additional regulators and mechanisms underlying chemoreceptor gene expression will lead to a better understanding of how animals modify their chemosensory behavior in response to changes in external and internal conditions.Other invertebrates and vertebrates alter chemoreceptor gene expression in response to environmental and developmental signals. For example, expression of olfactory receptor genes in the zebrafish E. coli OP50 as the primary food source unless indicated otherwise. The wild-type strain used was C. elegans variety Bristol, strain N2. Mutant strains used in this study were obtained from the Caenorhabditis Genetics Center (CGC) unless indicated otherwise: PR811 osm-6(p811), PR813 osm-5(p813), DA609 npr-1(ad609), RB1330 npr-1(ok1447), CX4148 npr-1(ky13); CX4057 npr-1(g320); VC2106 flp-18(gk3063), RB982 flp-21(ok889), VC461 egl-3(gk328), CB169 unc-31(e169), CB55 unc-2(e55), CB251 unc-36(e251), CB1370 daf-2(e1370), JT191 daf-28(sa191), CF1038 daf-16(mu86), LC33 bas-1(tm351), CB1112 cat-2(e1112), RB1161 tbh-1(ok1196), RB993 tdc-1(ok914), GR1321 tph-1(mg280), VC1262 osm-9(ok1677), CX4544 ocr-2(ak47), JY243 ocr-2(yz5), DA465 eat-2(ad465), DA1116 eat-2(ad1116), KM134 mef-2(gv1), PY1476 kin-29(oy38), CB139 unc-7(e139), CB101 unc-9(e101), MT6129 egl-19(n2368)gf, MT1212 egl-19(n582), VC223 tom-1(ok285), and DR476 daf-22(m130). Transgenic strains used in this study were: VDL3 oyIs56, VDL143 oyIs57, and BOL171 npr-1(ad609); lin-15 (RMG::npr-1) sre-1::GCaMP3Nematodes were grown at 20°C under standard conditions srh-234 and odr-10 coding sequences. Primer sequences for srh-234 are 5′-GGACAATTGAAATGCAACACA-3′ and 5′-GACGGGGACAATAAAGAGCA-3′. Primer sequences for odr-10 are 5′-GAGAATTGTGGATTACCCTAG-3′ and 5′-CTCAATATGCATTATAGGTCGTAATATG-3′.Total RNA was isolated from a growth-synchronized population of adult animals and reverse transcribed using oligo(dT) primers. Real-time quantitative reverse transcription–PCR (qRT–PCR) was performed with a Corbett Research Rotor-Gene 3000 real-time cycler, Platinum Taq polymerase (Invitrogen), and primers specific for srh-234p::gfp reporters were cultured and grown at 20°C on standard nematode growth media (NGM) plates seeded with E. coli OP50 as the bacterial food source unless indicated otherwise. Young adult animals were washed with M9 buffer (to remove any bacteria in the gut) and transferred onto plates with E. coli OP50 food (Fed) or without E. coli OP50 food for 12–24 hours (Starved) unless indicated otherwise. Levels of srh-234p::gfp expression in animals were measured under a dissection microscope equipped with epifluorescence as described gfp was scored as “bright” if levels of gfp fluorescence allowed visualization of one of the ADL cell bodies and dendritic process, and “dim” if gfp expression could not be detected or could be detected weakly at the same magnification. For more precise measurements, we mounted animals on a DM5500 compound microscope, and used Volocity analytical software to quantify gfp expression levels emanating from the srh-234p::gfp reporter. Statistical analyses of srh-234 expression were performed using either the two-sample t-test, or the χ2 test of independence to test for statistically significant differences between proportions in the categories “bright” and “dim” for different genotypes (d.f. 1). A proportion of 0% was set to a default of 1.Animals carrying srh-234p::gfp reporters to aztreonam-treated E. coli OP50 (inedible food). For generating inedible food, E. coli OP50 was treated with the aztreonam antibiotic as previously described E. coli OP50 was grown in LB to log phase at 37°C with shaking. Cultures were mixed with the aztreonam antibiotic (Sigma) to a final concentration of 10 µg/ml and incubated for an additional 3 hours at 37°C with minimal shaking to prevent bacterial shearing. Bacteria was spread onto NGM plates containing 10 µg/ml aztreonam and immediately dried and used the same day, since the septum inhibitory effects of aztreonam are short lived. Expression levels of srh-234p::gfp were measured and quantified following exposure to the different food conditions as described above.Different food conditions: We exposed fed and starved animals carrying srh-234p::gfp reporters to 1 ml of 30 mg/ml Illustra Sephadex G-50 DNA Grade Fine  suspended in water spread on NGM agar plates without bacterial food as described previously srh-234p::gfp expression levels. We confirmed that the sucrose floating procedure alone does not alter srh-234p::gfp expression levels.Sephadex-beads: We exposed starved wild-type animals carrying srh-234p::gfp reporters to NGM plates with or without 3 mg/ml octopamine-hydrochloride (Sigma) in the presence of food, and 1 mg/ml serotonin creatinine sulphate (Sigma) in the absence of food. For octopamine treatment, an overnight culture of E. coli OP50 in LB was spun down and resuspended in 1/20 volume of water. About 50 µl of the concentrated OP50 was spread on the assay plates and was left until the surface of the plates became dry. After 6 hours of exposure to serotonin and octopamine, srh-234p::gfp expression levels were measured and quantified.Monogenic amines: We exposed fed or starved wild-type animals carrying srh-234p::gfp reporters to low levels of crude dauer pheromone in the presence of UV-killed E. coli food as described previously Pheromones: We exposed fed wild-type animals carrying osm-6, npr-1, ocr-2, egl-19(gf), or cDNAs of daf-2 (daf-16a (this study), unc-7, unc-9 (this study), pkc-1(gf) and tetanus toxin (TeTx) , pMG2 sre-1p::npr-1 genomic::mCherry (ADL::npr-1), pMG3 sre-1p::ocr-2 genomic::mCherry (ADL::ocr-2), pMG4 sre-1::pkc-1(gf) cDNA (ADL::pkc-1(gf)), pMG5 sre-1p::tetanus toxin cDNA (ADL::TeTx), pVDL14 sre-1p::daf-2 cDNA SL2::mCherry (ADL::daf-2), pMG14 sre-1p::daf-16a cDNA SL2::mCherry (ADL::daf-16a), pMG24 sre-1p::unc-7 cDNA SL2::mCherry (ADL::unc-7), and pMG39 flp-21::unc-7 cDNA SL2::mCherry (flp-21::unc-7), pMG40 unc-31p::unc-7 cDNA SL2::mCherry , and pMG37 sre-1p::egl-19(gf) genomic SL2::mCherry (ADL::egl-19(gf)). Expression constructs rgef-1p::daf-2 (pJH664) , ges-1p::daf-2 (pJH668) (intestine:daf-2), and ges-1p::gfp::daf-16a (pJH2973) (intestine::daf-16a) are kind gifts from Mei Zhen npr-1p::npr-1 expression construct, we fused 2.5 Kb of the npr-1 regulatory sequence and the complete npr-1 wild-type genomic sequence to gfp as previously described unc-122p::dsRed (50–100 ng/µl) or the pRF4 rol-6(su1006) co-injection markers injected at 150 ng/µl. Expression constructs were injected at 20 ng/µl. For generating the cell-specific knock down constructs, we fused the sre-1 promoter to a 1 kb exon-rich genomic fragment of either unc-7 or unc-9 in the sense and anti-sense (sas) orientation as described ADL::unc-7(sas) or ADL::unc-9(sas) were mixed and microinjected together with the pRF4 rol-6(su1006) co-injection markers injected at 90 ng/µl. All amplified products in the generated constructs were sequenced to confirm the absence of errors generated via the amplification procedure.Expression vectors were generated by amplifying either the wild-type genomic sequences of srh-234p::gfp. Following brief laser exposure, the targeted dendrite was inspected for a visual break to confirm that is was severed. Prior to laser surgery of either the ADLL or ADLR sensory dendrite, fed animals were aestheticized on 2% agar pads with 3 mM Sodium Azide, removed post-surgery for recovery and returned to NGM agar plates containing E. coli OP50 for measuring and quantifying srh-234p::gfp expression levels 2, 4, 6 and 24 hours following surgery in fed animals. Expression of srh-234 in severed (“cut”) neurons was compared to controls (“un-cut”) neurons in the same animals that were aestheticized for imaging but received no laser surgery.Laser microsurgery was carried out as previously described GCaMP3 expressed specifically in ADL neurons under either fed or starved conditions, three L4 staged sre-1p::GCaMP3 integrated transgenic animals E. coli OP50 seeded plate for 1.5 days to obtain about 100 eggs. The eggs were grown until they became young adults at 20°C. The young adult animals were washed in 1× M9 buffer and divided and placed on two NGM plates . Images of GCaMP3 expression in the ADL neurons were captured under fixed exposure time (500 ms) with a Zeiss Axioplan microscopy using a 40× objective and Zeiss AxioCam HR camera at 0 hr, 6 hr, 12 hr or 24 hr in either fed or starved conditions (n = 40–60 for each). The fluorescence intensity of GCaMP3 in ADL was quantified using the Image J software (NIH).To measure fluorescence intensity of 2+ imaging experiments in the presence of C9  pheromone were carried out as previously described CaFigure S1srh-234 expression in starved animals. A) Percentage of starved wild-type L1 larvae (red line), and adults (black line) expressing srh-234p::gfp at wild-type levels at different time-points (hr) after they were placed on-food (E. coli OP50) plates. B) Percentage of starved animals expressing srh-234p::gfp at wild-type levels at different time-points (hr) after they were placed on-food (E. coli OP50) plates (black line), off-food plates (red line), or axenic-media without food (blue line). Error bars denote the SEP.Re-feeding restores (EPS)Click here for additional data file.Figure S2unc-7 and unc-9 does not suppress the npr-1-mediated reduction of srh-234 expression. Relative expression of srh-234p::gfp in the indicated genotypes compared to wild-type animals. For strains carrying ADL::unc-7(sas) and ADL::unc-9(sas) extrachromosomal arrays, data shown is for at least two independent transgenic lines. Animals (n = 10–25) were examined at 400× magnification for each genotype. * indicates values that are different from that of wild-type animals at P<0.001 using a two-sample t-test. n.s. indicates values that are not significantly different. (sas) indicates sense-antisense. Error bars denote the SEM.ADL-specific RNAi of (EPS)Click here for additional data file.Figure S3+ transients in the ADL neurons of well-fed or starved wild-type animals. (Left) Intracellular Ca2+ dynamics in GCaMP3-expressing ADL neurons upon addition of 500 nM C9   were observed in animals at 0 hr (A) or 6 hr (B) after they were placed on-food or off-food plates, respectively. (Middle) Scatter plot shows the peak percentage changes after C9 addition. Dotted lines indicate the median. (Right) The averages of the peak percentage change after C9 addition are shown. n≥10 neurons each. n.s. indicates the values between brackets that are not significantly different. Error bars denote the SEM.C9 pheromone-induced Ca2(EPS)Click here for additional data file.Figure S4+ sensor GCaMP3 specifically expressed in ADL of fed or starved wild-type animals. Fluorescence intensity (A.U.) was measure at 0, 6, 12 and 24 hr after they were placed on-food or off-food plates. # indicates the values that are different between the brackets at P<0.05 using a two-sample t-test. n≥40 animals for each. Error bars denote the SEM.Fluorescence intensity of the genetically encoded Ca2(EPS)Click here for additional data file.Figure S5srh-234 contains a putative MEF-2 and E-box sequence motif. Expression of gfp driven by 165 bp srh-234 regulatory sequence. Shown is a predicted E-box motif (stippled box) that drives expression of ADL-expressed genes The promoter sequence of (EPS)Click here for additional data file.Table S1a Expression of gfp gene fusions carried on extrachromosomal arrays, and arrays stably integrated into the genome, gmIs12[srb-6p::gfp] and oyIs56[srh-234p::gfp] were examined in adult animals in fed and starved conditions. b “+”, regulated, or “−” not regulated by fed and starved conditions. n = 150–250.Summary of ADL-expressed chemoreceptor genes examined as a function of feeding state. (DOCX)Click here for additional data file.Table S2srh-234p::gfp expression levels as a function of feeding state in different conditions and mutants. a Adult animals grown at 20°C in the presence of OP50 food were examined in all cases unless indicated otherwise. All strains contain stably integrated copies of oyIs56[srh-234p::gfp] fusion genes with the exception of bas-1 and unc-36 which contain integrated copies of oyIs57[srh-234p::gfp]. b Expression of oyIs56[srh-234p::gfp] was examined at 150× magnification as defined in c Expression of oyIs57[srh-234p::gfp] was examined at 400× magnification as defined in d Indicates values that are different from that of wild-type animals either in fed or starved conditions using a χ2 test of independence. e Compared to wild-type oyIs56[srh-234p::gfp when fed. f Compared to wild-type oyIs57[srh-234p::gfp] when fed. g Compared to wild-type oyIs56[srh-234p::gfp when starved. h Compared to kin-29(oy38) under same conditions. i Compared to unc-9(e101) under same conditions. j Compared to unc-7(e139) under same conditions. n = 150–350.Analysis of (DOCX)Click here for additional data file.


Self-rated health (SRH) has been demonstrated as a valid and appropriate predictor of incident mortality and chronic morbidity. Associations between lifestyle, chronic diseases, and SRH have been reported by various population studies but few have included data from developing countries. The aim of this study was to determine the prevalence of poor SRH in Malaysia and its association with lifestyle factors and chronic diseases among Malaysian adults.This study was based on 18,184 adults aged 18 and above who participated in the 2011 National Health and Morbidity Survey (NHMS). The NHMS was a cross-sectional survey (two-stage stratified sample) designed to collect health information on a nationally representative sample of the Malaysian adult population. Data were obtained via face-to-face interviews using validated questionnaires. Two categories were used to measure SRH: “good” (very good and good) and “poor” . The association of lifestyle factors and chronic diseases with poor SRH was examined using univariate and multivariate logistic regression.Approximately one-fifth of the Malaysian adult population (20.1 %) rated their health as poor (men: 18.4 % and women: 21.7 %). Prevalence increases with age from 16.2 % (aged 18–29) to 32.0 % (aged ≥60). In the multivariate logistic regression analysis, lifestyle factors associated with poor SRH included: underweight , physical inactivity , former smoker , former drinker , and current drinker . Chronic diseases associated with poor SRH included: asthma , arthritis , hypertension , hypercholesterolemia , and heart disease .This study indicates that several unhealthy lifestyle behaviours and chronic diseases are significantly associated with poor SRH among Malaysian adults. Effective public health strategies are needed to promote healthy lifestyles, and disease prevention interventions should be enhanced at the community level to improve overall health.The online version of this article (doi:10.1186/s12889-015-2080-z) contains supplementary material, which is available to authorized users. A variety of lifestyle factors and health-related behaviours such as smoking, alcohol consumption, physical activity, and dietary habits can affect a person’s health. An unhealthy lifestyle often results in a higher risk of chronic diseases such as heart disease, hypertension, hypercholesterolemia, and diabetes , 2. PrevUnderstanding the correlates of SRH can help public health professionals prioritise health-promotion and disease-prevention interventions. To date, studies that have investigated the determinants of SRH have focused primarily on Western countries, although we have found a amall number of studies that examine the topic from the perspective of developing countries in the Asian regions. Previous studies from Singapore , PakistaIn Malaysia, few studies have examined the topic of SRH. A recent study by Cheah  reportedThe NHMS 2011 was a nationwide, cross-sectional, population-based survey that used a two-stage stratified sampling design to select a nationally representative sample. The Malaysian states constituted the primary strata, with further stratification by urban–rural residence. The primary sampling units were enumeration blocks (EBs) provided by the Malaysian Department of Statistics from the 2010 census. Living quarters (LQs) represent the secondary sampling units. A total of 794 EBs  were systematically selected from all the Malaysian EBs  via a probability-proportional-to-size sampling technique. Subsequently, 12 LQs were randomly selected from the approximately 80 to 120 LQs in each EB. Finally, all households and eligible household members in the selected LQs were included in the sample. The survey methodology is described in greater detail by Fadhli et al. .Data collection for the NHMS 2011 was carried out from April to July 2011. Information on SRH, socio-demographic characteristics, lifestyle, and self-reported medically-diagnosed chronic diseases was obtained by trained interviewers via face-to-face interviews. The interviewers were extensively trained and conducted mock interviews prior to actual data collection. The interviewing process was closely monitored by field supervisors, who were health care personnel. A total of 18,231 eligible adults aged 18 years and older, living in the sampled households, were interviewed. Of these, 18,184 responded to the SRH questionnaire section of the NHMS 2011, giving a response rate of 99.7 %. To ensure a high response rate, unoccupied households were revisited up to three times. All of the eligible respondents provided written consent to be interviewed for the survey. The study protocol was approved by the Malaysian Medical and Research Ethics Committee (MREC), Ministry of Health Malaysia (NMRR-10-757-6837).Self-rated health was measured based on the question: “In general, how would you rate your health today?” There were five response categories: “very good”, “good”, “moderate”, “not good” and “very bad”. For purposes of analysis, the response categories were combined into two groups: “Good”, which included “very good” and “good”, and “Poor” which included “moderate”, “not good” and “very bad”. “Good” SRH served as the reference category in the logistic regression analyses.Socio-demographic variables included sex, age, ethnicity, residential area , marital status , educational level , current work status (working/not working) and monthly household income. Age was categorised into five groups: 18–29 years, 30–39 years, 40–49 years, 50–59 years, 60 years and above. Ethnicity was categorised into five groups: Malays, Chinese, Indians, other Bumiputeras , and “Others” . Monthly household income was grouped into four levels: less than MYR1000, MYR1000–MYR1999, MYR2000–MYR2999, MYR3000 and above [1 Malaysian Ringgit (MYR) ≈ 0.29 US Dollar (USD)].2). According to the World Health Organization (WHO 2000) guidelines [Lifestyle variables included body mass index (BMI), consumption of fruits and vegetables, physical activity, smoking, and alcohol consumption. Respondents’ weight and height were measured using the Tanita 318 digital weighing scale  and the Seca 206 Bodymeter measuring tape , to the nearest 0.1 kg and 0.1 cm, respectively. BMI was calculated as weight divided by height in meters squared , and a half cup of cooked chopped eggplant or tomato or carrot or leafy green vegetables. According to the WHO STEPS criteria [Consumption of fruits and vegetables was measured based on four questions: i) “In a typical week, how many days do you eat fruits?” ii) “Usually on the day you eat fruits, how many servings of fruits did you eat in a day?” iii) “In a typical week, how many days do you eat vegetables”, and iv) “Usually on the day you eat vegetables, how many servings of vegetables did you eat in a day?” Photographs of foods were used to help respondents recall the serving sizes of the fruits and vegetables they had consumed. The photographs showed single servings of commonly consumed fruits and vegetables such as the following: one medium-sized apple, one medium-sized banana, one slice of papaya, one slice of watermelon, eight medium-sized grapes, one cup of chopped raw leafy green vegetables or criteria , responscriteria , consumpThe level of physical activity was determined based on the short form of the International Physical Activity Questionnaire (IPAQ), the reliability and validity of which have been established . The oriSmoking status was divided into three categories: “never smoker” (never smoked in lifetime), “former smoker” , and “current smoker” . Alcohol consumption status was divided into three categories: “non-drinker” , “former drinker”  and “current drinker” .For chronic disease status, presence or absence of chronic diseases was based on self-reported information on chronic conditions diagnosed by a doctor or health care professional. Respondents were asked: “Have you ever been told by a doctor or Medical Assistant that you have __________?” followed by a list of chronic diseases included asthma, arthritis, diabetes mellitus, hypertension, hypercholesterolemia, and heart disease, to which the answer is “yes” or “no”.p = <0.25 in the univariate analysis were retained in the multivariate logistic regression model [p = <0.05 was considered to be statistically significant. The statistical analyses were performed using SPSS for Windows (version 20.0) statistical software . All analyses were conducted using a complex sampling design to ensure that sample weights and study design were taken into consideration.Frequency distributions of socio-demographic, lifestyle, and chronic disease variables were analysed by self-rated health status. Descriptive statistics were used to calculate the overall prevalence of poor SRH among Malaysian adults and the prevalence of poor SRH by socio-demographic, lifestyle and chronic disease variables. Associations of the measured factors with poor SRH were tested using chi-square tests. Univariate and multivariate logistic regression analyses were used to investigate the association between poor SRH, socio-demographic, lifestyle, and chronic disease variables, with “Good” SRH as the reference category. The results are presented as crude and adjusted odds ratios (ORs) with 95 % confidence intervals (CIs). Variables that were associated with poor SRH at n = 18,184). The sample included more women (53.4 %) than men (46.6 %). Among the 18,184 respondents, the distribution of self-rated health was as follow: “very good” 3,468 (19.4 %); “good” 11,031 (60.4 %); “moderate” 3,349 (18.2 %); “not good” 316 (1.8 %); and “very bad” 20 (0.1 %)  of participants reporting good SRH, and 20.1 % (n = 3685) reporting poor SRH. A higher proportion of women (57.5 %) than men (42.5 %) reported poor SRH. Similarly, a higher proportion of older adults (aged 60 years and above) reported poor SRH than younger adults (Table Table p = <0.001). Regarding lifestyle variables, individuals who were underweight, physically inactive, and former smokers had higher prevalence of poor SRH. Survey participants who had chronic diseases such as asthma, arthritis, diabetes mellitus, hypertension, hypercholesterolemia, and heart disease were found to have a higher prevalence of poor SRH, compared with their counterparts who did not have these diseases.Table p = <0.25 except for “Others” ethnic group, monthly household income, overweight BMI group, and current drinkers. In the final multivariate logistic regression model, we included all variables that were statistically significant in univariate analysis (p = <0.25) except for the “monthly household income” variable. After adjusting for all selected factors in the multivariate analysis, the odds of poor SRH were significantly higher for women compared with men, among older people (aged ≥60 years) compared with younger people (aged 18–29 years), and among people living in rural areas compared with urban areas. Malays, other Bumiputeras and “Others” ethnic groups had higher odds of poor SRH compared with the Chinese. Respondents who were underweight (p = <0.05), physically inactive (p = <0.001), former smokers (p = <0.01), current drinkers (p = <0.01), and former drinkers (p = <0.05) reported significantly higher odds of poor SRH compared with their respective counterparts. Self-reported medically-diagnosed chronic diseases that were significantly associated with poor SRH were asthma, arthritis, hypertension, hypercholesterolemia, and heart disease (p = <0.001 for each). Individuals with diabetes mellitus were not significantly associated with poor SRH in our study.Table The prevalence of poor SRH in Malaysia (20.1 %) is similar to that reported in neighbouring Singapore (23.2 %) , which mThe present study found a significant association between specific lifestyle factors and SRH. Individuals who are underweight, physically inactive, former smokers, and current drinkers were significantly associated with poor SRH. Previous studies have reported that obesity and underweight are significantly associated with poor SRH , 32, 33;total physical activity including different forms of physical activity . A recent study of physical activity using the same national survey data (NHMS 2011) reported that about two in five Malaysian adults were physically inactive, particularly women and the elderly [Previous studies have consistently shown that physical activity and exercise have positive effects on SRH –43. Our  elderly . In the In contrast to findings from the 2007 Behavioural Risk Factor Surveillance System (BRFSS) survey conducted in the United States  and the In our study, no statistically significant difference was found in poor SRH for current smokers compared with never smokers. This finding is contrary to previous studies , 13, 48,In line with previous studies , 53, 54 There is substantial evidence supporting the negative impact of chronic diseases on SRH , 34, 58.While this study emphasised the associations between lifestyle, chronic diseases, and SRH, several socio-demographic factors such as age, sex, ethnicity, and residence  were shown to have statistically significant influences on SRH in Malaysia. Older people were more likely to report poor SRH than younger people, which has been shown in other studies , 13, 35.The major strengths of this study are the representativeness and large sample size. The results can be extrapolated accurately and generalised to the Malaysian adult population. Furthermore, the participants were interviewed face-to-face by trained interviewers and the response rate was higher than reported for other studies. However, the study has several limitations that should be noted. The main limitation is the cross-sectional design of the study, which does not allow for determination of causal effect relationships. Another limitation is the possibility of recall bias associated with self-reported data on smoking, alcohol consumption, physical activity, and consumption of fruits and vegetables. Third, the “social desirability” of certain answer categories is a concern in face-to-face interviews. To project a favourable image, respondents may give answers that are deemed more “socially accepted” than their “true” answers. Finally, several other factors found to be associated with SRH in previous studies  were not investigated in this study.The prevalence of poor SRH among Malaysian adults is 20.1 %, which increases with age. Several negative lifestyle behaviours and chronic diseases were significantly associated with poor SRH among Malaysian adults. Additional public health strategies are needed to promote healthy lifestyles and disease prevention interventions should be implemented to improve personal and community health. More preventive measures and health care services targeting chronic diseases are urgently needed, particularly in rural communities. Further research that takes into account the influences of other potential confounders such as psychososial and environmental factors is needed to fully understand the correlates and determinants of SRH.


Sabellaria alveolata  is an important ecosystem engineer building reef structures which add to the topographic complexity in colonized areas. In Europe, the most extensive reef formation is located in the Bay of Mont-Saint-Michel (France). Since 2006, Sabellaria bio-constructions have developed on hard substrates along the west coast of the Cotentin Peninsula between Champeaux and Saint-Germain-sur-Ay on the northern part of the Bay of Mont-Saint-Michel. In this sector, two distinct types of bio-construction can be distinguished: platforms and reefs. The aim of this study is to analyse the patterns of the associated macrofauna on these platforms and reefs, as well as outside, and test for a correlation between the presence of Sabellaria bio-constructions and the richness of the benthic macrofauna. Univariate analyses are used to compare the macrofauna on four sites . The results show a higher taxonomic richness on the platform-type than on the reef-type structures, and also a higher taxonomic richness outside the bio-construction areas. This suggests that, on the examined sites, the presence of S. alveolata bio-constructions does not contribute to higher levels of benthic macrofaunal richness on hard substrates. Temporary bio-constructions along this coast exhibit reefs of interest at some sites as well as in very small zones which merit special attention.The polychaete  Sabellaria alveolata Reefs’ also benefits from a Biodiversity Action Plan in the UK (http://www.jncc.gov.uk/page-5155).Biogenic structures built by ecosystem engineers such as corals, molluscs and polychaetes provide favourable habitats for other benthic invertebrates and vertebrates; moreover, these structures can play an important role as nurseries for certain species of commercial interest and also represent an essential source of food for birds and fishes , and is also known in Europe, where it ranges from the Bristol Channel , and observed that TR is higher in degraded reef zones. The S. alveolata bio-constructions of the Sainte-Anne reef in the southern Bay of Mont-Saint-Michel have a high TR compared with the very low TR observed in the surrounding intertidal soft-bottom Macoma balthica community  favourable for marine macrofauna living in crevices are created during the phases of construction and destruction of a reef, i.e. growth, degradation, death and colonization Gruet , 1982. G. Anadon  describe. Anadon  describeS. alveolata living on the western coast of Cotentin are unlikely to participate in renewal of the populations. Therefore, along the west coast of Cotentin north of Champeaux, the temporary Sabellaria structures (established for probably less than 10 years) are associated with a southerly source of larvae coming from populations at Saint-Anne and Champeaux in the Bay of Mont-Saint-Michel. In certain years, Sabellaria larvae can reach the studied sites owing to south-westerly winds plus the lower velocity of nearshore tidal currents  of the mid-intertidal zone at four sites  located on the west coast of Cotentin, taking into account the northward expansion of the reefs in the years 2000.The aim of this study is to assess the macrofauna richness associated with three types of habitat  and Saint-Germain-sur-Ay (in the north), in a sector where the first recent 6 Delhay  Fig. 1)Sabellari6 Delhay , four saSabellaria reef formations were observed in the early 1960 s at a location north of Blainville-sur-Mer , which is also mainly parallel to the prevailing tidal current (i.e. NW–SW sector) as in the Bay of Mont-Saint-Michel . Cores were burrowed into the reefs or the platforms as deeply as possible (mainly between 0.10 and 0.30 m depending on the height of the bio-construction). For each site, sixteen cores were sampled; eight from a platform zone and eight from a reef zone, making up a total of 64 cores. The collected sediment was preserved in 10 % formalin.Sampling was carried out on the four sites between February and April 2014  and 17 April (SGSA), and 28 April (CHAM), by scraping to 0.05 m depth over a quadrat surface-area of 0.1 m2. For the three sites, ten quadrats representing a total area of 1 m2 were sampled from a reef zone and from outside the reef structure on hard substrate. Thus, a total of 60 quadrats were collected from the three sampling sites, the samples being preserved, sieved on 0.5-mm mesh and identified in the same way as for the core samples.A second sampling strategy was performed to compare the TR of areas with http://www.marinespecies.org; accessed on 15 December 2015] and Hill’s diversity numbers : ers Hill  as recomers Hill : N1 = exers Hill ; and N2 ers Hill .S. alveolata on reef and platform structures (core sampling);the spatial and temporal abundance patterns of the spatial and temporal patterns of associated macrofauna  on reef and platform structures (core sampling);the TR inside and outside reef structures .A Shapiro–Wilk normality test and a Bartlett’s test for homogeneity of variance are performed prior to each ANOVA with the R software to validate the assumptions of ANOVA. Then, ANOVAs are performed to assess:A Tukey Honestly Significant Difference test is used to determine differences between the sites, types of structure (reef or platform) and sampling dates., k-dominance curves are plotted for each substratum type and site and the associated species, excluding S. alveolata, are ranked in order of their dominance in terms of abundance.To compare the faunal TR in the three communities, i.e. from the platform, from the reef or outside the reef areaSabellaria alveolata is the most abundant species regardless of the site location or substrate type, apart from LING, where it does not appear among the dominant species and shows lower abundance on the degraded reef , but there is a significant difference in the densities of S. alveolata between different sites . The highest densities of S. alveolata are observed in the CHAM population (Tukey test) , when the maximum occurs, and summer (August)  (Tukey test).As observed with the spatial pattern, me Table ”. Howeveons Fig. ; a signiSabellaria alveolata does not differ significantly between platform and reef structures   Fig. .S. alveolata, it is noteworthy that their abundances are higher on the reefs than on the platforms.From the 64 cores, a total of 6731 individuals are identified (67 to species level) belonging to 80 taxa. The richest group is the polychaetes with 36 taxa. Although other species show much lower abundances than The taxonomic richness is significantly different between the reefs and platforms and also between different sites Table . MoreoveSite location is a significant factor influencing abundance, and the interactions between the two different structures and location are significantly different Table . The SGAS. alveolata, two groups dominate at these three sites . Arthropods are represented by 18 taxa, including two dominant species: the decapod Porcellana platycheles and the isopods Sphaeroma spp. Mollusca are represented by 18 taxa, with the gastropod Gibbula umbilicalis as dominant species  . The composition of the macrofauna on the platform structures appears more erratic between sites. Moreover, the dominance of species on platforms at SGSA and CHAM is more equally distributed than at the two other sites, with dominant species showing lower abundances.The SGSA site shows the highest TR, both on the platforms and on the reefs Fig. . Converse) Table . The LINSampling period and structure type are significant independent factors influencing the taxonomic richness Table , with a S. alveolata, are never the same. K-dominance curves reveal additional differences in species abundance between platform and reef  have a significant effect on the TR and total abundance Table . Becauseons Fig. a, with aons Fig. b, c, whium Table ”. The moK-dominance curves for species abundance inside and outside reefs at each site provide additional information on the structure of the communities  based on samples collected from four sites  found on these Sabellaria bio-constructions is of the same order of magnitude as that observed by Anadon , Nereidae (Perenereis spp.) and Serpulidae (Spirobranchus triqueter). On the Portuguese coast, the total number of taxa is 137, which appears higher than on the Cotentin coast. Polychaetes are the most abundant group on the Cotentin reefs, while the crustaceans dominate in the case of Ria de Vigo. Porras et al. . On the other hand, La Porta and Nicoletti , where the most abundant associated families are the Nereidae, the Phyllodocidae and the Serpulidae. Apart from the polychaetes, the most diversified zoological groups identified in the Sabellaria reefs of the western coast of Cotentin are the molluscs, including bivalves and gastropods, and the arthropods, including crustaceans  vagile epifauna such as the gastropod Gibbula spp. and (3) infauna species such as Venerupis corrugata, the cockle Cerastoderma edule and the Baltic tellin Macoma balthica (but only at the Champeaux site). The arthropods are dominated by the decapods Porcellana platycheles and the isopods Sphaeroma spp. and Gnathia dentata. The sipunculid Golfingia vulgaris is present in abundance at the four sampled sites. The fauna collected on the Sabellaria bio-constructions is in common with that of the surrounding hard-bottom and soft bottom substrates, especially the muddy sand and gravel sediments which favour the presence of infauna.Accounting for 36 taxa, polychaetes dominate the taxonomic richness on y Anadon  on the Ss et al.  determinicoletti  recordedSabellaria on the coast of France .Some taxonomic particularities have been highlighted by Gruet , 1982, wCorophium volutator, which is a typical species of intertidal estuarine mudflats. Finally, this author (op. cit.) stressed that TR was higher during the colonization and destruction phases than during the flourishing reef phase. Subsequently, Gruet  the fauna recorded on the reef is in common with the surrounding hard and soft-bottom substrates; (2) the TR decreases with increasing abundance of the engineer species Sabellaria alveolata, which shows very dense populations >20,000–60,000 ind m−2.Dubois et al.  describeUlva spp.) and the oyster Crassostrea gigas) on the TR of the Saint-Anne reef. These authors showed that the presence of the oyster had a significant positive effect on TR compared with areas lacking C. gigas and with algal-reef type structures.More recently, Dubois et al.  studied Sabellaria platforms at Champeaux. This author (op.cit.) stressed that the fauna was poor in terms of TR (26 taxa identified) and abundance, probably in relation to its location on the upper part of the mid-littoral zone with a long period of exposure of the Sabellaria reef. We identify 38 taxa in our samples from the Champeaux site, but our results are based on a more extensive sampling campaign.Moreover, Gruet  has provS. alveolata has developed as a flourishing reef monopolizing the area and volume by imposing a strong competition with other species. In this way, S. alveolata has occupied all the available space, where its density reaches and can exceed several thousands of individuals per square metre , but is increasing at CHAM (from 0.31 to 0.37 km2). There has been a marked decrease in the area covered by bio-constructions at the two other sites . At these two latter sites, there has also been an increase in the area covered by degraded reef. Destruction has been observed at BLSM since 2011  (Polychaeta: Sabellariidae) reefs has led to their classification as a remarkable natural habitat . Thus, Sabellaria alveolata reefs are characterized by their great importance for the conservation of natural heritage and marine biodiversity in Europe. The French national inventory of natural heritage (INPN) has recognized Sabellaria alveolata reefs . Consequently, the Champeaux and the Sainte-Anne reefs are included in this Marine Protected Area. Monitoring studies have been carried out on these reefs for two decades , which will involve the mapping and management of natural habitats including S. alveolata reefs. In this process, the presence of temporary S. alveolata reefs merits special attention.The Bay of Mont-Saint-Michel is classified as a Special Zone of Conservation and is a RAMSAR zone (Sabellaria bio-constructions (platform and reef types) on hard substrates along the west coast of the Cotentin Peninsula. Spatial analysis allows us to distinguish three groups among the studied sites. Champeaux is different from the other sites because the reef has developed on a soft substrate and the platform is located in the upper part of the mid-littoral zone. Lingreville is characterized by the presence of dead reef, which clearly reflects a degraded phase. The sites of Blainville-sur-Mer and Saint-Germain-sur-Ay are located in the north of the study area, and show similar patterns. The different study sites are found to display highly variable states ranging from a flourishing reef at Champeaux to a completely degraded reef at Lingreville. Some reef destruction is also observed at Blainville-sur-Mer. These results highlight a difference in TR between platforms and reefs, with platforms showing higher TR. Such variability supports the reef effect proposed in previous studies, i.e. a decrease in the TR of other resident macrofauna species in zones more intensely colonized by the honeycomb worm Sabellaria alveolata. Therefore, well-developed reefs in a flourishing state do not represent the most diversified habitats in terms of TR. This is because S. alveolata occupies all the space, inhibiting the creation of crevices, fissures and pits which offer the best refuges for TR in mosaic habitats developed during the construction and destruction phases of a reef. The project for the creation of a Natural Marine Park should be an opportunity to recognize and manage the natural heritage interest of such temporary reefs on hard bottom habitats along the western coast of Cotentin.The objective of this study is to estimate the macrofaunal TR associated with


RNA-RNA interaction plays an important role in the regulation of gene expression and cell development. In this process, an RNA molecule prohibits the translation of another RNA molecule by establishing stable interactions with it. In the RNA-RNA interaction prediction problem, two RNA sequences are given as inputs and the goal is to find the optimal secondary structure of two RNAs and between them. Some different algorithms have been proposed to predict RNA-RNA interaction structure. However, most of them suffer from high computational time.In this paper, we introduce a novel genetic algorithm called GRNAs to predict the RNA-RNA interaction. The proposed algorithm is performed on some standard datasets with appropriate accuracy and lower time complexity in comparison to the other state-of-the-art algorithms. In the proposed algorithm, each individual is a secondary structure of two interacting RNAs. The minimum free energy is considered as a fitness function for each individual. In each generation, the algorithm is converged to find the optimal secondary structure (minimum free energy structure) of two interacting RNAs by using crossover and mutation operations.This algorithm is properly employed for joint secondary structure prediction. The results achieved on a set of known interacting RNA pairs are compared with the other related algorithms and the effectiveness and validity of the proposed algorithm have been demonstrated. It has been shown that time complexity of the algorithm in each iteration is as efficient as the other approaches. Major successes have been achieved in the treatment of some cancers, including colon, breast and pancreatic by suppressing the gene expression involved in the development of these diseases using RNA-RNA interaction. The interaction between two RNAs is known as the newest and the most efficient method for gene silencing. It has been shown that the small interfering RNAs (siRNAs) can be used for silencing their target mRNAs . FurtherViennaRNA package ', respectively.A secondary structure of two interacting RNAs, R1 = CGGUUUGAGGUCCG and R2 = ACUACCGAAAAGUU be two RNA sequences. The SSIR of the two RNAs is shown as follows;Example. Let CGG and the reverse CCG (GCC). There are two hybrid regions between the sequences R1 and R2. The first one is produced by binding between UUU and the reverse AAA (AAA). The second one is generated by binding between GGU and the reverse ACC (CCA).In this example, each RNA has one stem. In the left hand RNA, one stem is found by the production of bonding between Genetic algorithm is an optimization method based on evolutionary biology that is widely used to solve search and optimization problems -22. In tR ' = r ' 1r ' 2 … r ' n (|R ' | = n) and R " = r " 1r " 2 … r " m (|R " | = m) are given as inputs. The two RNAs R ' and R " are converted to the sequence R = r1r2 … rl as follows;In the proposed algorithm, two RNA sequences N is an arbitrary character to distinguish between two sequences and l = m + n + 1.where R and reverse R,as follows;A dot matrix ri and rl-j+1  are the i-th and l-j+1-th nucleotides in the sequence R = r1r2 … rl, respectively. Each right-skewed consecutive value of 1’s which is parallel to the main diagonal in the dot matrix is selected as a sub-diagonal. Each sub-diagonal shows a possible stem in each RNA or hybrid region between two RNAs. Set DR shows all sub-diagonals in the dot matrix as follows;where i and j indicate the start position of the row and the column of a sub-diagonal with t+1 consecutive 1’s, respectively. Hence, each <i,j,t> is a set of consecutive base pairs as follows;where G-U pairs on our dataset approximated 14%. So, G-U pairs are removed from the sub-diagonals including more than 14% G-U pairs. For each d1 = < i1, j1, t1 > ∈ DR and d2 = < i2, j2, t2 > ∈ DR, d1 ∝ d2 is defined as;According to the prior knowledge, we know that Watson-Crick base pairs occur more than Wobble in RNA structures. In this regard, we compute the percent of d1 ∝ d2 represents all base pairs in d1 overlapping with d2.where P, a |DR| -tuple is randomly made as;For each individual xk (1 ≤ k ≤ |DR|) indicates the k-th sub-diagonal in I. In other words, individual P contains those sub-diagonals that their related x in I is equal to 1. Here, xk = 1 means sub-diagonal dk ∈ P, while xk = 0 points to dk ∉ P. Then, the individual P is constructed as follows;where Ck  is obtained as follows;where the set Ck ∈ P is a set of base pairs in dk without any common base pairs in the previous sub-diagonals, dt(1 ≤ t < k), of individual P. Finally, Ck is modified by removing the lonely base pairs from it as follows;Here, Notice that a set of the produced individuals creates an initial population.P, let S and H represent two RNAs secondary structures and binding sites between the two RNAs, respectively. Therefore, the fitness function is defined as follows;For each individual C ∈ {S, H}, MFE(C) denotes the minimum free energy of structure C. We apply RNAeval.exe [where for eval.exe  to compuCrossover operation is performed between the individuals with the rate of 0.9. The good and mediocre individuals are transferred to the next population. The remaining individuals are consecutively selected for crossover operation.P1 and P2 are selected as parents. For each individual Pi, 1 ≤ i ≤ 2, a |DR| -tuple is defined as follows:Let k, 1 ≤ k ≤ |DR|, is selected and I1 and I2 are crossed. Then, I ' 1 and I ' 2 are formed as follows:In this procedure, a random position P ' 1 and P ' 2 are generated from I ' 1 and I ' 2 similar to the described method in the initial population (refers to the Equation 1), respectively.In the following, two new individuals P is an individual selected for mutation. For the individual P, a |DR| -tuple is obtained by:Mutation operation is done with the rate of 0.1 on a few randomly selected individuals in each generation. Assume that xj, where xj = 1 is randomly selected and replaced to 0. Then, another xk (xk= 0) is replaced to 1. The new individual, P, is obtained from the changed I based on the proposed method in initial population (refers to the Equation 1). Finally, if Ck ∉ P , the other xi (xi = 0) is selected to replace with 1. This process continues until Ck ∈ P or the defined number of generations is reached. When mutation is performed on a number of individuals, they will be increasingly sorted based on their fitness values.An item The GRNAs algorithm terminates when the best individual in definite generations will not be changed or the defined number of generations be reached. After the termination of the algorithm, one of the best individuals is selected as the best folding of two RNAs and the best interaction between the two RNAs.O(l2) where l exposes sum of the length of the two RNAs. Let h and |P| be the number of individuals and the length of an individual P. The time complexity of creating the initial population is O(h. |P| 2). We set h = 40 and |P| = max{|R ' |, |R " |}, so h can be ignored. Sorting individuals based on their fitness values requires O(|P|. h. log h). Crossover and mutation operations take O(|P| 2) and O(|P|), respectively. Thus, the time complexity in each iteration in the proposed algorithm is O(h. |P|(|P| + log h)). The maximum number of iteration is at most I = 20. Therefore, the time complexity of the algorithm is O(I. h. |P|(|P| + log h) + l2) that is simplified with O(l2 + |P|).We have obtained the time and space complexity of GRNAs in each iteration. Making the dot matrix needs the complexity of h individuals of length |P| we need O(h. |P|) space complexity. Furthermore, the population in the algorithm uses both dot matrix and an array of sub-diagonals. Hence, the storage complexity of these two types is O(l2), where l denotes sum of the length of the two RNAs. Thus the total space complexity of GRNAs is O(h|P| + l2) which is simplified with O(l2 + |P|).On the other hand, for storing the 54-RepZ in the Escherichia coli bacteria [The GRNAs has been performed on a machine with two-Core Intel(R) Duo processor T6670 2.20 GHz and 4 GB RAM to predict the interaction structure between two RNAs. The proposed genetic algorithm is performed on two well-known datasets of RNA-RNA interactions. The first set contains: R1inv-R2inv, Tar-Tar*, DIS-DIS, CopA-CopT and IncRNAbacteria . The joiAlso, this algorithm is carried on the second set of datasets with their binding sites including some RNA pairs called: DsrA-Rpos, GcvB-argT, GcvB-dppA, GcvB-gltI, GcvB-livK, GcvB-livJ, GcvB-oppA, GcvB-STM4351, IstR-tisAB, MicA-ompA, MicA-lamB, MicC-ompC, MicF-ompF, OxyS-fhlA, RyhB-sdhD, RyhB-sodB, SgrS-ptsG and Spot42-galK . This daF) and Matthews Correlation Coefficient (MCC) [Sn) and positive predictive value (PPV). Assume that the number of correctly predicted base pairs, the number of false predicted base pairs and the number of unpredicted base pairs are indicated by TP, FP and FN, respectively. So, Sn, PPV, F, and MCC are defined as follows:To evaluate the prediction accuracy of the GRNAs, F-measure (nt (MCC)  are calcMCC evaluates the joint structure, i.e. both the binding sites between the two RNAs and the secondary structure of each single RNA. In two pairs MicA-ompA and OxyS-fhlA, PETcofold has the best MCC value and in other two pairs RyhB-uof-fur and RyhB-sodB, GRNAs has the highest MCC value.Table We also compared GRNAs with four state-of-the-art methods: inRNAs, IntaRNA, RNAup and RactIP. Table Our genetic algorithm randomly selects the sub-diagonals to make individuals. Therefore different individuals with variety sub-diagonals are constructed. Due to the nature of proposed genetic approach, some of the RNA-RNA interactions can be predicted more accurate than the other algorithms. For example the accuracy rate of Tar-Tar* is obtained 100%, while maximum accuracy of the other approaches is 90.9%.O(l2 + |P|), where l and |P| indicate sum of the length of the two RNAs and the length of an individual, respectively. Also, Space complexity of the proposed method is O(l2 + |P|).We compare the computational time complexity of GRNAs and state-of-the-are methods. The time and space complexity of several algorithms (TIRNA , App ap, 2 and 3In this paper, a new genetic algorithm was introduced for solving RNA-RNA interaction prediction problem. In this algorithm, all possible stems in each RNA and hybrid regions between two RNAs are extracted from a dot matrix showing all possible base pairs. Initial population is formed based on some stems and hybrid regions of the dot matrix. Minimum free energy is considered as a fitness function. Crossover operation is done between some consecutive individuals in the population. Mutation is taken on a few randomly selected individuals. Population generation continues until the minimum free energy of the best individual becomes minimal enough. Finally, one of the best individuals is selected to form RNA-RNA interaction structure. The proposed algorithm was tested on several RNA-RNA interaction datasets. The experimental results indicate a high accuracy of GRNAs. Furthermore, time and space complexity of GRNAs is as efficient as the other related studies.http://mostafa.ut.ac.ir/grnas.The program of GRNAs is available at The authors declare that they have no competing interests.SM participated in the design of the algorithm, performed the experiments and drafted the manuscript. FZM contributed in the design of the algorithm, performed the experiments, drafted the manuscript and supervised the project. NMC contributed in the design of the algorithm and drafted the manuscript. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.