Mesorhizobium. Here, we predicted putative sRNAs in the intergenic regions (IGRs) of M. huakuii 7653R by genome-wide comparisons with four related Mesorhizobial strains. The expression and transcribed regions of candidate sRNAs were analyzed using a set of high-throughput RNA deep sequencing data. In all, 39 candidate sRNAs were found, with 5 located in the symbiotic megaplasmids and 34 in the chromosome of M. huakuii 7653R. Of these, 24 were annotated as functional sRNAs in the Rfam database and 15 were recognized as putative novel sRNAs. The expression of nine selected sRNAs was confirmed by Northern blotting, and most of the nine selected sRNAs were highly expressed in 28 dpi nodules and under symbiosis-mimicking conditions. For those putative novel sRNAs, functional categorizations of their target genes were performed by analyzing the enriched GO terms. In addition, MH_s15 was shown to be an abundant and conserved sRNA.In bacteria, small non-coding RNAs (sRNAs) are critical regulators of cellular adaptation to changes in metabolism, physiology, or the external environment. In the last decade, more than 2000 of sRNA families have been reported in the Rfam database and have been shown to exert various regulatory functions in bacterial transcription and translation. However, little is known about sRNAs and their functions in  Small non-coding RNAs (sRNAs) in bacteria are usually 50–500 nucleotides (nts) in length and fulfill their riboregulations together with Hfq (an RNA chaperone) and ribonucleases (RNases) or RNase-containing complexes to mediate the expression and stability of target mRNAs , which can be scanned by using software such as QRNA, RNALfold and RNAz , and two megaplasmids, pMhu7653Ra  and pMhu7653Rb , with 7,205 protein-coding genes  symbiotic nodules and free-living cells of 7653R  that were treated with TAP. The depths of RNA-seq were shown in Tables A comparison of the analysis between the predicted sRNAs and RNA-seq products showed that, apart from 5 riboswitches, 19 functional annotated sRNAs and 15 of 16 putative novel sRNAs were detected in the sequencing dataset, and only one was not expressed in plant nodules or in free-living cultured cells Table . Of the 70-dependent transcription in alpha-Proteobacteria. In our sequencing results, 6S was strongly expressed in exponential-phase cells (OD600 = 0.8), and abundant transcripts were also detected in mature (28 dpi) and senescent (50 dpi) nodules. The expression of tmRNA (MH_S19), which rescues stalled ribosomes and tags incomplete polypeptides for degradation, was lower in both free-living and symbiotic conditions, and down-regulated in mature and senescent nodules compared with exponential-phase cells (>2-fold).There are four sRNAs, including 6S RNA, 4.5S RNA, bacterial RNase P class A, and tmRNA, that are highly conserved and regarded as housekeeping sRNAs in bacteria. In this study, these four housekeeping sRNAs were detected by both bioinformatic analysis and RNA-seq, and their expression profiles and expression levels are respectively shown in Figure M. huakuii 7653R possesses two symbiotic megaplasmids required for symbiosis and nitrogen fixation. In our results, the levels of ctRNA (MH_s1 in pMhu7653Ra and MH_s4 in pMhu7653Rb) were very high in free-living cells, approximately 3- to 4-fold higher in 28 dpi and 50 dpi nodules, respectively  is a highly conserved small RNA located in repB–repC intergenic regions. It regulates the replication and incompatibility of repABC-type plasmids  stimuli, indicating that a large number of sRNAs are required during rhizobial symbiotic nitrogen fixation. However, more studies are required to characterize the functions and mechanisms of action of these sRNAs.The expression and abundance of sRNAs were affected by various external stimuli, and these condition-specific expression patterns help to infer the functions of different sRNAs  was performed to analyze the conservation of all the detected sRNAs in s Figure . MH_s15 In order to better understand the potential function of the 16 new sRNA candidates identified in this work, the target genes of 16 putative novel sRNAs were predicted by using the webserver IntaRNA. These candidate target genes were subjective to GO annotation and enrichment analysis. The WEGO output of the target genes was shown in Figures M. huakuii 7653R genome. Based on the results of a comparative analysis of five related strains, we extracted 40 potential sRNAs and used RNA deep sequencing data to further identify their characteristics. Using this strategy, 24 annotated sRNAs with well-characterized functions in bacteria and 15 putative novel sRNAs were detected.Genome-wide screens for sRNAs have mainly used bioinformatic prediction, RNA-seq, and tiling array analyses. In general, the number of non-coding transcripts is large, reaching hundreds or thousands of species in an RNA-seq or tiling array experiment  and Smr7C so on, have not been discovered in 7653R. Hence, secondary structure conservation was adopted as a main predictive parameter to screen for the putative sRNAs in this study.Except for five cis-regulatory riboswitches, 34 of the 35 predicted sRNAs in 2O2, 2O2 has not been detected in free-living cells or bacteroids, but H2O2 accumulation was observed in the infection stages of the symbiotic interaction and in ultrathin sections of mature 6-week-old nodules. For this reason, H2O2 played an important signaling role in early symbiotic and senescence processes, and rhizobia have an efficient antioxidant defense system to detoxify H2O2 . The predictions of sRNAs were performed as previously described  in the tested genomes with E-values ≤ 1e−5. The alignments were interrogated using eQRNA version 2.0.4 (ftp://ftp.genetics.wustl.edu/pub/eddy/software/qrna.tar.Z) as described previously  was used to screen the conserved intergenic regions in these candidate genomic sequences, and overlapping sequences between eQRNA and RNAz were selected. Subsequently, pairwise alignments in both eQRNA and RNAz were calculated using RNAfold (ViennaRNA-2.1.1) to determine the stable secondary structures, with the stem-length options set to a minimum of 20 bp. The promoters and Rho-independent terminators were analyzed using Promoter 2.0, RNAMotif and Erpin to further assess the resulting sequences. Finally, the nucleotide sequences of potential sRNAs were further inspected according to the results of RNA-seq deep sequencing. Each predicted sRNA was used to query the Rfam database to capture any annotated RNA regulatory elements.The comparative analysis of IGRs was carried out among Rhizobium strains were cultured in tryptone yeast medium (TY) or AMS medium  at 28°C, and Escherichia coli strains were grown in Luria-Bertani (LB) at 37°C. When required, antibiotics were added at the following concentrations: ampicillin (Ap) 100 μg/ml; gentamicin (Gm) 20 μg/ml; streptomycin (Str) 20 μg/ml and kanamycin (Kan) 50 μg/ml.600 = 0.4–0.5). When required, the growing log cultures were subjected to the following stress conditions as described previously , salt stress (4 M NaCl for 30 min), acid stress (pH5 for 90 min), alkali stress (pH9 for 90 min), cold stress (20°C for 90 min) or heat stress (37°C for 90 min). For micro-oxygen stress, log phase cells growing in TY (OD600 = 0.4–0.5) were shifted to a low O2 environment by continuous sparging of the culture with purified N2 for 24 h (N2 ≥ 99%). Controls were non-stressed exponentially growing cultures. For symbiotic conditions, surface-sterilized seeds of A. sinicus L. cultivar XY202 were cultivated in pots filled with sterile sand and irrigated with nitrogen-free Fahraeus solution, and cultivation was performed in a green house with a 16 h light and 8 h darkness cycle at 22°C and 20°C, respectively. Root nodules were harvested 28 or 50 days after M. huakuii 7653R inoculation.In the Northern blotting analyses, free-living rhizobial cells were cultivated in TY at 28°C, and cells were harvested in the log phase , and immediately frozen in liquid nitrogen and stored at –80°C. Plant nodules were resuspended in TRIzol Reagent and frozen in liquid nitrogen, then immediately subjected to grinding using a baked mortar and pestle to crush the nodule tissues. Isolation of total RNA and Northern blotting were conducted according to the manufacturer's protocols. Crude RNA extracts were purified with DNaseI to remove potential genomic DNA, and the resulting RNA was stored at –80°C. Northern hybridizations were performed with end-labeled [γ−32P]ATP DNA probes  following the manufacturer's instructions to carry out the RNA deep sequencing. The RNA integrity number (RIN) of the total RNA samples was checked using an Agilent Bioanalyzer 2100 (Agilent). Qualified total RNA was further purified using an RNeasy micro kit (QIAGEN) and RNase-Free DNase Set (QIAGEN) according to the manufacturer's instructions.600 = 0.8, was isolated and separated into small (< 100 nt) and long (> 100 nt) fractions using a miRNeasy Mini Kit (Qiagen) or a mirVana miRNA Isolation Kit (Ambion), respectively, according to the manufacturer's instructions. Total purified RNA was then treated with tobacco acid pyrophosphatase (TAP) (2.5 U/5 μg RNA in 50 μl for 2 h) to maintain the start regions of the transcription products. The quantity and quality of the processed RNA samples were assessed using of a NanoDrop ND-1000 spectrophotometer and Agilent 2100 Bioanalyzer. Qualified RNA was ligated to 5′- and 3′-RNA adapters by PCR, then the free adapters were removed and the remaining cDNA was purified. A Qubit® 2.0 Fluorometer and Agilent2100 instrument were used to monitor the abundance and size of cDNA, respectively. Solexa sequencing was performed using an Illumina HiSeq 2500. After that, all sequencing data were mapped by using the genomic sequence of M. huakuii 7653R to obtain the effective reads.Total RNA, including small-sized RNAs from 28 dpi (days post-infection) nodules, 50 dpi nodules and free-living cells at ODhttp://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp). All of the encoded protein sequences of predicted target genes for each candidate sRNA were submitted to InterPro (http://www.ebi.ac.uk/interpro/sequence-search) for protein sequence analysis & classification, and the GO annotations (GO_ID) were extracted from the output. Further, the WEGO (Web Gene Ontology Annotation Plot) webserver (http://wego.genomics.org.cn/cgi-bin/wego/index.pl) was used to display the GO annotation results in histograms. The enriched GO terms of candidate small RNA target genes were summarized and demonstrated in a pie chart.In order to figure out the potential function of the 16 new sRNA candidates identified in this work, we predicted the target genes for each of these sRNAs by using the webserver IntaRNA (M. huakuii 7653R genome were performed using Promoter 2.0 (http://www.cbs.dtu.dk/services/Promoter/) and BPROM 2.4.3.1111 (http://www.molquest.com/). Predictions of Rho-independent terminators were implemented with RNAMotif 3.0.7 (http://casegroup.rutgers.edu/casegr-sh-2.5.html), TransTermHP 2.09 (transterm.cbcb.umd.edu/), FindTerm 2.4.3.1111 (http://www.molquest.com/) and Erpin 5.4 (http://tagc.univ-mrs.fr/erpin/). Searches for known functional RNA sequences among the predicted sRNAs were conducted within the Rfam database using Rfam, version 12.0 (http://rfam.xfam.org/search) . The target genes for the identified sRNAs were predicted using IntaRNA soft (http://rna.informatik.uni-freiburg.de/).Searches for promoters in the M. huakuii 7653R genome. WX and MB performed the functional analysis of the target genes. The article was written and revised by XF, ZW, and LY.XF and LY conceived and designed the experiments. ZW, ZJ and XF prepared RNA samples and conducted the analysis of the RNA-seq data. ZZ and HB predicted and identified the sRNAs in the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA-based analyses on microbial communities in deep-sea hydrothermal environments often encounter methodological difficulties with RNA extraction due to the presence of unique minerals in and the low biomass of samples. In the present study, we assessed RNA extraction methods for deep-sea vent chimneys that had complex mineral compositions. Mineral-RNA adsorption experiments were conducted using mock chimney minerals and Escherichia coli total RNA solution, and showed that detectable RNA significantly decreased possibly due to adsorption onto minerals. This decrease in RNA was prevented by the addition of sodium tripolyphosphate (STPP), deoxynucleotide triphosphates (dNTPs), salmon sperm DNA, and NaOH. The addition of STPP was also effective for RNA extraction from the mixture of E. coli cells and mock chimney minerals when TRIzol reagent and the RNeasy column were used, but not when the RNeasy PowerSoil total RNA kit was used. A combination of STPP, TRIzol reagent, the RNeasy column, and sonication resulted in the highest RNA yield from a natural chimney. This indirect extraction procedure is simple, rapid, inexpensive, and may be used for large-scale RNA extraction.RNA-based microbiological analyses,  Advances in sequencing technologies have increased the significance of culture-independent analyses in microbial ecology, in which high-throughput sequences are obtained directly from nucleic acids extracted from various microbial habitats. Many methods for nucleic acid extraction have been developed and some of them have been optimized for specific sample types and research goals , 16, 20.Deep-sea hydrothermal fields host diverse microbial communities of high biological productivity fueled primarily by chemolithoautotrophy , 33. Chee.g. a transcriptome analysis) have been applied to many microbial habitats, including soils  and barite (BaSO4) (3:2 [w/w]) with a mortar and pestle, followed by sterilization at 230°C for 30 min. Sulfide and sulfate minerals are major constituents of deep-sea vent chimney structures  and the RNeasy mini kit . When TRIzol reagent and the RNeasy column were used together, the aqueous phase after the TRIzol treatment was loaded into the RNeasy column after mixing with an equal volume of ethanol. RNA was eluted after washing processes according to the manufacturer’s instructions.A mock chimney was prepared by pulverizing pyrite , 100 μL of a potential adsorption inhibitor, and 10 mg of mock chimney powder on ice. We assessed each of the following potential inhibitors: sodium tripolyphosphate , deoxynucleotide triphosphates , salmon sperm DNA , and NaOH . dNTPs, salmon sperm DNA, and NaOH were previously assessed for nucleic acid extraction from various environmental samples -treated water was used as a control. After being incubated on ice for 0 h, 4 h, and 14 h, the mixture was vortexed for 2 s and centrifuged at 2,000×g for 20 s. The supernatant was recovered into a new tube, purified by the RNeasy column (Qiagen), and the RNA recovered was reverse transcribed into cDNA using random hexamers with the PrimeScript RT reagent kit with gDNA Eraser . E. coli 16S rRNA was quantified by qPCR  with the primer EUB338F-U533R  with the RNA 6000 pico chip kit (Agilent Technology). These experiments were run in triplicate.The RNA adsorption experiment was conducted by mixing 100 μL of the RNA solution  and 0.25 g of the mock chimney were mixed and then frozen at −80°C for 48 h. RNA was extracted using the RNeasy PowerSoil total RNA kit  or TRIzol reagent (Thermo Fisher Scientific) and the RNeasy column (Qiagen), in the presence or absence of 100 μL of 0.6 M STPP. RNA quality  was assessed using Bioanalyzer 2100 (Agilent Technology). 16S rRNA was quantified by qPCR as described above. All experiments were run in triplicate.Natsushima cruise NT15-13 (JAMSTEC) in August 2015. Immediately after its recovery onboard, the chimney structure was stored at −80°C until used.A chimney structure was obtained from the Noho site of the Sakai field  , Mid-OkiThe chimney sample was pulverized with a mortar and pestle to a fine powder of micron-size particles in liquid nitrogen. Total RNA was directly extracted from 1.79–1.92 g of the sample using TRIzol reagent (Thermo Fisher Scientific) and the RNeasy column (Qiagen), in the presence or absence of 100 μL of 0.6 M STPP. In addition, the mixture of the pulverized chimney structure and STPP was sonicated using TAITEC VP-050  at 10W for 20 s. After 30 min on ice, the supernatant was recovered, and RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) and the RNeasy column (Qiagen) (indirect extraction). STPP was used in the indirect RNA extraction procedure because the supernatant potentially includes tiny particles of the chimney structure and/or RNA accidentally released from cells by sonication. RNA quality and the 16S rRNA copy number were evaluated as described above.http://www.arb-silva.de/no_cache/download/archive/release_119/Exports). Raw sequencing data have been deposited in GenBank/EMBL/DDBJ under accession number DRA005630.Total RNA samples from the chimney structure were reverse transcribed to cDNA as described above. The V4–V5 regions of 16S rRNA cDNA were amplified and analyzed using Illumina sequencing (MiSeq) as previously described . SequencE. coli total RNA solution, and then quantified the amounts of dissolved 16S rRNA in supernatants. After 4 h of mixing, dissolved RNA decreased to approximately 40% of the initial amount   . This isey; n=3) . In contE. coli cells that was similar to that with the RNeasy PowerSoil total RNA kit without STPP. In addition, the quality of extracted RNA with STPP using TRIzol reagent and the RNeasy column was superior to that extracted using the RNeasy PowerSoil total RNA kit in the presence of STPP. The RNeasy PowerSoil total RNA kit resulted in RNA fragmentation and a lower RIN  and Fast RNA Pro Soil-Direct kit , generally take 1.5–2 h for RNA extraction. Furthermore, TRIzol reagent and the RNeasy column may reduce the cost of RNA extraction by approximately 40–80% from the commercial kits described above. TRIzol reagent and the RNeasy column may be easily used in larger scale experiments and the simultaneous extraction of RNA and DNA.When combined with STPP, TRIzol reagent and the RNeasy column provided efficient RNA extraction from the mixture of mock chimney minerals and ower RIN  , followed by the phylotypes of Methanococci (0.4–10.1%) and Deltaproteobacteria (0.8–6.1%)   . Membersl fields , 31, 32.reductor  in all treductor . Besidesdococcus , 35, werraction) , suggestin situ microbial diversity and function need to be monitored using a polyphasic approach. Although the STPP amount and sonication step need to be adjusted to each chimney sample, the indirect RNA extraction method developed in this study is simple, rapid, and cost-effective, and may be used for large-scale RNA extraction. This new method may extend analytical methods for microbial communities within deep-sea hydrothermal vent chimneys, and thus may further our understanding of microbial activities in deep-sea hydrothermal fields.There has been a growing interest worldwide in seafloor and subseafloor energy and mineral resources. Deep-sea vent ecosystems including microbial communities are faced with anthropogenic environmental disturbances, and temporal and spatial variabilities in


Epididymal Metastasis from a primary carcinoma of the prostate gland is a rare but recognised phenomena. We describe a case of such metastasis which, unlike previous reports, presents as a painful epididymal mass. Therefore it is important for urologists to consider epididymal metastasis as part of the differential diagnosis in a patient with known carcinoma of the prostate and a tender epididymal mass.


To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific transcription factors genes and snRNAs . We propose that Set1 modulates adaptive responses, as exemplified by the post-transcriptional inhibition of Ty1 retrotransposition.The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA  Highly conserved histone proteins undergo several types of covalent modifications including acetylation, methylation, phosphorylation, ubiquitylation, SUMOylation, citrullination and ADP-ribosylation . These mSaccharomyces cerevisiae, all H3K4 methylation is carried out by a complex called COMPASS (for complex of proteins associated with Set1) or Set1C (for Set1 Complex) ATP and then purified by Sephadex column  and gel elution method. For in vitro RNA electrophoretic mobility shift assay, reactions containing purified Set1C or individual subunits and 0.25 pmole of radiolabeled RNA in 20 μl reaction buffer (10 mM Tris-Cl (pH 7.5), 1 mM EDTA, 5 mM MgCl2, 50 mM K-glutamate, 5% glycerol, 1 mM DTT and two units of RNasin) were incubated at room temperature for 30 min. The samples were resolved by electrophoresis at 4 °C on 5% polyacrylamide gels in 1×TBE buffer and subjected to autoradiography. For in vitro HMT assay, 40 μl reactions containing 350 ng (histone amount) recombinant chromatin assembled as described in Kim et al. (2013) with H2Bub-containing histone octamer  prototrophs. Ty1 retrotransposition frequencies were defined as the mean of 3 experiments, each one performed with four independent clones.To estimate the frequency of Ty1mobility , overnigmobility , lacking


Heteroduplex oligonucleotides (HDOs), composed of a DNA/LNA gapmer and its complementary RNA, are a novel, promising candidates for antisense drugs. We previously reported oligodiaminogalactoses , designed to bind to A-type nucleic acid duplexes such as DNA/RNA and RNA/RNA duplexes. In this paper, we report oligodiguanidinogalactoses  as novel A-type duplex binding molecules. We aimed to study in detail applicability of ODAGals and ODGGals for additives to HDOs as an antisense drug. The effect of ODAGal4  and ODGGal3  on an HDO were evaluated by UV melting analyses, RNA degradation study by ribonuclease A (RNase A), and ribonuclease H (RNase H). Cleavage of a 13mer HDO by RNase A, which is considered to be the main cause of RNA degradation in serum, was effectively inhibited by the addition of only one equivalent of ODAGal4 and ODGGal3. In contrast, RNase H activity, which involves the cleavage of target RNAs by an antisense mechanism, was only slightly affected by the presence of the cationic oligosaccharides. These results suggest that ODAGal4 and ODGGal3 are useful because they could both stabilize the HDO and maintain RNase H activity of the gapmer. All these drugs are based on nucleic acids that have been chemically modified to be nuclease-resistant in vivo and have other favorable properties as drugs. The mechanisms of action of antisense drugs are mainly classified into two types5. In the first type, RNase H-dependent ASOs, which form a duplex structure with the complementary RNA and the RNA strand in the ASO/RNA duplex is cleaved by RNase H. The second type is steric blocker ASOs, which simply mask the function of the target RNA by duplex formation. In the former mechanism, the ASOs must contain consecutive DNA region for recognition by RNase H, as well as highly chemically modified nucleic acids to ensure the biological stability of the ASOs and the thermodynamic stability of the ASO/RNA duplex. One of the designs of effective RNase H-dependent ASOs is gapmer ASOs, which contain a central window of consecutive DNA and a wing region consisting of RNA-like nucleotides with high affinity for complementary RNA, such as 2′-OMe RNA and LNA6. Since gapmer type oligonucleotides was reported to show RNase H activity in 1980s8, they have been eagerly studied as nucleic acid drug candidates9 and the mipomersen is a successful one. Recently, novel gapmer-based nucleic acid drug candidates called heteroduplex oligonucleotides (HDOs) have been reported10. An HDO is composed of an LNA/DNA/LNA gapmer and its complementary RNA. The gapmer is also fully modified with phosphorothioate (PS); the complementary RNA contains partial PS and 2′-OMe modifications on a few nucleotides at each terminus, and the α-tocopherol moiety is anchored to the 5′-end for effective liver delivery.Antisense oligonucleotides (ASOs) are among the most successful nucleic acid candidates. Four antisense drugs have been approved: fomivirsen . In this study, we report the synthesis of β-(1 → 4)-linked 2,6-dideoxy-2,6-diguanidino-D-galactopyranose oligomers -linked 2,6-diamino-2,6-dideoxy D-galactopyranose oligomers , and showed that the ODAGal 4mer  could effectively bind to an siRNA and protect it almost completely against cleavage by RNase A11. First, ODAGal2 2 and ODAGal3 5 were guanidinylated using Goodman’s reagent14. In both cases, because ODAGals are hydrophilic molecules and compounds 3 and 6 are hydrophobic molecules, it was necessary to add 1,4-dioxane portionwise in parallel with the reaction progress. After guanidinylation, the Boc groups were removed under acidic conditions15 to afford ODGGal2 4 and ODGGal3 7 in yields of 7% and 12% yields, respectively. The low yields were mainly attributed to incomplete guanidinylation even under optimized conditions.Synthesis of the ODGGals was conducted according to Fig. 2, and the ∆Tm values in the presence of the ODGGal 2mer or ODGGal3 were determined, as shown in Entry 1. The ODGGal 2mer slightly increased the Tm value (2.3 °C), and the ∆Tm value in the presence of ODGGal3 was significantly high (13.2 °C). Next, Entries 2 to 5 show the Tm and ∆Tm values of other RNA/RNA duplexes, a DNA/RNA hybrid, and a DNA/DNA duplex in the absence and presence of ODGGal3. For the RNA/RNA duplexes, the ∆Tm values were greater than 10 °C in all cases although the ∆Tm value in Entry 3 could not be precisely calculated because the Tm value was too high. The base sequence of DNA/RNA in Entry 4 corresponds to the RNA/RNA duplex in Entry 2, and the ∆Tm value in Entry 4 (7.9 °C) was lower than that of the RNA/RNA duplex (11.9 °C). As shown in Entry 5, the ∆Tm value of the DNA/DNA duplex in the presence of ODGGal3 was relatively low (3.8 °C) compared to those of the RNA/RNA and DNA/RNA duplexes. While these tendencies the ODGGal3 were quite similar to those of the ODAGals in our previous report, all the ∆Tm values were significantly higher than not only those of the ODAGal3, but also those of the ODAGal411. These results suggest that the replacement of the amino groups by guanidino groups was effective to strengthen the thermostabilizing abilities of the ODAGals for A-type nucleic acid duplexes. We also conducted fluorescence anisotropy measurements to evaluate the binding affinity of ODGGal3 to these nucleic acid duplexes and the Kd values are shown in the Supplementary Information  in the absence and presence of ODGGal3 or ODAGal4, were measured. As shown in Table Tm values  were more similar to those of the RNA/RNA duplexes than those of the DNA/RNA duplexes in Table Tm values of the gapmer/RNA duplex  were almost the same as those of the HDO.Next, the 16. First, the RNase A resistance of the DNA/RNA hybrid was evaluated with DNA and FAM- and Dabcyl-modified RNA in the absence and presence of ODAGal3, ODAGal4, or ODGGal3. In these FRET systems, an increase in the fluorescence intensity reflects cleavage of the RNA strand; as shown in Fig. To evaluate RNase A resistances, a fluorescence resonance energy transfer (FRET) system was applied with a FAM group and a Dabcyl group11, in which an siRNA was almost completely protected against RNase A by the addition of only three equivalents of ODAGal4.Figure Next, we applied these systems to the HDO. 5′-Dabcyl-modified-gapmer and 3′-FAM-modified-RNA were used in these experiments. As shown in Fig. Furthermore, fluorescence-intensity increase rate became increasingly slow as the amount of ODGGal3 was increased, as shown in Fig. Finally, we evaluated the effect of ODAGal4 and ODGGal3 on the RNase H activity of the LNA-DNA-LNA gapmer in the HDO. In these experiments, after treatment of a duplex of the gapmer and the complementary RNA 13mer with RNase H for 5 to 60 min in the absence and presence of cationic oligosaccharides, the amount of intact RNA was evaluated by RP-HPLC analysis. The amount of intact RNA 13mer lost under each condition are plotted in Fig. We further conducted the RNA degradation experiments using the RNA in three times amount of the gapmer. Under the reaction conditions, the catalytic reaction efficiency could be evaluated because two-thirds of the RNA strand initially do not form a duplex. The reaction time was 1 h, which is enough to cleave the RNA strand which initially formed duplex to the gapmer  or DNA/RNA duplexes. From the UV melting analyses, the increases in the Tm values of the HDO upon addition of ODAGal4 or ODGGal3 were close to those of the RNA/RNA duplexes. We measured CD spectra of the HDO in the absence and presence of the cationic oligosaccharides and it was found that they were typical ones of A-type duplexes in all cases -PS-DNA/RNA duplex than a PO-DNA/RNA duplex. In the report, however, we used stereoregulated PS-DNA -PS DNA and an all-(Rp)-PS DNA)24. On the other hand, Arya et al. reported PS-linkage not much affected the binding ability of aminoglycosides and their stabilizing effect to DNA/RNA duplexes using stereorandom PS-DNA a year after our report25. In this study, we used the gapmer and Toc-RNA having stereorandom PS linkages and therefore the effect of the PS-modifications might be small in the UV melting analyses.RNA/RNA duplexes are known to form A-type duplex structures27. In the case of siRNAs, it was reported that a less thermodynamically stable duplex was more susceptible to RNase A cleavage; this may be because a partially dissociated region in the duplex can be recognized by RNase A26. Our previous report regarding siRNA-ODAGal4 complex supported this cleavage mechanism; the siRNAs were almost completely protected against RNase A even in anion-excess systems11. In DNA/RNA and HDO, cleavage of the RNA strand should occur by the same mechanism as that of siRNAs. In contrast, in the current study, the cleavage of the RNA strand in the DNA/RNA hybrid was not effectively inhibited even in the presence of large amounts of cationic oligosaccharides. Although the oligomer length and original Tm value were different in both cases, the difference in the degree of increase in RNase A resistance in the presence and absence of cationic oligosaccharides may arise from the thermodynamic stabilization effect of the duplex structure in the unstable terminus by the cationic saccharides in both cases. Therefore, discussion of RNase A resistance based on Tm values is important although the ∆Tm values may not completely correspond with RNase A resistance because the Tm value provides an index of the thermodynamic stability of the entire structure of a duplex. In the case of the HDO, effective inhibition of RNase A cleavage was observed even upon addition of one equivalent of ODAGal4 or ODGGal3 (i.e. the N/P ratios are under 0.4 in both cases). This effective protection may be at least partly due to significant thermodynamic stabilization of HDO by the cationic oligosaccharides. Furthermore, these results also suggest the formation of an HDO-ODAGal4 or HDO-ODGGal3 1-to-1 complex at a low concentration of 100 nM at 37 °C; this is promising for in vivo applications in the foreseeable future.RNase A is an endonuclease that cleaves single-stranded RNA. In addition, RNase A is known to cleave RNA strands in duplex structures; in previous studies, the cleavage of siRNAs in serum was reported to be mainly due to endogenous RNase A-like nucleases28. To explain our desirable results for nucleic drug applications, we should consider the binding mode of RNase H and cationic oligosaccharides to the gapmer/RNA hybrid. ODAGal4 and ODGGal3 are expected to bind to some phosphodiester or thiophosphodiester groups at the 5′-teminus that form the major groove as previously stated. In contrast, on the basis of crystal structures of RNase H-DNA/RNA complexes30 and studies of the RNase H cleavage sites in DNA/RNA hybrids31, phosphate groups closer to the 3′ terminus in both the DNA and RNA strands of DNA/RNA complexes have been suggested to be important in RNase H recognition. For this reason, binding of the cationic oligosaccharides and RNase H may be not competitive in the 13mer HDO. This hypothesis is also supported by finding that the kind of RNA fragments did not vary upon addition of cationic oligosaccharides.RNase H is an enzyme that specifically recognizes DNA/RNA hybrids and cleaves the RNA strand. In this study, the RNase H activity of the LNA-DNA-LNA gapmer was almost unchanged upon addition of ODAGal4 or ODGGal3. This is clearly a contrasting result, considering that these cationic oligosaccharides effectively inhibit RNase A cleavage. These results are promising for antisense drug application because it was demonstrated that ODAGal4 and ODGGal3 could increase the thermodynamic stability and RNase A resistance without the inhibition of catalytic cleavage of RNA strands by RNase H. In addition, it is interesting to compare these results with a previous report in which paromomycin, a naturally occulting amino sugar, was reported to inhibit RNase H rather than RNase A cleavage of DNA/RNA hybridsIn vivo assays and further experiments regarding nucleic drug applications are currently in progress.In this study, we evaluated the effects of artificial cationic oligosaccharides, ODAGal4 and ODGGal3. These oligosaccharides were demonstrated to effectively protect an HDO against RNase A cleavage without decreasing RNase H activity of the gapmer. These results suggest that ODAGal4 and ODGGal3 are promising additives for internal administration of HDOs. 1H NMR spectra were obtained on a 300 MHz Varian MERCURY 300 spectrometer with acetonitrile as an internal or external standard (δ 2.06) in D2O. Mass spectra were recorded on a Voyager 4327 system (Applied Biosystems) or a 910-MS FTMS system (Agilent Technologies). Purification by prep. TLC was conducted using Merck Kieselgel 60 F254 silica gel. The organic solvents were purified and dried using appropriate procedures. The yields of the compounds were estimated from the integral values in the NMR spectra compared with acetonitrile . DNA, RNA, and other nucleic acid oligomers were purchased from Japan Bio Services Co., Ltd., Hokkaido System Science Co., Ltd., or GeneDesign, Inc.1  and compound 2 (140 μmol) were dissolved in a solvent mixture of H2O and 1,4-dioxane . To this solution, triethylamine (150 μL) was added. After 16 h 1,4-dioxane (0.25 mL) was added; after an additional 73 h, more 1,4-dioxane (0.5 mL) was added. After 71 h, the solution was diluted with chloroform and washed with 10% aqueous citric acid solution. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude product was purified by prep. TLC ) to afford compound 3 containing some impurities not derived from sugar compounds (26.1 mg). 10 mg of the crude product was dissolved in a solvent mixture of TFA-dichloromethane . After 70 min, toluene (2 mL) was added and the solution was concentrated. The mixture was dissolved in 0.1 M hydrochloric acid (1.5 mL) and concentrated; this procedure was repeated four times. Then, the mixture was dissolved in water (1.5 mL) and concentrated; this procedure was repeated three times. The crude product was purified by precipitation (methanol-diethyl ether) to afford pure 4 .Goodman’s reagent 1H NMR  δ 4.80–4.74 , 4.48 , 4.17 , 4.01–3.95 , 3.90–3.79 , 3.72–3.40 , 3.31 .17H37N12O7 m/z [M+H]+: 521.29 Found: 521.29.ESI-MS: calcd for C1  and compound 5 (80 μmol) were dissolved in a solvent mixture of H2O and 1,4-dioxane . To this solution, triethylamine (200 μL) was added. After 17 h, 1,4-dioxane (0.25 mL) was added; after an additional 8 h, more 1,4-dioxane (0.25 mL) was added. After 22 h 1,4-dioxane (0.5 mL) was added; after an additional 74 h, more 1,4-dioxane (0.5 mL). After 7 d, the solution was diluted with dichloromethane and washed two times with 10% aqueous citric acid solution. The aqueous layer was back-extracted with dichloromethane two times; then, the combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by prep. TLC ) to afford compound 6 containing some impurities not derived from sugar compounds (33 mg). 17 mg of the crude product was dissolved in a solvent mixture of TFA-dichloromethane . After 2 h, toluene was added and the solution was concentrated. The mixture was dissolved in 0.1 M hydrochloric acid (1 mL) and concentrated; this procedure was repeated four times. Then the mixture was dissolved in water (1 mL) and concentrated; this procedure was repeated four times. The crude product was purified by precipitation (methanol-diethyl ether) to afford pure 7 .Goodman’s reagent 1H NMR  δ 4.80–4.74 , 4.49 , 4.16–4.11 , 4.01–3.94 , 3.88–3.78 , 3.71–3.53 , 3.50–3.26 .25H53N18O10 m/z [M + H]+: 765.42 Found: 765.50.MALDI-TOF MS: calcd for CAbsorbance versus temperature profile measurements were conducted with an eight-sample cell changer in quartz cells with a path length of 1 cm. All the experiments were conducted in 10 mM phosphate buffer containing 100 mM NaCl at pH 7.0. The concentration of the duplex was 5 μM. The UV absorbance at both 260 and 320 nm was monitored with the temperature. The samples containing oligonucleotides were first rapidly heated to 95 °C, maintained at this temperature for 20 min, and finally allowed to cool to room temperature at a rate of 0.5 °C/min. ODAGal or ODGGal was added to the solution; these samples were additionally cooled to 0 °C or 20 °C at a rate of 0.5 °C/min, left at this temperature for 10 min. Finally, the dissociation was recorded by heating the samples to 95 °C at a rate of 0.5 °C/min.All experiments were conducted using 100 nM DNA/RNA or HDO and 0 to 1000 nM of ODGGal or ODAGal in 10 mM Tris buffer containing 100 mM NaCl, pH 7.2 and 37 °C or pH 7.3 and 30 °C. To a solution of duplex with or without cationic oligosaccharides, 100 μg/mL of RNase A from bovine pancreas in Tris buffer was added to afford a final concentration of 0.50 μg/mL. 10 s after addition of RNase A, the fluorescence intensity was recorded  for 3600 s. The following instrument settings were used: Ex/Em, 490 nm/520 nm; response, 1 sec; band width (Ex), 1 nm; band width (Em), 3 nm.2 at pH 7.2 and 37 °C. To the solution of the gapmer/RNA hybrid with or without cationic oligosaccharides, 500 U/mL of RNase H from E. coli in Tris buffer was added to afford a final concentration of 40 U/mL. After 5, 10, 30, and 60 min, respectively, 30 μL of the solution was divided from the reaction mixture and heated at 95 °C for 3 min. Then the solution was cooled and analyzed by reversed-phase HPLC (RP-HPLC). RP-HPLC was conducted using a μ Bondasphere 5 μm C18 column, 100 Å, 3.9 mm × 150 mm with a linear gradient of 0% to 10% acetonitrile over 45 min followed by 10% to 40% acetonitrile over 15 min in 0.1 M TEAA buffer at 50 °C at a rate of 0.5 mL/min or a linear gradient of 0% to 10% acetonitrile over 30 min followed by 10% to 50% acetonitrile over 10 min in 0.1 M TEAA buffer at 50 °C at a rate of 0.5 mL/min.All experiments were conducted using 4 μM gapmer, 4 or 12 μM RNA and 4 μM ODAGal4 or ODGGal3 in 10 mM Tris buffer containing 100 mM NaCl and 0.5 mM of MgClSupplementary Information


Protein-RNA complexes formed by specific recognition between RNA and RNA-binding proteins play an important role in biological processes. More than a thousand of such proteins in human are curated and many novel RNA-binding proteins are to be discovered. Due to limitations of experimental approaches, computational techniques are needed for characterization of protein-RNA interactions. Although much progress has been made, adequate methodologies reliably providing atomic resolution structural details are still lacking. Although protein-RNA free docking approaches proved to be useful, in general, the template-based approaches provide higher quality of predictions. Templates are key to building a high quality model. Sequence/structure relationships were studied based on a representative set of binary protein-RNA complexes from PDB. Several approaches were tested for pairwise target/template alignment. The analysis revealed a transition point between random and correct binding modes. The results showed that structural alignment is better than sequence alignment in identifying good templates, suitable for generating protein-RNA complexes close to the native structure, and outperforms free docking, successfully predicting complexes where the free docking fails, including cases of significant conformational change upon binding. A template-based protein-RNA interaction modeling protocol PRIME was developed and benchmarked on a representative set of complexes. Structures of protein-RNA complexes are important for characterization of biological processes. The number of experimentally determined protein-RNA complexes is limited. Thus modeling of these complexes is important. Reliable structural predictions of proteins and their complexes are provided by comparative modeling, which takes advantage of similar complexes with experimentally determined structures. Thus, in the case of protein-RNA complexes, it is important to determine if similar proteins and RNAs bind in a similar way. We show that, similarly to the earlier published results on protein-protein complexes, such correlation of the protein-RNA binding mode and the monomers similarity indeed exists, and is stronger when the similarity is determined by structure rather than sequence alignment. The data shows clear transition from random to similar binding mode with the increase of the structural similarity of the monomers. On the basis of the results we designed and implemented a predictive tool, which should be useful for the biological community interested in modeling of protein-RNA interactions. About three quarters of the human genome could be transcribed into RNA, including 4,693 miRNAs  and 105,The principles of protein-RNA interaction are based on structural and physicochemical complementarity –22, and We performed such investigation on a representative set of protein-RNA complexes. The analysis of all-to-all alignments in the set revealed a transition point between random and correct binding modes. The results showed that structural alignment significantly outperforms sequence alignment in identifying good templates, suitable for generating protein-RNA complexes with the ligand RMSD from the native structure < 10 Å. A template-based protein-RNA modeling protocol was developed and benchmarked on a representative set of complexes. The study provides a way for protein-RNA structure modeling on a genome scale.BLASTClust [BLASTClust. Thus CD-hit package [bound set, NRBC90).Co-crystallized protein-RNA structures were downloaded from PDB . Structures with resolution better than 3.0 Å were retained. Multimeric complexes were split into binary ones, defined as one protein chain and one RNA chain. The minimal lengths of the protein and the RNA were 30 and 20 residues, respectively. The interface was defined by < 5 Å distance between any heavy atom of the protein and any heavy atom of the RNA. The minimal numbers of protein and RNA residues at the interface were 5 each. This resulted in 2,951 binary complexes, including 563 RNA chains and 2,721 protein chains. The RNA redundancy was removed by ASTClust  with seq package  was usedunbound set).The performance of the template-based and free docking was also tested on the protein-RNA docking benchmark set . To avoifasta35 with default parameters [needle in the EMBOSS package [In NRBC439 set all-to-all pairwise alignment was performed by three approaches. The first approach was local sequence alignment by rameters . Sequenc package , also wiTM-align [TM-align is TM-score, which varies from 0 for completely dissimilar structures, to 1 for identical structures. The output of SARA is a score, which depends on the RNA size. To establish a similar description of structural similarity of proteins and RNAs, the SARA score was normalized by the score value of the RNA aligned to itself, resulting in the score interval 0–1, similar to the protein alignment. As with the complex sequence identity, the complex structural score was defined as the minimum of TM-score and the normalized SARA score. The aligned atoms (Cα in protein and C3' in RNA) were used to calculate interaction RMSD (IRMSD) similarly to the one proposed for protein-protein complexes [The third approach was structural alignment. For the structure alignment of RNA we chose SARA , based oTM-align , followiTM-align , 40–44. omplexes , which nomplexes .The three alignment approaches were applied to NRBC439 to test the ability to detect a good template. Binary complexes in NRBC90 were queries for the template set NRBC349.TM-align and the transformation matrix was saved. The target RNA was superimposed on the template RNA by SARA. Since SARA does not output the transformation matrix, it was reproduced by superimposing the RNA from SARA's output onto the original query RNA.After a template was selected, the target protein was superimposed on the template protein by The ligand RMSD (RMSD of RNA C3' atoms) between the model and the native structure was calculated. The quality of the model was measured by the ligand RMSD. In protein-RNA docking, a prediction was defined as "acceptable"   and by the structure alignments.A previous study on template-based protein-protein docking determined strong dependence of the binding mode similarity on the structural similarity of the participating proteins, with the phase transition from dissimilar modes to the similar ones at TMm = 0.4 . In the As the figure shows, the transition to similar binding modes occurs near the complex sequence identity 0.3. The results of such comparison obtained by the structure alignment approach are shown in To distinguish the role of the protein in detecting a good template for a protein-RNA complex, the target/template similarity was also measured only for the protein component . This diStructural similarity vs. sequence identity of the protein-RNA complexes is plotted in A structure alignment-based docking was implemented in a procedure PRIME (Protein-RNA Interaction ModEling). Benchmarking of PRIME suggests that 65% of target models can be built successfully Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Caenorhabditis elegans has emerged as a valuable model for understanding the molecular and cellular basis of neurological disorders. The worm offers important physiological similarities to mammalian models such as conserved neuron morphology, ion channels, and neurotransmitters. While a wide-array of behavioral assays are available in C. elegans, an assay for electroshock/electroconvulsion remains absent. Here, we have developed a quantitative behavioral method to assess the locomotor response following electric shock in C. elegans. Electric shock impairs normal locomotion, and induces paralysis and muscle twitching; after a brief recovery period, shocked animals resume normal locomotion. We tested electric shock responses in loss-of-function mutants for unc-25, which encodes the GABA biosynthetic enzyme GAD, and unc-49, which encodes the GABAA receptor. unc-25 and unc-49 mutants have decreased inhibitory GABAergic transmission to muscles, and take significantly more time to recover normal locomotion following electric shock compared to wild-type. Importantly, increased sensitivity of unc-25 and unc-49 mutants to electric shock is rescued by treatment with antiepileptic drugs, such as retigabine. Additionally, we show that pentylenetetrazol (PTZ), a GABAA receptor antagonist and proconvulsant in mammalian and C. elegans seizure models, increases susceptibility of worms to electric shock.The microscopic nematode  Caenorhabditis elegans has begun to emerge as a powerful in vivo model for research on neurological conditions including neurodegenerative diseases and neurodevelopmental disorders [C. elegans possesses a relatively simple nervous system, but has important conserved features of nervous system function at the level of ion channels and neurotransmitters. Of particular relevance to this study is the conservation of gamma-aminobutyric acid (GABA) and GABA receptors in inhibitory neurotransmission, and acetylcholine (ACh) and acetylcholine receptors in excitatory neurotransmission [C. elegans has a fully mapped connectome, is small and inexpensive to maintain and preserve, has a short three-day generation time, and is conducive for large-scale screening [isorders –7. In cosmission –11. In acreening –17.C. elegans as a convulsion model have largely focused on mutant backgrounds that are susceptible to the GABAA receptor antagonist, pentylenetetrazole (PTZ), a chemical proconvulsant commonly used to induce acute seizures in mammals [C. elegans [lis-1, unc-49, unc-47, or unc-25 mutants [In the past, studies investigating  mammals , 18, 19. mutants . When thC. elegans following electric shock. This approach has similarities to convulsion models previously established for fruit flies [Here, we developed an electroshock assay in which we quantitatively monitor paralysis duration and convulsions in it flies –23. In mit flies , 25. We unc-25 and unc-49 mutants, which lack inhibitory GABAergic neurotransmission, had delayed recovery following shock. To complement these findings, we tested three anticonvulsant drugs commonly used as antiepileptic therapy in humans, retigabine (RTG), sodium valproate (VPA), and levetiracetam (LEV) [C. elegans lack voltage-gated sodium channels, we did not select AEDs with Nav as their primary target. In all cases tested, these anticonvulsant compounds improve recovery following electric shock. Interestingly, previous researchers treated C. elegans with VPA and observed extended lifespan, but the effects of these compounds have not been tested on behavior, or in a worm seizure model [C. elegans seizure model has not been evaluated.The widespread use of PTZ in other models of seizure prompted us to investigate the effects of PTZ in our worm electroshock model , 27. PTZam (LEV) –30. Sincre model . A largeC. elegans are susceptible to an electric shock method of inducing paralysis and convulsions, similar to well-established methods in other model systems [Here we have demonstrated that  systems –23, 26. C. elegans were maintained on standard NGM agar plates seeded with OP50 E. coli. L4 worms were picked and transferred the evening prior to testing and maintained overnight at 25°C. C. elegans used in these experiments were Bristol N2 strain, CB156 unc-25 (e156), CB382 unc-49 (e382). C. elegans strains were ordered from the Caenorhabditis Genetics Center .The experimental setup consisted of a Grass SD9 stimulator, Grass SD44 stimulator (used as 3 second timer), dissecting microscope with a camera (Hitachi model KP-D20BU), a twelve-inch television monitor, and an HDD and DVR recorder (Magnavox model MDR535H/F7). A schematic of the setup is shown in C. elegans were selected and placed on an NGM agar plate with OP50 E. coli and stored at 25°C. The tubing utilized was Tygon® microbore tubing with an inner diameter of 0.040”, outer diameter of 0.070”, and wall thickness of 0.015” (Taylor Scientific product number 13-9124-20). The tube was cut into 9mm segments and filled with 15μL of M9 solution or M9 + drug treatment. Thirty minutes prior to stimulation (Grass SD9 stimulator), approximately ten 1-day old adult C. elegans were picked directly off an NGM plate and transferred to the Tygon tube using a platinum wire pick. , the experiments were visually analyzed. The time from the beginning of the stimulus to the time when each individual animal resumed a sinusoidal wave-like swimming motion was recorded. It should be noted that speed of the sinusoidal wave was not taken into consideration when considering recovery, only the wave motion itself. As a result of electrolysis, peripheral bubbles formed on either end of the stimulation tube. Additionally, as seen in seizures . We examDrugs were dissolved directly into M9 solution and approximately 15μL of solution was aliquoted into the clear plastic tubing. Drugs of interest were dissolved directly into M9 and worms were incubated for a total of 30 minutes prior to electric shock. The drugs tested were PTZ, RTG, VPA, and LEV, which were obtained from Sigma-Aldrich, St. Louis, MO, USA.post-hoc Multiple Comparisons test (HS = Holm-Sidak) and Student’s t-test using SigmaPlot 11.0 . All bar graphs represent mean ± SEM and asterisks denote significance between bars with all P≤ 0.05, *≤0.05, ***≤0.001, and ns = not significant. “n” is defined as recovery time for one animal where n≥10 and the minimum number of experimental trials per treatment was six. All relevant materials are available by request without restriction.Data was analyzed using One-Way ANOVA followed by a C. elegans using electric shock to induce convulsions similar to practices in other systems [We set out to develop a model of seizure in  systems , 25. To C. elegans.Shown in C. elegans moves by generating repeated sinusoidal body bends in a given direction. While somewhat of an oversimplification, generally body bends are generated by excitatory cholinergic motor neurons stimulating contraction of body wall muscles on one side of the animal, while inhibitory GABAergic motor neurons trigger relaxation of body wall muscles on the opposing side of the animal [unc-25 mutants, which are unable to synthesize GABA. A strong loss-of-function allele of unc-25, e156 [unc-25 mutants which recover in 89.5±105 seconds  = -4.681, P<0.001, unc-49, the GABAA receptor on muscles, also delayed recovery  = -2.35, P≤0.05, C. elegans are susceptible to PTZ, as previous studies did not detect responses in wild-type animals to even PTZ concentrations as high as 145mM (approximately 20mg/mL) [unc-25 mutants were exposed to 72mM PTZ they did not recover suggesting survival might be lost in these animals after electric shock  = -3.324, P<0.05). Similar to results with higher concentration of PTZ, a cocktail of 1 mM PTZ and 1mM RTG significantly decreased time to recovery  = 2.534, P<0.05). These pharmacological results complement our genetic experiments, and re-enforce the concept that reducing GABAergic motor neuron function increases susceptibility to electroshock.To pharmacologically assess how altering GABAergic transmission affects recovery from electric shock, we treated wild-type animals with PTZ. In wild-type animals, a dose response curve suggested a concentration of 72mM was enough to significantly slow recovery compared to sham controls (Student’s 20mg/mL) . The effs of PTZ . A dose C. elegans   The ion (lf) , and phaunc-25 and unc-49 mutants, loss of inhibitory, GABAergic transmission to muscles significantly slowed recovery to normal sinusoidal body motion following electroshock compared to wild-type animals  which shows there is no significant difference in recovery from electric shock in concentrations up to 0.5% DMSO  was somewhat surprising. We tested a strong loss-of-function allele, e156, for the only known GABA synthesis gene in C. elegans, unc-25. unc-25 (lf) showed a large enhancer effect when exposed to several different concentrations of PTZ. Previous studies suggest that e156 is potentially a complete null for unc-25 and GABA synthesis [unc-25 (e156) mutants should not have increased behavioral responses to PTZ. However, our experiments demonstrate that delayed recovery of unc-25 mutants following electroshock is dramatically enhanced with PTZ treatment. There are several potential explanations for this result. First, there is evidence that PTZ might block not only GABA receptors, but also reduce calcium channel selectivity and depolarize the cell membrane [unc-25 may not be a null, but a strong hypomorph for GAD, in which case very low, but physiologically critical, concentrations of GABA would be present but not detected by immunohistochemistry [unc-25 (lf) could result from both unc-25 being a hypomorph, and PTZ having effects on GABA receptors as well as calcium channels.The effects of PTZ, a GABA receptor antagonist, on wild-type animals provides further pharmacological evidence that reduced GABAergic transmission affects responses to electroshock. Our results demonstrate that wild-type animals respond to PTZ in a dose dependent manner. The concentrations of PTZ selected for analysis of wild-type animals were based on prior studies; however, in these studies, PTZ was mixed into the NGM agar plates whereas our protocol fully submerges the worms in PTZ dissolved in M9. Exposure on plates could alter vulnerability to PTZ, which could explain why wild-type worms were not susceptible to PTZ in previous plate-based assays, but are susceptible to PTZ in our electroshock assay. Alternatively, our electroshock assay might be particularly sensitive to the ratio of excitatory to inhibitory input to muscles making it highly sensitive to PTZ. Doses of PTZ used in combination with RTG were selected based on worms being able to recover from the convulsions, but taking longer than sham treated animals. For wild-type worms, 72mM PTZ was used whereas 1mM was selected for ynthesis , 9, 19. membrane . Second,hemistry . In thisC. elegans. Because it is inexpensive, rapid and has shown relevance with existing antiepileptic drugs, our C. elegans electroshock assay has the potential to become an initial screening tool for human seizure therapeutics. Further, our assay could act as a complement to other approaches that alter cellular excitability in worms, such as aldicarb sensitivity [C. elegans has the potential to provide molecular and cellular insights that are complementary to other whole organism systems, such as flies and rodents. Additionally, since worms lack voltage gated sodium channels, our electroshock assay is ideally suited for identifying novel AEDs that do not target sodium channels. This characteristic, along with the low cost and high throughput potential of our assay, could provide significant benefits for AED drug discovery over other more established models of seizure.Electroshock is one of the most common models of acute and chronic seizure in mammals. We have now developed a similar electroshock assay for sitivity . Thus, tFuture experiments will be needed to further validate our electroshock assay as a model of seizure. For example, optogenetic and further pharmacological manipulation of the excitatory and inhibitory inputs to muscles could be analyzed for impacts on electroshock recovery. This could provide further support for our proposed model that the response to electroshock is impacted by excitatory to inhibitory transmission balance on muscles. Further, genes and pathways that affect fly seizure models might be tested in order to understand the relevance of our electroshock assay to other existing seizure models –23, 52.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 VideoExamples of the experimental setup, followed by raw data video of wild-type worms in control conditions. There are five worms in this experimental tube and recovery times for all five are recorded.(MP4)Click here for additional data file.S2 Videounc-25(e156) in control conditions, the middle five worms are considered for analysis since the worm on the far right of the video is not clearly visible. Of the five worms, three do not completely recovery; therefore, only two recovery times are recorded from this video.Mutant worms deficient in GABA display slightly altered locomotion patters compared to wild-type. In this raw data example of (MP4)Click here for additional data file.


C. elegans LIN-41. Here we employ an integrative approach to dissect the RNA specificity of LIN-41. Using computational analysis, structural biology, and in vivo studies in worms and human cells, we find that a positively charged pocket, specific to the NHL domain of LIN-41 and its homologs (collectively LIN41), recognizes a stem-loop RNA element, whose shape determines the binding specificity. Surprisingly, the mechanism of RNA recognition by LIN41 is drastically different from that of its more distant relative, the fly Brat. Our phylogenetic analysis suggests that this reflects a rapid evolution of the domain, presenting an interesting example of a conserved protein fold that acquired completely different solutions to RNA recognition.RNA-binding proteins regulate all aspects of RNA metabolism. Their association with RNA is mediated by RNA-binding domains, of which many remain uncharacterized. A recently reported example is the NHL domain, found in prominent regulators of cellular plasticity like the  C. elegans LIN-41 and its homologs, including human TRIM71/LIN41, contain the RNA binding NHL domain. Here the authors combine computational analysis, structural biology and in vivo studies, to explain how these proteins bind RNA and how rapid evolution of NHL domains resulted in different solutions to RNA recognition.The  Highlighting their importance, mutations in the human TRIM-NHL proteins have been associated with various diseases, including cancer and neurological disorders4. The term “TRIM” refers to a tripartite motif consisting of RING finger, B-box(es), and coiled-coil domains, and the “NHL” to the so-called NHL repeats, named after the proteins NCL-1, HT2A, and LIN-415. Some TRIM-NHL proteins additionally contain a filamin domain between the TRIM and NHL domains4. The Caenorhabditis elegans TRIM-NHL protein LIN-41 (here referred to as CeLIN41) is widely known as a prototypic target of the let-7 miRNA and a key factor in the heterochronic pathway, which regulates developmental transitions in the soma7. In addition, the germline-expressed CeLIN41 controls reprogramming into pluripotency during the oocyte to embryo transition8. Also the human TRIM71/LIN41 (HsLIN41) has been implicated in cellular plasticity, facilitating the reprogramming of differentiated cells into pluripotent cells9. In both worms and mammalian cells, LIN41 function has been connected to messenger RNA regulation. By associating with specific mRNAs, LIN41 targets them for either degradation or translational repression11. However, what determines the specificity of LIN41 toward its targets has remained unknown.RNA-binding proteins (RBPs), by controlling different aspects of RNA metabolism, are central to gene expression. The TRIM-NHL family of RBPs includes key regulators of cell proliferation and differentiation14. The NHL domain, which folds into a WD40-like β-propeller, is an example of a recently reported RNA-binding fold16. However, how exactly the NHL domain binds RNA remains largely unexplored. Thus far, the RNA-binding specificity has been explained only for the NHL domain of the Drosophila melanogaster protein Brat (DmBrat), which associates with single-stranded RNA (ssRNA) in a sequence-specific manner18. In contrast to this binding strategy, we find that LIN41 evolved a very different solution to RNA binding. By combining computational analysis with in vivo studies and crystallography, we find that a positively charged pocket, specific to the NHL domain of LIN41, recognizes a short stem-loop (SL) element, LRE (LIN41 response element), whose shape dictates the binding specificity. By analyzing predicted structure models of NHL domains from various proteins and species, we propose that the distinct RNA-binding mechanisms of LIN41 and Brat reflect a rapid evolution of the NHL domain.Different types of RNA-binding domains (RBDs), such as K homology, RNA recognition motif (RRM) or zinc finger among others, are used by RBPs to associate with specific RNA elements. Recent studies suggest that the currently understood types of RBP–RNA interactions are but the tip of an iceberg, implying the existence of many uncharacterized RBDs19. A large-scale RNAcompete study of 205 RBPs reported that the vast majority of RBPs bind to ssRNA20. Although a handful of those proteins showed statistically significant evidence for binding secondary structures, the structures were not absolutely required. Since then, additional studies using RNAcompete on diverse RBPs were published23, opening the possibility to revisit the question of preference of RBPs for RNA sequence versus structure. This experimental platform (GPL16119) uses RNA oligonucleotides ranging from 30 to 41 nucleotides, potentially allowing assessment of compact RNA structures. Similar to previous analyses, we quantified sequence preferences by calculating enrichments of sequence 7-mers  in the RBP-bound fraction over the unbound fraction. To capture potential structure preferences in a comparable fashion, we in silico folded all RNA sequences by RNAfold24 and obtained RNA secondary structure in a dot-bracket format. In this format, a dot represents an unpaired nucleotide and a bracket a paired nucleotide. To roughly match the total number of combinations for sequence and structure n-mers, we set the length of the dot-bracket n-mers to 11, which resulted in a total of 9020 combinations. Henceforth, binding preferences for sequence 7-mers or structure 11-mers were calculated identically: for each RBP experiment, we asked whether there exists a small number of sequence 7-mers, or structure 11-mers, which show substantially higher binding enrichments than any other 7-mer or 11-mer, respectively  versus structured elements (X axis) for every RBP. In accordance with previous findings20, we found that the majority of RBPs displayed preference for sequence over structure. However, a handful of RBPs deviated drastically from that trend. Those RBPs were not part of the original large-scale study20, which may explain why they were not previously reported as having preference for secondary structure. The top three outliers  and calculated enrichments for structure 11-mers, as described in Fig. n-mer, we compared its overall occurrence in the RNA pool with its enrichment in the bound RNA fraction , and lin-29A, which associates via the first exon of its 5′-UTR, for simplicity lin-29A 5′-UTR10. Using programs to predict RNA secondary structure, we observed a number of SLs with tri-loops in those UTRs (Supplementary Note mab-10 and lin-29A UTRs). Interestingly, in the mab-10 3′-UTR, the tri-loop SLs were present in the regions previously shown to bind CeLIN41 in gel-shift experiments10 , suggesting possible functional similarity. Hence, we tested those SLs as candidate CeLIN41 binding sites  restored the binding  under the control of different 5′ or 3′-UTRs. To test SLs in the 3′-UTR, we created a synthetic “condensed” mab-10 3′-UTR, packing closer the five candidate SLs by deleting the intervening sequences. This 3′-UTR variant was sufficient to bind CeLIN41 in vitro and the binding required the SLs  under the control of mab-10 3′-UTR variants and, on the other hand, a control plasmid expressing firefly luciferase (FL)  binds structured RNA Fig. . TherefoFL) Fig. . MammaliUTR Fig. . This reons Fig. . Thus, bDanio rerio (zebrafish) protein (DrLIN41), which bound the SL elements of mab-10 3′-UTR with similar specificity as the worm (CeLIN41) protein , the nucleotide is flipped out, due to the C2′-endo conformation of the preceding – 1 uracil moiety, leading to placement of this base onto the rim outside of the central RNA-binding cavity. Bases at loop positions II and III (C/U(II) and A/A(III) in lin-29A and mab-10, respectively), on the other hand, stack and complement the continuous base stacking of the helical stem at the 3′-side . This is considerably longer than hydrogen bonds found in other base pairs of the hairpins, which have a standard length of 2.8–2.9 Å.Next, to determine how LIN41 binds RNA SLs, we determined structures of the DrLIN41 protein in complex with two 13 nucleotide-long RNAs corresponding to SL-containing fragments of 3 Å Fig. . The RNAThe basic NHL cavity interacts mainly with the sugar-phosphate backbone of the RNA SLs, predominantly via electrostatic interactions Fig. . The cavlin-41 alleles mg181 and mg1826). Similarly, the Tyr941Ala mutation (658 in DrLIN41) failed to rescue the developmental defects in lin-41 mutant worms8.Comparing the LIN41 NHL domains from different species, the majority of residues involved in RNA SL binding are identical  and the two adjacent positions in the stem (positions – 1 and + 1), revealed by the crystal structure, we decided to examine all possible variations in these positions; all combinations of nucleotides in the positions I, II, and III (64), and all possible base-pairing combinations of nucleotides in the positions – 1 and + 1 (6), leading to a total of 384 RNA variants Fig. .Fig. 6Th24, we calculated pairing probabilities and grouped them into seven bins . We then combined structural and sequence features into a total of 64 × 6 × 7 = 2688 unique RNA recognition patterns . This revealed that the residues in the – 1/ + 1 stem positions, the pairing probability, as well as the 3-mer loop, were all critical for binding  bound to CeLIN41 with a dissociation constant (Kd) of 1.322 µM, whereas a control 13-mer SL with five nucleotides in the loop did not show any detectable binding . The RNAs were examined by RNA-seq (RIP-seq) and enrichment for all transcripts was calculated . This resulted in a total of 177,179 predicted binding sites in 5′-UTRs (n = 6888), CDS , and 3′-UTR  with varying strengths (Supplementary Table r = 0.158). Second, the predicted binding sites in the 3′-UTR contributed the most to CeLIN41 binding, suggesting that CeLIN41 regulates the majority of its targets via their 3′-UTRs.Regulatory RBPs typically associate with the 3′-UTRs of mRNAs. However, in addition to binding 3′-UTRs, CeLIN41 associates with at least one mRNA target via its 5′-UTR27, the DrLIN41 NHL domain is most similar to the NHL domain of DmBrat, with an overall r.m.s.d. of 1.9–2.1 Å, for PDB entries 1Q7F, 4ZLR, and 5EX7, respectively28. However, the upper (RNA-binding) surfaces of these NHL domains are structurally very different . The key protein determinant of this mode of interaction is a mixed hydrophobic and positively charged surface of high structural complexity, which specifically accommodates individual bases, as well as increases binding energy via electrostatic interactions with at least some parts of the RNA sugar phosphate backbone. The surface of the DmBrat NHL domain features cavities and channels, which “scan” individual bases for sequence specificity. The three bases on the 3′-side of the consensus motif reside in a deep channel and position the fourth flipped out G(3) base in another cleft  was shown to induce translational repression, whereas the binding to the 3′-UTR (mab-10) results in mRNA decay10. As in both cases the recruitment of CeLIN41 is mediated by LREs, the mRNA regulation is presumably dependent on co-factors recruited to RNA by CeLIN41. The same may apply to HsLIN41, which, when artificially tethered to reporter RNAs, mediates both translational repression and mRNA degradation11. In addition to the somatic function, CeLIN41, and particularly its NHL domain, is also critical in the germline, where putative targets remain to be evaluated32. For example, the regulation of one putative target, cdc-25.3 mRNA, depends on both CeLIN41 and other RBPs, OMA-1, and OMA-230. However, whether this regulation is direct and mediated via LREs remains to be tested.LIN41 proteins have emerged as important regulators of proliferation vs. differentiation decisions in progenitor cells. In the 34. However, unlike CeLIN418, HsLIN41 has E3 ubiquitin ligase activity, regulating protein turnover37. Thus, which HsLIN41 functions are related to mRNA binding remains to be seen, and the LRE model is expected to help evaluating it. Although the model is not sufficiently predictive to determine which mRNAs are LIN41 targets, our data indicate that the targets tend to contain LREs. Thus, scanning candidate mRNAs for LREs should help in selecting best candidates for functional validation. The same applies to DmWech, which functions in muscles, particularly in the regulation of integrin-cytoskeleton connection38. Our finding suggest that at least some of these functions may involve mRNA regulation. Finally, although we focus here on mRNAs, it is possible that LRE-like SLs mediate the association of LIN41 also with other types of RNAs. For example, a recent study of proteins involved in miRNA processing showed that HsLIN41 specifically binds the apical loop of specific miRNA precursors, such as pre-miR29a and pri-miR-1-239. Interestingly, when we used structure prediction programs to examine possible secondary structures of these two pre-miRNAs, we noticed that their apical loops could fold into an LRE-like SL (our unpublished observation), potentially explaining why these specific miRNAs, but not others, are regulated by HsLIN41.Vertebrate LIN41 proteins are essential for different aspects of development, e.g., pluripotency, limb development, and neural tube closure42. Our phylogenic analysis of proteins carrying the NHL domain suggests that the different RNA-binding strategies of LIN41 and DmBrat reflect a rapid evolution of the RNA-binding surface of the domain. One example is DmWech, whose NHL domain, despite presumably binding LREs, displays a quite different distribution of the electrostatic potential. The NHL domains of other related proteins discussed here have diverged much further, to the point where predicting their RNA-binding preference (sequence versus structure) becomes highly speculative. Thus, whether other proteins containing NHL domains bind RNA, and whether their binding mechanisms are reminiscent of Brat-like recognition of ssRNA or LIN41-like recognition of SLs, are exciting questions for future research.The mechanism of RNA recognition by LIN41 is very different from that of DmBrat, cautioning against generalizations about RNA-binding modes derived from few studies. The RRM is another example of a protein fold that can recognize RNA sequence or structure; it binds predominantly ssRNA but, in some proteins such as U1A, RBMY, and TAF15, it recognizes RNA SLsC. elegans strains used in this study. To obtain synchronous worm populations, embryos were extracted from gravid adults with a bleaching solution (30% (v/v) sodium hypochlorite (5% chlorine) reagent , 750 mM KOH), and were incubated overnight in the absence of food at room temperature in M9 buffer . Hatched, arrested L1 larvae were plated on food and incubated at 25 °C for the desired number of hours. For RNA interference (RNAi) experiments, arrested L1 larvae were plated on RNAi-inducing NGM agar plates containing Escherichia coli HT115 bacteria carrying plasmids targeting genes of interest43.Supplementary Table www.ncbi.nlm.nih.gov/geo/). The respective accessions were GSE41235, GSE73000, GSE75554, GSE60498, GSE96990, and GSE93949. We downloaded the data for GSE41235 from hugheslab.ccbr.utoronto.ca/supplementary-data/RNAcompete_eukarya/ and the rest from GEO (Series Matrix File). All the data were then combined into a single table containing the binding scores from the original authors. As a precautionary measure, we then removed 11.9% of the 241,357 oligonucleotides that showed a median intensity lower than 6 (log2 space) over the full set of 244 experiments (hugheslab.ccbr.utoronto.ca/supplementary-data/RNAcompete_eukarya/raw_data.txt.gz), arguing that those are probably less reliable 24. In this case, we used a motif length of 11 to roughly match the number of structure combinations (n = 9020) to the number of 7-mer sequence combinations . Theoretically, there are 311 = 177,147 dot-bracket strings of length 11 but most of those do not represent valid secondary structures.We downloaded publicly available data from six studies  that used the RNAcomplete assay based on the platform GPL16119 (20. We thus downloaded a large compendium of such experiments (hugheslab.ccbr.utoronto.ca/supplementary-data/RNAcompete_eukarya/raw_data.txt.gz) and created an input sample by calculating the median intensity for every oligo on the array. When comparing this input with LIN-41, TRIM71, and Wech , we noticed sample-specific shifts for oligonucleotides with very low intensity levels likely to be caused by technical limitations: in the case of LIN-41, oligonucleotides with a low intensity in the input showed no substantial enrichment in the pulldown. This could be explained by low efficiency oligonucleotides that do not measure the intended target but cross hybridize to produce some low level of intensity. We thus removed those oligonucleotides using an intensity cutoff , and extracted the raw intensity values from the respective channels on the two color arrays. Unlike in most setups using two color array, where samples of interest and control samples are run on the same array, in the RNAcompete setup each channel contained a different RNAcompete experiment. The original authors calculated enrichment values for a particular RBP by comparing a particular sample with a compendium of other RBPs24 using the option -p to calculate the partition function. From the resulting base pairing matrix, we extracted all the values representing nucleotide pairs with a distance of exactly four. We then log2 transformed the pairing probabilities (after adding a pseudo count of 0.0001) and grouped the values into seven bins. We then combined the structure preferences with the sequence preferences by enumerating all possible binding patterns based on the two sequence features I,II,II (64 combination) and – 1,1 , and the single structure feature (7 combinations). This resulted in 2688 (64 × 6 x 7) binding patterns. We inferred the contribution of each binding pattern to LIN41 binding by calculating the mean LIN41 binding enrichment of all the oligonucleotides on the array that contained the respective binding pattern. Due to the large number of RNAs with low enrichment values, any systematic sequence bias can potentially dominate the model output. Indeed, when inspecting the binding enrichment heatmap . For downstream analysis, we considered the group “minimal” as a control, for which we would expect close to no binding.Based on the observation that LIN41 binds an RNA stem loop with a loop length of exactly three nucleotides, we devised a model to characterize the detailed binding specificity. We incorporated two sequence features, namely the loop region  and the two nucleotides surrounding the loop (positions – 1 and 1). To capture the tendency of a given RNA to form a SL in the thermodynamic ensemble, we calculated pairing probabilities between positions – 1 and 1 based on RNAfold (2.1.5)C. elegans, we used transcript annotations from WormBase (WS259) and extracted 5′-UTR, CDS, and 3′-UTR information from the file c_elegans.PRJNA13758.WS259.annotations.gff2.gz.We used the same scanning procedure as for the RNAs in the RNAcompete assay but replaced the RNA folding program RNAfold (2.1.5) by RNAplfold (2.1.5), using the parameter -c 0 to report also very low pairing probabilities. The change to RNAplfold allowed us to efficiently fold not only short RNAs but also the full-length transcripts. For lin-41(rrr3) animals expressing a single copy rescuing FLAG-GFP-LIN-41 transgene. Worm pellets were prepared by harvesting worms (grown in two different weeks on enriched peptone plates and fed E. coli OP50 bacteria) and freezing in liquid nitrogen. Lysates from two biological replicates were prepared by grinding the frozen worm pellets using mortar and pestle, and dissolving in lysis buffer , supplemented with Complete EDTA-free protease inhibitors  and 200 U/ml RNase inhibitor . Lysates were cleared by centrifugation at 20,000 × g for 30 min at 4 °C. IPs were performed by incubating lysates equivalent to 9 mg total protein with 20 μl of anti-FLAG M2 magnetic beads  for 3 h at 4 °C while rotating. Beads were washed with lysis buffer supplemented with 300 mM NaCl and then bound RNP complexes were extracted by adding 100 µl of freshly prepared elution buffer , 1% SDS). RNA was extracted from the eluate using picopure RNA isolation kit (Thermofischer Scientific KIT0204) according to the manufacturer’s instructions. Library preparation and sequencing was performed as described44.RNA co-immunoprecipitation (RIP) was performed on total lysates from 45 using the C. elegans genome assembly ce10 and gene annotation from WormBase (WS220). After normalization for library size, log2 expression levels were calculated after adding a pseudocount of 8 (y = log2[x + 8]). To account for nonlinear scaling between the samples, we quantile-normalized the data using the function normalize.quantiles from the R package preprocessCore. IP enrichments were then calculated by subtracting the input samples from the respective IP samples. For the comparison with the predicted CeLIN41 sites, non-expressed genes (expression < 5 in the input) were removed.Gene intensities were quantified as described previously46. All of the secondary structures that were predicted within 10% of free energy of the most stable variant were considered.The RNA secondary structures were predicted using RNA Structure software version 5.8.110. Each binding experiment was performed two to three times with freshly trasncribed RNA. Briefly, 1 µl of 5 µM protein was pre-incubated with 4 µl of 2 × gel-shift buffer . The reaction was made up to 7 µl with sterile water, incubated for 10 min at room temperature, following which 1 µl of RNA probe  was added. The reaction was incubated for 30 min and loaded onto the gel, electrophoresed at 25 mA, dried, and auto-radiographed.Radioactively labeled probes for electrophoretic mobility shift assays (EMSAs) were transcribed from PCR products with T3 RNA polymerase. Templates for probe synthesis were generated by PCR with an extended phage T3 RNA polymerase promoter (5′-AATTAACCCTCACTAAAGGGAGAA-2′), appended to the 5′-end of the forward primer, and gel-purified. EMSA assay was performed as described earlierY = B + A × (K + L + X – ((K + L + X)2 – 4 × L × X)0.5)/(2 × L)] to determine the equilibrium dissociation constant (K) using GraphPad Prism7.To measure the binding affinity of the LIN41–LRE complex, a series of binding reactions were set up with varying concentrations of protein and a fixed amount of 5′-Cy5 conjugated RNA (100 nM) in the assay buffer  (Supplementary Table 47. Modified 5′-UTRs and 3′-UTR fragments were ordered as gBlocks® Gene Fragments (Integrated DNA Technologies) and cloned into Entry clones using Gibson assembly48. Supplementary Data ttTi5605 locus on chromosome II, using the protocol for injection with low DNA concentration49.The GFP reporter plasmids were cloned with the MultiSite Gateway Technology (Thermo Fisher Scientific) and the destination vector pCFJ150lin-4150. Worms were mounted on a 2% (w/v) agarose pad with a drop of 10 mM levamisole solution and imaged on a Zeiss LSM 700 confocal microscope driven by Zen 2012 Software. Before acquiring images of representative worms, the GFP signals for at least ten worms were observed to verify that they were comparable among different worms in each worm line and for each condition. Fluorescent and differential interference contrast images were acquired with a × 40/1.3 oil-immersion objective . Selections of representative regions and processing of images was performed with Fiji51. Identical worm lines grown on mock or lin-41 RNAi bacteria were imaged and processed with identical settings.Synchronized L1 larvae were grown at 25 °C on RNAi-inducing plates with HT115 bacteria for 20 h and then subjected to confocal imaging. The HT115 bacteria either contained the insert-less L4440 parental RNAi vector (denoted “mock RNAi”) or an RNAi vector with an insert targeting 11. The 3′-UTRs were cloned into pRLTK (RL) plasmid using the XbaI and NotI sites. Primers are listed in Supplementary Data 52. Five hundred nanograms of pCl-neo-TRIM71 and 100 ng each of pRLTK and pFLTK (FL) plasmids were transfected using Effectene transfection reagent , following the manufacturer’s instructions. Transfection was done in three independent biological replicates of cells. Luciferase assay was done using dual-luciferase reporter assay system , following the manufacturer’s instructions. For each measurement, average of three technical replicates was used for further analysis.pCl-neo-TRIM71 (HsLIN41) plasmid is a kind gift from Loedige et al.g. The supernatants were transferred to fresh Eppendorf tubes and protein concentrations determined by nanodrop. Two hundred micrograms of protein was mixed with SDS lysis buffer  and boiled for 5 min at 95 °C. Proteins were separated by SDS-PAGE (loading: 50 µg protein extract per well) and transferred to polyvinylidene difluoride membranes by semi-dry blotting. The following antibodies were used: monoclonal rat anti-HA clone 3F10  and monoclonal rat anti-Tubulin [YL1/2] . Detection was performed with a horseradish peroxidase-conjugated secondary antibody , ECL Western Blotting Detection Reagents , and an Amersham 600 chemiluminescence imager . Western blotting was performed for all the three biological replicates. One representative blot is shown in the results.HEK293 cell lysates previously used for the dual luciferase assay were thawed on ice and cleared by centrifugation at 4 °C for 10 min at 16,000 × D.rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization. This DNA template was cloned into pOPINF using In-Fusion cloning53 to yield pOPINF_DrLIN41 (435–824). The translated sequence corresponds to Uniprot E7FAM5 (amino acids 435–824) with an N-terminal 3C cleavable histidine tag. Protein expression was carried out in Sf9 insect cells using pOPINF_DrLIN41 (435–824) in the FlashBAC baculovirus system. His-tagged DrLIN41 filamin-NHL protein was extracted from a baculovirus-infected Sf9 cell pellet by thoroughly re-suspending the cells in ice-cold nickel lysis buffer , freshly supplemented with Complete EDTA-free protease inhibitors  and Benzonase . After 30 min on ice, the lysate was centrifuged at 30,000 × g for 30 min at 4 °C. The clarified soluble lysate was incubated in batch mode with Ni-NTA Superflow  and then transferred into a 10 ml Econo-Pac column  for washing with nickel wash buffer . Elution was performed with nickel wash buffer containing 125 mM imidazole. The eluate was concentrated and separated on a HiLoad 16/600 Superdex 200  gel filtration column equilibrated in 20 mM Tris pH 8.0, 200 mM NaCl, 2 mM TCEP, and 0.02 % NaN3. His-tagged DrLIN41 protein fractions were pooled, concentrated to 4.6 mg/ml, frozen in liquid nitrogen, and stored at – 80 °C.The DNA sequence encoding 54 to incorporate an N-terminal Strep tag followed by a TEV cleavage site. Protein expression and lysis was done as described above except the lysis buffer used was 100 mM Tris pH 8.0, 150 mM NaCl, 2 mM TCEP, 2 mM MgCl2, 0.2% Tween-20, 0.02% NaN3, freshly supplemented with Complete EDTA-free protease inhibitors  and Benzonase . Clarified lysate was loaded onto a 5 ml StrepTrap HP column . The column was washed in three 10 ml steps using binding buffer , high-salt wash buffer (binding buffer containing 1 M NaCl) followed by binding buffer. Elution was done using 10 ml binding buffer containing 2.5 mM desthiobiotin. The subsequent gel filtration, concentration, and storage steps were as described above.For the CeLIN41 filamin and NHL domains (isoform B of Q9U489), a DNA insert encoding amino acids 691–1147 was cloned into pAc8D. rerio LIN41 protein consisting of the filamin and NHL domains (residues 435–824) was crystallized using the sitting-drop vapor diffusion method at 20 °C with a Phoenix nano-liter dispensing robot (Art Robbins). One hundred nanoliters of DrLIN41 protein at 4.6 mg/ml in protein buffer  was mixed with 100 nl of crystallization buffer  PEG 8000, 0.1 M Na3 Citrate). Long rectangular plate-type crystals measuring ca. 300 × 35 μm were obtained after 10 days. Crystals were cryo-protected in crystallization buffer containing 30% ethylene glycol followed by cryo-cooling in liquid nitrogen. For the DrLIN41–RNA complexes, it was necessary to screen different RNA targets and sequence lengths to obtain good diffracting crystals. RNA was chemically synthesized, purified by PAGE, deprotected to the 2′-hydroxyl form, desalted, and lyophilized (Dharmacon). Stock concentrations of RNA (30 mM) were made in 0.1 M Tris pH 8.0. To promote the formation of SLs the RNA was heated to 90 °C for 5 min and then rapidly cooled on ice for 30 min. The RNA was then added in a tenfold molar excess to the freshly purified His-tagged D. rerio LIN41 filamin-NHL protein (107 µM) and incubated for 1 h on ice. The RNA–protein complex was passed through a 0.1 µm Ultrafree-MC filter (Millipore) before setting up crystallization experiments as described above. D. rerio LIN41 filamin-NHL with lin-29A RNA (5′-GGAGUCCAACUCC-3′) crystallized overnight in Morpheus HT-96  condition G10 . D. rerio LIN41 filamin-NHL with mab-10 RNA (5′-UGCAUUUAAUGCA-3′) crystallized after 13 days in similar conditions from the same crystallization screen, condition G2 . Crystals were harvested after 27 days growth at 20 °C and cryo-cooled in liquid nitrogen.His-tagged 12121 with two molecules per a.u. Crystals of DrLIN41 (filamin-NHL) in complex with lin-29A/mab-10 RNAs belonged to space group P3121 with one complex per a.u. and diffracted to 1.9 Å and 2.35 Å, respectively. Diffraction data were integrated and scaled using the XDS program package55 and the DrLIN41 filamin-NHL unbound structure was solved by the molecular replacement method with PHASER56 using truncated homology models of the filamin and NHL domain as search models. DrLIN41 (filamin-NHL) RNA complex structures were solved by molecular replacement using filamin and NHL domains of the unbound structure as search models. Clear mFo-DFc electron density for the missing RNA part allowed unambiguous building of the hairpin. This was further supported by a phased anomalous difference Fourier map calculated from a separately collected highly redundant data set  = 23.9) at longer wavelength  for the lin-29A complex which featured clear peaks for all phosphorus atoms of the RNA sugar phosphate backbone. All structures were then manually completed and further improved by the crystallographic simulated annealing routine followed by individual B-factor refinement in PHENIX57. Structures were then finalized by several rounds of manual rebuilding in COOT58 and refinement in BUSTER59 (unbound) and PHENIX (RNA complexes). Final structures were validated using tools implemented in COOT. Structural images for figures were prepared with PyMOL (http://pymol.sourceforge.net/).X-ray data collection was carried out at SLS PX-II/III beamlines in Villigen, Switzerland, at 100 K. DrLIN41 filamin-NHL protein crystals diffracted to 2.6 Å and belonged to space group P2D. rerio (DrLIN41) sequences as queries, against the non-redundant protein sequence database. This led to the identification of a large group of 3762 closely related sequences. Partial sequences, highly identical sequences from the same organism and smaller isoforms were removed. Based on a preliminary multiple sequence alignment, the NHL domain was extracted. Eventually, 71 representative sequences of NHL domains were selected from NHL-domain-containing protein in higher eukaryotes (mostly model organisms with fully sequenced genomes) and a few prokaryotes for further analyses.To address the question of differential RNA-binding properties of the NHL domain-containing proteins, a comprehensive bioinformatics analysis of Brat, LIN41, and other known NHL domain containing RBPs was carried out. Sequence database searches were carried out with the DmBrat and the LIN41 protein from 60, which takes into account both sequence and structural information. We performed the analysis iteratively, first by calculating the alignment of NHL domain sequences with a support of experimentally determined structures of DmBrat and DrLIN41, and later refined it using also predicted 3D structures of other subfamily members identified by phylogenetic analysis. To elucidate the phylogenetic relationships of NHL domains, the Minimum Evolution phylogenetic tree was calculated on the basis of alignment of the NHL domain sequences of the 71 selected proteins, including DmBrat and DrLIN41. The robustness of branches was assessed by calculating 1000 bootstrap replicates.A multiple sequence alignment of selected NHL domains was calculated using PROMALS3DC. elegans (CeLIN41), DmWech, CeNHL1, and TRIM3 from Homo sapiens (HsTRIM3) and DmMei-P26 proteins. To this end, a series of alignments to proteins of known structure were obtained through the GeneSilico meta-server61 using various protein fold recognition methods. The meta-server consistently reported the structure of DmBrat as the potentially best template for modeling of the NHL domains, and we used the variant in complex with RNA (PDB code: 5EX7) for the modeling of the CeNHL1, HsTRIM3, and DmMei-P26 proteins. The DrLIN41 structure is obtained during this study and was not available in the database for the Fold Recognition analysis. However, it was used as the template for the modeling of CeLIN41 and the DmWech proteins, due to their closer phylogenetic relationship. Comparative modeling as well as structure analysis was performed using SwissPDBViewer and SwissModel62. Evolutionary sequence conservation among the various subgroups of the NHL domain-containing proteins were analyzed using ConSurf63 and were mapped on the NHL domain structure of DrLIN41 , 6FQ3 (DrLin41-lin-29A complex), and 6FQL (DrLin41-mab-10 complex). All RNA-sequencing data generated in this study have been deposited in the NCBI Gene Expression OmnibusSupplementary InformationPeer Review ReportDescription of Additional Supplementary InfoSupplementary Data 1