TMX2 is a thioredoxin family protein, but its functions have not been clarified. To elucidate the function of TMX2, we explored TMX2-interacting proteins by LC-MS. As a result, importin-β, Ran GTPase (Ran), RanGAP, and RanBP2 were identified. Importin-β is an adaptor protein which imports cargoes from cytosol to the nucleus, and is exported into the cytosol by interaction with RanGTP. At the cytoplasmic nuclear pore, RanGAP and RanBP2 facilitate hydrolysis of RanGTP to RanGDP and the disassembly of the Ran-importin-β complex, which allows the recycling of importin-β and reentry of Ran into the nucleus. Despite its interaction of TMX2 with importin-β, we showed that TMX2 is not a transport cargo. We found that TMX2 localizes in the outer nuclear membrane with its N-terminus and C-terminus facing the cytoplasm, where it co-localizes with importin-β and Ran. Ran is predominantly distributed in the nucleus, but TMX2 knockdown disrupted the nucleocytoplasmic Ran gradient, and the cysteine 112 residue of Ran was important in its regulation by TMX2. In addition, knockdown of TMX2 suppressed importin-β-mediated transport of protein. These results suggest that TMX2 works as a regulator of protein nuclear transport, and that TMX2 facilitates the nucleocytoplasmic Ran cycle by interaction with nuclear pore proteins. TMX proteins are integral transmembrane proteins that localize predominantly in the ER, but are also found in mitochondria-associated membranes.2 TMX1, TMX3, and TMX4 have a conserved active site, Cys-X-X-Cys (CXXC), for oxidoreductase activity in a thioredoxin-like domain5, and regulate protein quality in the ER7, ER-mitochondria Ca2+ flux, platelet function8, eye development9, and so on. On the other hand, TMX2 has a single cysteine in the conserved SXXC motif and lacks oxidoreductase activity. TMX2 cDNA was first isolated in 2003, and TMX2 mRNA has been found in a variety of human tissues, with the highest levels in the heart, brain, liver, kidney, and pancreas10. TMX2 protein was found in the supernatant of cultured bovine periosteal cells11. A recent TMX2 knockout experiment in primary mouse cortical neurons showed the protective function of TMX2 from ER stress12. Apart from these findings, however, there is no information about TMX2, and its target proteins and functions remain unknown. Though PDI family proteins, ERp44 and ERp29, also lack the CXXC motif or oxidoreductase activity, these proteins are known to serve as a scaffold or coat protein for protein transport; ERp44 has a role in the transport of immature proteins from the Golgi apparatus to the ER for the quality control of secretory proteins13, and ERp29 regulates ER membrane penetration of the polyomavirus coat protein to access the cytosol14.Thioredoxin-related transmembrane proteins (TMXs) are protein disulfide isomerase (PDI) family members and possess a transmembrane region. TMX1 was first identified in 2001 by Matsuo 16. In the nucleus, the binding of importin-β and the GTP-bound form of Ran (RanGTP) releases the cargo. RanGTP is also essential for the nuclear export of cargo proteins with Chromosomal Maintenance 1 (CRM1), which is also known as Exportin-1, and for recycling importin-β from the nucleus to the cytosol17. Hence, levels of both the total Ran protein and GTP-bound Ran are abundant in the nucleus while small amounts are present in the cytoplasm, and this nucleus/cytosol Ran gradient helps properly drive the cargo transport cycle. It has been shown that impairment of the transport cycle accompanying disruption of the Ran gradient induces cell senescence19, and may be related with diseases such as Hutchinson-Gilford Progeria Syndrome21.In this study, we searched for TMX2-binding proteins, and identified several nuclear transport proteins including importin-β and the Ras-related small nuclear GTPase, Ran. Nucleocytoplasmic trafficking through the nuclear pore complex is a fundamental cellular process in eukaryotic cells. Importin-β family proteins serve as adaptors of cargo proteins that have a nuclear localization signal (NLS) with importin-α, or directly bind to cargo proteins and transport them through the nuclear pore complex into the nucleusIn the present study, we found new functions of TMX2 in the maintenance of the Ran protein gradient and regulation of importin-β-dependent cargo transport.23. We prepared TMX2, importin-β, or Ran fused to the fragment of Venus protein, and confirmed that these fusion proteins do not emit fluorescence by itself , which encodes 296 amino acids, was isolated from Hep3B cells. To identify the TMX2-interacting protein, Flag-TMX2 was overexpressed in HEK293 cells, and immunoprecipitated with anti-Flag antibody. The cell lysates were analyzed by SDS-PAGE and silver staining. Protein bands exhibiting differential immunoprecipitation patterns between mock and Flag-TMX2 fractions were analyzed by mass spectrometry, and the protein band indicated by the arrow was identified as Karyopherin Subunit Beta 1 (KPNB1/importin-β) Fig. . Importiile Fig. , suggestWe found that in addition to the TMX2 isoform 1 (NP_057043.1), the mRNA of TMX2 isoform 2, which encodes a protein (NP_001137484.1) (258 amino acids) with deletion of amino acids 84–122 of isoform 1 (WT), was expressed in HEK293 cells. However, the levels of protein and mRNA of TMX2 isoform 2 were relatively less abundant than those of isoform 1 Fig. . Isoform24, while Tween20 permeabilizes all cellular membranes. Insertion of Flag tag into any position of TMX2 did not affect its localization in the nuclear envelope as indicated by the signal of Flag after permeabilization by Tween20. The fluorescence signals of all Flag tag-inserted TMX2 proteins were detectable by polyclonal anti-TMX2 antibody, which recognizes an epitope in full-length TMX2, even with digitonin treatment, suggesting that part of TMX2 faces the cytoplasm. By permeabilization with digitonin, the signal for Flag tag was detected in Flag(N)-TMX2, TMX2-Flag(C), 16Flag-TMX2, or 80Flag-TMX2, suggesting that the Flag tags within these TMX2 proteins face the cytoplasm. However, 56Flag-TMX2 was not detected, or was detected only partially in the nuclear envelope by anti-Flag antibody with digitonin. We confirmed that digitonin can partially permeabilize the outer membrane of the nuclear envelope, not only the plasma membrane, based on the result that the signal of Flag tag which faces the perinuclear space in emerin-Flag protein was not detected, or detected only partially in the nuclear envelope  and either an importin-α/β-dependent classical NLS of SV40, or an importin-β-dependent NLS of PTHrP was expressed with si-RNA targeting TMX2. Although knockdown of TMX2 did not affect transport of the cargo protein with SV40 NLS , and 213–222 AA (LTKQLPTLIL), respectively, in the TMX2 sequence. We have demonstrated that the localization of TMX2 was not affected by the insertion of a mutation into the sequence of NLS or NES of TMX2 35. Thus, the interaction between the importin-β-RanGTP complex and RanGAP-RanBP2 is necessary for the recycling pathway of importin-β and Ran for the next round of transport. BecauseTMX2 preferentially interacted with RanQ69L (the GTP-bound form), and RanGAP and RanBP2 have also been identified as TMX2-interacting proteins as well as importin-β and Ran, TMX2 may facilitate each event or series of events: namely, GTP hydrolysis of Ran, importin-β-Ran disassembly and the sumoylation of Ran on TMX2 in the cytoplasmic surface of the nuclear envelope. Consequentially, TMX2 will maintain the Ran gradient by driving the reentry of Ran into the nucleus by NTF2, and also accelerates the recycling of importin-β for the import of the cargo into the nucleus.We found that localization of TMX2 in the nuclear envelope was important to maintaining the nucleocytoplasmic Ran protein gradient. Ran is predominantly located in the nucleus, and most nuclear Ran is in the GTP-bound form due to the activity of nucleotide exchange factor (RCC1), which replaces the GDP bound to Ran with GTP36. These findings suggest that the efficiency of the nuclear import of these cargoes reflects the Ran sensitivity of the transport pathway.Indeed, TMX2 was important for maintaining the importin-β-dependent cargo import. However, TMX2 knockdown inhibited the transport of cargo with PTHrP NLS, which is directly recognized by importin-β, but not the classical NLS of SV40, which is imported by importin-α/β. Many reports have indicated that disruption of the Ran gradient leads to impairment of importin-β-dependent cargo import, but it has been shown that disruption of the Ran gradient in Hutchinson-Gilford Progeria syndrome (HGPS) fibroblasts did not affect transport of the classical SV40 NLS-cargo protein, despite its alteration of the transport of the Tpr protein, which has bipartite NLS6, TMX34, and TMX45 are not induced by heat or ER stress; however, oxidative stress by H2O2 treatment did induce TMX2 expression. It was previously found that H2O2 treatment alters the Ran distribution from the nucleus to the cytosol37. We confirmed this phenomenon, and treatment of cells with oxidizing agent, diamide, or hypoxia-reoxygenation also decreased nuclear Ran levels , DMEM/Ham’sF-12, anti-DYKDDDDK (Flag) antibody and anti-GFP(VC) antibody (mFX75) were purchased from Fujifilm Wako Pure Chemical Corporation . Fetal bovine serum (FBS), penicillin–streptomycin solution, and digitonin were purchased from Sigma Chemical , DAPI solution from Dojindo , anti-Ran antibody from Bethyl Laboratories Inc. , anti-HA antibody (M180-3) from MBL , and protein A Sepharose and glutathione Sepharose 4B from GE Healthcare .GGATCCGAAAAGATGGCGGTCTTG -3′ and 5′- AAGAATTCTTATTTATCCTTCTTGTTTTCCCCA -3′, and inserted into 3xFlag-pcDNA4 with BamHI and EcoRI. For TMX2-Venus constructs, the Venus sequence was inserted into pcDNA3.1( + ) with NotI and XbaI, and cDNA of TMX2 WT or isoform 2 without the stop codon was inserted with BamHI and EcoRI. TMX2 WT cDNA was also inserted into a pET21a vector or pGEX-5 × -1 vector for protein expression in E. coli. A TMX2 cDNA-encoding region  was inserted into a pGEX-5 × -1 vector. For analysis of the topology of TMX2, a 1xFlag tag was inserted at the N- or C-terminus, or at amino acid position 16, 56, 80, or 90 of TMX2 in pcDNA3.1( + ). For BiFC assay, TMX2 cDNA without the stop codon was inserted into pBiFC-VN155(I152L) vector . For preparation of the importin-β-mCherry construct, human importin-β was amplified with the primers 5′- AAGGATCCTCCGCCATGGAGCTGATC -3′ and 5′-TTATTATTGCGGCCGCCAGCTTGGTTCTTCAGTTTCCTCAGT-3′, and inserted with BamHI and NotI into pcDNA3.1( + ), in which mCherry sequence had been inserted with NotI and XbaI. For preparation of importin-β-VC155 construct, linker + VC155 sequence was amplified from pBiFC-VC155 vector (Addgene) and inserted into the above vector with NotI and XbaI, instead of mCherry sequence. Importin-β cDNA was also inserted into 3xFlag-pcDNA4 and pQE80L vector. For the emerin-Flag construct, a 3xFlag tag sequence was inserted into pcDNA with NotI and XbaI; then, human emerin cDNA was amplified with the primers 5′-AAGGATCCCCCGCCATGGACAACTACGCAGATCTTTC-3′ and 5′- ATTATTATGCGGCCGCCGAAGGGGTTGCCTTCTTCAGCCTGC-3′, and inserted into the 3xFlag-pcDNA with BamHI and NotI. Human Ran cDNA was amplified with the primers 5′-AAGGATCCATGGCTGCGCAGGGAGAGCCCC-3′ and 5′-TTTCTCGAGTCACAGGTCATCATCCTCATCCGGG-3′ and inserted into 3xFlag-pcDNA4 with BamHI and XhoI. Ran cDNA was also inserted into pQE80L for protein expression in E. coli, and into pBiFC-VC155 or pBiFC-VN155(I152L) vector. Human importin-α cDNA was amplified with the primers 5′-AAGGATCCATGTCCACCAACGAGAATGCTAA-3′ and 5′-TTGAATTCCTAAAAGTTAAAGGTCCCAGGAG-3′, and inserted into 3xFlag-pcDNA4 with BamHI and EcoRI. Human CRM1 cDNA was amplified with 5′-AAGGATCCTCTATGCCAGCAATTATGAC-3′ and 5′-ATAATAATGCGGCCGCTTAATCACACATTTCTTCTGGAA-3′, and inserted into 3xFlag-pcDNA4 with BamHI and NotI. For preparation of NES-2xVenus-NLS, the Venus sequence without the stop codon was inserted into pcDNA with HindIII and BamHI, and BamHI and EcoRI. The oligonucleotides for SV40 NLS with the stop codon, 5′-GGCCGCGCTCACCTAAGAAGAAGAGGAAGGTTGAATAAT-3′ and 5′-CTAGATTATTCAACCTTCCTCTTCTTCTTAGGTGAGCGC-3′, were annealed and inserted into 2xVenus/pcDNA with NotI and XbaI. Human PTHrP cDNA encoding amino acids 102–130 containing the NLS was amplified with the primers 5′-AATAATAAGCGGCCGCAAAGATACCTAACTCAGGAAACTAACA-3′ and 5′-AATCTAGATTAGGGCTTGCCTTTCTTTTTC-3′, and inserted into 2xVenus/pcDNA with NotI and XbaI. Then, the oligonucleotides for NES of human cAMP-dependent protein kinase inhibitor alpha (PKI), 5′-AGCTTATGAATGAATTAGCCTTGAAATTAGCAGGTCTTGATATCAACAAGACAA-3′ and 5′-AGCTTTGTCTTGTTGATATCAAGACCTGCTAATTTCAAGGCTAATTCATTCATA-3′, were annealed and inserted into 2xVenus-NLS/pcDNA with HindIII.The entire coding regions of human TMX2 WT (isoform1) (Accession No. NM_015959.4) and TMX2 isoform 2 (Accession No. NM_001144012.2) were amplified from Hep3B cDNA with the primers 5′- AA2 and 95% air.Hela cells were cultured in DMEM containing 10% (v/v) FBS, 100 units/ml penicillin and 100 μg/ml streptomycin, and HEK293 cells were cultured in DMEM/Ham’sF-12 with 10% FBS and penicillin streptomycin. Cells were maintained at 37 °C in 5% COTMA Plus (Wako) following the manufacturer’s instructions.siRNA against TMX2 was obtained by Qiagen . The target sequence of si-TMX2_1 (cat. No. SI00112735) is CUAGAUUUAACCCUAAGGUAA, that of si-TMX2_2 (cat. No. SI00112742) is AAGGUGGAUGUUGGACGCUAU, and that of si-TMX2_3 (cat.No. SI02777509) is UUCGUUUAUGGUCUUCAUUAA. AllStars Neg. Control  was used as the control siRNA. The siRNA was transfected into the cells with ScreenFect42.Cells were washed with PBS and lysed in buffer (50 mM Tris–HCl (pH 7.5) containing 150 mM NaCl and 0.25% NP40) with a protease inhibitor cocktail . Genomic DNA was fragmented by passing the lysed suspension through a needle attached to a syringe, and stored on ice for 30 min. After centrifugation at 14,000 × g for 15 min, the supernatant was incubated with antibody overnight at 4 °C. Protein A-Sepharose was then added, and the mixture was incubated for 1 h at 4 °C. The immunocomplexes were precipitated and washed with buffer (50 mM Tris–HCl (pH 7.5) containing 150 mM NaCl and 0.05% NP40), then analyzed by immunoblotting. Nuclear extracts were prepared from HEK293 cells as described previously43. E. coli, BL21 (DE3) or DH5α, was transformed with TMX2/pET21a or importin-β/pQE80L, respectively, and the proteins were expressed. The expressed proteins were purified by a Ni-NTA agarose column and used for the preparation of the antibody in rabbits. All experiments were conducted in accordance with guidelines on the welfare of experimental animals and with the approval of the Ethics Committee on the use of animals of Kwansei Gakuin University.The antibodies against human TMX2 and importin-β were prepared in rabbits using the method described previouslyProtein extracts of HEK293 cells or cells overexpressing Flag-TMX2 were immunoprecipitated with anti-TMX2 or anti-Flag antibody, separated with SDS-PAGE and stained with a SilverQuest™ Silver Staining Kit . Protein bands were excised from the gel and digested in-gel with trypsin. The digested proteins were loaded onto nano-HPLC capillary columns NTCC-360  containing 15 cm of 3 μm particle-size C18 reverse-phase column material  with a gradient of 0 to 40% buffer B (100% acetonitrile and 0.1% formic acid) for 30 min, followed by a gradient of 40 to 100% buffer B for 5 min, and 100% buffer B for 8 min at a flow rate of 300 nL/min . Eluted peptides were analyzed by an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). The peptide spectral data were searched against the UniProt database using SequestHT.Cells were fixed with 4% paraformaldehyde at 4 °C for 20 min, permeabilized with 0.1% Tween20 and blocked with 0.1% BSA in PBS for 1 h. For digitonin treatment, cells were fixed and incubated with 40 μg/mL digitonin for 3 min, followed by blocking with 0.1% BSA in PBS for 1 h. Then, cells were incubated with specific primary antibodies in blocking solution overnight, and incubated with a secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (H + L)  or anti-mouse IgG (H + L)-Affinity Pure Dylight 594 conjugate  in blocking solution with DAPI for 1 h. Images were obtained by confocal microscopy, Nikon A1  or TCS SP8 . For BiFC assay, TMX2 fused to VN155  was co-expressed with importin-β-VC155  or HA-Ran-VC155 in HEK293 cells. Cells were fixed with 4% paraformaldehyde, and the overexpression of each protein was confirmed by anti-TMX2 antibody, and anti-VC or anti-HA antibody, followed by secondary anti-rabbit IgG conjugated with Alexa Fluor 647 and anti-mouse IgG with Dylight 594. Venus fluorescence was detected as BiFC signal.E. coli BL21 was transformed with TMX2 /pGEX-5 × -1, and GST-tagged TMX2 proteins were expressed. E. coli was suspended in binding buffer  and sonicated. The proteins were solubilized by addition of 1% NP40 for 1 h, and the supernatant was collected after centrifugation. Equilibrated glutathione Sepharose 4B was added to the supernatant and incubated for 1 h. The beads were washed with the binding buffer, and the TMX2-packed beads were incubated with HEK293 cell lysates or purified His-tagged importin-β or Ran protein in the binding buffer with 0.25% NP40. The complex was washed and analyzed by SDS-PAGE followed by western blotting.p values < 0.05 were considered statistically significant. For multiple comparisons, one-way ANOVA followed by a Bonferroni/Dunn post-hoc test was used.Statistical analysis for single comparisons between means was carried out with Student’s t test, and Supplementary data


Peronospora destructor (Berk.) is an important biotrophic oomycete that causes downy mildew on onion (Allium Cepa L.) worldwide, especially in humid and temperate regions. The disease attacks bulb and seed production of onion, resulting in losses in yield and quality of bulbs. Epidemiological studies have increased our understanding and control of downy mildew on onion; however, little is known about the molecular aspects of P. destructor behavior during infection. Here, we isolated RNA from four samples of sporangia and sporangiophores of P. destructor, which were maintained by spore inoculation onto onions in a growth chamber. We then used an Ion PGM next generation sequencer to acquire and assemble the RNA sequences of P. destructor. By transcriptome shotgun assembly, we obtained 2335 contigs ; mean length, 881.6 nt). The data are accessible at NCBI (BioProject PRJNA391849). Our data resource will facilitate further studies of the molecular events during P. destructor infection. Specifications tableValue of the data•of P. destructor.This dataset represents RNA sequences from sporangia and sporangiophore formation •P. destructor infection.This dataset will provide information about the molecular events occurring during •Peronospora species.The dataset presented here provide reference sequence data for further genetic studies and will be useful for comparative studies among 1P. destructor maintained by spore inoculation onto onion shoots in a growth chamber. The raw data were deposited into the NCBI׳s Sequence Read Archive (SRA)  under the BioProject PRJNA391849. The RNA sequencing data are summarized in P. destructor transcriptome because they were highly homologous to the known sequences of other related oomycetes  as P. destructor H-YUNI1 isolate. Four samples of sporangia suspension were prepared and used for separate RNA extractions.Healthy onion bulbs were planted in a nursery and cultivated for a month under greenhouse conditions at daytime and nighttime temperatures of 25 °C and 18 °C, respectively. The onion seedlings developed from the bulbs were then spray inoculated with the collected spores described above. Sporangia production was carried out by the following three steps: First, the inoculated seedlings were moved to a KCSLPH-1400CT incubation box  and incubated in the dark for 12 h at 17 °C under highly humid conditions (100% relative humidity), as previously described 2.2de novo by transcriptome shotgun assembly . Because no genome sequence data of P. destructor is available in public databases, the RNA contigs were subjected to NCBI BLAST analysis and contaminants including contigs annotated with regard to plant and bacteria were manually removed.Total RNA was extracted from each of the four sporangia samples by using an RNeasy Plant Mini Kit . RNA templates were prepared with an Ion Total RNA-Seq Kit v2  and an Ion PGM Hi-Q OT2 Kit on an Ion OneTouch 2 System (Thermo Fisher Scientific), and then sequenced using an Ion PGM Hi-Q Sequencing Kit and a 318 Chip Kit v2 on an Ion PGM System next generation sequencer (Thermo Fisher Scientific), according to all the manufactures׳ recommendations. Assembly of sequence data and analysis were carried out using CLC Genomic Workbench software . For quality control, raw reads with a Phread quality score less than 20 and adaptors were removed. RNA contigs were assembled


Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax. Finally, we demonstrate effective detection of the small RNA directly from serum, without the need for pre-processing, with a single step RT-qPCR assay. Our findings identify a species-specific trypanosome small RNA that can be detected at high levels in the serum of cattle with active parasite infections. This provides the basis for the development of a cheap, non-invasive and highly effective diagnostic test for trypanosomiasis.Human and animal African trypanosomiasis  remain a significant health and economic issue across much of sub-Saharan Africa. Effective control of AAT and potential eradication of HAT requires affordable, sensitive and specific diagnostic tests that can be used in the field. Small RNAs in the blood or serum are attractive disease biomarkers due to their stability, accessibility and available technologies for detection. Using RNAseq, we have identified a trypanosome specific small RNA to be present at high levels in the serum of infected cattle. The small RNA is derived from the non-coding 7SL RNA of the peptide signal recognition particle and is detected in the serum of infected cattle at significantly higher levels than in the parasite, suggesting active processing and secretion. We show effective detection of the small RNA in the serum of infected cattle using a custom RT-qPCR assay. Strikingly, the RNA can be detected before microscopy detection of parasitaemia in the blood, and it can also be detected during remission periods of infection when no parasitaemia is detectable by microscopy. However, RNA levels drop following treatment with trypanocides, demonstrating accurate prediction of active infection. While the small RNA sequence is conserved between different species of trypanosome, nucleotide differences within the sequence allow generation of highly specific assays that can distinguish between infections with  African trypanosomes cause significant disease in humans and animals across sub-Saharan Africa. For both human and animal infections diagnostics that can accurately identify an active infection are lacking–this is particularly the case in animal disease where most diagnosis is based upon clinical signs, which is not a specific or sensitive means of detecting infection. There is therefore a significant unmet need for a pathogen marker of active infection that accurately indicates whether an animal or human is currently infected. Through analysing the blood of cattle infected with trypanosomes, we identified a short sequence of RNA that was present at very high levels. This small RNA derives from the trypanosome genome, and we could identify its presence in the genome of all three species that are responsible for human and animal disease. We were able to design species-specific tests, and showed that in samples from infected animals the assays were more sensitive than the traditional microscope-based detection, importantly the signal disappeared relatively quickly after successful treatment, and when treatment failed, the assay was able to accurately identify when infection persisted. We also demonstrated that the causative agent of human trypanosomiasis secretes the marker at similar levels to that seen in the animal-infective trypanosomes. Therefore, we have discovered a marker of trypanosome infection that is present at high levels in the blood of infected animals, disappears quickly upon successful treatment, but is effective at detecting instances of unsuccessful treatment and persistent infection. This represents a potentially powerful diagnostic tool for human and animal trypanosomiasis. Glossina species), cause Human African Trypanosomiasis (HAT) and Animal African Trypanosomiasis (AAT) across sub-Saharan Africa. AAT, caused by Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei, infects approximately 70 million and kills 3 million cattle per year, and is one of the most significant infectious disease constraints upon agriculture in the region ). Reverse transcription was carried out using a commercial cDNA Reverse Transcription Kit , replacing the random primers with the aforementioned TaqMan assay primer. Typically, 100 ng RNA was used per 15 μL reaction. The following thermocycling conditions were applied for the RT reaction: 16°C for 30 minutes, 42°C for 30 minutes and 85°C for 5 minutes to inactivate the reverse transcriptase.A species-specific 7SL-derived small RNA stem loop primer-probe detection assay was optimised, using custom primer and probe mixes made by Life Technologies, based on specific sequences  per RT reaction.Subsequent to the RT reaction, a qPCR was performed using a commercial kit , according to manufacturer’s instructions. At this stage, 1 μL custom prime-probe was also added to the qPCR reaction, along with 1.5 μL RT reaction. The qPCR cycling profile was as follows: 50°C for 2 minutes, 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and a probe detection step of 60°C for 1 minute.When serum was used as a substrate for RT-qPCR, samples were heat treated at 65°C for 15 minutes and 6 μL of the serum used per RT reaction. Single step RT-PCR reactions were performed according to manufacturer’s guidelines . Single step RT-PCR reactions were performed using the TaqMan small RNA assay primer and primer-probe mixes mentioned previously.T. congolense IL3000 genome , using Novoalign , with the following parameters: “–l 20 –t 30 –h 60 –m–o SAM”. To quantify the alignments, the python tool HTSeq-count was used with default parameters [T. congolense IL3000 transcript reference file in .gff format downloaded from TriTrypDB (v9.0) [Raw RNA deep-sequencing data were subject to quality control using FastQC (v0.11.5) . Adapterrameters  with theB (v9.0) . Data wain vivo infections, total RNA was extracted using Trizol LS from serum samples obtained from two cattle experimentally infected with the livestock trypanosome T. congolense , serum from an uninfected control cow, and an in vitro-derived T. congolense cell pellet (approximately 4 x 107 cells). The RNA was submitted for small RNA deep sequencing, selecting for RNAs between 20- and 39-bp long. The resulting reads were aligned to the T. congolense genome (TriTrypDB v9.0) using novoalign with strict parameters  aligning uniquely to the T. congolense genome. Reads were also aligned to a bovine genome  and 1.3%  were uniquely mapped to the rus, UMD   and 58.9%  unique and multimapped reads aligning to the T. congolense genome, respectively. The relatively high number of unmapped reads  in this sample with respect to the T. congolense genome is explained by the comparatively incomplete assembly and annotation of the reference T. congolense genome when compared to, for example, the genome of T. brucei.A total of 9,439,764 reads were generated from RNAseq of the T. congolense genome aligned to ribosomal RNA (rRNA) loci, the sequences of which are known to be deeply conserved in eukaryotes [T. congolense genome were generated using HTSeq-count, resulting in total read counts mapping uniquely to annotated features of 42,302 and 18,230 for the two infected samples, 322,385 for the pellet sample and 524 for the uninfected sample. Read counts were normalised for library depth . Strikingly, after eliminating reads associated with rRNA loci, the majority of reads from both infected serum samples originated from one specific 26-bp sequence   , and is .8.2861) . There wT. congolense was aligned to the genome assemblies of several species of African trypanosome to determine whether related trypanosome species encode for the 7SL-sRNA species. Sequences corresponding to the 7SL-sRNA were clearly identifiable in the genomes of all related trypanosome species examined, suggesting expression of 7SL-sRNA may be a common feature of African trypanosomes, and indeed, related trypanosomatids. Whilst no sequence variation was observed across any of the T. brucei subspecies , there were several nucleotide polymorphisms relative to the T. brucei sequence in both the T. vivax and T. congolense sequences, raising the possibility that specific assays could be designed to distinguish between the three primary livestock trypanosome pathogens  the T. brucei RT-qPCR assay resulted in positive detection of the 7SL-sRNA, highlighting the potential of the sRNA for diagnostics in human disease (The 7SL-sRNA sequence of athogens . To inveathogens . Each practivity . Importa disease .in vitro cultures of both T. brucei (Lister 427) and T. congolense (IL3000). Cells were seeded at 5 × 104 cells/mL (n = 2), and density was periodically counted by haemocytometer and supernatant samples were taken simultaneously for RT-qPCR analysis , as calculated relative to the zero hour time point. Furthermore, relative 7SL-sRNA levels appeared to increase correlating with cell density   , an initT 2/133) . This obT 2/133) . Indeed,Interestingly, in one case , 7SL-sRNA was detected after 42 days, suggesting a relapse. Almost 15 days later, live trypanosomes were observed by microscopy, after which the infection was once again cleared . A similin vivo, as successful drug treatment rapidly leads to the loss of signal, suggesting active infections are required to sustain the high abundance of the 7SL-sRNA. This further highlights the potential of 7SL-sRNA as a diagnostic for active trypanosome infections, rather than simply exposure such as is observed using antibody-based serological tests.Importantly, these results indicate that 7SL-sRNA is short-lived T. vivax (STIB 719) were also tested using the T. vivax-specific RT-qPCR assay  over a period of 85 days post-infection. Treatment was administered after peak parasitaemia was observed by microscopy, typically after ~14 days. For the T. vivax trials, rescue treatment was administered if the trial compound failed.Samples from a second clinical trial involving 21 cattle experimentally infected with CR assay . As withT. congolense, 7SL-sRNA was detected by the first available time point, and once again mirrored parasitaemia observed by microscopy. In most cattle, T. vivax was cleared post-treatment , and the 7SL-sRNA signal was absent at the next sampling timepoint .As demonstrated for However, in several cases, the presence of 7SL-sRNA was detected in time points where no parasites were observed by microscopy, again highlighting the increased sensitivity exhibited by the RT-qPCR compared to microscopy. Indeed, in these cases, such as animals 498, 524 and 527, 7SL-sRNA appeared to indicate cyclical changes in parasitaemia commonly associated with trypanosome infections .The above theory was further strengthened when investigating several animals that suffered from relapse of infection due to treatment failure, as confirmed by microscopy  . In animTherefore, by RT-qPCR analysis of a highly abundant secretory/excretory small RNA, infection status was confirmed more accurately than by microscopy. Further analysis is required, ideally with time courses that include frequent sampling of host serum pre- and post-treatment, including at subtherapeutic doses, in order to accurately determine both the kinetics of decay of 7SL-RNA signal after successful treatment and the association between treatment failure, parasite dynamics and 7SL-RNA signal.Whilst our data suggests that the 7SL-sRNA presents a realistic target for development of a molecular diagnostic for both HAT and AAT, in reality for diagnostic assays to be field-applicable in the settings in which both diseases occur, an assay requires several attributes: high on this list are two related aspects—low cost and minimal number of processing steps. A two-step RT-qPCR involving a lengthy RNA extraction protocol is therefore not desirable. We therefore investigated whether a one-step RT-qPCR would simplify the assay and if the RNA could be detected directly from serum samples without the requirement for RNA extraction .T. brucei infected animal 6632  were re-tested using the single step assay . Therefore, sensitive and specific markers of active infection are still required for both HAT and AAT.To meet the challenges of both elimination of HAT and management of AAT, improved diagnostic techniques are crucial. Current methods of diagnosis are suboptimal, particularly for AAT, and this is hindering progress on reducing the disease burden , 4. TradT. b. gambiense, at physiologically relevant abundances both in vivo and in vitro. Crucially, the RNA, termed 7SL-sRNA, is indicative of active infection and is detected even when parasitaemia is below levels detectable by microscopy. Furthermore, successful drug treatment results in rapid loss of detectable 7SL-sRNA signal, thereby exhibiting a key characteristic of a marker that correlates with active infection.In this study we identified a species-specific small RNA excreted/secreted by all three AAT relevant species, as well as the main causative agent of HAT, T. b. gambiense (ELIANE) grown in vitro, and the level of signal was the same as observed with T. b. brucei. Developing a diagnostic for HAT that is able to detect both T. b. gambiense and T. b. rhodesiense would be greatly beneficial, as currently there is no molecular test that is widely used in the field for T. b. rhodesiense diagnosis [Whilst this study was able to utilise samples from multiple experimental animal trials, human blood or CSF samples that would be suitable for testing could not be identified, although we hypothesise the 7SL-sRNA would also be present at similar levels in these types of clinical samples in human patients–we tested culture supernatant from iagnosis .Whilst the 7SL-sRNA is detectable using laboratory PCR machines, these assays are clearly not field applicable in their current state, and further work must be carried out to adapt the assays  platform; see below) to be field applicable. However, there have also been considerable efforts to exploit differential host small RNA levels as biomarkers of disease states in human medicine, in particular miRNAs in cancer, which has led to several other potentially field-applicable diagnostic techniques that could be adapted to detect 7SL-sRNA.Several other technologies have the potential to be alternative platforms suitable for small RNA detection. In particular, the LAMP assay, which has been previously developed for all three livestock trypanosome species based on gDNA targets , 40, 41 Leishmania spp. [Trypanosoma cruzi, a closely related pathogen that causes Chagas’ disease in South America [The discovery of a small RNA secreted/excreted by African trypanosomes at high abundance also raises interesting and potentially important biological questions. There is currently a great deal of interest in the ability of pathogens to communicate with each other and to manipulate host functions through the delivery of small RNAs . Communinia spp.  and Tryp America . HoweverLeptomonas collosoma [T. brucei [In addition to the potential biological role of 7SL-sRNA, how the small RNA is processed and released from trypanosomes is yet to be determined. The existence of a potential passenger strand could suggest a role for DICER in processing the larger 7SL RNA, as this type III endonuclease has previously been shown to process mammalian 7SL RNA . Intriguollosoma , sRNA-76. brucei ). The tRT. brucei, T. congolense and T. vivax during infections. Species-specific RT-qPCR assays were developed, and we have shown that there is good correlation between 7SL-sRNA levels and parasite numbers, but importantly 7SL-sRNA can be detected both before patent parasitaemia and during phases of infection when parasitaemia becomes subpatent (both chronic infection and treatment failure), and critically the 7SL-sRNA signal decays rapidly after successful chemotherapy. Therefore, 7SL-sRNA represents a marker of active infection, and is a novel and viable target for the development of much needed diagnostics for both HAT and AAT, and may also provide insights into important host-pathogen interactions.In summary, we have detected a trypanosome small RNA (7SL-sRNA), derived from the non-coding 7SL RNA of the SRP, which is excreted/secreted at high levels by S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 FigT. congolense KONT2/133, and subsequently divided into four groups depending on a treatment regimen with a candidate trypanocide. Data for the remaining 13 cattle are shown. Parasitaemia scores (right axis) were measured by microscopy every two to three days, indicated by grey bars; approximate equivalent parasitaemia (parasites/mL): 1 = 1 x 102; 2 = 1 x 103; 3 = 1 x 104; 4 = 1–5 x 105; 5 = 5 x 105–5 x 106; 6 = >5 x 106; grey bars measuring zero (red line) indicate where parasitaemia was measured but not detected, and no bar indicates that parasitaemia was not measured. Plasma samples were obtained at longer intervals (approximately weekly) from which RNA was extracted; 7SL-RNA RT-qPCR results (left axis) are shown by the black line graph, and and were calculated by normalising to an uninfected serum control; green diamond indicates day when animal was treated with a rescue drug (isometamidium chloride or diminazene aceturate) and red diamond indicates when test drug was administered.Twenty-one cattle were challenged with (PDF)Click here for additional data file.S2 FigT. vivax, and subsequently divided into four groups depending on a treatment regimen with a candidate trypanocide. Data for the remaining 13 cattle are shown. Parasitaemia scores (right axis) were measured by microscopy every two to three days, indicated by grey bars; approximate equivalent parasitaemia (parasites/mL): 1 = 1 x 102; 2 = 1 x 103; 3 = 1 x 104; 4 = 1–5 x 105; 5 = 5 x 105–5 x 106; 6 = >5 x 106; grey bars measuring zero (red line) indicate where parasitaemia was measured but not detected, and no bar indicates that parasitaemia was not measured. Plasma samples were obtained at longer intervals (approximately weekly) from which RNA was extracted; 7SL-RNA RT-qPCR results (left axis) are shown by the black line graph, and and were calculated by normalising to an uninfected serum control; green diamond indicates day when animal was treated with a rescue drug (isometamidium chloride or diminazene aceturate) and red diamond indicates when test drug was administered.Twenty-one cattle were challenged with (PDF)Click here for additional data file.


The RNA Characterization of Secondary Structure Motifs, RNA CoSSMos, database is a freely accessible online database that allows users to identify secondary structure motifs among RNA 3D structures and explore their structural features. RNA CoSSMos 2.0 now requires two closing base pairs for all RNA loop motifs to create a less redundant database of secondary structures. Furthermore, RNA CoSSMos 2.0 represents an upgraded database with new features that summarize search findings and aid in the search for 3D structural patterns among RNA secondary structure motifs. Previously, users were limited to viewing search results individually, with no built-in tools to compare search results. RNA CoSSMos 2.0 provides two new features, allowing users to summarize, analyze and compare their search result findings. A function has been added to the website that calculates the average and representative structures of the search results. Additionally, users can now view a summary page of their search results that reports percentages of each structural feature found, including sugar pucker, glycosidic linkage, hydrogen bonding patterns and stacking interactions. Other upgrades include a newly embedded NGL structural viewer, the option to download the clipped structure coordinates in *.pdb format and improved NMR structure results. RNA CoSSMos 2.0 is no longer simply a search engine for a structure database; it now has the capability of analyzing, comparing and summarizing search results.http://rnacossmos.comDatabase URL:  A deep understanding of the primary, secondary and tertiary structures of RNA and the relationship between these levels of structure is essential to understand and predict how RNA folds, gain knowledge of its biological functions and design potential therapeutics. A variety of approaches have been taken in predicting RNA 3D structure from sequence, with template-based approaches resulting in a particularly high level of accuracy . A templMC-Search  database was created through a manual classification and inspection of RNA structures , 3. The C-Search , 12. MC-e motifs , 12. SubAnnotate , 12 was ractions , 12. The*.pdb format and (vi) the addition of the model numbers of NMR results to the database. The RNA CoSSMos website has been moved to a faster webhost and is now available at http://rnacossmos.com.Since the RNA CoSSMos release in 2011, the RNA-containing structures in the PDB have increased from 2156 to over 3909, leading to subsequent growth of the RNA CoSSMos database. An RNA CoSSMos search returns results which can be viewed individually on the website. For searches that return a large number of results, individual viewing can become a daunting task. Additionally, studies that search for tertiary structural patterns among secondary structure motifs, such as those performed on single mismatches , have shIn addition to viewing search results individually, users can now further explore their search results in the ‘Summarize Results’ tab. Two features have been added to the website to allow users to obtain a summary of their search results: (i) an average structure feature and (ii) a results summary feature. These features (described below) can be chosen together or independently, depending on the needs of the user. Users can further customize their summary by selecting which structures in their search results should be included in the summary calculation. If the user’s search contains ≤350 results, they can select the desired results using check boxes. Alternatively, the user can download the search results as a delimited spreadsheet, edit the file to select for results to include in the summary and then upload the file to the RNA CoSSMos website. An option is also available to include all of the search results in the summary calculations.*.pdb file. In addition to the average structure, the root mean square deviation (RMSD) of each result structure compared to the average structure is calculated, and the result that has the smallest RMSD to the average structure is reported as the representative structure. The representative structure can also be viewed on the website and downloaded as a *.pdb file  phosphate atoms only, (ii) phosphate-sugar backbone atoms or (iii) phosphate-sugar backbone and bases. If users select the ‘phosphate-sugar backbone and bases’ option, all of the atom coordinates from the backbone and three atom coordinates from each base  are used in the calculation to allow users to calculate average structures for results with differing sequences. To calculate the average structure, the coordinates are superimposed using the Kabsch algorithm  to calcupdb file .MC-Annotate results into tables that report percentages of sugar puckers, glycosidic linkages, hydrogen bonding patterns and stacking interactions within the loop and closing base pairs across all of the selected RNA CoSSMos search results. These percentages are reported for individual features (e.g. the sugar pucker of the first base in the loop) and combinations of structural features (e.g. the residue conformations of the first and last bases in the loop and hydrogen-bonding interactions between these bases). The tables utilize the residue numbering system described in this paper and on the RNA CoSSMos FAQ wiki page (http://rnacossmos.com/faq2.php). For user convenience, the tables can be expanded and collapsed so that users can focus on the structural annotations of greatest interest  contained a hairpin of seven nucleotides in residues A1387–A1395, a hairpin of three nucleotides in residues A1388–A1392 and a bulge of two nucleotides in residues A1387–A1388 and A1392–A1395 . To redu folding . With thIn order to compare the structures and structural features of the results, a common residue numbering system was necessary. The residue ID numbers within each result were renumbered as follows . For haihttp://nglviewer.org) which does not require the use of Java or plugins. The NGL molecular viewer has replaced the Jmol viewer on the individual detailed results pages and is also utilized to view the average and representative structures. Previously, users could download a snapshot of the Jmol viewer. Now, users can download a clipped *.pdb file of the motif structure in which the residue ID numbers have been renumbered according to the residue numbering system described in this paper. This will allow users to view and manipulate the clipped structures on their own computers using their preferred software in addition to 3D structure viewing on the RNA CoSSMos website. Additional viewing options allow users to see individual motifs and representative structures embedded within the global structure found in the *.pdb file.RNA CoSSMos previously utilized a Jmol applet  for 3D sModel numbers corresponding to those in the original PDB structure have been added to the RNA CoSSMos 2.0 database for ensemble structures solved by NMR. For each NMR ensemble within each motif, the model that is closest to the average of the ensemble results is selected as the representative model for the ensemble. The representative models are denoted in the database with an ‘*’ after the model number. Users can view the detailed results of the individual models or can utilize the new summary features to learn about structural information within the ensemble or across multiple selected ensembles. When a user wishes to utilize the ‘Summarize Results’ tab with results that contain NMR structures, the user is given the option to include all of the models within the ensemble or only the representative model in the summary calculations.Hairpins that contain four bases in the loop are commonly referred to as tetraloops. Tetraloops with the sequence GNRA  often form an unusually stable 3D fold that is well-documented , 22. To The new RNA CoSSMos database continues to provide users with a unique online tool to search for 3D structure characteristics of RNA secondary structure motifs using a simple and intuitive graphical user interface but now allows its users to explore and compare structural features and patterns among their customized set of search results in addition to viewing the results individually. Additional updates not described here to improve ease of use, user customization and database completeness and accuracy have been made to ensure that the database remains a reliable and valuable tool for the scientific community. In 2014, the PDBx/mmCIF file format became the standard PDB format to allow for large structures that cannot be represented in the *.pdb file format. Currently, ~6% of RNA containing structures are only offered in the PDBx/mmCIF file format. Future versions of the RNA CoSSMos database will incorporate the PBDX/mmCIF file format, and future directions of the database will be driven by feedback from the users. As the PDB continues to grow, so will the RNA CoSSMos database and its capabilities.http://rnacossmos.com. We will maintain the redirection from the previously used URL (http://cossmos.slu.edu) for the foreseeable future, but users are encouraged to update their links accordingly.The RNA CoSSMos 2.0 database is freely available online and is now hosted at a new URL


Long non-coding RNAs (lncRNAs) are emerging as important regulators of cellular processes and are extensively involved in the development of different cancers; including leukemias. As one of the accepted methods of lncRNA function is affecting chromatin structure; lncRNA binding has been shown for different chromatin modifiers. Histone lysine methyltransferases (HKMTs) are also subject of lncRNA regulation as demonstrated for example in the case of Polycomb Repressive Complex 2 (PRC2). Mixed Lineage Leukemia (MLL) proteins that catalyze the methylation of H3K4 have been implicated in several different cancers; yet many details of their regulation and targeting remain elusive. In this work we explored the RNA binding capability of two; so far uncharacterized regions of MLL4; with the aim of shedding light to the existence of possible regulatory lncRNA interactions of the protein. We demonstrated that both regions; one that contains a predicted RNA binding sequence and one that does not; are capable of binding to different RNA constructs in vitro. To our knowledge, these findings are the first to indicate that an MLL protein itself is capable of lncRNA binding. Long non-coding RNAs (lncRNAs) are transcribed RNA molecules longer than 200 nucleotides that do not code for translated proteins. The human genome is estimated to code for about 58,000 lncRNAs , that arThe physiological processes where lncRNA regulation have been suggested involve cell cycle regulation, epithelial mesenchymal transition (EMT) , cancer Various lncRNAs are shown to have altered expression levels in different leukemias, resulting in a crucial influence on cellular transformation , chromosAs lncRNAs are also considered valuable drug targets, it is essential that the molecular details of their functions are uncovered.Polycomb repressive complex (PRC2) is the most studied histone modifier that relies on lncRNA binding in its function, being able to bind several lncRNAs including HOTAIR, Xist, RepA, Braveheart, MALAT1 and MEG3 . In vitrApart from PRC2, other histone lysine methyltransferases (HKMTs) or HKMT complex components also appear to bind lncRNAs with a relevant physiological outcome.LncRNA EZR-AS1 enhances EZR expression through recruiting SMYD2 to the upstream region of its promoter region and elevating the activating H3K4 methylation .G9a interacts with lncRNA PARTICLE to regulate MATA2 expression upon mild irradiation . The intWDR5, a component of the MLL1-4 and SET1a/1b complexes was proven to interact with lncRNAs NeST and HOTTIP with an effect on microbial susceptibility through the enhancement of interferon-γ expression . FurtherThe family of mammalian MLL (Mixed Lineage Leukemia) proteins consist of Set1a, Set1b and four MLL proteins, MLL1, MLL2, MLL3 and MLL4. They work in COMPASS-like complexes and catalyze H3K4 mono-, di- or tri-methylation, each complex having different specificity and methylase activity . MLL3 anIn a previous work  we suggeSince multiple evidence point in the direction that leukemic processes are fundamentally affected by lncRNAs and MLL complexes are involved in this regulation, we concentrated on MLL proteins. Taken the analogy of the PRC2 complex, where more than one complex subunits are capable of lncRNA binding, we aimed at testing the ability of MLL4 to bind different RNA molecules.As a first step, we mapped the predicted RNA binding motifs on the sequence of four MLL proteins. We used DisoRDPbind, an RNA interaction prediction tool specifically designed to find RNA interaction sites in the disordered regions of proteins. Results shown in A comparison with our earlier studies  revealed440) that encompasses the 300 nucleotides already known to be involved in protein-RNA interactions and also the nucleotides that has not been studied yet. Since there is no information available about the region of MEG3 that is able to bind proteins, we used the full length MEG3 for our experiments.As for binding RNAs, we opted to test two different lncRNA constructs, both having been reported to play a role in leukemias. The first is HOTAIR, that has the ability to bind EZH2 (PRC2). The 5’ 300 nucleotides of HOTAIR are thought to mediate its binding to PRC2 complex subunits, but the latest annotation in the NCBI database contains an additional 140 bases at the beginning of HOTAIR sequence, compared to the one reported earlier. Therefore, we prepared the longer version  of the samples resulted in the loss of fluorescent signal in a protein concentration-dependent manner  experiments confirmed the findings of the MST measurements  as both and MEG3 A,B RNAs.and MEG3 C, result3500–3630:HOTAIR440 interaction observed in MST was seen in the EMSA experiments as well, since at high protein:RNA ratios the samples obtained a highly viscous quality and completely remained in the wells during the electrophoretic run. Successful experiments could only be carried out by lowering the applied protein concentration, but the interaction was clearly observable even under these circumstances.The anomalous behavior of the MLL43500–3630, which contains a predicted RNA binding region presented higher affinities to RNAs than the other MLL4 segment, indicating the validity of the prediction. On the other hand, binding of MLL44210–4280 could also be detected in all cases, raising the possibility of the existence of RNA binding sequences differing from the already described interaction motifs. EZH2, a known RNA binding HKTM also interacts with RNAs through a region [In all of the tested interactions, MLL4a region  that hasHistone methylation is one of the most studied and best-characterized histone modifications that drive the regulation of complete genetic programs in the cells. However, many details of the regulation and targeting of the enzyme complexes mediating histone methylation remain elusive and a subject of debate . One pos4210–4280 bound MEG3 stronger than HOTAIR440 or the 50 nt random RNA, even though the determination of the exact Kd-s was not successful in all cases.Our expectation was that the isolated small regions of the MLL4 protein would bind RNAs in a nonspecific manner, such as was observed for the isolated PRC2 complex components . Surpris3500–3630 region that showed dramatically different behavior with the different RNAs. Binding to MEG3 gave a Kd of 0.722 μM, while the binding to the 50 nt random RNAs proved to be so weak that a Kd calculation was not successful. Binding to HOTAIR440 seemed to be the strongest with an apparent Kd of 0.1 μM, but it led to the aggregation of the protein-RNA complex. The aggregation was dependent on protein-RNA ratio and could be detected through a wide protein concentration range. The same aggregation could not be observed with a shorter HOTAIR construct that consisted of 300 bases  [http://biomine.cs.vcu.edu/servers/DisoRDPbind/) [Disorder and disordered binding site predictions were performed with the IUPred2A online prediction tool (lte.hu/)  which inDPbind/) . Cancer-DPbind/)  and COSMDPbind/) .HOTAIR: Gene ID: 100124700MEG3: Gene ID: 55384MLL1: Uniprot: Q03164MLL2: Uniprot: Q9UMN6MLL3: Uniprot: Q8NEZ4MLL4: Uniprot: O146863500–3630 and MLL44210–4280. DNA sequences coding for each protein were cloned into pET22b cloning vector. Induction was done for 4 h at 28 °C by 0.1 M IPTG, cells were pelleted by centrifugation  then lysed by sonication in lysis buffer , cell debris was removed by centrifugation . The supernatant was filtered through 0.2 μm nitrocellulose filter then purified over HisTrap HP column on an AKTA Explorer system using a gradient elution of two buffers . Representative purification results are shown on 4210–4280 region was highlighted by its appearance at a larger size than its actual molecular weight (17 kDa vs. 7 kDa). Elution fractions containing sufficiently pure proteins were dialyzed against distilled water then lyophilized and stored at −20 °C. Lyophilized proteins were dissolved before use in ultrapure water or the appropriate assay buffer. The identity of the purified proteins was confirmed by mass spectrometry.The same methods of protein overexpression and purification were used for both protein constructs, MLL4HOX transcript antisense RNA (HOTAIR):300 (140–440 nt) and HOTAIR440 (1–440 nt) DNA sequences cloned into pEX-A128 vector were purchased from Eurofins Genomics . After 2 h digestion with EcoRV restriction enzyme at 37 °C, the gel-purified, linearized DNA templates were used to synthesize RNA by in-vitro transcription.HOTAIRMaternally Expressed 3 (MEG3) lncRNA:pCI-MEG3 was a gift from Anne Klibanski  . PrimersT7 RNA promoter region followed by:MEG3 forward primer:TAATACGACTCACTATAGGGGCAGAGAGGGAGCGCGCCTTGGMEG3 reverse primer:GATATCTTTTTGTTAAGACAGGAAACACATTTATTGAGAGC50 nt RNA:50 nucleotide RNA was an artificial randomized RNA sequence.DNA templates were T7 promoter region followed by:50 nt forward oligo:TAATACGACTCACTATAGAAGAATGGCCTCGCGGAGGCATGCGTCATGCTAGCGTGCGGGGTACTCTT and50 nt reverse oligo:AAGAGTACCCCGCACGCTAGCATGACGCATGCCTCCGCGAGGCCATTCTTCTATAGTGAGTCGTATTATranscribed RNA:GAAGAAUGGCCUCGCGGAGGCAUGCGUCAUGCUAGCGUGCGGGGUACUCUUAll primers and oligonucleotides were purchased from Sigma-Aldrich Ltd. .® RNA Clean-up XS Kit . The quality and intactness of the purified transcription products were analysed by native and formaldehyde agarose gel electrophoresis.Tested RNAs were synthesized by in vitro transcription carried out with New England BioLabs HiScribe™ T7 Quick High Yield RNA Synthesis Kit . Fluorescein-labelled, single-stranded RNA probes were generated by using Roche  Fluorescein RNA Labeling Mix (11685619910) and NEB 10× T7 reaction buffer (#B2041A). After transcription, remaining DNA templates were eliminated with DNaseI treatment. RNA sample purification was carried out using Macherey-Nagel NucleoSpinBiotinylation of the RNAs was performed using Pierce™ RNA 3′ End Biotinylation Kit  according to the instructions of the manufacturer. Overnight incubation at 16 °C was applied for the ligation of the biotin label. Final RNA concentrations were determined using NanoDrop™ 1000 Spectrophotometer .Purified RNAs were stored −80 °C until usage in the presence of RNAINH-RO Roche Protector RNase Inhibitor (20U).http://bestsel.elte.hu).CD measurements were performed in quartz cells of 0.1 mm pathlengths using a Jasco J-810  spectropolarimeter. Far-UV CD spectra were recorded in the range of 180–260 nm with a scanning speed of 20 nm/min, bandwidth of 1 nm and integration time of 4 s. 6 scans were accumulated. Thermal denaturation was recorded in a 1 mm cell at 220 nm from 10 to 100 °C with scanning rate of 120 °C/h. The temperature was controlled using a PTE Peltier unit. The thermal denaturation profile was fitted according to the Gibbs-Helmholtz equation assuming a two-state model, which is represented by a sigmoidal curve . CD specRNA-protein binding assays were carried out on a Microscale Thermophoresis system . Standard treated capillaries (Cat. Number: MO-K002) were used for measurements. Instrument settings are presented in Normalized fluorescence values after 1.25 s after turning on the IR laser were used as T-jump values.RNA concentrations were set to give an initial raw fluorescence between 300 and 1000 counts and varied between 30 and 100 nM. All experiments were done at room temperature. DEPC-treated PBS buffer containing 0.05% NP-40 was used as assay buffer.® Chemiluminescent RNA EMSA Kit  was used for the EMSA experiments. Assay control was performed according to the instructions of the manufacturer with the control reaction provided with the kit. In short, 6.25 nM biotin-labeled IRE RNA was incubated with 2 μg of cytosolic liver extract with or without 1 μM of unlabeled IRE RNA. The result of the assay control is presented on ® Turbo™ Transfer System  and crosslinked to the membrane by UV-light crosslinking. After proper washing and blocking, biotin labeled RNA was detected by chemiluminescence using Streptavidin-Horseradish Peroxidase Conjugate.LightShift


Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs ( Virus-induced diseases cause significant reductions in both crop quality and yield worldwide ,2. The gvsiRNAs) of 21 and 22 nt, respectively  . RISC-meesiRNAs, act antivirally and support RNA slicing . Cytoplasmic extract (BYL) was prepared from the evacuolated cells as described  ribozyme right downstream of the TBSV cDNA. This was done by PCR amplification of the ribozyme-encoding cDNA from plasmid pWNVRepliconHDVr (Cauliflower mosaic virus (CaMV) 35S promoter module into the BsaI-cut binary vector pVM-BGW (To generate a cDNA clone encoding TBSV (−)-strand, the complete TBSV (T100) cDNA was PCR-amplified  with a reverse primer containing an upstream T3 promoter sequence and a forward primer that introduced an additional XbaI site adjacent to the TBSV sequence. By ApaI/SmaI cloning, the original TBSV cDNA sequence including the T7 promoter was removed from plasmid A and replaced by the PCR fragment generating plasmid B. To generate GFP mRNA fragments containing complementary sequences of TBSV siRNAs, we first replaced the target site of gf698 siRNA in plasmid pGFP-C1 by two BpiI sites. This was done by inserting two PCR fragments between Eco72I and BamHI. The cDNA fragments (double-stranded oligonucleotides) encoding the respective TBSV target site sequences were then inserted into the BpiI-digested plasmid. To generate a binary vector for the expression of TBSV genomic RNA in plants, we first cloned a sequence encoding the iconHDVr , followe pVM-BGW .AGO mRNAs were synthesized in the presence of monomethylated cap analog m7GP3G  from SwaI-linearized plasmid constructs (in vitro transcription in the presence of 0.5 μCi/μl [α-32P]-CTP (3000 Ci/mmol). dsRNAs were produced by mixing equimolar amounts of sense and antisense transcripts in STE buffer , heating for 2 min at 94°C and decreasing the temperature to 25°C within 30 min. The ds nature of the annealed RNAs was confirmed by treatment with 0.05 U/μl RNase T1 (Thermo Fisher Scientific) for 15–60 min at 37°C and subsequent gel analysis.nstructs ,28 usingnstructs . TBSV  BYL, using conditions previously described  BYL under the above-described conditions. Samples were mixed with an equal volume of immunoprecipitation buffer  NP-40, 1 mM DTT) and 20 μl anti-FLAG M2 affinity gel . Following overnight incubation at 4°C with gentle agitation, the resin was washed four times with IPB and once with IPB containing 300 mM NaCl. RNA was isolated and analyzed as described above for the DCL assay. Data were obtained from two different AGO1 and three different AGO2 experiments.To generate siRNA-programmed AGO/RISC https://github.com/najoshi/sickle). The remaining and truncated reads were then mapped onto a combined reference of the tobacco genome (Ntab-BX_AWOK-SS.fa) and the TBSV sequence (TBSV.fa) using the bowtie aligner (v1.1.2) (http://www.R-project.org/) and the R package Rsamtools (http://bioconductor.org/packages/release/bioc/html/Rsamtools.html). The set of mapped reads were partitioned into six sets depending on the read-lengths  and the mapped strand (positive or negative), and the starting positions of the mapped reads were counted as peak expression. Reads were stored in 5′ to 3′ orientation in the Sequence Alignment Map (SAM) format, so the first (last) nucleotide was counted for reads mapped onto the positive (negative) strand. All statistical analyses were performed using custom R scripts executed in R Studio Server. Each of these error bars was calculated as standard error of the mean applying the std.error function of the plotrix (Raw reads were adaptor clipped using cutadapt (v1.12)  and qual(v1.1.2)  allowing(v1.1.2)  (-view - plotrix  package.The control siRNA gf698 siRNA targeting the GFP mRNA was described earlier . RNA oliN. benthamiana AGO1L or AGO2 mRNA (32P-labeled target RNA (20 fmol) were added, and the cleavage reaction performed for 15 min. Total RNA was isolated by treating the reaction with 20 μg proteinase K in the presence of 0.5% SDS for 30 min at 37°C, followed by chloroform extraction and ethanol precipitation. RNAs from assays with full-length TBSV RNA as target were separated on 1.5% denaturing agarose gels; all other RNA samples were separated on 5% TBE polyacrylamide gels containing 8 M urea. 32P-labeled target RNAs and cleavage products were visualized by phosphor-imaging .AGO/RISC programmed with a specific siRNA were generated as described above in a 20 μl reaction containing 50% (v/v) BYL, 0.5 pmol GO2 mRNA ,34 and 1A. tumefaciens strain GV3101. Individual colonies were grown overnight in selective medium and subsequently used to inoculate 30-ml induction medium . Cultures were grown overnight at 30°C until they reached an OD600 of 0.5–0.7, harvested by centrifugation for 10 min at 1780 g and re-suspended in an appropriate volume of infiltration medium  to obtain an OD600 of 1.0. Suspensions were incubated at room temperature for at least 3 h and 2 ml were infiltrated into two leaves of 4- to 6-week-old N. benthamiana plants using a 1 ml needleless syringe. Forty-eight hours later, each of the two leaves was infiltrated with two times 50 μl A. tumefaciens carrying a binary vector containing the full-length TBSV (T100) cDNA downstream of a CaMV 35S promoter, followed by a Hepatitis delta virus ribozyme and a 35S terminator sequence. Bacterial suspensions were prepared as described above; shortly before infiltration, they were 1:1000 diluted with cultures containing an mCherry ORF instead of TBSV cDNA. After infiltration, plants were grown in a chamber  for 14 h at 23°C, 90–100 μmolm−2s−1 light (at shelf level) and for 10 h at 21°C in the dark. Plants were daily monitored for symptom development for 5 weeks (25 days in case of the RDR6i plants). Mechanical inoculation of TBSV to N. benthamiana plants was performed by using infectious in vitro transcripts. Prior to the application of the viral RNA, the leaves were dusted with carborundum powder. TBSV RNA (2 ng/μl) was mixed with an equal volume of inoculation buffer  and 2.5 μl two times rubbed onto the surface of the previously infiltrated leaves. The treated leaves were rinsed with water and the plants grown and monitored as described above.To express selected siRNAs as artificial microRNAs in plants, the corresponding sequences were introduced into the binary vector pMDC32B-AtMIR390a-B/c  and the esiRNAs, we used an in vitro system of cytoplasmic extracts from N. tabacum BY-2 protoplasts (in vitro-translated AGO protein of choice (in vitro-generated RISC can be tested in a ‘slicer assay’ with a chosen target RNA (Tomato bushy stunt virus (TBSV)   as a modin vitro transcription and annealing of RNA molecules corresponding to the full-length (+)-strand TBSV genome (4778 nt) and the complementary (−)-strand replication intermediate .Considering its capability of acting as a silencing inducer and its homogeneous composition consisting of equal quantities of (+)- and (−)-strand RNA, we performed all of the experiments described below with TBSV dsRNA molecules. We generated the dsRNAs by in vitro translation of proteins in the extract , these data support the idea that GC-rich sequences are moderately preferred DCL target sites.In the subsequent analyses, we focused on the 21 nt siRNAs as the most important antiviral siRNA species ,39,40. Iet al.  with Turin vitro assembled AGO/RISC can be isolated from BYL . Next, we isolated the generated siRNA-associating (‘programmed’) AGO1/RISC via immunoprecipitation using an anti-FLAG affinity gel and demonstrated it to be functional in a slicing assay with the GFP mRNA -strand viral RNA molecules are accessible to RISC-mediated degradation, whereas the (−)-strand intermediates are not . This obIn order to gain initial insights into the properties of NGS-associated (-)vsiRNAs, we first classified them according to the following criteria Table . In a fiesiRNAs, we tested the two or three top candidates of each dataset  attempt to identify sites in the TBSV genome that are particularly susceptible to RISC and to (ii) attempt to identify those vsiRNAs that target these sites. Since in vitro slicing experiments with the full-length TBSV genome have been found to be too insensitive for the detection of cleavage products produced by individual species of a vsiRNA pool  in the guide strand, and a complementary A (instead of a U) in the passenger strand. Similarly, we modified the vsiRNA179 (AGO1-selected) by changing the 5′U of the guide strand to an A and the corresponding A of the passenger strand to a U. By changing the 5′ nucleotide, which is known to have no effect on target interactions of the siRNAs , the AGOesiRNA209 was functional with AGO1, and the 5′A-modified esiRNA179 was functional with AGO2. However, when we applied ‘non-cognate’ 5′ nucleotide constellations of esiRNA179 with AGO1 or AGO2 and esiRNA209 with AGO1, we detected considerably lower slicing activities. The scenario was different with esiRNA209: in tests with AGO2, this RNA was very active, regardless of the 5′ nucleotide selected, which suggested a higher flexibility of AGO2 in the uptake of siRNAs with different 5′ termini  and AGO2-specific RNAs (esiRNA209 and esiRNA3243) and that we observed to be the most effective in the in vitro slicer assays , about 90% of the plants ‘vaccinated’ with esiRNA209 remained unaffected. Correspondingly, esiRNAs 3243, 3939 and 179 yielded protection rates of 76%, 53% and 41%, respectively. The protection rate of the vsiRNA mixture was comparable to that of the individual esiRNA209  of the viral RNA . Other methods identified functional siRNAs from mapping analyses of RISC-cleavage sites in viral RNAs isolated from infected plants  plants are replaced by a rapid assay procedure. The BYL imitates the plant’s silencing response rather effectively and besides reproducing the DCL-mediated RNA processing, the system enables specific screening with defined assembled AGO/RISC species, with pools or with single small RNAs and with target RNAs of choice. By limiting the screen to (-)vsiRNAs, we were able to further focus the esiRNA identification procedure on those candidates that target the silencing-accessible parental and/ or progeny viral (+)-strand RNA genomes . The particular accessibility of the TBSV 5′-end to RISC may also be explained by the involvement of this region in translation initiation. For example, it is conceivable that the long-range RNA–RNA interactions of a stem–loop structure close to the immediate TBSV RNA’s 5′-terminus with the CITE  in the 3′UTR that enables translation initiation and/or ribosome movement  were almost completely protective and that the protective potential was not further increased by the use of an esiRNA mixture . A conceivable, alternative form of application is to generate and transiently apply short dsRNA molecules that consist almost exclusively of a few esiRNA sequences. Following topical application and uptake, which may be achieved in different ways . However, future experiments will have to show whether 21 nt or perhaps 22 nt esiRNAs in this or other forms of application will also allow longer-lasting protection against viral pathogens than is possible in current transient approaches.A very obvious way to generate virus-resistant plants would be the transgenic expression of identified ent ways , these desiRNA identification screen, all types of RNA applications may now be quickly adapted to modified virus forms. In particular, we expect that the use of combinations of esiRNAs not only increases the speed and flexibility of transient treatments, but also significantly improves cross-protection against rapidly changing virus populations in transient as well as transgenic applications. Apart from the identification of esiRNAs that target viral RNAs, we anticipate this approach to be broadly applicable to a wide range of different functional RNAs. For example, it is conceivable that esiRNAs directed against mRNAs of nematodes, fungi or other pests can be identified in a similar way and used in plant protection measures.Importantly, with the established NGS datasets have been deposited in GEO under accession number GSE121644.gkz678_Supplemental_FilesClick here for additional data file.


Escherichia coli host factor that was essential for replication of the bacteriophage Qβ, the Hfq protein is a ubiquitous and highly abundant RNA-binding protein in many bacteria. With the assistance of Hfq, small RNAs in bacteria play important roles in regulating the stability and translation of mRNAs by base pairing. In this study, we want to elucidate the Hfq-assisted sRNA-mRNA regulation in Yersinia pestis. A global map of Hfq interaction sites in Y. pestis was obtained by sequencing cDNAs converted from the Hfq-bound RNA fragments using UV cross-linking coupled immunoprecipitation technology. We demonstrate that Hfq could bind to hundreds of sRNAs and the majority of mRNAs in Y. pestis. The enriched binding motifs in sRNAs and mRNAs are complementary to each other, suggesting a general base-pairing mechanism for sRNA-mRNA interaction. The Hfq-bound sRNA and mRNA regions were both destabilized. The results suggest that Hfq binding facilitates sRNA-mRNA base pairing and coordinates their degradation, which might enable Hfq to surveil the homeostasis of most mRNAs in bacteria.Discovered in 1968 as an  in vivo binding nature remains elusive. Here we reported genome-wide Hfq-bound RNAs in Yersinia pestis, a causative agent of plague, by using cross-linking immunoprecipitation coupled with deep sequencing (CLIP-seq) approach. We show that the Hfq binding density is enriched in more than 80% mRNAs of Y. pestis and that Hfq also globally binds noncoding small RNAs (sRNAs) encoded by the intergenic, antisense, and 3′ regions of mRNAs. An Hfq U-rich stretch is highly enriched in sRNAs, while motifs partially complementary to AGAAUAA and GGGGAUUA are enriched in both mRNAs and sRNAs. Hfq-binding motifs are enriched at both terminal sites and in the gene body of mRNAs. Surprisingly, a large fraction of the sRNA and mRNA regions bound by Hfq and those downstream are destabilized, likely via a 5′P-activated RNase E degradation pathway, which is consistent with a model in which Hfq facilitates sRNA-mRNA base pairing and the coupled degradation in Y. pestis. These results together have presented a high-quality Hfq-RNA interaction map in Y. pestis, which should be important for further deciphering the regulatory role of Hfq-sRNAs in Y. pestis.Hfq is a ubiquitous Sm-like RNA-binding protein in bacteria involved in physiological fitness and pathogenesis, while its IMPORTANCE Discovered in 1968 as an Escherichia coli host factor that was essential for replication of the bacteriophage Qβ, the Hfq protein is a ubiquitous and highly abundant RNA-binding protein in many bacteria. With the assistance of Hfq, small RNAs in bacteria play important roles in regulating the stability and translation of mRNAs by base pairing. In this study, we want to elucidate the Hfq-assisted sRNA-mRNA regulation in Yersinia pestis. A global map of Hfq interaction sites in Y. pestis was obtained by sequencing cDNAs converted from the Hfq-bound RNA fragments using UV cross-linking coupled immunoprecipitation technology. We demonstrate that Hfq could bind to hundreds of sRNAs and the majority of mRNAs in Y. pestis. The enriched binding motifs in sRNAs and mRNAs are complementary to each other, suggesting a general base-pairing mechanism for sRNA-mRNA interaction. The Hfq-bound sRNA and mRNA regions were both destabilized. The results suggest that Hfq binding facilitates sRNA-mRNA base pairing and coordinates their degradation, which might enable Hfq to surveil the homeostasis of most mRNAs in bacteria.Author Video: An author video summary of this article is available. Both subclasses of sRNAs are capable of causing translation inhibition, mRNA cleavage, or degradation  sequences typical of rho-independent terminators, with uridine stacked in pockets between neighboring monomers around the central pole , 15–18. 15–19–22–nd mRNAs –29.Gammaproteobacteria is RNase E, which recognizes its substrates via two different modes of action    and anotLIP-seq) . ConsideY. pestis cells, we used two strains expressing a FLAG epitope. The experimental Hfq strain expressed Hfq-FLAG from a plasmid in a hfq deletion (Δhfq) genetic background (Hfq-FLAG). A wild-type (WT) strain with Flag epitope (WT-FLAG) was used as a control. Another control by transforming pHfq into a Δhfq strain (Hfq) was also obtained. A previous study has shown that the exogenously expressed Hfq-FLAG was functionally competent (R > 0.98) of these three strains  PAGE gel electrophoresis of cellular Hfq on Hfq-FLAG strain under nondenaturing conditions followed by Western blot analysis. 25 mM Tris-HCl (pH 8.0) was used as a control. (B) Gene expression correlation analysis between Hfq-FLAG and WT-FLAG by Pearson correlation. (C) Experimental procedure of CLIP-seq and RNA-seq. (D) Western blot of Hfq-RNA complex separated by SDS-PAGE by using anti-FLAG monoclonal antibody (left) and electrophoresis visualization of PCR products amplified from cDNA before (middle) and after (right) gel-cutting purification. (E) Enriched functional KEGG pathways of Hfq-bound and -unbound genes under Copyright © 2019 Han et al.2019Han et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Y. pestis 91001 genome  of all mRNA genes were enriched in the Hfq-FLAG strain. Transcriptome sequencing data from the two experimental controls . The CLIcontrols . Hfq-boucontrols . These rcpxP mRNA. The RNA-seq reads were spread throughout the coding region, while CLIP-seq reads peaked at the 3′ untranslated region (3′UTR) corresponding to the CpxQ sRNA  experiment (See methods). After coimmunoprecipitation by anti-FLAG using Hfq-FLAG and WT-FLAG cell lysates, we used SsrA sRNA as an unbound control to validate 15 randomly chosen Hfq-bound sRNAs and mRNAs, including Ffs, BtuB, CysT, FadB, FimA3, KatA, KdsA, SecE, SmpB, Tuf, YapA, YP-PMT054, and YP-PMT117. All of these Hfq-bound and unbound sRNAs and mRNAs were validated .To more globally validate the CLIP-seq results, we performed two independent sets of Hfq RIP-seq experiments with the same strains and growth conditions. A total of 9,186,327 and 26,971,805 clean reads were obtained from the Hfq-FLAG strains. The mapping features of RIP-seq data were similar to those of the CLIP-seq data . Moreove10.1128/mSystems.00245-19.2FIG S2Y. pestis genes from CLIP-seq, RIP-seq, and RNA-seq experiments. (B) Hfq binding of the intergenic and antisense regions. Bar plot presentation of the results from analysis of CLIP-seq and RIP-seq 2nd data. (C) Read density illustration of Y. pestis known sRNAs in CLIP-seq, RIP-seq, and RNA-seq experiments, including Spf, SsrS, CsrB, Ffs, MicF, and RnpB. A plus or minus sign behind experiment represents the detection state of sRNA in each experiment. Download FIG S2, TIF file, 4 MB.Sample correlation and sRNA prediction of CLIP-seq and RNA-seq data. (A) Three-dimensional correlation of the read density mapped onto all Copyright © 2019 Han et al.2019Han et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In order to better understand the length features of Hfq-bound intergenic and antisense RNAs revealed by CLIP-seq data, longer Hfq-bound RNA segments (with mean insertion size of 150 nucleotides [nt]) were selected for sequencing in RIP-seq experiments shown in this study. Without RNase T1 digestion, RIP-seq methodology preferred longer transcripts and selected against short sRNA transcripts. We found that 67.5% intergenic regions and 70.3% antisense regions showed Hfq-bound evidence from CLIP-seq data, while 49.1% and 30.4% corresponding regions obtained Hfq-bound evidence from one set of RIP-seq data . We alsoY. pestis. Compared to the 5′ leader regions . A total of 2,511 and 1,518 peaks were recovered from CLIP-seq and RNA-seq data, respectively . The dom10.1128/mSystems.00245-19.7TABLE S3Table S3, DOCX file, 0.02 MB.Peak summary and classification by region. Download Copyright © 2019 Han et al.2019Han et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Y. pestis transcriptome.Theoretically, CLIP-seq peaks indicated the Hfq-bound regions, while RNA-seq peaks indicated the steady-level transcripts. The latter is expected to cover the former. We then selected Hfq-bound peaks in which CLIP peaks containing fourfold-more CLIP-seq reads than RNA-seq reads, resulting in 1,499 qualified peaks. These peaks were defined as strong peaks, and other peaks from CLIP-seq were defined as weak peaks. Among these peaks, 1,168 overlapped the known genes, 131 overlapped the antisense strands, and 200 overlapped in the intergenic regions , showingY. pestis were conserved in short motif sequence composition but quite flexible in motif organizations. For example, the top motif AAUAA was highly represented in mRNAs, intergenic RNAs, and antisense RNAs  is a symbol of the Hfq-bound sRNAs. We found that the Uic peaks . As expend peaks , left. Nnd peaks , left. Ae 3′ end , right. nse RNAs . It is n stretch  to D. Th10.1128/mSystems.00245-19.3FIG S36 stretch frequency and position in antisense peaks of three samples, including CLIP-seq, RIP-seq, and RNA-seq (top). (B) U5 stretch frequency and position in intergenic peaks of the three samples. (C) U5 stretch frequency and position in antisense peaks of the three samples. (D) U5 stretch frequency and position in genic peaks of the three samples. (E) Venn diagram of the overlap among three different studies  U studies –49. (F) Copyright © 2019 Han et al.2019Han et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Y. pestis and E. coli showed that the above motif patterns were true for all mRNAs . Compared to the RNA-seq sRNA peaks, Hfq-bound sRNA peaks were closer to canonical transcription terminators, and most of them were located downstream of the predicted terminators .To further explore the above hypothesis, we separated sRNA peaks into three different classes. Stable non-Hfq-bound sRNA peaks (type I) have RNA-seq peaks only and have 156 sRNA members. Unstable Hfq-bound sRNA peaks (type II) have Hfq-bound peaks only and have 361 members. Stable Hfq-bound sRNA peaks (type III) have both Hfq-bound and RNA-seq peaks that overlapped by at least one nucleotide and have 93 members . Please 10.1128/mSystems.00245-19.4FIG S410P value). Download FIG S4, TIF file, 4.4 MB.Characterization of sRNAs bound by Hfq. (A) Violin plot of the length distribution of CLIP-seq and RNA-seq peaks. Peaks were divided into three classes: CLIP-specific, RNA-specific, and Overlap. (B) Read distribution and Northern blot of three type sRNAs classified by Hfq binding. The top left three panels represent Hfq-unbound stable sRNAs (type I), the top right three panels represent Hfq-bound unstable sRNAs (type II), and the bottom two panels represent Hfq-bound stable sRNAs (type III). Please note that Northern blot analysis of sRNA level was performed on total RNAs from the Hfq+ (WT) and Hfq− (Hfq deleted) strains. (C) Enrichment of intergenic (left) and antisense (right) Hfq-bound motifs in three different types of peaks, including strong Hfq-bound peaks, all Hfq-bound peaks, and RNA-seq peaks. Bar height represents the enrichment of motifs . To. ToY. pe E. coli  and the  E. coli . The two E. coli , insets.E. coli and we detected three dominant 5′ sites  polymerase I and is used for substrate recognition by binding to rho-independent terminators  showed Hfq binding density above background. These results lead to a hypothesis that Hfq might control the stability of most mRNAs with its sRNA partners.Hfq is known to play a role in mRNA degradation in minators . It is blmonella , 61, 62,In vitro studies have revealed that Hfq uses its proximal face to bind poly(U) sequence in sRNAs and its distal and rim surfaces to contact A-rich and UA-rich sequences in sRNA and mRNAs  were added before the TAA termination codon of the Hfq gene to construct the C-terminally Flag-tagged plasmids. A fragment covering a region of 298 nucleotides (nt) upstream, the entire 10.1128/mSystems.00245-19.10TABLE S6Table S6, DOCX file, 0.01 MB.Oligonucleotide primers used for plasmid construction. Download Copyright © 2019 Han et al.2019Han et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Y. pestis wild-type strain 201 belongs to a newly established Y. pestis biovar, microtus, which is avirulent in humans but highly lethal in mice. The hfq deletion strain (Δhfq) was generated by λ-Red homologous recombination methods as previously described  broth (Difco) supplemented with appropriate antibiotics overnight at 26°C with shaking at 200 rpm until exponential growth phase . Bacterial growth was stopped by centrifugation for 6 min at 5,000 rpm at 4°C. The pellets were frozen into liquid nitrogen and stored at −80°C until the cells were lysed. Western blotting was performed by using monoclonal FLAG antibody (Sigma) to detect the FLAG-tagged proteins.escribed . The Y. Y. pestis Hfq-FLAG, WT-FLAG, and Hfq strains mentioned above by using TRIzol reagent (Invitrogen). For CLIP-seq, two strains with FLAG grown under the same conditions were collected and resuspended in 10 mM Tris-HCl (pH 8.0). The pellets were dispersed on a petri dish and irradiated uncovered with 400 mJ/cm2 of UV 254 nm to form the cross-linked RNA-protein complex. Bacterial cells were collected and lysed in RIP lysis buffer  and subjected to coimmunoprecipitation (Co-IP). Co-IP was conducted to isolate the FLAG-bound and Hfq-FLAG-bound RNA by using FLAG antibody according to the manufacturer’s instructions for RNA-binding protein immunoprecipitation kit (Millipore). Briefly, the lysate was centrifuged at 12,000 × g at 4°C for 10 min. The clear lysate was incubated with 1.0 ml of bead-antibody complex in RIP immunoprecipitation buffer, followed by incubation at 4°C for 3 h on a rotator. The immunoprecipitation tubes were centrifuged briefly and placed on the magnetic separator, and the supernatant was discarded. The anti-FLAG beads were then washed a total of six times with 0.5 ml of RIP wash buffer and digested by RNase T. The immunoprecipitated RNA fragments were radiolabeled using PNK and separated by SDS-PAGE. The bands corresponding to the equivalent size of Hfq protein were cut out and purified. The cross-linked RNA-protein complexes were digested with proteinase K at 55°C for 30 min. RNA was extracted using TRIzol and phenol-chloroform, followed by isopropanol precipitation. The purified RNA was treated with DNase I (Promega) and sequenced using the Illumina/Solexa RNA-sequencing protocol.For RNA-seq, total RNAs were extracted from For RIP-seq, 500 μl lysate was incubated with 10 μg anti-Flag antibody or control IgG antibody overnight at 4°C. The immunoprecipitates were further incubated with protein A Dynabeads for 1 h at 4°C. After applying to magnet and removing the supernatants, the beads were sequentially washed with lysis buffer, high-salt buffer , and PNK buffer  two times in each buffer. The immunoprecipitates were eluted from the beads with elution buffer , and the RNA was purified with TRIzol reagent (Life Technologies).Purified RNAs were iron fragmented at 95°C followed by end repair and 5′ adaptor ligation. Then reverse transcription was performed with reverse transcriptase (RT) primer harboring 3′ adaptor sequence and randomized hexamer. The cDNAs were purified and amplified, and PCR products corresponding to 200 to 500 bp were purified, quantified and stored at −80°C until used for sequencing.For high-throughput sequencing, the libraries were prepared following the manufacturer’s instructions and applied to Illumina GAIIx system for 80 single-end sequencing by ABLife Inc., Wuhan, China.A260)/A280 using SmartSpec Plus (Bio-Rad). RNA integrity was further verified by 1.5% agarose gel electrophoresis. A total of 2.5 μg of total RNA was used for cDNA library preparation with the adaptors in accordance with Illumina protocol. Briefly, RNAs were ligated to 3′ and 5′ adaptors sequentially and reverse transcribed to cDNA and then PCR amplified . The whole library was applied to 10% native polyacrylamide gel electrophoresis, and bands corresponding to 80- to 380-bp RNA insertion were cut and eluted. After ethanol precipitation and washing, the purified RNA libraries were quantified with Qubit fluorometer (Invitrogen) and used for cluster generation and 36-nt single-end sequencing analysis by using the Illumina GAIIx  according to the manufacturer’s instructions.Total RNA was treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm  by using FASTX-Toolkit (version 0.0.13). After that, filtered reads (>13 nt) were aligned to the e genome  by bowtie genome  with no Y. pestis sRNA and Hfq binding site, we developed a window-based algorithm to detect peaks from alignment results among intragenic, intergenic, and antisense regions. A 5-bp window size was chosen as the default window size. The peak starting site was defined as the end of one window whose median depth is lower than one quarter depth from any one of the eight downstream adjacent windows. The peak terminal site was defined as the start of one window whose median depth is lower than one quarter depth from any one of the eight upstream adjacent windows. After obtaining the original peaks, we then filtered the peaks according to the following three thresholds: (i) the length of peaks should range from 40 bp to 500 bp; (ii) the maximum height of one peak should be no less than 60; (iii) the medium height of one peak should be no less than 20 nt. After peak definition, we classified the peaks into three different classes according to their locations: (i) intragenic peaks were defined as peaks whose locus overlapped with known mRNA genes on the same strand; (ii) antisense peaks were defined as peaks whose locus overlapped with known mRNA genes on the opposite strand; (iii) intergenic peaks were defined as neither intragenic nor antisense peaks. Antisense and intergenic peaks were defined as sRNAs both in RNA-seq and CLIP-seq samples.To have an exact prediction of We performed the peak calling method for both CLIP-seq and RNA-seq. According to the peak location, we classified peaks into three types. Stable Hfq-unbound peaks (type I) refer to those having RNA-seq transcript peaks only. Unstable Hfq-bound peaks (type II) refer to those having Hfq-bound peaks only. Stable Hfq-bound peaks (type III) refer to those with both Hfq-bound and transcript peaks that overlapped at least one nucleotide (93 members) .The strategy of identifying strongly Hfq-bound and -unbound mRNAs and sRNAs was described as follows. Briefly, peaks identified from the above strategy were the candidate bound and unbound site from Hfq-FLAG CLIP and Hfq-FLAG RNA samples. The total number of mapped reads within each peak was calculated. The sequencing data were displayed with the relative depth of mapped reads at each position of nucleotides on the global-genome scale. A fourfold threshold of total base in each peak between CLIP-seq and RNA-seq was used to define the Hfq-bound or -unbound peaks. The corresponding peak genes were defined as the Hfq-bound or non-Hfq-bound genes.http://emboss.sourceforge.net/apps/release/6.2/emboss/apps/fuzznuc.html), and motif distribution was plotted by the normalized lengths of peaks.To illustrate the binding nucleotide pattern of Hfq, we searched the RNA motif enrichment by Homer software . Then wein vitro transcription using T7 RNA polymerase were added. The membranes were then hybridized overnight at 68°C in a DIG Easy Hyb according to the manufacturer’s protocols. RNA was immunologically detected and exposed to X-ray film. Multiple exposures to X-ray film were taken to achieve the desired signal strength.A DIG Northern Starter kit (Roche) was used to perform Northern blotting according to the manufacturer’s protocol as previously described . Pure baTaq Mix (Thermo Fisher Scientific), and PCR products were electrophoretically analyzed on agarose gels.Based on the immunoprecipitation, purified RNAs were treated at 65°C for 5 min. Then reverse transcription was performed with RT primer harboring 3′ adaptor sequence and randomized hexamer. The cDNAs were amplified by 2× Dream Y. pestis to the KEGG database. Then we used hypergeometric test to calculate the enrichment of a given gene set, and all genes were regarded as background. R software was used to perform statistical significance analysis, including all the hypothesis testing types in the article.In-house script (sogen) was used for visualization of next-generation sequence data and genomic annotations. Circos software  was usedGSE77555.The RNA-seq, CLIP-seq, and RIP-seq data reported in this paper have been deposited in NCBI GEO under accession number


Zea mays L. cv. Silver Queen). Plants were grown under elevated CO2 in both field based open top chambers (OTCs) and indoor growth chamber conditions [1]. After 20 days after radicle emergence, intact leaf section of maize was exposed for 2 hours to SHS treatment. Samples were stored in liquid nitrogen immediately and used thereafter for metabolism and transcriptomes determinations. Metabolism consisting of 37 targeted metabolites together with corresponding reference standard were determined by gas chromatography coupled to mass spectrometry (GC-MS). Total RNA was extracted using TRIzol® reagent according to the manufacturer's instructions . RNA integrity was assessed using RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system . Transcriptomes were determined by Illumina Hiseq 4000 platform. Further interpretation and discussion on these datasets can be found in the related article entitled “Elevated CO2 concentrations may alleviate the detrimental effects of sudden heat stress on photosynthetic carbon metabolism in maize” [1].This data article describes the analysis of sudden heat stress (SHS) induced transcriptomes and metabolism in SQ maize cultivar ( Experiments were conducted in both fields-based open top chambers (OTCs), and indoor conditions. The location of field is at Beltsville Agricultural Research Center (BARC), USDA-ARS . The designed 4/4 random blocks for the experiment are as displayed in 2, as described elsewhere [SQ Corn seeds were supplied by the maize germplasm information resources from the United States of America, USA (GRIN: 2 (380 μmol mol−1) or high CO2 (560 μmol mol−1) concentrations, as described earlier [−2 s−1 and 12/12 h, respectively. Local air humidity was 60% during the day time.For indoor chambers, plants were grown under either ambient CO2.22 as mentioned above. The marked part of the whole intact leaves is placed in a water jacketed leaf chamber  via software SPSS 10.0  was applied to identify significant differences between heat stress and CO2 treatments for specific metabolite in SQ maize cultivar leaves.Leaves from six different plants around 20-day old were used D for metescribed . The squescribed . Organicescribed . Derived2.4Total RNA was extracted using TRIzol® reagents, following manufacturer's instructions . Quality and purity of RNA were determined by 1% of agarose gels and nano-drop , respectively. RNA integrity was evaluated via Agilent Bioanalyzer 2100 system . The total amount of RNA per sample was normalized to 1.5 μg, which was used as an input for RNA sequencing. Sequencing libraries were generated using NEBNext® UltraTMRNA Library Prep Kit for Illumina® . Sequencing libraries was featured by Illumina Hiseq 4000 platform with 150bp pair-read was generated . The ori