O-methyl, requiring additional processing steps before linker additions during cloning processes; due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. Here we present a new strategy to clone 5′ modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. The 7-h cloning process only needs ∼1 h of labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA; the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, the all-liquid-based reaction can be performed in an automated manner.High-throughput sequencing has become a standard tool for analyzing RNA and DNA. This method usually needs a cDNA/DNA library ligated with specific 5′ and 3′ linkers. Unlike mRNA, small RNA often contains modifications including 5′ cap or triphosphate and 2′- High-throughput sequencing has become a revolutionary technique for analyzing DNA/RNA. As the cost has significantly decreased over the past decade, it is becoming a standard tool for gene analyses in biomedical fields. Therefore, any technical advance in this technique will have a broad impact. Since a single sequencing run is usually sufficient for analyzing multiple samples, individual samples are usually cloned with specific barcodes, pooled, and sequenced as a single library for cost-sharing, and then debarcoded to obtain sample-specific sequences . AlthougUsually DNA, mRNA, and small RNA sequencing libraries are constructed with distinct linkers and/or chemistry, forcing customers to purchase different kits . Lack ofCaenorhabditis elegans 22G-RNA (22G) bears a 5′ triphosphate, which is incompatible with 5′ ligation . Unlike the previously reported methods, these treatments are coperformed in the linker ligation reactions, avoiding the steps for enzyme removal/inactivation and/or buffer exchange. All the steps are performed inside a PCR tube in an all-liquid-based manner, significantly reducing labor time. This method is able to construct a small RNA library using as little as ∼20 ng of total RNA, a level much lower than the amount required by available commercial kits. Since it is all-liquid-based, it can be adapted to automation. The cost is minimal since the method only needs a few common enzymes, which can be purchased or easily purified using a single His-tag purification. In all, our strategy is more sensitive, convenient, versatile and cost-effective than most available methods.We have developed a unified strategy for constructing high-throughput small RNA/mRNA libraries. Since the linkers are derived from a strategy for making high-throughput DNA libraries, the barcoded PCR primers here can be used to amplify high-throughput DNA libraries too. Our strategy is capable of cloning modified and unmodified small RNAs using the enzymatic treatments including dephophorylating RNA by 5′GCUCUUCCGAUCU-OHOH-ACACUCUUUCCCUACACGAC is an RNA oligo with 5′OH blocking the RNA ligase-mediated activation (adenylylation with ATP) (5′AGATCGGAAGAGCACACGTCTGAACTCCAGTCAddCApp- is an activated DNA oligo (adenylylated) which allows for ligation without ATP, that is, serving as a ligation donor with the 3′ ddC (dideoxyC) blocking its usage as a ligation acceptor  of the 5′ linker and the first 13 nt of the 3′ linker ligated to each target DNA strand form double-stranded DNA, a commonly used strategy required for DNA-based ligation . Since with ATP) . Therefoacceptor . This de linkers B.Supplemental Material .A complete list of the ligation linkers, RT primer, and PCR primers including 64 barcoded 3′ primers are provided in the Supplemental Table S2, small RNA is first ligated with 0.5 µM activated 3′ linker using 0.5 µM truncated T4 RNA ligase 2 in 10 µL buffer containing 50 mM Tris (pH 7.5), 10 mM DTT, and 10 mM MgCl2. Since the 3′ linker is 3′ ddC-modified primarily for blocking 3′ linker self-ligation, it can only be ligated to the 3′OH of target RNA. This strategy also avoids the activation of the 5′ phosphate of target RNA since (i) no ATP is used and (ii) the truncated T4 RNA ligase 2 does not possess the adenylylation activity  denaturing the truncated T4 RNA ligase 2; and (ii) annealing with ∼0.5 µM RT oligo to the 3′ linker either ligated or unligated by adding 0.5 µL of a 10 µM RT oligo. And then 0.4 µM 5′ linker, 0.5 mM ATP, 0.25 µM RNA ligase 1, and water are added to the reaction reaching a final volume of 20 µL for the 5′ ligation. T4 RNA ligase 1 activates the 3′-ligated target RNA if it contains or has been enzymatically treated to expose 5′ monophosphate (5′p). In contrast, the 5′ linker lacking 5′p cannot be activated. Therefore, the 5′ linker can only serve as a ligation acceptor ligated to the 3′-ligated and 5′-activated target RNA. The annealing of the RT oligo with the 3′ linker may reduce the ligation of the free 3′ linker, if any are left after the 3′ ligation, with the 5′ linker, since T4 RNA ligase 1 prefers single-stranded substrates. This strategy may significantly reduce the formation of 5′ linker-3′ linker ligation especially when the RNA substrate is much less than the 3′ linker, generating excessive 3′ linker, as discussed below in the single worm RNA cloning.A 30-minute RT step follows the 5′ ligation with addition of ∼0.2 µM Superscript II or III (Invitrogen), ∼4 mM additional DTT, ∼0.4 mM dNTP, and ∼2 µL 10× RT dilution buffer (0.25 M Tris pH 8.8 and 0.75 M KCl) reaching a final volume of ∼24 µL. Then the enzymes are inactivated at 85°C for 5 min. The obtained cDNA is amplified and extended to obtain the full-size linkers by PCR. 8-nt barcodes are inserted in the 3′ PCR primers for labeling individual samples. A typical 50 µL PCR reaction is composed of 1× PFU buffer, 15 mM tetramethylammonium chloride for reducing primer dimmer, 0.1 mM dNTPs, 0.1 µM 5′ and 3′ primers, 5 µL RT reaction, and 1× PFU polymerase. The PCR is first amplified for 5 cycles  and then amplified for 11 cycles . Additional 0.6 µM 5′ and 3′ primers are added and the PCR is amplified for 2 more cycles .Supplemental Table S2.In all, the whole cloning process is performed in a single PCR tube with liquid components added sequentially and can be easily finished within ∼7 h including ∼ 1-h labor time. A formulated working protocol is presented in the C. elegans adult worms, and using purified C. elegans small RNA of size less than 200 nt, which contain miRNA, 22G-RNA (siRNA), 21U-RNA (piRNA), tRNA, and 5.8S/5S rRNA  or other byproducts. The result clearly indicates that our method is able to clone small RNA at fmole level from single worms and deliver high reliability at this level, as the two individual worm samples exhibited consistent small RNA profiles D.C. elegans PIR-1 works at 20°C, it is a perfect candidate for the desired activity. We obtained a recombinant C. elegans PIR-1 of homogeneity from E. coli . To examine its dephosphorylation activity, we coapplied this enzyme in the 3′ ligation step with the truncated T4 RNA ligase 2, generating 3′ ligated and 5′ p-RNA using the same reaction. Then the RNA was further 5′ ligated. The high-throughput sequencing analysis confirmed that this strategy worked efficiently for cloning ppp-RNA. As shown in Small ppp-RNA cannot be directly ligated at the 5′ end. To clone it, ppp-RNA is usually treated with commercial RNA polyphosphatases, generating 5′ monophosphorylated RNA (p-RNA), which is compatible with 5′ ligation . HoweverE. coli and examined its decapping activity in the ligation buffer. hDcp2 worked well in the 5′ ligation step with T4 RNA ligase 1  or promoter-associated small RNA is generated during transcription initiation and bears a 5′ G cap with size <200 nt . The capligase 1 B, as theligase 1 B or circreported .In our condition, hDcp2 does not work well when added in the 3′ ligation step (data not shown). Since the buffer conditions for the 3′ and 5′ ligations are almost the same, we suspect that the hDcp2 we purified may be contaminated with a low level of RNA phosphatases, which dephosphorylated the p-RNA generated by hDcp2 in the 2-h 3′ ligation step, making it incompatible for the following 5′ ligation. In contrast, when hDcp2 is added in the 5′ ligation, the ligase may add a linker to the 5′p of the decapped RNA before dephosphorylation by any RNA phosphatase.In addition to cloning small RNA, we aimed to use the same method to clone mRNA. In general for mRNA cloning, mRNA is fragmented to small RNA, ligated with linkers, and converted to cDNA. However, a partial digestion of mRNA either chemically or enzymatically usually generates small RNA with 5′OH and 2′p or 3′p or cyclic phosphate at the 3′ end. For ligation-based cloning methods, these ends have to be converted to 5′p and 3′OH. To avoid these conversion steps, we utilized nuclease P1, an enzyme cutting single-stranded RNA or DNA, generating small RNA or DNA with 5′p and 3′OH . A potenSupplemental Fig. S2A) and the individual gene expression levels correlate well between the two samples . Moreover, our method was capable of cloning at least 1 ng mRNA (data not shown), a sensitivity level sufficient for most routine work.We then constructed a library with poly (A)-containing mRNA fragmented using nuclease P1. The obtained mRNA profile is similar to that of a previous one made using alkali hydrolysis. For example, the reads spread randomly along the length of mRNA  and BB is ∼0.01% (0.01%/[98%+0.01%]). Therefore, if perfectly matched products are selected, abundant DNA species are overrepresented, and if bulged products are selected, less abundant DNA species are overrepresented.To solve the overcycle issue, theoretically both perfectly matched and bulged products can be purified together. However, PCR products often contain primer dimers and undesirable products, which form bulged products with desirable PCR products once PCR reactions are overcycled, basically making this purifying-all strategy ineffective. To solve this issue, we used to examine the PCR product at cycle number 12, 16, 20, and 24 from the same PCR reaction on a PAGE gel, obtain the maximal PCR number without overcycle, and use this number to mass produce the product . AlthougA high-throughput sequencing library usually contains multiple barcoded samples for cost-sharing. To obtain a specific composition, individual samples are purified, quantified and then mixed as a pooled sample. Since samples may have primer dimers and/or other byproducts including cDNA derived from rRNA and tRNA, a gel purification is required for obtaining target cDNA of specific sizes. This process is tedious and time consuming . To reduOur major goal is to design a simple, convenient and cost-effective method for cloning small RNA. For this purpose, we have developed an all-liquid-based multistep reaction in a single PCR tube. The whole procedure takes ∼ 7 h with only ∼ 1 h labor time and the rest primarily used for increasing ligation efficiency of modified RNA. It can be shortened to ∼4 h for cloning unmodified miRNA . Like the commercial kits, our method starts with total RNA, a strategy providing convenience but generating byproducts derived from tRNA and rRNA. In addition, the whole procedure involves no gel purification or DNA precipitation . Our method is much more sensitive than the commercial kits, working with as little as 16 ng total RNA. We speculate that we could start with even less total RNA if using one gel purification to enrich target cDNA amplicons while removing primer dimmers and linker-linker ligation byproducts, followed by a second round of PCR amplification for mass producing target cDNA amplicons.We prefer ligation reactions for adding 5′ linkers to target RNA over other techniques, for example, cDNA circularization or template switching via reverse transcriptases. Since most degraded RNA bears a 5′OH, it cannot be cloned using ligation, which requires a 5′p. Therefore, not only does the 5′ ligation add a linker for cDNA application and sequencing, but also serves as a selection mechanism for enriching authentic small RNAs. Due to this selection, our method works well with samples heavily contaminated with degraded RNA including some immunoprecipitated RNA samples. At the same time, our method can be easily adapted to clone RNA bearing a 5′OH simply by adding T4 Polynucleotide Kinase (PNK) in the 5′ ligation step (data not shown).Our method only needs a few common enzymes such as T4 RNA ligases, reverses transcriptases, DNA polymerases and PIR-1 and hDcp2. The last two are required for processing 5′ modified RNA but not for p-RNA including miRNA, Dicer-dependent siRNA, and piRNA. We easily obtained RNase-free enzymes using a single His-tag-mediated purification. The first three enzymes above are also commercially available. However, for some reasons, when tested by other labs, the protocol did not work well with some commercial enzymes, but worked well with our purified enzymes. We believe that the failure may be caused by the commercial T4 RNA ligase since commercial SuperScript III and Taq worked in our method. We speculated that the commercial T4 RNA ligases used may be contaminated with a trace amount of RNases or phosphatases. We estimate that the cost per library construction is negligible if using home-made enzymes and activated 3′ linkers, and less than $10 if using commercial enzymes and 3′ activated linkers.O-methyl at the 3′ end, which decreases the 3′ ligation efficiency. This inhibitory effect is overcome by addition of 25% PEG-8000   . csRNA i22G-RNA) . To enri22G-RNA) . 5′ OH-RWe aimed to develop a versatile method for cloning both mRNA and small RNA and to use the same sets of PCR primers to amplify libraries derived from DNA and RNA. Since the 5′ and 3′ linkers are derived from the DNA cloning system, the PCR primers definitely work with DNA libraries. Actually, we used these primers to amplify DNA libraries constructed using a commercial kit (data not shown) without purchasing the expensive barcoded primers. For mRNA cloning, we first used alkali-hydrolysis to fragment mRNA, generating small RNA with 5′OH and 3′ cyclic phosphate. Then we used PNK to fix the ends in the 3′ ligation step by: (i) removing the 3′ cyclic phosphate at room temperature for ∼5 h; and (ii) phosphorylating the 5′ end with addition of ATP at 37°C for 1 h (data not shown). Although it works well, the 3′ ligation step takes ∼6 h, making it impossible to finish the cloning process within 1 d. This prompts us to use nuclease P1, which generates ligation-compatible ends. The cost of nuclease P1 is close to alkali-hydrolysis, basically nothing. Nuclease P1 works well at 60°C, significantly reducing the effect of RNA secondary structures on the digestion pattern and efficiency. Moreover, our partial digestion condition is insensitive to the RNA amount so that there is no need to optimize the enzyme/RNA ratio for getting fragmented RNA of desired size.All in all, our method is convenient, sensitive, and versatile. Any laboratory with basic molecular techniques can establish an integrated system to clone small RNA and mRNA.RNA was extracted using TRI reagents (Sigma T9424) with a tissue tearor according to the manufacturer's protocol. Worms were grown on OP50 at 20°C for ∼70 h. To prepare single worm RNA, individual worms were transferred to a PCR tube and incubated with 0.4 µg/µL proteinase K in 10 µL buffer containing 40 mM Tris (pH 7.5), 10 mM EDTA, 0.3 M NaCl, 0.5% NP-40 and 0.5% SDS at 65°C for 10 min. The resulting RNA was phenol/chloroform extracted and coprecipitated with 20 µg glycogen.C. elegans PIR-1 gene, T23G7.5a.1, was integrated into pET-28a between the NdeI and BamHI restriction sites. The resulting plasmid was transformed into the BL21 (DE3)-RIL strain for protein expression. To express the recombinant PIR-1, a single colony was grown in 5 ml Terrific Broth (TB) medium containing 50 µg/mL Kanamycin and 20 µg/mL chloramphenicol at 37°C for 8 h; the whole culture was inoculated into 1 L TB media containing 50 µg/mL Kanamycin and 20 µg/mL chloramphenicol to grow ∼24 h at room temperature, reaching OD600 0.5; and the protein expression was induced using 0.5 mM IPTG at 16°C for 15 h. The cells were pelleted and resuspended in 25 mL lysis/wash buffer containing 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM 2-mercaptoethanol, 5% glycerol, 10 mM imidazole, 0.01% NP40, and 1 mM PMSF. The cells were sonicated on ice using 30 cycles of 20-sec sonication followed by 40-sec pause. The resulting solution was centrifuged at 20,000g at 4°C for 10 min and the supernatant was mixed with 2.5 mL HisPur Ni-NTA (Thermo Fisher Scientific) beads, which were prewashed three times with the lysis/wash buffer without PMSF. After 1-h nutation at 4°C, the beads were washed with 200 mL lysis/wash buffer in a 20 mL column and the protein was eluted using 10 of 0.5 mL lysis/wash buffer containing 0.4 M imidazole. The fractions 5–7, which contained the majority of the protein, were pooled and loaded onto a HiPrep Sephacryl S-100 HR column and fractionated with the imidazole-free lysis/wash buffer using the NGC Quest 100 Plus Chromatography System (Bio-Rad). The FPLC fractions contained a recombinant PIR-1 of high homogeneity . The pooled fractions were dialyzed for storage at −80°C using a buffer containing 20 mM Tris (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.01% NP-40, 0.1 mM EDTA, and 50% glycerol.The coding sequence of T4 DNA ligase was used to adenylylate a 3′ linker oligo 5′p-AGATCGGAAGAGCACACGTCTGAACTCCAGTCA/ddC/, which together with 5′ACGGCATACGAGGGAAG/ddC/ was annealed to 5′CTCTTCCGATCTGCTTCCCTCGT A/ddC/ at 95°C for 2 min followed by a slow cooling (0.1°C/sec) to room temperature, forming a nicked double-stranded DNA at 10 µM concentration in 10 mM Tris pH 7.5 and 30 mM KCl. Then 2.5 µM of the annealed DNA was incubated with 2.5 µM of T4 DNA ligase in 1× DNA ligation buffer at 37°C for 1 h, followed by phenol extraction, DNA precipitation and purification using a 15% PAGE/7M urea gel.2 at 60°C for 10 min, generating RNA fragments with a median size of ∼150 nt.A total of 8–2000 ng total RNA was partially digested with 0.01 unit of nuclease P1 in a buffer containing 50 mM sodium citrate (pH 7.0) and 10 mM MgClC. elegans analyses, reads were mapped to the genome (WormBase release WS215) and the Generic Genome Browser was used to visualize the alignments  scripts and Bowtie 0.12.7 . For C. ignments . The sofSupplemental material is available for this article.


This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5′ untranslated regions (5′UTRs) of 20–60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5′UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5′UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with  RNAs frequently misfold into stable but biologically inactive structures especially under stresses, while RNA chaperones interact with various RNAs to prevent the structures that may cause premature transcriptional termination or pausing, and affect mRNA decay and translation. This work for the first time reports that an archaeal RNA chaperone TRAM0076 globally affects the transcription of methanogenic Archaea through posttranscriptional actions. TRAM0076 also binds many cellular mRNAs, possibly at the 5′-untranslated regions. This work uncovers an important regulatory element of ancient life in the RNA world. RNA chaperones impact mRNA metabolism including transcript synthesis, processing and degradation as well as translation. They play an important role in controlling gene expression, especially in response to environmental perturbations such as cold shock . The distbp and tfb genes, so this general mechanism of transcriptional regulation appears to be restricted to just a few groups. While homologs of some of the bacterial transcription regulatory proteins have been identified in methanogens, these genes are not abundant in many methanogens and archaea in general [Methanococcus maripaludis S2 possesses only 29 homologs of bacterial transcription factors, and the function of most of these genes is unknown. This suggests that transcription regulation may not be prevalent in methanoarchaea. Noticeably, genome-wide transcription start site mapping indicates that long 5′ un-translation regions (5′UTR) are common in methanogenic archaea [In methanogens as well as other Archaea, transcription of messenger as well as stable RNAs employs a homolog of the eukaryotic RNA polymerase II . Transcr general . For ins archaea –22. In b archaea , 24. The archaea , 21, andin vitro studies indicated that these TRAM proteins exhibit typical properties of RNA chaperones. A cold-responsive standalone TRAM protein from the cold-tolerant methanogenic archaeon Methanococcoides burtonii called Ctr3 was reported to preferentially bind tRNAs and 5S rRNA [Methanolobus psychrophilus R15 possesses four TRAM proteins that can replace the cold shock proteins in E. coli and exhibit nonspecific RNA binding and duplex RNA melting activity in vitro [TRAM is an archetype domain found in many proteins involved in RNA modifications, such as TRM2 methylases and methylthiotransferase RimO, as well as the translation initiation factor IF-2β subunit and ribosomal protein S2 . Protein 5S rRNA . The psyin vitro . HoweverM. maripaludis was selected to interrogate the biological significance of archaeal standalone TRAM proteins and their regulatory role in gene expression. M. maripaludis has only a single TRAM-encoding gene (MMP0076) and is genetic tractable. Through genetic, physiological, biochemical and transcriptomic studies, we demonstrate that the standalone TRAM protein MMP0076 (named TRAM0076 hereafter) from M. maripaludis acts as an RNA chaperone and is required for the normal expression of more than half of the genome and normal growth. TRAM0076 controls the expression of some mRNAs through unfolding the kinetically trapped RNA structures in the 5′UTR elements. Thus, the archaeal standalone TRAM protein appears to play a fundamental role in transcription.In this study, MMP0076, which encodes a standalone TRAM protein (TRAM0076), was replaced with the pac cassette for puromycin resistance in M. maripaludis S0001 as described by Tumbula et al. [MMP0076::pac deletion mutant (Δ0076). The growth rate of the mutant (0.065 ± 0.003 h-1 vs 0.15 ± 0.001 h-1 of strain S0001) was markedly reduced at the optimal growth temperature of 37°C. To confirm that the growth reduction phenotype was not due to a mutation at a second site, the Δ0076 mutant was complemented by expressing TRAM0076 from the replicative plasmid pMEV2-MMP0076 carrying the MMP0076 gene (MMP0076-com strain). Growth of strain MMP0076-com was completely restored to wild-type levels , an ATP-dependent RNA-helicase (MMP1141), an RNA binding S1 domain protein (MMP1127), and translation initiation factor IF-2α (MMP1707)  , implyin complex . This coE. coli was tested. MMP0076 was cloned into the pINIII expression vector and transformed into the E. coli mutant BX04, which has a quadruple deletion of cspA, B, G and E and does not grow at low temperatures [E. coli mutant BX04 at low temperatures  were tested as substrates in RNA electrophoretic mobility shift assay (rEMSA). These RNAs are about 100 nt in length and possess all possible 5-nt combinations (1024) . Each ofout 1 μM . TogetheE. coli was measured. Strain RL211 of E. coli carries a cat (chloramphenicol resistance) gene immediately downstream of a strong trpL transcriptional terminator  at the 3′ terminus, were used as substrates  increased the fluorescence intensity to about 54%  assay  was thenbstrates . Oligodebout 54%  of the dE. coli Csps, aromatic and positively charged amino acid side chains play important roles in unfolding RNA structures by base stacking and electrostatic compensating interactions [E. coli RL211  of which were 20–60 nt in length , transcription terminator-like hairpin structures were common in the 5′UTRs of this length  transcript, 116 nt in the F420-non-reducing hydrogenase subunit  transcript and 104 nt in a peptide methionine sulfoxide reductase (MMP0848) transcript  whose abundances were reduced in the Δ0076 mutant and possessed potential hairpin structures were examined in the molecular beacon system  in the replicative vector and transformed into the Δ0076 and parental strains of M. maripaludis. Expression of the mCherry reporter gene was assayed by measuring the fluorescence intensity of whole cells during growth. Compared to the parental strain, the expression of mCherry in the Δ0076 mutant was reduced by approximately 50% for MMP1515 and MMP1697   were also examined. Sensitivity to ribonucleases confirmed that the RIP collected nucleic acid was RNA. The RNA abundance in the Flag-tagged TRAM0076 precipitant was much higher than that of the mock control  . The Fla control . Compare control . Flag-ta control  as a thrMMP1407) and its RNA component, two histones (MMP0386 and MMP1347), translation initiation factors (MMP1406), elongation factor 1α and β subunits (MMP1370 and MMP1401), RNA polymerase subunits RpoN and RpoF (MMP1326 and MMP0092), ribosomal proteins , some methanogenesis genes , and an ArsR family transcriptional regulator (MMP1442). Of note, the TRAM0076 transcript was also highly enriched, suggesting a potential self-regulation of its own expression. In contrast, some RNAs were conspicuously unenriched, including rRNAs and tRNAs. These stable RNAs are either highly structured or tightly associated with proteins.Among the RNAs precipitated by Flag-tagged TRAM0076, some were highly enriched. These included the messages encoding RNase P protein , a RNAP subunit (MMP1360), and three downregulated transcripts  were enriched in the Flag-tagged TRAM0076 precipitant by 6.8 to 100-fold higher than in the mock precipitant. These experiments confirmed that TRAM0076 bound a large number of transcripts in vivo.Precipitation of some of the highly enriched RNAs and downregulated methanogenic genes in the ΔCR qPCR; . While tRNA chaperones, through RNA binding and structured RNA unfolding activities, ensure the correct folding of targeted RNAs and enable them to adopt their functionally active states, thereby playing important roles in control of gene expression , 38, 39.in vivo and in vitro, and unfolds RNA hairpins and other secondary structures. Importantly, mutations of the amino acid residues essential for RNA unfolding also eliminate the biological effects of TRAM0076 in Methanococcus, demonstrating that its biological role depends on its structured RNA unfolding activity. Further, the methanococcal TRAM0076 facilitated transcription of some genes by unfolding the 5′UTR structures, presumably to prevent premature transcriptional termination or pausing.The methanococcal TRAM0076 displays the defining properties of an RNA chaperone. It binds RNAs without obvious sequence specificity both E. coli Csps, which exert roles mainly when the organism encounters cold shock or lower temperatures [Methanococcus as deletion of the gene impaired the growth at both optimal and lower temperatures. Accordingly, absence of TRAM0076 causes changes in the expression of 55% of the total genes, much more than the gene numbers that are affected by Csps [0076 mutant possess a 5′UTR of 20–60 nt, and both in vivo and in vitro experiments demonstrate that TRAM0076 unfolds 5′UTR structures. Therefore, one of the mechanisms that TRAM0076 facilitates archaeal transcription would be prevention of 5′UTR structure formation by unfolding the kinetically trapped stem-loop structures and transcription antitermination activity, similar to that of the bacterial Csp proteins [Although TRAM0076 shares the RNA chaperone activities of the bacterial cold shock proteins, unlike the eratures , TRAM007 by Csps . Thus, tproteins .MMP0076 also causes increased expression of about one third of the total genes, including a DEAD/DEAH RNA helicase (MMP0457) and a SAM-dependent methyltransferase (MMP0874). Transcripts of the two genes were precipitated by the Flag-tagged TRAM0076, evidence that they were bound by TRAM0076 in vivo. Moreover, the transcripts of MMP0457 and MMP0874 possess long 5′UTRs of 223 nt and 84 nt, respectively. As the two genes are upregulated in the Δ0076 mutant, TRAM0076 must act on them by some mechanisms other than transcriptional antitermination. TRAM0076 also bound the transcript of an AsrR family transcription regulator. By controlling the expression of other regulatory proteins, TRAM0076 could exert an indirect role in regulation of gene expression. It is worthy to note that, unlike the in vitro experiment results of the cold-responsive TRAM protein Crt3 from M. burtonii [in vivo assay, though the two proteins have highly similar sequences  medium with shaking.The microbial strains used in this study and their characteristics are listed in M. maripaludis strains were cultured in pre-reduced DSMZ medium 141 containing 40 mM formate at 37°C under a gas phase of N2/CO2  as previously described [escribed , 47. GroMMP0076 gene of M. maripaludis S0001 was deleted by replacement with the pac cassette, which encodes puromycin resistance, through homologous recombination using the integration vector pIJA03 and polyethylene glycol (PEG)-mediated transformation [MMP0076 was PCR amplified and inserted into the shuttle vector pMEV2 at the NsiI/XbaI site to generate pMEV2-0076. After verifying the sequence, pMEV2-0076 was transformed into the Δ0076 mutant to construct MMP0076-com strain, and the transformants were screened in the presence of 1 mg/ml neomycin. As a control, the empty vector pMEV2 was transformed into both the wild-type strain and the Δ0076 mutant, respectively.Plasmids used for genetic manipulations are listed in ormation , 48. ForE. coli BX04 and RL211 strains, the open reading frame of MMP0076 was PCR amplified from the genomic DNA using primer pairs P3/P4 (E. coli cspA and cspE and Mpsy_3066 (encoding TRAM3066) of M. psychrophilus R15 [For expressing TRAM0076 in rs P3/P4  and thenilus R15  were clo6-tagged recombinant TRAM0076, MMP0076 sequence was amplified by primer pairs P11/P12 plysS. Expression of the 6×His tagged proteins were induced by IPTG  and purified as described previously [2+-nitrilotriacetatic acid-agarose columns (Novagen), HiTrap Q HP anion exchange columns and gel filtration chromatography according to the manufacturer’s protocols .To produce recombinant TRAMs, plasmids p28a-3066, p28a-0076 and its mutants, p28a-cspA and p28a-cspE were each transformed to Using the approach of Zhang et al. , rEMSA wE. coli RL211. The transformants were cultured overnight in LB complemented with 100 μg/ml ampicillin and then diluted a 100-fold into the fresh medium. When the culture grew to an OD600 nm of 0.4–0.6, 1 mM IPTG was added, and cultivation continued for another 2 h. Next, six μl of 10-fold diluted cultures were spotted on LB plates containing 100 μg/ml ampicillin, 1 mM IPTG, with or without 30 μg/ml chloramphenicol, and grown for 2–3 days.To determine the nucleic-acid unfolding activity of TRAM0076 and its mutants, plasmids pIN-0076 and its derivatives were each transformed into M. maripaludis, another replicative vector pMEV4-mCherry-neo carrying a neo cassette (neomycin resistant) and a mCherry reporter gene was used. Each of the three 5′UTRs  was inserted upstream the mCherry gene in the vector, resulting plasmids pMEV4-0127PUO, pMEV4-1515PUO, and pMEV4-1697PUO, and then transformed into the Δ0076 mutant and the wild-type strain, respectively. The pMEV4-1515PUOMT was constructed by the site-directed mutation of the pMEV4-1515PUO described above. Expression of the mCherry reporter gene was assayed by detecting the fluorescence at 575 nm (excitation) and 610 nm (emission) for the cultures during growth.To detect the role of TRAM0076 to facilitate transcription in et al. [MMP0127, MMP1515 and MMP1697  CspA and CspE were included as positive controls. Fluorescence was measured in 96-well plates using a Synergy H4 hybrid Reader  at 460 nm for excitation and 515 nm for emission.A fluorescent molecular beacon system developed by Nakaminami et al.  was used MMP1697  at the 5M. maripaludis S0001 and the Δ0076 mutant were harvested from three independent cultures. The total RNA was extracted using TRIzol (Ambion), and the whole-transcript cDNA libraries, high-throughput sequencing and quality control (QC) were performed as previously described by Li et al. [M. maripaludis S2 reference genome . The differential expression analysis of transcripts between wild-type and Δ0076 mutant was performed by using the DESeq algorithm with the normalized mapped read counts, and a threshold of the adjusted P value Padj <0.05 was considered as a significant differential expression [2) among the three biological repeats ranged 0.889 to 0.989.Cultures at the mid-exponential phase of i et al. . Uniquelpression , 31. TheM. maripaludis S0001 as described in [Genome-wide transcription start sites (TSS) were mapped using the differential RNA-sequencing (dRNA-seq) approach as described previously . After Tribed in .MMP0076 gene tagged with a C-terminal his6 or a his6 plus 3×Flag-tag was inserted into pMEV4 that carries a neo cassette by using the Gibson assembly method [0076 mutant to construct MMP0076-his6 and MMP0076-his6-3×Flag strains. Cells at the late exponential phase were collected, and the pellet was washed with 1 mL of buffer A (50 mM Tris-HCl (pH7.5), 500 mM NaCl, 10% (w/v) glycerol), snap-frozen in liquid nitrogen, and stored at -80°C. RNA co-immunoprecipitation (RIP) was then performed as described previously [2, 150 mM NaCl, 0.5% Triton X-100 supplemented with cOmplete mini protease inhibitor (Roche) and 10 U/ml RNasin (Promega) by repeated pipetting. Prior to immunoprecipitation, 1% of the supernatant was used to prepare total RNA as input. The remainder was pre-cleared by mouse IgG-agarose (Sigma), and then incubated with 40 μl of Anti-Flag M2 Magnetic Beads (Sigma) at 4°C and pre-blocked by incubation in 1% BSA. The Co-IP anti-Flag beads were washed three times with 1 ml buffer B, and two times with 1 ml buffer B plus 150mM NaCl. RNA-bound to the Flag-tagged TRAM0076 was eluted using the FLAG Peptide (Sigma), and RNA was extracted and precipitated with ethanol with NaOAc and glycogen as carrier. Extracted RNAs were quantified by Quant-iT RiboGreen RNA kit (invitrogen).y method  and traneviously , 50, 51 M. maripaludis S2 genome . Read counts were normalized to the Fragment Per Kilobase of transcript per Million fragments mapped (FPKM).The immunoprecipitated RNAs were treated with DNase I, and cDNA libraries were constructed using NEBNextH UltraTM Directional RNA Library Prep Kit for IlluminaH (New England Biolabs) as described previously . SequencQuantitative RT-PCR (qPCR) was performed to verify RIP-seq data. cDNAs were generated from 2 μg of input RNA and 10 μl of immunoprecipitated RNA by RT-PCR with random primers using Moloney murine leukemia virus reverse transcriptase (Promega). qPCR amplification was performed using the corresponding primers  at MasteE. coli BX04 [E. coli BX04 and 10-fold serial dilutions of the transformant were spotted on LB plates containing 100 μg/ml ampicillin and incubated for 2–5 days at 37°C, 22°C and 18°C.Using the same approach as described previously , TRAM007oli BX04  was perfS1 FigThe tree was constructed using MEGA7.0, and the bar represents 10% sequence difference. The archaeal species and their encoded TRAM proteins are indicated, and numbers following each species are the TRAM genes copies.(TIF)Click here for additional data file.S2 FigE. coli BX04 carries a deletion of the cspABGE operon, and its growth at low temperature is retarded. (A) Overnight liquid cultures of the E. coli strains that overexpress MMP0076, Mpsy_3066, cspA or cspE were diluted to OD600 of 0.9 with the fresh medium and then 10-fold serially diluted to 10−5 and spotted on LB plates supplemented with ampicillin. The plates were incubated at 18°C, 22°C and 37°C for 2-5 days with or without IPTG, as indicated. pINIII indicates the cultures carrying the empty plasmid vector. + and–, cultured in the presence or absence of IPTG, respectively. pINIII, strain carrying the empty plasmid pINIII. (B) SDS-PAGE confirmation of TRAM protein expression in strain BX04. Left: PAGE of 2.4 μg of purified CspA, CspE, TRAM3066 and TRAM0076. Right: Cell extracts from strain BX04 overexpressing cspA, cspE, Mpsy_3066 or MMP0076 were electrophoresed on 5% SDS-PAGE. + and–, cultured in the presence or absence of IPTG, respectively. pINIII, strain carrying the empty plasmid pINIII. Asterisks indicate the predicted migration of the overexpressed proteins.(TIF)Click here for additional data file.S3 FigE. coli CspA and TRAM3066 are included as references. Calculated the equilibrium dissociation constant KD values are shown for each pair of protein-RNAs. The protein concentrations for each reaction are color coded and labeled in the bottom row. The x axis indicates the reaction time in seconds, and the y axis shows arbitrary resonance units (RU), where 1000 RU corresponds to a surface density of 1 ng/mm2.Two RNA Pentaprobes were used as substrates and immobilized to the SPR chip, and the RNA binding affinity was tested by addition of the indicated concentrations of TRAM0076. (TIF)Click here for additional data file.S4 FigMMP0076 (PMEV2-0076WT) was included as the reference. Growth was measured at 37°C for triplicate cultures. Averages and standard deviations are shown.Complementation of the wild-type (TIF)Click here for additional data file.S5 FigThe box indicates the range from the lower to the upper quartile, and the line inside each box refers to the median length of 5′UTRs. Extreme outliers are depicted by dots.(TIF)Click here for additional data file.S6 Fig0076 mutant  predicted by Mfold software  were shown.Hairpin structures like transcription terminator in the 5′UTRs of some down-regulated transcripts in the Δ(PDF)Click here for additional data file.S7 FigThe middle exponential 37°C cultures of the 5′UTR-mCherry reporter strains as used in (TIF)Click here for additional data file.S8 FigAgarose gel detection of the RNAs co-immunoprecipitated by Flag-tagged TRAM0076 (RIP1 and RIP2) and un-tagged TRAM0076 (mock) (A), and that from the Input samples (Input1 and Input2) (B).(TIF)Click here for additional data file.S9 FigFPKM. Extreme outliers are depicted by dots.The box indicates the range from the lower to the upper quartile, and the line inside the box refers to the median value of (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Dataset(XLS)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.


Coral reefs worldwide face unprecedented cumulative anthropogenic effects of interacting local human pressures, global climate change and distal social processes. Reefs are also bound by the natural biophysical environment within which they exist. In this context, a key challenge for effective management is understanding how anthropogenic and biophysical conditions interact to drive distinct coral reef configurations. Here, we use machine learning to conduct explanatory predictions on reef ecosystems defined by both fish and benthic communities. Drawing on the most spatially extensive dataset available across the Hawaiian archipelago—20 anthropogenic and biophysical predictors over 620 survey sites—we model the occurrence of four distinct reef regimes and provide a novel approach to quantify the relative influence of human and environmental variables in shaping reef ecosystems. Our findings highlight the nuances of what underpins different coral reef regimes, the overwhelming importance of biophysical predictors and how a reef's natural setting may either expand or narrow the opportunity space for management interventions. The methods developed through this study can help inform reef practitioners and hold promises for replication across a broad range of ecosystems. Beyonet al. [To date, descriptions of alternative reef regimes have predominantly addressed benthic community structure, with an emphasis on shifts from coral to algal dominance –9. Changet al.  addresseIn the face of escalating human impacts, such as overfishing, reduced water quality and effects from climate change, there is growing awareness surrounding the multi-causality of reef regimes  and poteThe main Hawaiian Islands—the populated portion of the Hawaiian archipelago, hereafter referred to as the Hawaiian Islands for brevity—span gradients in both environmental conditions  and huma2.(a)et al. [et al. [Situated in the middle of the Pacific Ocean, the Hawaiian Islands consist of eight high volcanic islands with varying human population density and exposure to natural forces . The stuet al.  identifi [et al. .Figure Kyphosus hawaiiensis and Naso unicornis), high turf and macroalgae cover, and low hard coral cover. Regime 3 exhibits high fish biomass and turf algae cover, no macroalgae and moderate hard coral cover. Regime 5 displays moderate fish biomass, less turf algae and higher hard coral cover, commonly comprised of the coral Porites compressa. For detailed methodology and description of the regimes, see Donovan et al. [Sites classified into regime 1 show the characteristics of a degraded reef, i.e. low fish biomass, low hard coral cover and high algae cover . Regime 2 is characterized by rugose habitat with high fish biomass et al. [We reviewed all continuous spatial layers of anthropogenic and environmental drivers compiled by Wedding et al.  for coasThe selection of anthropogenic predictors expanded on a human dimensions framework that identified the primary human impacts mediating coral reef condition . It incla (as a proxy for phytoplankton biomass and thus primary production), irradiance and wave power. Five climatological metrics were available for each predictor: long-term mean, standard deviation of the long-term mean, maximum monthly climatological mean, maximum anomaly and frequency of anomalies [Biophysical predictors were derived from time series of variables known to be major drivers of coral reef ecosystems: sea surface temperature, chlorophyll-nomalies ,20. Choinomalies . Dependiet al. [For a majority of the datasets, the temporal range represented approximately a 10 year average, which matched the temporal spread of the biological surveys used to identify regimes  and provet al. .(c)ggBRT are available on GitHub (https://github.com/JBjouffray). We used boosted regression trees (BRTs) [All statistical analyses were conducted using R v. 3.3.2 . Statists (BRTs)  to examis (BRTs) .gbm.step routine [dismo package v. 1.1-4 [The classification of sites into different regimes was converted to presence-absence of each regime  and mode routine  in the dv. 1.1-4 . Trees wv. 1.1-4 ,25. VariI coefficients from the model residuals [Model performance was evaluated by 10-fold cross-validation that allows to test the model against withheld portions of the data which are not used in model fitting . We lookesiduals .We calculated the relative importance of each predictor based on the number of times a variable was selected for splitting, weighted by the squared improvement to the model as a result of each split and averaged over all trees ,28. To aPartial dependency plots with 95% confidence intervals obtained from 1000 bootstrap replicates  were useInput data for the predictor variables had different native spatial resolutions . For ins3.(a)I between 0.02 and 0.04). The pattern of predictors' contributions differed among regimes, with regimes 1 and 2 displaying a few strongly influential predictors, while regimes 3 and 5 were best explained by a broader, but less influential, set of variables (a). This was also reflected by the number of predictors having a relative influence above what could be expected by chance: five for regime 1, six for regime 2 and nine for regimes 3 and 5 (a). The regimes distributed along a continuum of biophysical and anthropogenic influence (b), with an overwhelming contribution of biophysical variables in predicting the occurrence of regimes 3, 2 and 5 . Regime 1, on the other hand, was most effectively predicted by anthropogenic variables (57%).BRT models performed well for all four regimes , with deviance explained from 37% to 41%, high predictive performance (AUC scores between 0.88–0.91) and minimal spatial autocorrelation a). Topographical complexity of the seafloor was the second strongest predictor (20.1%), suggesting regime 1 is more likely to occur in areas with low structural complexity. Depth (8.8%) and effluent (5.5%) both displayed positive relationships. Interaction patterns reflected the influence of the most important predictors with the probability of regime 1 occurring being greatest when fishing catch was high and structural complexity was low . Cooler maximum monthly climatological sea surface temperature (9.3%), higher complexity (8.5%) and high variation of temperature (7.8%) all increased the probability for regime 2 to occur, while depth (8.7%) showed a slightly negative relationship. The two most important interactions , indicating a higher occurrence of this regime in wave-exposed sites b. Coolerractions  revealedc). Occurrence probability was higher in places with low maximum monthly climatological chlorophyll-a concentration (13.6%)—but positively correlated with a higher frequency of anomalous chlorophyll-a events (7.7%). Regime 3 was more likely at depths shallower than 25 m (8.9%) and in wave-sheltered environments (8.2%). The interactions were weak, with no clear interaction pattern apparent . It was also associated with higher levels of new development along the coastline (9%) and, to a lesser extent, increased catch from non-commercial shore spearfishing (6.9%). The most important interaction involved variation of irradiance and observed presence of invasive algae , increased topographical complexity of the seafloor (10.1%) and lower variation of irradiance (9.5%) d. It wasve algae . However(c)Repeating the analysis at multiple standardized grain sizes  yielded largely similar results to the ones described above for all four regimes in terms of influential predictors and shape of the relationships. There was no significant difference across grain sizes with regard to model performance, or relative contribution of anthropogenic versus biophysical variables .4.Identifying the underlying drivers of different coral reef ecosystem regimes has great value for managers seeking viable strategies to avoid, or reverse, regime shifts. Drawing on an unprecedented compilation of data, this study presents, to our knowledge, the first attempt at quantifying the relative importance of anthropogenic and biophysical drivers in predicting reef ecosystems defined by both fish and benthic communities. As such, it offers novel insights into coral reef dynamics that can inform management strategies, as well as a promising analytical approach that might be applied in other ecosystems. Our findings provide empirical evidence that dealing with alternative regimes is inherently a social–ecological issue and that designing effective management interventions requires both focusing on prominent human drivers while accounting for the natural bounds set by the local biophysical environment.The overwhelming influence of biophysical predictors in explaining the occurrence of three out of four regimes is striking. Only the most degraded regime, characterized by low fish biomass, few corals and high turf cover, was primarily predicted by anthropogenic variables (i.e. fishing and effluent). This confirms a large body of literature highlighting the detrimental effects of high fishing pressure and effluent discharge on reef ecosystems ,32. Studa in an otherwise rather oligotrophic background. This could illustrate how a pulsed delivery of oceanic-derived nutrients from physical processes such as internal waves or current-driven upwelling [Our findings also highlight the critical role of wave power and suggest that it drives the occurrence of a specific regime (i.e. regime 2), characterized by exposed sites with high fish biomass but limited coral cover. By contrast, regime 3, which displays substantial coral cover, occurs most commonly in sheltered environments with small pulses of chlorophyll-pwelling ,38 may bpwelling .Depth and complexity appeared almost systematically among the five most influential predictors, regardless of regime type. Both variables have been identified as key features influencing the structure of reef communities and offering potential for recovery from disturbances . Areas with complex reef structure, for instance, provide refuge from predation and often harbour higher fish abundance and diversity . While tet al. [Defining ecological regimes allows capture of a considerable level of complexity of reef ecosystems ,12. The et al.  improveda, used here as a proxy for oceanic primary production [Although different reef regimes were explained by a broad range of anthropogenic and biophysical variables, the particularly strong influence of the latter warrants further consideration. First, it may be specific to Hawaiian reefs. The archipelago is one of the most isolated in the world, is located at subtropical latitudes and experiences large oceanic forcings ,19. Someoduction , can alsoduction ,46. In aCoral reef managers are often faced with the challenge of where to allocate their limited resources and what management options to prioritize. Recent studies have shown the potential of fisheries regulations to facilitate reef recovery ,48 and bIn the wake of the 2014–2016 coral bleaching event, the State of Hawai‘i pledged to effectively manage 30 per cent of its nearshore waters by 2030. Our analyses, together with our publicly available database, represent valuable resources to assist managers and policy-makers in this process. Ultimately, however, addressing the challenges coral reefs are facing globally will also require identifying distal drivers of change  and recognizing that leverage may lie far away from the reef ,53. Only


Mature spermatozoa contain a whole repertoire of the various classes of cellular RNAs, both coding and non-coding. It was hypothesized that after fertilization they might impact development, a claim supported by experimental evidence in various systems. Despite the current increasing interest in the transgenerational maintenance of epigenetic traits and their possible determination by RNAs, little remains known about conservation in sperm and across generations and the specificities and mechanisms involved in transgenerational maintenance. We identified two distinct fractions of RNAs in mature mouse sperm, one readily extracted in the aqueous phase of the classical TRIzol procedure and a distinct fraction hybridized with homologous DNA in DNA-RNA complexes recovered from the interface, purified after DNase hydrolysis and analyzed by RNA-seq methodology. This DNA-associated RNA (D RNA) was found to represent as much as half of the cell contents in differentiated sperm, in which a major part of the cytoplasmic material has been discarded. Stable complexes were purified free of proteins and identified as hybrids (R-loops) on the basis of their sensitivity to RNase H hydrolysis. Further analysis by RNA-seq identified transcripts from all the coding and non-coding regions of the genome, thus revealing an extensive wave of transcription, prior to or concomitant with the terminal compaction of the chromatin. The presence of significant amounts of RNA in the transcriptionally inert spermatozoon has been observed ,2. On thThe experiments described herein were carried out in compliance with the relevant institutional and French animal welfare laws, guidelines and policies. Sperm biopsies of animals (C57BL/6 genetic background) were kindly provided. The experiments were approved by the French ethic committee .Mature mouse spermatozoa were isolated after swim-up from the cauda epididymides from individual mice (6 and 14 months old). These samples did not contain any somatic cells, as estimated by the most simple tests of spreading on slide, Giemsa staining and microscope checking. On the basis of this routine test, we can safely say that the level of contamination by somatic cells and germinal precursors is less than 1 cell in 10,000. Such a low estimate was confirmed by the absence of the 5, 18 and 28S ribosomal RNAs characteristic of mature sperm ,6,10,11 www.zymoresearch.com). The free-RNA (R) fraction was separated and columns that retained the DNA fraction were treated first with RNase A and then with DNase. The RNA fraction that remained bound to the DNA was released and further purified. Gel electrophoresis and staining with ethidium bromide revealed a smear distribution from long to short oligonucleotides (not shown). RNase H treatment before Dnase treatment displaces the majority of the signals, while RNase A has no effect [Extraction of total nucleic acids was performed by the separation of DNA and RNA with the standard TRIzolTM protocol . TRIzol o effect  . See above extraction of DNA-bound RNA. After DNase digestion, 10–100 ng of RNA was recovered and sent to Eurofins Genomics Eurofins Medigenomix GmbH, Ebersberg, Germany) for high-throughput sequencing on an Illumina HiSeq 2500 or Illumina MiSeq Genome Analyzer. RNA libraries representing biological replicates of sperm free-RNA and DNA-bound RNA fractions, each pooled from two males, consist of two technical replicates.In total, 10 to 20 × 10To characterize the transcripts in mouse sperm, we generated approximately 66 , 69 , 77  and 87  million reads mapped to unique sites in the wild-type mouse genome from D and R sperm samples. The average fragment length of the total reads was ~100 bp. More than 50% of reads mapped uniquely to the mouse genome. All the primary sequence characteristics for sample libraries of the D and R sequences of germ cells are summarized in the RNA-seq analysis covers the whole genome with both fractions; however, the distribution of the transcripts was not completely identical to their identity and level. Variations were observed in all classes of RNAs from D to R fractions.p-values were adjusted using the Benjamini and Hochberg method [p-value (Padj) of 0.05 was set as the significant differential transcript threshold. Plots were generated using the ggplot2 R package (version 3.1.0) [p-value of less than 0.05 were considered significantly enriched. For heatmaps, log2  was used. RNA-seq reads were also quantified for tRNA-derived fragments and piRNA. An index of tRNA and piRNA sequences was constructed using tRNA sequences from GtRNAdb [Quality control of the RAW sequencing reads was performed using FastQC 0.11.7. RNA-seq reads were processed by cutadapt v1.16 trimming adaptor sequences on the 3′ end with a 10% error rate. Based on the FastQC results, 10 nucleotides were cropped from the 5′ end of each read, and reads with a length of less than 20 bp were discarded. Samples were then directly quantified using Salmon v0.12.0  on an ing method . An adjun 3.1.0) . Hisat2 n 3.1.0) . Visualin 3.1.0) . Gene onn 3.1.0) . The res GtRNAdb  and piRNSamples used for quantitative real-time PCR (RT-PCR) validation of the RNA-seq results were extracted as indicated above. The ten selected significant differential transcripts  were confirmed by RT-PCR analysis. R and D extraction from sperm were implemented as indicated above. All primers  were dest-test. Data are considered to be statistically significant with p-value < 0.05.Data are presented as means ± SEM. Group means comparisons are performed using Student’s Padj 0 < 0.05) compared to the R fraction samples. Further quantitative comparison of protein-coding from D to R samples, however, showed the significant differential load of protein-coding transcripts. Comparison of D to R samples demonstrates that generally lower amounts of transcripts are found in D fractions  between D and R sperm samples . Exon profiles of sperm samples were highlighted for free sperm RNA compared to DNA-associated RNAs. D and R sperm samples demonstrate more closed percent identity in exon features to protein-coding and lncRNA transcripts. On the other hand, D fraction shows a greater abundance of exon features than R fractions .Several classes of small non-coding transcripts were also present within the DNA–RNA hybrid sperm libraries relative to R. Hierarchical clustering of differentially detected tRNA-derived fragments and piRNA in D compared to R revealed that R1 and R3 samples are not clustered together again for small non-coding transcripts, which may reflect the difference in age of the animal used .Padj < 0.05) were assessed with DAVID Bioinformatics Resources [p-value <0.05)  . Enrichre <0.05) .Hisat2 was used to align trimmed RNA-seq reads to the reference genome (mm10), then aligned reads to the whole genome are visualized in D samples versus R using IGV, the differences between R and D might reflect the better preservation of the transcripts in the D fraction . Among dp < 0.05). Furthermore, we also measured these transcripts in somatic cells, such as the brain and liver and a comparison of the means ± SEM of the D fraction in sperm to the D fraction in somatic cells showed a significant difference between most of them (p < 0.05); the level of transcripts was higher in sperm than somatic cells  in D fractions, described to be involved in the development, were measured using quantitative real-time PCR. qPCR analysis (see below) confirmed the results of the RNA-seq. The relative transcripts of ten selected RNAs   in D fractions versus R were confirmed by quantitative real-time PCR (qPCR) using R and D extractions obtained from the sperm of wild-type mice. The transcript quantities were normalized to GAPDH as a reference gene . The traic cells . DetailsOur initial motivation to study the sperm transcriptome was the discovery that RNAs transferred during fertilization may act in the control of gene expression and establishment of specific phenotypes in the progeny ,28,29,30RNA-seq analysis led us to conclude that the transcripts identified correspond to the entirety of the genome, including both coding and non-coding regions. The relative levels of a series of the abundant transcripts previously identified in spermatid RNAs  were assessed  all tranTFAP2A), the sequence-specific DNA-binding protein that regulates transcription and binding to the consensus sequence 5′-GCCNNNGGC-3′ and activates genes involved in development [Free transcript profiling in mature sperm represents a subset of RNAs from the round spermatid stage . Sperm celopment . In addiIn the present study, we report that the open region by RNA is maintained along the chromosomes in a silent sperm genome. The essential process that occurs during the first day of the embryo’s life is the replication of the two genomes. Accordingly, insertions of the transcripts are not compatible with the progress of the replication fork, and RNAs are to be removed to complete the first embryonic division. With the evidence for the associations of RNAs in compact sperm DNA, we now confirm that the formation of DNA–RNA hybrids is part of the normal genome life. These results provide an intriguing piece of information on how cells package genomes for transmission. Together, our findings provide a way to identify signals transferred to the oocytes in complex mammalian systems ,31,33,34


Gut microbiome is increasingly recognized as a regulator of development, inflammation, host metabolic processes, and xenobiotic metabolism. However, there is little known regarding whether the gut microbiome modulates lncRNA gene expression in various host metabolic organs. The goals of this study were to 1) characterize the tissue-specific expression of lncRNAs and 2) identify and annotate lncRNAs differentially regulated in the absence of gut microbiome.Long non-coding RNAs (lncRNAs) are increasingly recognized as regulators of tissue-specific cellular functions and have been shown to regulate transcriptional and translational processes, acting as signals, decoys, guides, and scaffolds. It has been suggested that some lncRNAs act in n = 3 per group). RNA-Seq was conducted and reads were mapped to the mouse reference genome (mm10) using HISAT. Transcript abundance and differential expression was determined with Cufflinks using the reference databases NONCODE 2016 for lncRNAs and UCSC mm10 for PCGs. Although the constitutive expression of lncRNAs was ubiquitous within the enterohepatic (liver and intestine) and the peripheral metabolic tissues (fat and muscle) in conventional mice, differential expression of lncRNAs by lack of gut microbiota was highly tissue specific. Interestingly, the majority of gut microbiota-regulated lncRNAs were in jejunum. Most lncRNAs were co-regulated with neighboring PCGs. STRING analysis showed that differentially expressed PCGs in proximity to lncRNAs form tissue-specific networks, suggesting that lncRNAs may interact with gut microbiota/microbial metabolites to regulate tissue-specific functions.Total RNA was isolated from various tissues  from adult male conventional and germ-free mice  contains supplementary material, which is available to authorized users. Integrity of total RNA was confirmed using an Agilent 2100 Bioanalyzer , and samples with RNA integrity values of about 7.0 were used for sequencing. The complementary DNA (cDNA) libraries were constructed from total RNA samples using a TruSeq RNA Sample Prep Kit with poly-A tail selection . Sequencing was performed on an Illumina HiSeq2000 sequencer using a 50 bp paired-end multiplexing strategy at the Kansas University Medical Center Genome Sequencing Facility. These procedures were described in previous publications , 43, 85.The raw RNA-Seq data was deposited in Gene Expression Onmibus (GEO) database (accession numbers: GSE79848 and GSE102867). As shown in Fig. https://manticore.niehs.nih.gov/pavis2/) was used to identify lncRNAs proximal to PCGs, including 5 kb upstream of the transcription start site (TSS), intronic, exonic, 5′-untranslated region (UTR), 3’-UTR, and up to 1 kb downstream of the transcriptional termination site (TTS). A lncRNA and PCG are considered paired in a certain tissue if 1) the lncRNA overlaps with or is within 5 kb upstream of TSS or 1 kb downstream of TTS of any PCG and 2) both the lncRNA and the proximal PCG were differentially expressed between CV and GF mice (FPKM > 1 in CV or GF mice and FDR-BH < 0.05). Gene structure and relative genomic location of the lncRNA-PCG pairs were visualized using Integrated Genome Viewer .To annotate and visualize the genomic location of lncRNAs relative to the closest PCGs, the web-based tool peak annotation and visualization  [For each organ, the differentially expressed PCGs that paired with a differentially expressed lncRNA were submitted for STRING  analysis  .http://www.noncode.org/download.php) and mouse protein-coding transcript sequences were retrieved from Ensembl Biomart. LncTar Version 1.0, a command line tool for predicting lncRNA-RNA interactions, was used to generate a tab-delimited file of lncRNA and PCG pairs based on predicted free-energy associations. Specifically, we predicted the interactions between lncRNA and the paired PCG nascent transcript. A threshold of − 0.08 ndG was set because it is the lowest suggested threshold and could detect all possible lncRNA-mRNA interactions.Mouse lncRNA sequences were retrieved from the NONCODE 2016 database  for both lncRNAs and the paired PCGs are shown. Table S3. Regulation of intergenic lncRNAs in liver by the absence of gut mirobiome. Table S4. Regulation of intergenic lncRNAs in duodenum by the absence of gut mirobiome. Table S5. Regulation of intergenic lncRNAs in jejunum by the absence of gut mirobiome. Table S6. Regulation of intergenic lncRNAs in illeum by the absence of gut mirobiome. Table S7. Regulation of intergenic lncRNAs in colon by the absence of gut mirobiome. Table S8. Regulation of intergenic lncRNAs in BAT by the absence of gut mirobiome. Table S9. Regulation of intergenic lncRNAs in WAT by the absence of gut mirobiome. Table S10. Regulation of intergenic lncRNAs in skeletal muscle by the absence of gut mirobiome. (XLSX 279 kb)Additional file 2:Figure S1. Two-way hierarchical clustering dendrograms of differentially expressed lncRNAs by lack of gut microbiota. Figure S2-S8. Other examples of the expression of lncRNA-PCG pairs that were differentially regulated by lack of gut microbiota in liver , duodenum , jejunum , ileum , colon , and BAT . Figure S9. Genomic locations of lncRNA-PCG pairs described in Additional file 2: Figures S2-S3 and S5-S8. Figure S10-17. All differentially regulated PCG networks , enriched Biological Process (GO), Molecular Function (GO), Cellular Component (GO), and KEGG Pathways in liver , duodenum , jejunum , ileum , colon , BAT , WAT , and skeletal muscle . Figure S18. RT-qPCR validation of selected lncRNAs from the RNA-Seq data. (PDF 143690 kb)


The widely observed RNA-DNA differences (RDDs) have been found to be due to nucleotide alteration by RNA editing. Canonical RNA editing  mediated by the adenosine deaminases acting on RNA (ADAR) family and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) family during the transcriptional process is considered common and essential for the development of an individual. To date, an increasing number of RNA editing sites have been reported in human, rodents, and some farm animals; however, genome-wide detection of RNA editing events in sheep has not been reported. The aim of this study was to identify RNA editing events in sheep by comparing the RNA-seq and DNA-seq data from three biological replicates of the kidney and spleen tissues.BLCAP and NEIL1 genes are in line with those in previous reports on the porcine and human homologs, suggesting the existence of evolutionarily conserved RNA editing sites and they may play an important role in the structure and function of genes.A total of 607 and 994 common edited sites within the three biological replicates were identified in the ovine kidney and spleen, respectively. Many of the RDDs were specific to an individual. The RNA editing-related genes identified in the present study might be evolved for specific biological functions in sheep, such as structural constituent of the cytoskeleton and microtubule-based processes. Furthermore, the edited sites found in the ovine Our study is the first to investigate RNA editing events in sheep. We screened out 607 and 994 RNA editing sites in three biological replicates of the ovine kidney and spleen and annotated 164 and 247 genes in the kidney and spleen, respectively. The gene function and conservation analysis of these RNA editing-related genes suggest that RNA editing is associated with important gene function in sheep. The putative functionally important RNA editing sites reported in the present study will help future studies on the relationship between these edited sites and the genetic traits in sheep.The online version of this article (10.1186/s40104-019-0331-z) contains supplementary material, which is available to authorized users. According to the central dogma, it is assumed that the sequence of mRNA truthfully reflects that of the DNA template. However, we know that the RNA sequences are not coded one-to-one by their corresponding DNA sequences. RNA modifications during co- and post-transcriptional processes contribute to the complexity of alternative splicing, and sequences and structures of the mature RNA molecules. One of the most important mechanisms is RNA editing, which results in RNA–DNA differences (RDDs), e.g., codon changes leading to protein variants . It has The most commonly observed RNA editing event in human is A-to-I editing . The genes of the adenosine deaminases acting on RNA (ADAR) family were investigated to mediate the A-to-I editing process by binding the double-stranded (ds) RNA secondary structure . AnotherAccording to the Rigorously Annotated Database of A-to-I RNA Editing (RADAR) database , only a To the best of our knowledge, there is no report on genome-wide scanning of RNA editing sites in sheep. In the present study, for the first time, we applied DNA and RNA resequencing data to identify potential editing events in sheep. We restricted our search for RNA editing sites only to homogenous genomic DNA (gDNA) sequences, but heterogenous RNA sequences as putative RDDs were also included to minimize false positives. In addition, crucial thresholds were set for the detection of RDDs for RNA and DNA mapping and genotype quality in order to eliminate uncertain reads and single-nucleotide variants (SNVs). Furthermore, we excluded the RDDs that are present on repetitive regions, which may result in mis-mapped reads and variant call errors , 14. OveThe kidney and spleen tissues, and blood samples were obtained from three adult (2 years old) male Lanping sheep (a Chinese indigenous sheep breed) from the same population in Yunnan Province of China. These animals were unrelated according to their pedigree records. DNA was extracted from blood samples with the routine phenol–chloroform extraction method. The total RNA was extracted and purified using the RNA Sample Total Kit . The quality of gDNA and RNA was evaluated using NanoDrop 2000 (Thermo Fisher Scientific) and by agarose gel electrophoresis (1.2% agarose gel).For each DNA sample, a whole-genome sequencing library was built using the Illumina TruSeq DNA Sample Preparation Kit  with an insert size of ~ 350 bp. The libraries were sequenced on the Illumina HiSeq 2000 platform. The paired-end reads of 100 bp were generated for each fragment.The total RNA from each sample was used as input for the TruSeq RNA Prep Kit  and by means of indexed adapters; a sequencing library was created according to the manufacturer’s instructions. RNA sequencing was conducted on the Illumina HiSeq 2500 platform, resulting in paired-end 100-bp reads. The insert length of the RNA-seq library ranged from 300 to 400 bp. The DNA- and RNA-seq data have been deposited to the National Center for Biotechnology Information (NCBI) SRA Database (accession number: SRP133430).The raw reads of both DNA- and RNA-seq data were first checked using the FastQC tool  and trimFor the RNA-seq data, the reads were mapped to the sheep reference genome using the two-pass mapping strategy in STAR (v2.6) , and onlJoint variant calling was conducted on the BAM file using Samtools/Bcftools , 20 to cIn the detection of RNA–DNA differences, only positions supported by at least four genomic reads and completely homozygous were retained. The following types of mismatches were also discarded according to a previous study : 1) suppftp://ftp.ncbi.nlm.nih.gov/genomes/Ovis_aries/GFF/) to extract the RDDs located in the transcriptional regions, and then identified their types with the help of the strand information of each transcript.Due to our RNA-seq data are not strandness specific, we utilized the gene annotation file (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer sequences are listed in Additional file To validate the homozygous/heterozygous status of the putative RDDs, we used PCR to amplify RDDs on 13 putative editing sites. The PCR products of DNA and cDNA samples were sequenced by Sanger sequencing. Primers were designed using the NCBI Primer-BLAST tools (ftp://ftp.ncbi.nlm.nih.gov/genomes/Ovis_aries/GFF/). The genes with mismatches located in the exonic regions were extracted for the functional enrichment analysis. Gene Ontology annotation were computed using the DAVID web tool [Transcriptome quantification and differential expression analyses were performed using Cuffdiff . ANNOVARweb tool .https://www.ensembl.org/index.html).To analyze the conservation of upstream and downstream sequences of an editing site, alignment of genomic sequences among different species was conducted using the alignment tool, which is available on Ensembl . Transcriptome-wide screening revealed 84,361 and 97,295 high-quality heterozygous SNVs in the kidney and spleen tissues of sheep, respectively.In the present study, SNV sites that were homozygous at the DNA level and heterozygous at the transcriptome level were extracted as putative RNA editing sites. After comparing the DNA-seq and RNA-seq reads generated from the same individuals and applying additional filters for removing RDDs in the splicing region, a total of 18,644 and 25,407 RDDs in the kidney and spleen were obtained, respectively. In the three kidney tissues replicates, 7,869, 6,036, and 7,779 RDDs were retained, respectively. Furthermore, 10,391, 9,253, and 10,702 RDDs were obtained from the three spleen tissue samples  are shown in Additional file We validated 13 RDDs by Sanger sequencing method, 9 of the 13 sites were confirmed as the true edited sites. The sites on chr11:41753132 were considered false positives by comparing their gDNA and cDNA sequencing chromatograms. At the two predicted editing sites of P< 0.01) higher in the spleen than in the kidney  and spleen (346/994) was found in the intergenic regions; the proportion of exonic DNA-RNA mismatches was both close to 15% in the kidney (106/607) and spleen (144/994). In the intronic regions, the mismatches in the kidney (177/607) and spleen (346/994) were made up of 27–28% RDDs in total. Especially, additional 15 and 52 mismatches were detected in ncRNAs in the kidney and spleen, respectively  type of mismatches between DNA and RNA in transcriptional regions , respectively. Eighty-three genes were found annotated in both the kidney and spleen RDDs. In the kidney, 44 RDDs (21 genes) in the kidney and 63 RDDs (33 genes) in the spleen were found to be nonsynonymous mutations resulting in amino acid changes or stop–gain mutations  and the previously reported RNA editing sites, revealed that bladder cancer associated protein (BLCAP) and Nei endonuclease VIII-like 1 (NEIL1) have non-repetitive RDD sites.The type and location of the two editing sites on genomic reads and RNA reads were shown by IGV tools in Fig. BLCAP and NEIL1 were also found in previous studies in chicken [Interestingly, exploration of the human RNA editing database RADAR   on exon 1 of BLCAP and NEIL1 genomic sequences were performed using the Ensembl online alignment tool. The edited sites detected in BLCAP and NEIL1 were both located in conserved genomic regions with 100% sequence similarity among these species , and in the UTRs of genes , 29, 30.Generally, among all the RDD types, A-to-I and C-to-U are presumed to be canonical editing sites; the other 10 non-canonical editing sites were usually presumed to be false-positive results due to the current sequencing technology and analysis limitations .APOBEC1 is not expressed in the two tissues investigated, but APOBEC2 and APOBEC3F were found to be expressed at a low level , providing a clue that the Y/C editing site in BLCAP is associated with the function of BLCAP and the development of cancer [NEIL1 gene, which codes for a DNA repair enzyme, has also been reported in multiple tissues in human [he brain  and fat he brain , and thef cancer . Exon 6 in human  and pig in human . Additioin human .These editing events occur in the protein coding region can directly alter the protein sequences and structures, and then affect the biological function. These results suggest a specific regulatory mechanism for gene function derived by RNA editing.BLCAP and NEIL1 are conserved in pig and human, suggesting that evolutionarily conserved RNA editing sites may be important for gene function and the development of an individual. Our study provides some putative functionally important RNA editing sites in sheep, and it is important to conduct further studies to reveal the functional changes in RNA editing event genes and their roles in economically beneficial traits and diseases in sheep.To the best of our knowledge, our study is the first to detect RNA editing in sheep by comparing RNA-seq and DNA-seq data from three biological replicates of the kidney and spleen tissues. Under stringent sequence data filters, a total of 607 and 994 common RDDs were identified in the ovine kidney and spleen as the reliable RNA editing sites. Further,164 and 247 genes were annotated to the kidney and spleen RDDs. The amino acid conversion edited sites found in the ovine Additional file 1:Figure S1. Shared RDDs of the Three Biological Replicates in the Kidney and Spleen. Figure S2. Conservation Analysis of BLCAP. Figure S3. Conservation Analysis of NEIL1. Table S1. Validation of RNA Editing Sites by Sanger Sequencing. Table S2. FPKM (mean ± SD) of APOBEC3F and APOBEC2 in the Kidney and Spleen. Table S3. PCR Primers Used in Sanger Sequencing Validation. Table S4. Nonsynonymous Common Editing Sites in the Kidney of Sheep. Table S5. Nonsynonymous Common Editing Sites in the Spleen of sheep. Table S6. Functional Annotation Results of RNA Editing Genes in the Kidney of sheep. Table S7. Functional Annotation Results of RNA Editing Genes in the spleen of sheep. (DOCX 1050 kb)Additional file 2:Putative RDDs in Sheep Individual1. (XLSX 1098 kb)Additional file 3:Putative RDDs in Sheep Individual2. (XLSX 880 kb)Additional file 4:Putative RDDs in Sheep Individual3. (XLSX 1115 kb)


FGFR3 and FGFR4 in the recurrent tumor compared with the treatment-naïve tumor, indicating a pivotal role for FGFR signaling in cancer cell survival through CIRT. Inhibition of FGFR using the clinically available pan-FGFR inhibitor LY2874455 sensitized multiple cancer cell lines to carbon ions at 3 Gy , the daily dose prescribed to the patient. The sensitizer enhancement ratio was 1.66 ± 0.17, 1.27 ± 0.09, and 1.20 ± 0.18 in A549, H1299, and H1703 cells, respectively. Our data indicate the potential usefulness of the analytical pipeline employed in this pilot study to identify targetable mutations associated with resistance to CIRT, and of LY21874455 as a sensitizer for CIRT-resistant cancers. The results warrant validation in larger cohorts.Radiotherapy is an essential component of cancer therapy. Carbon ion radiotherapy (CIRT) promises to improve outcomes compared with standard of care in many cancers. Nevertheless, clinicians often observe in-field recurrence after CIRT. This indicates the presence of a subset of cancers that harbor intrinsic resistance to CIRT. Thus, the development of methods to identify and sensitize CIRT-resistant cancers is needed. To address this issue, we analyzed a unique donor-matched pair of clinical specimens: a treatment-naïve tumor, and the tumor that recurred locally after CIRT in the same patient. Exon sequencing of 409 cancer-related genes identified enrichment of somatic mutations in  Radiotherapy is an essential component of cancer therapy . Carbon To explore targetable biological properties in CIRT-resistant cancers, we analyzed the genetic profile of tumors collected from a patient with uterine cervical cancer who experienced local recurrence after CIRT . The patPIK3CA (E545K) were identified in both T1 and T2. These mutations were validated by Sanger sequencing  on 27 November, 2013. Written informed consent was obtained from the patient. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.Tissue sample collection was performed as previously described . The preDNA preparation was performed as previously described . In brieSequencing was performed as previously described . Forty nSomatic mutations were determined as previously described . In briehttps://www.alliancegenome.org), GO ribbons for the genes of interest were obtained. The color intensity for the annotations shown in the GO ribbons was interpreted as scores with a range of 0–4. The GO enrichment score was calculated by multiplying the color intensity score with the subtraction of T1 variant frequency from T2.Using the Alliance of Genome Resource . A


First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Pankaj Dubey and Tushna Kapoor are co-first authors on ‘ Tushna KapoorWhat is your scientific background and the general focus of your lab?TK- I completed my BSc (Honours) in Biochemistry from Sri Venkateswara College, University of Delhi, and then joined the integrated MSc-PhD program at the Tata Institute of Fundamental Research (TIFR). My research training at TIFR has been in core cell biology and microscopy.Drosophila. We explore the role of Kinesin-2 in development and function of cilia and axonal transport. A few years back, we stumbled upon the role of Kinesin-2 in maturation of sperm. Since then, the lab has also been studying the role of the cytoskeleton, adhesion and signaling in Drosophila spermatogenesis. We employ genetic tools, biochemistry and imaging to understand the cell biology of each project.Our lab focuses on the cellular biology of the cytoskeleton, motors and transport in Pankaj DubeyHow would you explain the main findings of your paper to non-scientific family and friends?PD- This is bit of a tricky one; the non-scientific community always find it amusing that we work on insect sperm release. I explain to them that we are looking at how two cells imprison sperm and we found the ‘lock’ of this prison. When we break the lock, sperm are released early, affecting the ability of insects to produce more insects. It makes an interesting prison-break story.TK- I tell them that a group of sperm cells develop within an enclosure formed by two covering nurse cells. This specialized environment is essential for sperm development. Thus, this enclosure needs to be maintained as the sperms divide, elongate to form a tail and subsequently mature. Therefore, a ‘glue-like’ or ‘sticking’ protein is required for the two covering cells to stick with each other and maintain the enclosure through all these developmental processes. The work in this paper describes the maturation and role of these proteins (septate junctions) through different stages of sperm development.“The non-scientific community always find it amusing that we work on insect sperm release […] it makes an interesting prison-break story.”What are the potential implications of these results for your field of research?PD- It was believed that two somatic cyst cells are attached to each other throughout spermatogenesis. Our data systematically provide evidence of the existence and maturation of junctions that may keep them together throughout spermatogenesis. We explored the hypothetical implication of destroying these junctions at the final stages of spermatogenesis. Our data will compel the field to study the role of these junctions during the middle stages of spermatogenesis and exploration of more such structures. Although analog structures – tight junctions – are present during mammalian spermatogenesis, the kind of cell biological insight we can get using Drosophila is somewhat difficult in mammals. We hope our results further the cause of the essential role of cell adhesion in various developmental contexts.TK-Drosophila has been an underappreciated model system to study spermatogenesis. It is, however, strikingly similar to mammalian spermatogenesis, along with the ease of genetic manipulation and imaging. One such example is presented here with the junctions imparting barrier and signaling functions during the early stages, and a structural role during the release stages.This paper also illustrates the remodeling, maturation and roles of septate junctions in a system other than a typical epithelial layer, where they are most commonly studied.What has surprised you the most while conducting your research?PD- In general, working with Drosophila spermatogenesis, I get fascinated by the way system is so beautifully organized. In contrast to mammals, insect sperms are huge – almost as long as the length of their testis. Hence, the containment of growing spermatids becomes essential for spermatogenesis. Having a cell enclosure stitched by septate junctions makes up an elegant solution to avoid the mess that otherwise would have happened with hundreds of 1.8 mm long spermatids floating in Drosophila testis. The septate junctions are not known for extensive changes once they are formed and we were surprised to see the dynamic reorganization of the junctions as the spermatid matured.TK- I experienced first-hand how, even with limited techniques and lack of live imaging, scientists could make accurate observations and predictions on dynamic processes. Previous electron microscopy (EM) work has shown that the mature junctions are assembled only in later stages of spermatogenesis, and it was proposed that the position of this junction moves over time and with the developmental stage. We were able to reaffirm these results by live confocal imaging of fluorescently tagged proteins. It was pleasantly surprising.“I experienced first-hand how, even with limited techniques and lack of live imaging, scientists could make accurate observations and predictions on dynamic processes.”What, in your opinion, are some of the greatest achievements in your field and how has this influenced your research?PD- There are three important achievements that have driven the spermatogenesis research in Drosophila. Along with other great researchers, the late Dr Tokuyasu's meticulous characterization of Drosophila spermatogenesis stages in the 1970s using transmission EM was an extraordinary achievement. The six seminal papers from Tokuyasu are essential reads even today for everyone working on Drosophila spermatogenesis.TK- I think the collective body of work over many years that show that sites of cell adhesion are not just that, but also hubs of signaling and polarity, have been great findings. Also, recently there have been a flurry of papers that show that, apart from their conventional role of providing a permeability barrier, tight/septate junctions also provide structural and mechanical integrity to cells/tissues. This is exciting as it shows that there is room to explore even in seminal discoveries and that the boundaries are always blurring in science. Both these aspects of septate junctions have been highlighted in our paper as they were shown to have different roles at different stages of spermatogenesis.What changes do you think could improve the professional lives of early-career scientists?PD- I think good mentorship, a cheerful lab atmosphere and space to make mistakes is key to start developing independent thinking. I think is it also important to remind early scientists to keep their personal lives in focus while getting immersed in experiments.TK- Funding is a crucial factor. Both at the level of research, as well as pay-scales of early-career scientists. Many people drop out of science because of the poor pay versus the physical, emotional and mental effort that goes into this career. Also, I think the scientific community needs to inculcate a greater respect for people's lives outside the lab – be it their personal or family lives.“I think good mentorship, a cheerful lab atmosphere and space to make mistakes is key to start developing independent thinking.”What's next for you?TK- I am currently working on another paper where we show the role of an actin-based structure in preventing abnormal sperm release. Apart from that, I also have another project. I plan to complete my PhD within the next 18 months and then move on to do a post-doc within the fields of cell adhesion-cytoskeleton-membrane dynamics.