Beta macrocarpa. Within this ‘core’ sequence resides a conserved “coremin” motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3) possessing “coremin” at its 5′ end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt) or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S.cerevisiae ribonuclease mutants identified the 5′-to-3′ exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA). Substitution of the BNYVV-RNA3 ‘core’ sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle.The RNA3 species of the beet necrotic yellow vein virus (BNYVV), a multipartite positive-stranded RNA phytovirus, contains the ‘core’ nucleotide sequence required for its systemic movement in  In eukaryotic cells, the RNA decay machinery plays a major role in determining both the quantity and quality of gene expression. Nuclear RNA surveillance complexes remove aberrant and unprocessed transcripts, while the cytoplasmic RNA decay machinery coordinates with translation to determine the half-life of mRNAs . Most mRThe process of subverting the host RNA decay machinery can also lead to the generation of new viral RNA species. Arthropod-borne flaviviruses use a knot-like three helix junction structure at the beginning of the 3′ untranslated region of their genomic RNAs to stall Xrn1 and generate a stable RNA decay intermediate ,11,12,13Nicotiana benthamiana [NbXRN4 enhances viral accumulation in local infections of tobacco mosaic virus (TMV) and accelerates its systemic movement in the plant [Animal, insect, and yeast Xrn1 homologs are cytoplasmic RNA decay enzymes that ensure proper homeostasis and cell growth . These ethamiana . Furtherhe plant , while ahe plant .Benyviridae family and is the type member of the Benyvirus genus [Polymyxa betae and causes the sugar beet Rhizomania disease. The BNYVV genome is composed of four to five linear, positive-sense, single-stranded, capped, and polyadenylated RNAs. The small viral genomic RNAs are required for the natural infection of β species and for transmission of the virus, but are dispensable for laboratory host mechanical infection since BNYVV-RNA1 and -RNA2 are necessary and sufficient for viral replication in rub-inoculated laboratory host plants [β species. Systemic infections of β macrocarpa absolutely require a 20 nt long “coremin” sequence that is embedded in the RNA3 ‘core’ region [Beet necrotic yellow vein virus (BNYVV) belongs to the us genus ,28. BNYVt plants . RNA1 ent plants , acting t plants . The ncRt plants , is proct plants  and is e’ region ,34,35. ISaccharomyces cerevisiae to show that ncRNA3 production can be achieved by the action of either the yeast exoribonuclease Xrn1 or plant XRN4. The coremin motif embedded within a 55 nt region is sufficient to stall 5′-to-3′ exoribonucleases in vitro and in insect cell extracts similar to the sfRNA structural motif. The ‘core’ domain substitution with the sfRNA sequence allows the accumulation of ncRNA species in yeast and in vitro. However, such substitution surprisingly prevents the production of ncRNA in the viral context as well as long-distance movement of the chimeric virus in plants. These data suggest that the original “coremin” sequence itself, rather than simply stalling Xrn-mediated RNA decay, is mandatory for viral systemic movement in Beta macrocarpa. Virus-induced gene silencing (VIGS) of NbXRN4 downregulated BNYVV-RNA accumulation, a situation that recalls bamboo mosaic virus behavior [Here, we use genetic approaches in behavior  without EcoRI restriction site (position 377) of the full-length cDNA clone of BNYVV-RNA3 (pB35) was filled in with a Klenow fragment then self-ligated to produce pB36. pB36 served for site-directed mutagenesis [EcoRI site at the 5′ terminus of the genome to produce a pB37 infectious clone The resulting cDNA copy of RNA3 was then cloned into EcoRI-HindIII-digested p426GPD vector to produce p426GPD37. The fragment from pB35E containing the mutated “coremin” sequence [BamHI-HindIII restriction sites to create the p426GPD37E vector.The sequence  was subcEcoRI and HindIII restriction sites. BN3 and 1R to 9R constructs contained an EcoRI and a T7 promoter sequence followed by the BNYVV-RNA3 3′ end cDNA sequence starting from BamHI (position 750) to HindIII . BN3sf/pk1 differed from BN3 by the nt 1147–1477 replacement with nt 10,422–10,728 from the flaviviral sequence (accession number AY274505). The vectors BN3sf and BN3pk1 served for a BamHI-HindIII sequence switch with p426GPD37 and pB35 to produce p426GPD-RNA3sf/pk1 and pB35sf/pk1, respectively.A BN3sf cDNA clone and its BN3pk1 variant were obtained as synthetic genes products ordered at Genscript®  and delivered into pUC57 between PstI and HindIII of pGEM-4 . The plasmid used to produce the RNA containing the upstream portion of the DENV 3′ UTR has been described previously [HindIII. The plasmid was linearized with EarI to generate competitor RNAs. The BNYVV variant B-1 and B-2 RNAs were generated from plasmids linearized with HypCH4III and Hyp1881, respectively. Control RNAs were generated from pGEM-4 linearized with either EarI (to generate 168 base cold competitor RNA) or with HindIII . The DENV 3′ UTR plasmid was linearized with EarI to generate the DENV 3′ UTR competitor RNA.A plasmid encoding the BNYVV-55 element (sequences 1222–1277 from BNYVV RNA3) was generated by inserting oligos containing the indicated BNYVV sequence (NBCI reference sequence: NC_003516) into the eviously . Internaeviously  and puriArabidopsis thaliana XRN4 (ATXRN4) was obtained by Gateway cloning between pDONR207 containing A. thaliana XRN4 gene (http://www.addgene.org/vector-database/2393/) and the destination vector pAG423GPD-ccdB HA (https://www.addgene.org/14246/), using LR Clonase™ enzyme mix  following the manufacturer’s recommendations.Yeast vectors pRS415 (empty backbone), pAJ152 (expressing Xrn1), and pRDK307  were provided by Lionel Benard . Yeast vYeast strains were cultured on YPD complete media and transformed using the method described by Gietz and Schiestl . TransfoIn vitro production of ncRNA3 was from a pUC19 construct containing the ncRNA3 sequence (nt 1234 to 1774 followed by 15A) downstream of a T7 promoter. Amplicons allowing the synthesis of transcripts possessing 3′ shortened ends were from T7 promoter-containing primer starting position 750 together with primers complementary to RNA3, ranging from position 1739 to 1308 . The linearized full-length BNYVV clones of RNA1 (pB15), RNA2 (pB21), RNA2Δp14 (pB2-3722), RNA2BA2 (pB2-BA2) .Chenopodium quinoa and B. macrocarpa or for the electroporation of C. quinoa protoplasts as described before [C. quinoa local lesions and protoplasts. RNAs were extracted using “Polysomes” buffer [RNA1 and RNA2 transcripts supplemented or not with wild-type (wt), mutated, or chimeric RNA3 served for the mechanical infection of d before ,41,42. R” buffer  followed” buffer .Agrobacterium tumefaciens cells (GV3101 or C58C1) were prepared as described previously [N. benthamiana (4–5 weeks-old seedling). XRN4 gene silencing was performed as described [Cultures of eviously  for agroescribed  and VIGSTM Reverse Transcriptase (Promega) following the manufacturer’s instructions. cDNAs were submitted to qPCR using LightCycler® 480 SYBR Green Master mix  and a LightCycler® 480 instrument (Roche). Xrn4 5′ and 3′ sequences flanking the VIGS-targeted sequence were assayed using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Protein phosphatase 2A (PP2A) as internal references.RNAs were treated with RQ1 DNAse  before reverse transcription using oligo(dT) primer  and Improm-IIHindIII-linearized pB35sf, pB35pk1, BN3, BN3sf, and BN3pk1 or PsiI-linearized pB35 and pB35E plasmids served to produce in vitro run-off transcripts without adding a Cap analog [p analog . The DNAKluyveromyces lactis Xrn1 (residues 1–1245) [Escherichia coli BL21 cells. For Xrn1 decay assays performed to identify decay intermediates, approximately 85 fmol of each radiolabeled input RNA was used. For RNA competition assays, ~85 fmol of a pGEM-4 reporter RNA was incubated with a 20-fold molar excess of the pGEM-4 control competitor or the viral-derived DENV 3′UTR or BNYVV 55mer core RNAs. These competitor RNAs were lightly radiolabeled with 32P-UTP to allow for easy quantification and follow their fate following incubation. RNA reaction products were separated on 5% denaturing polyacrylamide gels and visualized by phosphorimaging. The average of the percent RNA remaining in each time point from three independent experiments was used to generate graphical data. Significance was determined using a two-way ANOVA and p-values were generated using Tukey’s multiple comparisons test as a post-hoc test. The * represents a p-value <0.001 at both time points for the viral 3′ UTR compared to the control.In vitro 5′-to-3′ Xrn1 decay assays and competitions were performed essentially as described  using C6 1–1245)  purifiedSaccharomyces cerevisiae FY4 strains were transformed with plasmid constructs expressing either RNA3 deleted of its 5′ UTR  were implicated in BNYVV-RNA3 processing, leading to ncRNA3 accumulation in a heterologous, non-viral wheat germ extract system . To furts 5′ UTR , wt RNA3s 5′ UTR  in whichs 5′ UTR A. The ems 5′ UTR . In the s 5′ UTR B, lane 1s 5′ UTR . We perfs 5′ UTR C, lane 1sequence C, lane 2S. cerevisiae cellular machinery recapitulates accumulation of ncRNA3 observed in planta, we screened a collection of viable nuclease mutants to identify gene products responsible for RNA3 processing. Four strains were selected, FY4∆rnh1, ∆rrp6, ∆rex3, and ∆xrn1, and then transformed with the RNA3 construct. Rnh1 is a nuclease protein implicated in RNA degradation from RNA-DNA duplexes [xrn1 accumulated full-length RNA3 and RNA3E (RAT1 gene product) on nuclear primary transcripts. Unfortunately, the ∆xrn2 mutant is not available and the double ∆xrn1-∆xrn2 mutant is not viable and could thus not be tested [xrn1 yeast strains by co-transforming them with plasmids expressing wt or mutated yeast Xrn1 . In the presence of an empty vector, only traces of ncRNA3 species were detected , A. thadetected B, lane 6dcp2, ∆xrn1, and ∆dcp2-∆xrn1 [Taken together, these results demonstrate that ectopic expression of yeast Xrn1 or plant XRN4 can functionally complement 5′-to-3′ processing of RNA3, leading to ncRNA3 accumulation in vivo. The 5′ extremity of the ncRNA3 species produced by XRN4 complementation in yeast was found to be identical to those characterized in planta, in wild-type FY4, and in Xrn1-complemented yeast strains. Similar experiments were performed using yeast strain W303-1A and mutants ∆p2-∆xrn1 . Only wiS. cerevisiae FY4 strain  as compared to the published sfRNA 5′ extremity, which has been determined previously by primer extension [xrn1 together with vectors expressing Xrn1  genomic RNA, including the Kunjin virus strain, provokes an Xrn1 stalling event leading to sfRNA accumulation. Such sfRNA species promote pathogenesis due to their complex secondary folding structure ,9,12. Th4 strain B, left arn genes B, right.rn genes A, chimerrn genes B, lane 1rn genes B, lane 4xtension . The ncRing Xrn1 B, lane 7g vector B, lane 8We confirmed Xrn1 function in ncRNA production using in vitro transcripts and purified enzymes by adapting a biochemical assay described for flaviviruses ,18. FullUsing chimeric sfRNA substrates (full-length RNA3sf and truncated BN3sf species), we reproduced the accumulation of ncRNA species of expected size , 44 (B-2), or 55 (B-3) nucleotides downstream in an Xrn1 decay assay performed using a short time course with an amount of highly active recombinant escribed . As seenescribed .Previous work indicated that in addition to stalling Xrn1, structures from other viruses can also serve as reversible inhibitors and transiently but significantly repress Xrn1 activity ,37,53. TB. macrocarpa. We inoculated B. macrocarpa leaves with RNA1 and 2 helper strain to ensure replication, encapsidation, cell-to-cell movement, and suppression of post-transcriptional gene silencing functions [While ncRNA3 and ncRNA3sf are produced in a similar way in vivo and in vitro, we pursue a translational approach and test whether the production of ncRNA3sf could support the long-distance movement of BNYVV in unctions ,33 aloneunctions  or to chB. macrocarpa inoculated leaves were similar to those presented for C. quinoa leaves or protoplasts . The effect of this KD on replication of BNYVV after inoculation was investigated bearing in mind a mixed infection context. While exoribonuclease XRN4 appears to be required for the production and accumulation of ncRNA3 species and ncRNA3 acts in synergy with BNYVV VSR [N. benthamiana plants were silenced using recombinant TRV constructs [Both tobacco rattle virus (TRV) and BNYVV infect NYVV VSR , we testNYVV VSR . TherefoNYVV VSR . N. bentnstructs  carryingnstructs  supplemeViral RNAs were detected in 6 inoculated leaves out of 10 in XRN4 KD plants A and 9 aB. macrocarpa host requires the cis-active RNA sequence named ‘core’ and is not based on RNA3 p25 expression [B. macrocarpa species. Indeed, mutations introduced within “coremin” prevented ncRNA3 species accumulation and viral systemic movement on Beta species [A. thaliana, such a plant is unfortunately not a host for BNYVV and therefore not suitable to set up genetic screens. Furthermore, B. macrocarpa is far from being genetically characterized. Therefore, other approaches consist in the use of heterologous genetic systems able to recapitulate one or more biological functions. We expressed BNYVV-RNA3 in S. cerevisiae outside a context of viral infection. We succeeded in the detection of ncRNA3, indicating the presence of yeast cellular factor recapitulating plant processes. Using S. cerevisiae KO mutants, we identified Xrn1 exoribonuclease as the main actor for ncRNA3 production from ectopically expressed RNA3 species, which was confirmed by complementation experiments of the S. cerevisiae ∆xrn1 mutant strain with plant XRN4 or yeast Xrn1 . We susVirus-induced gene silencing (VIGS) knockdown is a well-described method for functional validation of cellular pathways required in biological processes. This approach requires a systemic plant infection with a non-related virus possessing a sequence derived from the target gene. Reduction of XRN4 expression was not sufficient to alleviate the production of the ncRNA3 species. The ncRNA3 was still detected (N. benthamiana with TRV and BNYVV. XRN4 KD lowered BNYVV RNAs accumulation, suggesting a role of the nuclease in the replication steps of BNYVV.Our experiments demonstrate the role of the “coremin” sequence and 3′ proximal sequences in 5′-to-3′ exoribonuclease stalling leading to the accumulation of ncRNA species and to long-distance movement of the virus. In the context of VIGS mixed infection, no viral long-distance movement was observed even when a PDS control was used. Such absence of systemic movement is not explained and could be the result of the coinfection of Our results can be correlated to those obtained with bamboo mosaic virus, whose replication complex hijacks the XRN4 ribonuclease to ensure an efficient amplification of the virus . West Ni


The potential damage caused by an environmental threat increases with proximity to the body, so animals perform more effective and stronger defensive responses when threatening stimuli occur nearby the body, in a region termed the defensive peripersonal space (DPPS)  Bufacchi and Iannetti show that gravitational cues affect the shape of the defensive peripersonal space. They come to this conclusion by measuring how the magnitude of the eye-defensive blink reflex changes as a function of the position of external threats and participants’ body posture. In Experiments 1 and 2, participants were upright . In ExpeIf the hypothesis that the DPPS vertical asymmetry is determined by gravitational cues is correct, the shape of the DPPS should remain asymmetric along the gravity axis, regardless of head orientation. Alternatively, the DPPS could remain asymmetric along the head vertical axis, regardless of head orientation with respect to gravity. To distinguish between these two possibilities, we altered body posture relative to the direction of gravity, and derived the geometry of the DPPS. In Experiment 3, participants lay supine. In Experiment 4, participants lay on their left side  and S1. A 5 x 2 repeated-measures ANOVA on the data pooled from Experiments 1 and 3 showed significant effects of ‘hand-position’  and a significant ‘hand-position’ x ‘body-position’ interaction . This interaction arose from a larger HBR magnitude in hand position ‘far-high’  when participants were upright. So when an individual is supine, the DPPS shape is no longer elongated equally far above eye level, but becomes less asymmetrical along the head vertical axis .A 5 x 2 repeated-measures ANOVA on the data pooled from Experiments 2 and 4 showed significant main effects of ‘hand-position’  and ‘body-position’ . This latter main effect arose from a larger HBR magnitude in hand position ‘right’  when participants were lying on their side. Therefore, when individuals lie on their left side, although the HBR magnitude remains largest in position ‘middle’, the HBR increase in position ‘right’ indicates that the DPPS shape becomes asymmetrical: it has a larger extent on the right side — opposite to the direction of gravity . All staTo formally test the effect of gravity on DPPS shape, we used three versions of a geometric model of the DPPS Taken together, these results clearly support the notion that the brain uses a malleable DPPS representation, and continuously updates the threat value of stimuli based on gravitational cues, automatically inferring the effects of physical laws. The ability of the nervous system to adjust the DPPS shape based on gravitational cues has a clear survival advantage. Gravity causes all objects to fall: in natural environments a threatening object is more likely to cause damage when it is above the body than when it is below — a fact obviously independent of body posture. Therefore, heightened defensive responses to above-body threats, which have greater hit probability, would maximally mitigate harm. There are a few hints of a vertical asymmetry in the threat value assigned to environmental events. Vertical asymmetries in visual perception are well documented Adjustment of the HBR magnitude results from a top-down cortical modulation of the excitability of brainstem interneurons The concept that a change in harm probability — determined, in the current experiments, by different body postures relative to gravity — causes a change in DPPS shape is in line with previous findings, and provides an overarching narrative. For example, when the hit probability of objects in front of the body increases because of locomotion R.J.B. and G.D.I. designed the study, analysed and interpreted the data, and wrote the paper. R.J.B. collected the data.


Background: The development of new antiviral drugs based on nucleic acids is under scrutiny. An important problem in this aspect is to find the most vulnerable conservative regions in the viral genome as targets for the action of these agents. Another challenge is the development of an efficient system for their delivery into cells. To solve this problem, we proposed a TiO2·PL–DNA nanocomposite consisting of titanium dioxide nanoparticles and polylysine (PL)-containing oligonucleotides.Results: The TiO2·PL–DNA nanocomposites bearing the DNA fragments targeted to different conservative regions of (−)RNA and (+)RNA of segment 5 of influenza A virus (IAV) were studied for their antiviral activity in MDCK cells infected with the H1N1, H5N1, and H3N2 virus subtypes. Within the negative strand of each of the studied strains, the efficiency of DNA fragments increased in the direction of its 3’-end. Thus, the DNA fragment aimed at the 3’-noncoding region of (−)RNA was the most efficient and inhibited the reproduction of different IAV subtypes by 3–4 orders of magnitude. Although to a lesser extent, the DNA fragments targeted at the AUG region of (+)RNA and the corresponding region of (−)RNA were also active. For all studied viral subtypes, the nanocomposites bearing the DNA fragments targeted to (−)RNA appeared to be more efficient than those containing fragments aimed at the corresponding (+)RNA regions.Conclusion: The proposed TiO2·PL–DNA nanocomposites can be successfully used for highly efficient and site-specific inhibition of influenza A virus of different subtypes. Some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs were found. The (−)RNA strand of IAV segment 5 appeared to be more sensitive as compared to (+)RNA. The fundamental scientific problem of life sciences, especially modern molecular biology and fundamental medicine, is the development of approaches to the directed action on the genetic material of cells. Fragments of nucleic acids  can be used for this purpose because they are known to site specifically interact with certain regions of nucleic acid (NA) targets. The development of antisense technology for the creation of drugs that can gene specifically affect the genes responsible for infectious, hereditary, cancer, and cardiovascular diseases is an important task of modern medicine and pharmacology. The selective recognition of molecular targets by NA-based therapeutics may minimize negative side effects compared to conventional pharmaceuticals that typically have less specificity.Influenza A viruses (IAVs) are prominent in infectious diseases of humans and animals and periodically cause epidemics and epizootics. At present, the development of new antiviral drugs based on native or chemically modified nucleic acids is under scrutiny. Researchers all over the world explore the therapeutic potential of NA-based drugs to fight against the genetically variable viruses including IAV.Many attempts to produce effective antiviral drugs against IAV have not yet been successful because of the low ability of NA-based drugs to penetrate into cells. In the last few years, there were a large number of publications describing different delivery systems of nucleic acid fragments into cells –3, incluAnother important problem of using NA-based drugs is to select a proper target in the IAV genome. The choice of the most efficient antiviral NA-based drugs to affect IAV replication may contribute to the creation of therapeutic preparations, the activity of which would be less dependent on mutations. The IAV genome contains eight single-stranded RNA segments of negative polarity and belongs to the Ortomyxoviridae family. All eight segments are important for the viral replication. After infection of cells, the viral (−)RNA segments are transcribed into (+)mRNA and replicated into (+)cRNA . The IAVDifferent regions of segment 5 have been studied for their sensitivity to the action of NA-based drugs of various nature –16. Howe2·PL–DNA system [2 nanoparticles through the polylysine (PL) linker. It was shown that TiO2·PL–DNA nanocomposites can penetrate into cells without any additional treatment . It was demonstrated that these nanocomposites exhibited a low toxicity and very high activity against IAV in the cell culture [To deliver antisense oligonucleotides into cells, we previously proposed the TiOA system , where o culture –19. Only2·PL–DNA nanocomposites, targeted to conservative regions of (−)RNA and (+)RNA of different IAV subtypes .In this work, we examined the antiviral activity of DNA fragments in the proposed TiO5’AGUAGAAACAAGG3’and 5’CCUGCUUUUGCU3’, respectively) [The choice of the most suitable regions in nucleic acids for oligonucleotide-based agents is of great importance. In order to inhibit the different subtypes of viruses, these regions should have conservative nucleotide sequences and be accessible for the interaction with complementary oligonucleotides. As many other researchers have –16, we hctively) –21.The region including the AUG codon in (+)RNA ,14 and t12As mentioned above, after infection of cells viral (−)RNA are transcribed into (+)mRNA and replicated into complementary (+)cRNA . To affe2 nanoparticles (≈5 nm in diameter) in the anatase form [2 surface. This affinity can be explained in all likelihood by the electrostatic interaction between the positively charged amino groups of PL and the negatively charged TiO2 surface at neutral pH [2 nanoparticles.The synthetic DNA fragments were conjugated with polylysine (PL), and the resultant PL–DNA conjugates were noncovalently immobilized on TiOase form  due to tutral pH . Since tutral pH , the maj2·PL–DNA nanocomposite was used to inhibit the reproduction of IAV in cell culture. TiO2 nanoparticles (of ≈5 nm in diameter) are known to penetrate through cell membrane [2 nanoparticles may also play a protective role. In addition, the PL linker attached to the 3’-end of the DNA fragment in PL–DNA also protects oligonucleotides from cellular nucleases. Thus, native DNA fragments with phosphodiester internucleotide bonds can be used without any additional modification.The resultant TiOmembrane . It was membrane –18 that membrane . We thinWe investigated the antiviral action of eight nanocomposites of this type bearing DNA fragments complementary to eight conservative regions of RNA segment 5 of negative and positive polarity , Table 1The inhibiting effect of the chosen DNA fragments on the virus reproduction was examined in MDCK cells infected with one of three different subtypes of influenza A virus .50/cell and at 5 µg/mL concentration of the nanocomposites, which corresponded to a 0.1 µM concentration for the DNA fragment. This nanocomposite concentration was much lower than TC50/mL  with the studied nanocomposites in comparison with the control without any samples. In addition, the results are demonstrated in 50/mL.The antiviral activity of the nanocomposites was studied in the postinfection assays, that is, the cells were initially infected with the virus and then incubated with nanocomposites. The experiments were carried out at a multiplicity of infection (MOI) of 0.1 TCID1/DNA5 pair (3/DNA7 pairs (5) or very low (DNA3) activity. There is a small difference  between the counterparts when they both exhibit either the low or high activity .First of all, it should be noted that the studied nanocomposites containing the DNA fragments targeted to (−)RNA of IAV segment 5 were more efficient against the virus as compared to those targeted to (+)RNA (compare rows 1–4 and 5–8 in 8 to DNA5). Thus, the DNA5 fragment, which inhibited the virus reproduction by up to 3–4 orders of magnitude (row 5), appeared to be the most active while the DNA8 fragment targeted to the opposite 5’-end of (−)RNA (row 8) showed low activity.It can be seen that the nanocomposites bearing the DNA fragments targeted to (−)RNA became more potent as they move along the (−)strand from the 5’ to the 3’ end (rows 8 to 5) (2·PL–DNA2 bearing the DNA fragment targeted to the AUG region of (+)RNA. And yet, the antiviral activity of this fragment was lower than that of the corresponding DNA fragment targeted to (−)RNA  with regards to the antiviral effect, and the difference between DNA4 and DNA5 fragments is low . Thus, in the case of the (+)RNA strand, there is no regular increase or decrease in the activity of the DNA fragments as they move along this strand.Only one of the studied nanocomposites containing the DNA fragment complementary to (+)RNA was quite active , which was TiO2·PL–DNA5, It is not surprising that for all studied subtypes, the difference between the nanocomposites bearing the site-directed DNA fragments and those containing the random sequence was significant. The maximal difference of 2–3 orders of magnitude was observed for the most efficient nanocomposite RNA and (−)RNA of IAV segment 5 were the most vulnerable to the action of the DNA fragments in the proposed nanocomposites. Specifically, the AUG region of (+)RNA and the corresponding region of (−)RNA, the region in (−)RNA complementary to the coding region in (+)RNA, and especially the 3’-end of (−)RNA RNA of IAV segment 5 is more vulnerable to the action of DNA fragments than the (+)strand may indicate that the negative strand is attacked immediately after its release from the complex with the viral proteins (nucleoprotein and polymerases) before transcription into (+)mRNA and replication into complementary (+)cRNA.5 oligonucleotide directed to the 3’-noncoding region of (−)vRNA contains the conserved sequence of 12 nucleotides common for all eight RNA segments of various IAV strains. Most likely, the observed strong effect of this fragment is partially explained by its simultaneous action to the other segments of the viral genome.The most efficient DNA2·PL–DNA nanocomposites leads to the more efficient inhibition of the IAV replication as compared to the data of other researchers who used NA-based agents against IAV segment 5 [It should be noted that the use of the proposed TiOegment 5 –16.8). They showed that this DNA fragment was efficient against various strains of IAV. The best IC50 and SI50 values were 2.22 µM and 394, respectively. Unfortunately, the authors did not indicate the level of the virus inhibition.Transfection of the IAV-infected MDCK cells with phosphorothioate oligonucleotide (4 µM) targeted to the internal coding regions of the (+)strand of the NP gene  in the presence of lipofectamine led to a slight reduction of the virus titer by 0.93–1.23 log . Duan et5, DNA1, DNA8, and DNA4 were examined against the NP segment [1 << DNA8 < DNA4 ≤ DNA5 . This order differed from our case (DNA4 << DNA1 ≈ DNA8 ≤ DNA5) but the most efficient DNA fragment was the same, although its efficiency in the virus inhibition was still significantly lower even at higher concentration than in our case (3–4 orders of magnitude at 0.1 µM concentration). Morpholino oligonucleotides targeted at the NP gene with the sequence as our DNA5 and DNA2 fragments showed high inhibitory activity in cells infected by influenza A H7N7 virus  [4 morpholino oligonucleotide displayed very low activity in these experiments. It was shown [Morpholino oligonucleotides with the same sequences as our DNA segment . To ensuigomers) . The DNAas shown  that a 1The above literature data confirm the idea about the usefulness of NA-based agents as antiviral drugs. Our results along with the literature data show that the 3’-nontranslated and AUG-containing conserved regions are most vulnerable for the antiviral action of NA-based compounds.5 fragment inhibited the reproduction of virus of the H1N1, H5N1, and H3N2 strains by factors of ≈1000, ≈3500, and ≈9000, respectively  [It should be emphasized that our experiments were carried out at a relatively high infection of cells (0.1 MOI) and at a low concentration of unmodified DNA fragments (0.1 µM) in the nanocomposites. The most active DNAectively , which wmL value 00 µg/mL 2·PL–DNA nanocomposites capable of penetrating without transfection reagents or physical impact. DNA fragments in the TiO2·PL–DNA nanocomposites were targeted to conservative regions of (−)RNA and (+)RNA of influenza A virus of the H1N1, H5N1, and H3N2 subtypes.For the effective delivery of oligonucleotides into cells, we used TiO2·PL–DNA5 nanocomposite was highly active against all three strains , with the H3N2 strain being the most susceptible to the antiviral effect of the proposed nanocomposites. The results of this work may be taken into account when choosing suitable targets in the IAV genome for the action of nucleic acid-based drugs.We found some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs, where these patterns were the same for all tested subtypes. The (−)RNA of segment 5 appeared to be a more vulnerable strand of IAV as compared to the corresponding (+)RNA strand. The most sensitive sites in the IAV segment 5 to the action of the proposed agents were found to be the AUG-containing site in (+) RNA, the corresponding region in (−)RNA, and especially the 3’-noncoding region of (−)RNA. The most efficient TiOIt should be noted that the effect of the most efficient nanocomposites on the viral reproduction (inhibition of the IAV replication by 3–4 orders of magnitude) was higher than that of other NA-based compounds described in literature . It is important to emphasize that we used the most affordable, inexpensive, unprotected phosphodiester oligodeoxynucleotides.2·PL–DNA nanocomposites offer great potential to serve as platforms for drug development against a broad array of diseases involving nucleic acids that spans from infectious diseases to hereditary disorders.Owing to their remarkable antiviral activity, the proposed TiO2 nanoparticles were synthesized in the crystal form (anatase) as described in [All chemicals were obtained from commercial suppliers: RPMI-1640 medium; antibiotics ; trypsin, L-glutamine; PBS buffer ; and fetal calf serum . TiOribed in . Сhicken2·PL–DNA nanocomposites were prepared by mixing PL–DNA and anatase nanoparticles (≈5 nm diameter) at room temperature for 20–30 min. The yield of the immobilization was ≈95%, with the capacity of the nanocomposites for oligonucleotides being ≈20 nmol/mg. A more detailed description of the nanocomposite preparation can be found elesewhere [Oligonucleotides were synthesized by the phosphoramidite method on an ASM-800 DNA synthesizer  using phosphoramidite monomers . The polylysine (PL)-containing oligonucleotides (PL–DNA) were synthesized as described in . The TiOesewhere .2, and 100% humidity. The cells at ≈80% confluence were initially infected with one of the IAV subtypes, which was added into each well in RPMI-1640 medium (100 μL) containing trypsin (2 μg/mL) at a multiple infection of 0.1 TCID50/cell. The control sample was RPMI-1640 medium (100 μL) containing trypsin (2 μg/mL). After 1 h of virus adsorption at room temperature, the virus-containing medium was removed, and the cells were rinsed with RPMI-1640 medium without trypsin. The studied samples of the nanocomposite taken in RPMI-1640 medium without trypsin (100 μL/well) at a concentration of 5 μg/mL were applied to the infected MDCK cells, followed by incubation for 4 h at 37 °С, 5% CO2, and 100% humidity. After incubation for 4 h at room temperature, the medium containing the sample was removed, the cells were rinsed with RPMI-1640 medium without trypsin, and the same medium containing trypsin was added into each well (100 μL). After incubation for 48 h, serial 10-fold dilutions (from 10−1 to 10−8) of the culture virus-containing liquid from each well were applied to MDCK cells for 48 h to evaluate the virus titer. The presence of the virus was visually determined under a microscope by the cytopathic action and in the hemagglutination reaction with a 1% suspension of chicken erythrocytes. The virus titer was expressed in terms of TCID50/mL or log TCID50/mL  in 96-well plates (100 μL/well) and incubated at 37 °С, 5% CO


Firstly, lectin microarray was used to screen glycoforms and Phaseolus vulgaris Leucoagglutinin (PHA-L) reactive structure  was found to be increased significantly in HCC patients with metastasis compared with those with non-metastasis. Then, PHA-L affinity glycoproteins were enriched followed by N-glycosite occupancy measurement with strategy of tandem 18O stable isotope labeling. 11 glycoproteins with significantly changed N-glycosite occupancy were identified, they were associated with cell migration, invasion and adhesion through p38 mitogen-activated protein kinase signaling pathway and nuclear factor kappa B signaling pathway. Quantification of N-glycosite occupancy for PHA-L reactive glycoproteins could help to discover important glycoproteins of potential clinically significance in terms of HCC etiology. Also, understanding of N-glycosite occupancy alterations will aid the characterization of molecular mechanism of HCC metastasis as well as establishment of novel glycobiomarkers.Hepatocelluar carcinoma (HCC) is one of the most common malignant tumors with high incidence of metastasis. Glycosylation is involved in fundamental molecular and cell biology process occurring in cancer including metastasis formation. In this study, lectin microarray, lectin blotting, lectin affinity chromatography and tandem  Hepatocellular carcinoma (HCC) as the major primary liver cancer is the third leading cause of cancer-related death and account for 70–85% of the liver cancers worldwide  and microheterogeneity (glycan structure) are urgently needed for understanding the functions of glycans in HCC. Especially, N-glycosite occupancy is associated with the enzymatic activity and the physical stability of glycoproteins  could quantify N-glycosite occupancy by measuring the intensity ratios of 18O/16O for glycosylated (6 Da) and for non-glycosylated (4 Da) peptides , and the total volume of pooling sera was 60 μL. Albumin and IgG were depleted by ProteoExtract® Albumin/IgG removal kit  from pooling sera according to the manufacturer's description.One microgram proteins were biotinylated by Lightning-Link Biotin Labeling Kit . A lectin microarray was produced using 50 lectins . The name and the binding specificity of 50 lectins were provided in Table Twenty microgram proteins for identification were separated by SDS-PAGE and transferred onto PVDF membranes . After blocking, the membranes were incubated with biotinylated Datura Stramonium Agglutinin (DSA), Maackia Amurensis Lectin-I (MAL-I), PHA-L and Wheat Germ Agglutinin (WGA) , respectively. The membranes were washed with 0.1% TBS-Tween20  and then incubated with Streptavidin Horseradish Peroxidase (HRP) Conjugate . Amersham ECL prime western blotting detection reagents  were used to detect the bands on the membranes.2, 1 mM MgCl2). Then, 3 mg proteins from different assemblages (non-metastatic or metastatic) were added into PHA-L agarose and incubated at 4°C overnight with a round shaker. Lectin-binding solution was used to wash the agarose and the bound fraction was eluted by 200 mM N-acetyl-D-(+)-glucosamine. The eluted fraction was separated by SDS-PAGE and stained by PhastaGel™ Blue R. The gels containing all bands were cut and processed for in-gel digestion.PHA-L agarose was washed and resuspended with the lectin-binding solution -phosphine hydrochloride  and iodoacetamide , respectively. Subsequently, the gel pieces were re-dehydrated with 100% ACN, and then digested in trypsin solution (5 ng/μL) at 37°C overnight. TOSIL strategy was performed as described previously  connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive)  equipped with an online nano-electrospray ion source. The Q-Exactive mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra  were acquired with a mass resolution of 70 K, followed by 10 sequential high energy collisional dissociation (HCD) MS/MS scans with a resolution of 17.5 K. In all cases, one microscan was recorded using dynamic exclusion of 30 s.18O (Asn), 18O C-term and oxidation (Met); peptide tolerance, 10 ppm; MS/MS tolerance, 0.05 Da. The relative quantities of N-glycosylated and its parent protein levels were obtained simultaneous by measuring the intensity ratios of 18O/16O for glycosylated (6 Da) and for non-glycosylated (4 Da) peptides from the same proteins respectively. A comparison of these two ratios can be utilized to evaluate the change of N-glycosite occupancy between HCC patients with metastasis (18O labeling) and those with non-metastasis (16O labeling) by the Equation as follows:The deglycosylated glycopeptides and non-glycosylated peptides were searched against SWISS-PROT human database using the MaxQuant 1.5.3.30, a quantitative proteomics software package. The parameters for searching were set: enzyme, partial trypsin; missed cleavages allowed, two; fixed modification, carboxyamidomethyla-tion (Cys); variable modifications, deamidation (Asn), deamidation plus Fold changes >1.5 or <0.667 were considered to be significant. Fold changes between 1.2 and 1.5 (1.2–1.5) or between 0.667 and 0.833 (0.667–0.833) were considered as minor.http://www.omicsbean.com). Ingenuity Pathway Analysis (IPA) analysis  was used to investigate biological interactions. Motif extractor  was used to identify overrepresented glycosylation motifs. Proteins annotated in International Protein Index (IPI) human proteome database were used as background and the significance value was set as 0.000001  and we divided protein-lectin binding intensities of them into 3 grades: weak binding (5>S/B ≥2), medium binding (15>S/B≥5) and strong binding (S/B≥15). In non-metastatic HCC samples, Caragana Arborescens Lectin (CAL), Euonymus Europaeus Lectin (EEL), MAL-I, Maackia Amurensis Lectin-II (MAL-II) were weak binding; Erythrina Cristagalli Lectin (ECL), Galanthus Nivalis Lectin (GNL) and Lens Culinaris Agglutinin (LCA) were medium binding; DSA, Lycopersicon Esculentum Lectin (LEL), Naja Mossambica Lectin (NML), Phaseolus Coccineus Lectin (PCL), PHA-L, Solanum Tuberosum Lectin (STL), and WGA were strong binding. While, in metastatic samples, EEL, MAL-I, MAL-II were weak binding; CAL, ECL, GNL, and LCA were medium binding; DSA, LEL, NML, PCL, PHA-L, STL, and WGA were strong binding.Among these 31 lectins, 14 lectins had statistical significance n, Sia and β1,6-GlcNAc branched N-glycan were increased significantly in HCC patients with metastasis; while, α-1,3Gal and α-1,3mannose were decreased significantly.Quantitative results of S/B and specific binding abilities of the 14 lectins were shown in Figures Lectin blotting was performed to validate changed glycoforms using biotinylated lectin DSA, MAL-I, PHA-L, and WGA. Coomassie brilliant blue staining showed similar global abundance of serum proteins in HCC patients with metastasis and those with non-metastasis. GlcNAc (which binds to DSA), α-2,3Sia or β-1,4Gal (which binds to MAL-I), β1,6-GlcNAc branched N-glycan (which binds to PHA-L) and (GlcNAc)n or multivalent Sia (which binds to WGA) were increased in HCC patients with metastasis compared with those with non-metastasis, which were consistent with the results of lectin microarray Figure .Among them, β1,6-GlcNAc branched N-glycan was significantly changed. This structure was catalyzed by UDP-N-acetylglucosamine: α-6-D-manno-side β1–6-N-acetylglucosaminyltransferase (EC2.4.1.155) which was known as GnT-V. Expression levels of β1,6-GlcNAc branched N-glycan and GnT-V were associated with metastasis in human digestive cancers such as colorectal carcinoma and gastric cancer . The average (five replicates) measured ratios of N-glycosite occupancy was 1:1.19 (fold change = 1.19), which indicated the cutoff of fold change was 1.19. Considering complexity of sera, the cutoff was set as 1.5 (data not shown). Among these deglycosylated glycopeptides, there were 6 deglycosylated glycopeptides displayed significant changes in N-glycosite occupancy  and 7 deglycosylated glycopeptides with minor changes . Figure Then, PHA-L affinity chromatography was chosen to enrich serum N-glycoproteins and a total of deglycosylated glycopeptides from 14 glycoproteins were quantified in HCC patients with metastasis compared with those with non-metastasis Table . The cutEN#LTAPGSDSAVFFEQGTTR from ceruloplasmin and ALGFEN#ATQALGR from galectin-3-binding protein were reduced in N-glycosylated and parent protein levels, but they displayed increasing trends in N-glycosite occupancy. CATPHGDN#ASLEATFVK from fibulin-1 were up-regulated in N-glycosylated and parent protein levels, but N-glycosite occupancy of it was down-regulated. It was noteworthy that different N-glycosite from the same parent protein had the different occupancy, for example, AAIPSALDTN#SSK and ALGFEN#ATQALGR from galectin-3-binding protein, had fold changes of 0.747 and 1.213 in N-glycosite occupancy, respectively. In our previous study, N-glycosite occupancy of VVLHPN#YSQVDIGLIK was changed significantly in HCC patients compared with patients with hepatitis B virus infection (HBV) and liver cirrhosis (LC) , protein activation cascade (p = 1.37e-09), defense response (p = 4.89e-09), negative regulation of protein metabolic process (p = 5.05e-09), and so on. In molecular function annotation, the 11 N-glycoproteins were main associated with peptidase regulator activity (p = 9.62e-09), protein binding (p = 6.02e-04), glycoprotein binding (p = 3.47e-04) and scavenger receptor activity (p = 2.96e-02).In GO annotation, 11 serum N-glycoproteins with changed N-glycosite occupancy were categorized using OmicsBean according to their cellular components, biological processes, and molecular functions Figure . The annMotif-X was used to extract overrepresented motif of amino acids for the 13 deglycosylated glycopeptide with different N-glycosite occupancy from the 11 N-glycoproteins. Setting up “N” as central character, a conserved glycosylation motif was enriched by the created logo-like representations Figure .Corresponding network of these 11 N-glycoproteins were obtained using IPA analysis Figure . Based oGlycoproteomics was critical to discover altered glycoproteins and glycans in occurrence and development of disease. Lectin microarray technology has been widely used for glycosylation studies in recent years . CP was a copper-binding glycoprotein synthesized by the liver, had ferroxidase activity and would be an iron-regulatory protein. Occurrence, development and metastasis of HCC were consistently characterized by the lack of iron accumulation  was found to be increased significantly in HCC patients with metastasis compared with those with non-metastasis. Then, 11 PHA-L reactive glycoproteins with significantly changed N-glycosite occupancy were identified, which were associated with cell migration, invasion and adhesion through p38 MAPK and NF-κB signaling pathway. Alterations in N-glycosite occupancy were also related with HCC metastasis. β1,6 GlcNAc branching of N-glycans might be a metastasis-promoting glycoform and we believe quantification of changes in N-glycosite occupancy for PHA-L reactive glycoproteins in HCC metastasis serum could help to discover important glycoprotein of potential clinically significance as well as characterization of molecular mechanism of HCC metastasis.Study design: SZ, YL. Data acquisition and analysis: TL, SS, WL, XQ, LS. Interpretation of data: TL, SS, WL, LS, SZ, YL. Drafting and revising the work: TL, SS, SZ, YL. Final approval: TL, SS, WL, XQ, LS, SZ, YL. Agreement to be accountable of all aspects of the work: TL, SS, WL, XQ, LS, SZ, YL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNAs are well-suited to act as cellular sensors that detect and respond to metabolite changes in the environment, due to their ability to fold into complex structures. Here, we introduce a genome-wide strategy called PARCEL that experimentally identifies RNA aptamers in vitro, in a high-throughput manner. By applying PARCEL to a collection of prokaryotic and eukaryotic organisms, we have revealed 58 new RNA aptamers to three key metabolites, greatly expanding the list of natural RNA aptamers. The newly identified RNA aptamers exhibit significant sequence conservation, are highly structured and show an unexpected prevalence in coding regions. We identified a prokaryotic precursor tmRNA that binds vitamin B2 (FMN) to facilitate its maturation, as well as eukaryotic mRNAs that bind and respond to FMN, suggesting FMN as the second RNA-binding ligand to affect eukaryotic expression. PARCEL results show that RNA-based sensing and gene regulation is more widespread than previously appreciated in different organisms. Riboswitches recognize and respond to specific metabolites by altering gene expression. Here, the authors developed a high-throughput method (PARCEL) to experimentally identify RNA aptamers across transcriptomes. As such, comprehensive mapping of regulatory networks in microbes under different environmental conditions is crucial to understanding their biology. While many of these regulators have been extensively studied at the protein and DNA levels, RNA’s role as direct sensors and responders remains relatively under-explored. One class of cellular RNA sensors, known as riboswitches, can recognize and respond to specific metabolites by altering gene expression2. Upon binding to their ligands, riboswitches undergo conformational changes that result in the regulation of gene expression through diverse means, such as changes in transcription, translation and decay2, informing the host of its environmental conditions. The modularity of riboswitches also allows them to be transplantable to different systems, broadening their use as biological sensors.Microorganisms are constantly sensing their environment for changes in temperature, pH, metabolites, and nutrients so as to regulate their gene expression programs to best adapt to different signals for growth, survival, and virulence3, the ability to comprehensively identify natural RNA aptamers from transcriptomes would expand our toolbox for synthetic biology and deepen our understanding of RNA -based gene regulation in vivo. Currently, potential natural RNA aptamers are mostly identified through computational determination of sequence and structural homology to known riboswitches4. However, as RNA can adopt different folds for binding to the same ligand and organisms can diverge greatly in sequence content, computational means of searching for riboswitches through sequence homology have limited scope5, and strategies that allow direct experimental detection are highly desirable. One recent strategy, Term-seq, utilizes high-throughput sequencing to detect differential transcription termination events in bacteria under different conditions6. However, complementary strategies for detecting riboswitches that act through other mechanisms (such as translation inhibition), as well as riboswitches that bind to metabolites whose intracellular concentrations are not easily altered, need to be developed.While in vitro selection methods, such as SELEX, have been applied with variable success to generate new synthetic RNA aptamersHere, we report an in vitro method for experimentally identifying RNA aptamers in transcriptomes by detecting ligand-induced RNA structural changes using high-throughput sequencing Fig. . This me7. Strategies using double-strand or single-strand specific nucleases , as well as in-line probing which probes nucleotide flexibility8, were tested for their abilities to detect structural changes using high-throughput sequencing  versus S1 nuclease libraries . We then extracted total RNA from the pooled bacteria, performed ribosomal depletion to enrich for mRNAs, and did structure probing, followed by deep sequencing. We had two biological replicates for each experiment and obtained more than 7 million reads per replicate .As B. subtilis and P. aeruginosa, including 4/5 known TPP riboswitches, 2/2 FMN riboswitches, 9/11 SAM riboswitches in B. subtilis, as well as 1/1 TPP and 1/1 FMN riboswitches in P. aeruginosa11, highlighting the high sensitivity of the method  in 9. Indeed, we observed the strongest structural change in TPP riboswitches upon binding to TPP, followed by thiamine and then oxythiamine . The newly identified elements exhibit lower folding energies than their dinucleotide shuffled controls, indicating that they are more structured . PARCEL identified 23 new RNA aptamers that exhibited structural changes in the presence of the metabolite pool  as S. cerevisae is known to take up exogenous FMN, unlike C. albicans15. Both transcript and protein levels of FLAG-tagged C. albicans RPS31 and ATP1 were measured, following growth of the integrated strains under varying FMN concentrations. We found that while transcript levels of FLAG-tagged C. albicans RPS31 and ATP1 in fmn1∆ did not change with increasing FMN concentrations  to disrupt RPS31 RNA structure without altering its protein sequence. As expected, codon-optimized RPS31 mRNA is structurally different from wildtype RPS31 mRNA, and does not show structure changes in the presence and absence of FMN  or stationary phases (OD600 > 2). Total RNA from P. aeruginosa was extracted using Trizol reagent (Thermo Fisher Scientific). Total RNA from B. subtilis was extracted by first incubating B. subtilis in 4 mg per mL of lysozyme for 15 min before using Trizol LS reagent. Ribosomal depleted RNA, Ribo(−) RNA, was obtained by using Ribo-Zero rRNA Removal Kit (Epicenter) according to manufacturer’s instructions. S. cerevisiae S288C was grown in YPD to exponential phase (OD600 = 0.6–0.8). C. albicans strain SC5314 was grown in YPD or GMM (yeast nitrogen base without amino acids and with 2% glucose) to exponential (OD600 = 0.6–0.8) or stationary phases (OD600 > 2). Total RNA from S. cerevisiae or C. albicans was extracted using a slightly modified protocol that uses hot acid phenol17. Poly(A) + RNA was obtained by using the Poly(A) Purist MAG kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Poly(A)+ or Ribo(−) RNA were then structure probed in the presence and absence of metabolites.fmn1∆ mutant was created by replacement of FMN1 in BY4741 strain with KanMX using homologous recombination18. RPS31 from C. albicans, with a C-terminal FLAG-tag (GATTACAAGGACGACGATGACAAG), was integrated together with URA3 at the ura3∆ site to generate the RPS31::FLAG knock-in strains. ATP1 from C. albicans, with a N-terminal FLAG-tag, was integrated together with URA3 at the ura3∆ site to generate the ATP1::FLAG knock-in strains.2) and metabolites to the RNA. The RNA pool was slowly brought to 37 °C for 30 min and structure probed using RNase V1  or S1 nuclease  at 37 °C for 15 min. The nuclease reactions were inactivated using phenol chloroform extraction and ethanol precipitated. In-line probing reactions were performed in 50 mM Tris-HCl (pH 8.3), 20 mM MgCl2, and 100 mM KCl at 25 °C for 40 hours8. The in-line probed RNA was phosphorylated using T4 polynucleotide kinase (PNK) in T4 PNK buffer and 1 mM ATP to capture the cleavage sites.Briefly, 250 ng of Poly(A)+ or Ribo(−) RNA was heated to 90 °C for 2 min and cooled on ice for 2 min before adding 10X RNA structure buffer . As fragmentation results in 5′OH, and is hence ligation incompatible, it does not interfere with the downstream library preparation process. Fragmented RNA was then purified using RiboMinus concentration module (Life Technologies), using the modified protocol for RNAs that are <200 bases. The RNA was eluted in 12 µl of nuclease free water and concentrated to 2 µl using a vacuum centrifuge. The RNA was then ligated to 5′ adapter from NEBNext Multiplex Small RNA Library Prep Set for Illumina using T4 RNA ligase1  at 16 °C overnight. The 5′ adapter ligated RNAs were then purified through a 6% TBE urea PAGE gel and size selected for 50–200 bases. The RNA was then ligated to 3′ adapter, reverse transcribed, and PCR amplified using the NEBNext Multiplex Small RNA Library Prep Set (New England Biolabs) for Illumina using manufacturer’s instructions. Eighteen cycles of PCR amplification were typically performed for each library.19.Cleavage and modification sites along structure probed RNA were identified using primer extension. Briefly, a primer located ~30–50 bases downstream of the structure probed region was labeled with ɣP32 ATP using T4 PNK kinase. The labeled primer was then purified using a 15% TBE urea PAGE gel. The labeled primer was incubated with the RNA at 65 °C for 5 min, followed by 35 °C for 5 min, and then cooled at 4 °C. To detect the structure probed sites, we add 3 µl of enzyme mix (4:1:1 of first-strand buffer: DTT: NTP) to the reaction, incubated at 52 °C for 1 min, and Superscript III was added to the reaction at 52 °C for 10 min. To generate a sequencing ladder for the RNA, we added 1 µl of ddNTP (5 mM) to the reaction after the enzyme mix, and before adding Superscript III. 4 M sodium hydroxide was added to the reaction to denature the RNA before the samples were loaded onto a 7 M TBE-Urea PAGE sequencing gel. Gel images were quantified using the software Semi-automated footprinting analysis (SAFA)20 with default parameters.RNA secondary structure predictions were generated using RNA footprinting data with RNase V1, S1 nuclease, and NAI as constraints, using the program RNAfoldfmn1∆ mutation were supplemented with 10 mM FMN and 200 µg per mL G418 in the cultures. The overnight cultures (1:100 dilution of OD600 2.0) were used to seed 50 mL YPD cultures supplemented with 2.5, 5.0 or 10.0 mM FMN. Cells were harvested after 4–6 h of growth at 30 °C, with shaking (when OD600 reaches 0.4). The cultures were split for RNA extraction and Western blotting, pelleted, and washed once with PBS. The resultant cell pellets were frozen and stored at −80 °C.The RPS31::FLAG and ATP1::FLAG strains were inoculated from single colonies into 2 mL SC-ura media and grown overnight at 30 °C, with shaking. Strains with the TM DNase (ThermoFisher Scientific)17. We made cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and qPCR was performed using SYBR Green Master Mix (Roche) on a Light Cycler 96 instrument (Roche). Primers used are listed as below. The RPS31 and ATP1 primers are specific for the knock-in C. albicans RPS31 and ATP1, and do not amplify the endogenous S. cerevisiae RPS31 and ATP1. Normalized fold changes were calculated by normalizing against actin (ACT1) and the respective strain cultured at 10 mM FMN.RNA was extracted from frozen yeast pellets using the hot acidic phenol method and treated with TURBOTM, Mini, EDTA-free [Roche] in 2.5 mL of lysis buffer). An equal volume of glass beads  was added and cells were lysed in a Mini-Beadbeater-96 (Biospec Products) for two cycles of 15 s with a two minute interval. Cell lysates were centrifuged and supernatants were run on 4–20% Mini-Protean TGX Stain-Free protein gels (Bio-Rad). To determine the relative levels of total protein (loading), the gels were first imaged on a ChemiDoc MP System (Bio-Rad) using the stain-free technology21. Following wet transfer and blocking with 5% milk in PBST, RPS31::FLAG and ATP1::FLAG were detected using mouse anti-FLAG M2 primary antibody  and sheep anti-mouse IgG, HRP-linked secondary antibodies . All images were taken using the ChemiDoc MP System (Bio-Rad) and analyzed with ImageJ22. Corresponding uncropped images of blots (in main figures) can be found in supplementary figures.The frozen cell pellet was resuspended in 80 µL of lysis buffer  with proteinase inhibitor 23. In the case of S. cerevisiae S288C and C. albicans SC5314, we extracted the UTR annotation from Bruno et al.25 and integrated them into their corresponding transcriptomes before alignment by bowtie2. In both cases, only uniquely mapped reads were used for subsequent analysis.Short reads from PARCEL libraries  in For each position along the genome or transcriptome, we counted the number of reads whose first mapped base was one base downstream of the inspected position. Higher counts suggest greater accessibility to V1 nuclease, and are more likely to be associated with a double-stranded conformation. In all expressed transcripts, positions with zero count could either be associated with a single-stranded conformation, or come from a heavily folded region that is inaccessible to V1 nuclease.26, where we compared samples treated by one specific metabolite  to samples from all other conditions. We focused on positions that were generally accessible to the V1 nuclease by applying a minimum abundance threshold  and then computed a score si for each passed position i based on edgeR-generated p-values : si = ln(0.1) − ln .Since RNA structural changes should typically span across multiple bases, we looked for regions that exhibit differential V1 counts to increase the sensitivity/specificity of detecting RNA aptamers. We first evaluated the significance of differential V1 counts at each nucleotide position using the edgeR packagesi, the more likely that differential V1 cutting was observed at that specific position. Accordingly, positions that failed to pass the abundance threshold were assigned a penalty score of −10. We then looked for segments of contiguous positions  with the highest aggregate score i, by applying the Kadane algorithm . We then determined the significance of these high-scoring segments based on Karlin–Altschul statistics, similar to the approach used in BLAST27.The higher the score, 27, the expected value  of high-scoring segments with an aggregate score of at least S is given by the formula:As described by Karlin and AltschulS to estimate the two parameters required i.e., K and λ. Specifically, λ can be calculated from the formula: pi is the corresponding probability of the scoresi. As si = ln(0.1) − ln and pvali approximately follows a uniform distribution ) due to the assumption that the majority of nucleotide positions do not undergo any structural changes, the equation λ = 0.862871. The parameter K is bounded between δ is the smallest span of si, K is bounded between C* is defined by the formula:Therefore, we examined the extreme value distribution of the aggregate score Sk is the random variable representing the sum of k independently chosen si i.e., Here, C* can be solved to take the value of 0.0809635, and the upper and lower bounds of K, K−, and K+, both equal C*, i.e., K = 0.0809635. We then calculated Ev for high-scoring segments by applying equation  and low bonferroni-corrected p-value .Taken together, equation . SegmentWe evaluated the distribution of RNA aptamers across operons in bacteria, and along transcripts in fungi (including a 500 bp window on either side when UTRs were not specified). We plotted the histogram of all RNA aptamer positions, with operons in bacteria and coding regions in fungi being scaled to 1 kbp. There are cases where the same position can be considered as belonging to multiple classes and in such cases, we preferentially assigned positions to the 5′ UTR, then to the operon or CDS, and lastly to the 3′ UTR.28, we chose fairly divergent, and yet not too divergent species (median Ks ranges from 0.1 to 0.4) for this analysis. The orthologous riboswitches, 3′ UTR, 5′ UTR, and protein coding regions were identified using blastn, for non-coding, or genblastG29, for coding sequence, in other species, respectively. To identify orthologous noncoding sequences in other organisms with high sensitivity, we changed the default blastn parameters as follows: “-e 1e-5 -word_size 7 –gapopen 2 –gapextend 1”30. We aligned the noncoding sequences using MUSCLE31, and the coding sequences using MACSE32, to construct the multiple sequence alignment. The nucleotide substitution rate of riboswitches, 3′ UTR and 5′ UTR were calculated using Kimura’s 2-parameter method33. The synonymous and non-synonymous substitution rates was calculated using Kakscalculator34 with the LPB method.We estimated the sequence conservation of identified RNA aptamers by measuring nucleotide substitution rate of these regions to their blastn-identified orthologous sequences. If the identified aptamer regions were shorter than 200 bases in length, we extended them on both sides to a maximum of 200 bases. As highly divergent and highly similar sequences would result in an unreliable estimate of nucleotide substitution rateB. subtilis, P. aeruginosa, and C. albicans across the Bacillus, Pseudomonas, and Candida genus using blastn (with parameters: “-e 1e-5 -word_size 7 –gapopen 2 –gapextend 1”). The species that were used in each genus are: B. subtilis XF-1, B. subtilis BSn5, B. malacitensis CR-95, B. natto BEST195, B. licheniformis DSM13, B. subtilis subsp. spizizenii W23, and B. cereus ATCC-14579 for Bacillus; P. aeruginosa PAO1, P. aeruginosa PA7, P. mendocina 1267_PMEN, P. knackmussii B13, P. oryzihabitans USDA-ARS-USMARC-56511, P. pseudoalcaligenes KF707, P. stutzeri A1501, P. stutzeri A1501, P. mendocina ymp, P. entomophila L48, P. putida F1, P. fluorescens SBW25, and P. syringae pv. tomato str. DC3000 for Pseudomonas; C. albicans SC5314, C. albicans WO-1, and C. dubliniensis for Candida. We then built multiple species alignments for each RNA aptamer region using MUSCLE31. We used the program Alifoldz12 to calculate the energy and structural stability of the consensus structure. For each RNA aptamer alignment, a shuffled alignment was generated as a control using the “shuffle.pl” script from the Alifoldz package.We searched for orthologous sequences of RNA aptamers identified in All relevant data are available from the authors upon request. Data has been deposited under GEO accession number GSE106133.Supplementary information(PDF 12834 kb)Peer Review File(PDF 137 kb)


Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365 nm wavelength  and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3 days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol.RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in  Historically, UV light of 254 nm wavelength has been used to crosslink RNA-protein interactions RNA-binding proteins (RBPs) display key functions in the cell such as RNA regulation, transport, translation or degradation by formation of dynamic ribonucleoparticles (RNPs) to regulate RNA fate and are thus fundamental for post-transcriptional gene regulation PTGR, . The neeCaenorhabditis elegansArabidopsis thalianaRecently, the RNA interactome capture method has been developed to identify RNA-binding proteins associated to eukaryotic polyadanylated (poly(A)) RNAs Saccharomyces cerevisiae is a prominent model organism for studying genetics and eukaryotic cell biology such as the cell cycle due to its smaller proteome and transcriptome and well-established tools for gene editing. RBPs have been identified in yeast before, utilising in vitro techniques such as protein arrays combined with fluorescently labeled RNA probes in vivo crosslinking and poly (A) selection Mitchell et al. The unicellular yeast S. cerevisiae before in vivo using UV crosslinking. The resulting covalently crosslinked RNA-protein hybrids can then be purified under stringent wash conditions that remove secondary binders such as protein-protein interactions in RNPs. Here, we present a comprehensive description of the method itself, provide background for the individual steps and discuss the potential and limitations of the protocol.We used RNA interactome capture in yeast in combination with Orbitrap mass spectrometry to determine the mRNA-bound proteome of 22.1We included a step-by-step protocol in the All buffers should be prepared using double distilled or ultra pure water, followed by autoclaving or sterile filtration.2.1.1S. cerevisae strain BY4741 for all experiments. Cells were grown in standard YPDA (yeast peptone dextrose adenine) medium or using a synthetic complete (SC) medium , synthetic defined medium lacking uracil , 120 μM Uracil, 1% glucose). Liquid yeast cultures were inocluated with an OD600 of 0.01–0.05 and grown at 30 °C while shaking at 160 rpm. For RNA interactome capture experiments, 5 ml YPDA preculture was inoculated from a single colony on an agar plate and grown over night. The next day, a 1 L culture of SC-medium120μM Ura was started with the preculture. Because we generally include a non-irradiated sample as negative control, for each experiment we have at least two yeast cultures.We used 2.1.2120μMUra medium were grown to an OD600 of 0.5 and 4-thiouracil  was added to a final concentration of 500 μM using a 50 mM 4tU stock solution . We used 4-thiouracil (4tU) and not 4-thiouridine (4SU) which is routinely used for in vivo RNA labeling in mammalian cell culture experiments (see discussion). Note also that YPDA medium can be used as previously shown 2O (ddH2O). From this moment on, cells were kept on ice whenever possible.Yeast in SC2.1.32 which roughly corresponded to 3 and 30 min or irradiation time, respectively. Every 10 min, irradiation was interrupted and the dishes were carefully rocked for 30 s to allow cooling of the cells and a redistribution of cells in the liquid layer. Finally, cells were collected from both dishes using a 20 ml glass pipet by carefully washing off the cells with the liquid in the dish in a 50 ml tube on ice. The non-crosslink control was kept on ice during the irradiation of the other samples.Next, we spread the cells from one sample evenly onto two standard Petri dishes (10 cm diameter). The dishes were placed on a tray with ice and Ultraviolet crosslinking was performed in a Spectrolinker device  emitting UV light at 365 nm wavelength (PAR-CL). The cells were placed in ∼ 5 cm distance to the UV bulbs. We used energies from 0.72 to 7.2 J/cm2.1.4Cells were pelleted by centrifugation  and the pellet was dissolved in 2 ml lysis buffer ; 1 protease inhibitor mix ; 1 mg/ml RNasin; 200 mM vanadyl-ribonucleoside complex, VRC). The mixture was distributed over several 2 ml screw-cap tubes containing an equivalent of 300 μl acid washed glass beads and cell lysis was performed using a Fast Prep device  in a cold room. The lysate was cleared of cell debris by centrifugation  and the supernatants were transferred to a 50 ml tube on ice. At this point, the lysate can be snap-frozen and stored at −80 °C for up to one week before continuing the protocol.2.1.5Oligo d(T) beads (NEB S1419) were pre-equilibrated with lysis buffer: we washed 1 ml of beads 3 times with 1 ml of lysis buffer using a magnet to remove the supernatant. The yeast lysate in the 50 ml tube was filled up to 25 ml with lysis buffer and an aliquot (100–500 μl) was saved at -20 °C as input sample. We added an equivalent of 1 ml oligo d(T) beads (pre-equilibrated) per litre of starting culture. Binding of the beads to RNA was then performed in an overhead rotating device in the cold for 1 h using a rotation of 20 rpm. Separation of the magnetic beads was also at 4 °C for until the supernatant was clear  and the supernatant was stored on ice for a second round of binding. All subsequent steps were carried out in a 15 ml tube at room temperature but buffers were kept on ice. The beads were then washed in 10 ml lysis buffer by rotating overhead for 5 min and magnetic separation until the supernatant was clear. We then repeated this wash step again two times in wash buffer 1 , two times in wash buffer 2  and two times in low salt buffer  while discarding supernatants inbetween. An important hallmark of the experiment can usually be observed when performing washes with wash buffer 2: the UV-irradiated sample only should form a transparent “halo” in the colour of the beads surrounding the opaque oligo d(T) beads after magnetic separation indicating successful crosslinking of proteins to the selected poly (A) RNA. Finally, beads were resuspended in 500 μl elution buffer , transferred to a 1.5 ml tube and incubated at 55 °C for 10 min. After magnetic separation, the supernatant (eluate) was collected and stored at -20 °C. The used beads were recovered in lysis buffer and washed 3 times to re-activate and pre-equilibrate the beads for a second round of interactome capture using the supernatant from the first oligo d(T) binding. Due to loss of magnetic beads during the first round, we routinely added another 200 μl freshly pre-equilibrated beads and repeated the selection procedure exactly as in the first round. The second eluate was then added to the first (the majority of poly (A) RNA and proteins are recovered already in the first round). We did not observe a significant increase in RBP yield when performing a third round of interactome capture in yeast.2.2At this point, eluates from the oligo d(T) selection consist of crosslinked proteins (RBPs), crosslinked poly (A) RNA and non-crosslinked poly (A) RNA from yeast.2.2.1To be able to analyse proteins, removal of the covalently bound RNA is highly advisable (e.g. for western blotting or proteomics applications). We perform RNA digestion by adding 250 μl of 5× RNase buffer , 200 U RNase A  and 200 U RNase T1 . Digestion was done for 1 h at 37 °C before placing the sample on ice. We then concentrated the RBPs using an Amicon Ultra centrifugal filter  according to the manufacturers instructions, resulting in 25–30 μl of RBPs. These can be directly subjected to SDS-PAGE, mass spectrometry sample preparation, etc. Note however that the two RNases are retained in the sample in relatively high amounts; however because of their small size and the subsequent limited number of peptides derived from the these after trypsin treatment, RNases do not interfere with the downstream mass spectrometric analysis, at least under our experimental conditions. SDS PAGE and silver staining were performed using standard techniques as described before 2.2.2When investigating the RNA after oligo d(T) capture, Proteinase K digestion can be used to remove the crosslinked polypeptides. We performed enzymatic proteolysis by adding 250 μl of 5 x PK buffer  and 0.4 mg/ml proteinase K (Roche) at 50 °C for 1 h. We then performed RNA extraction using either the single step method Finally, I would like to highlight that both strategies, removal of proteins or RNA, will result in samples that still contain covalently crosslinked residues from the partner molecule; neither RNase nor protease digestion completely removes the crosslinked RNA or peptide(s), respectively. This should be taken into consideration when planning downstream analyses such as (q)RT-PCR which is sensitive to such crosslinking sites 2.2.3in vitro transcribed using T7 RNA polymerase (Thermo Fisher 18033-019) using the protocol provided by the manufacturer, 5′-end labeled using T4 polynucleotide kinase  and gamma 32P ATP . After purification, the RNA was added as spike-into whole cell lysates generated with lysis buffer +/− LiDS. After magnetic separation, flowthrough of each intermediary step of the interactome capture protocol was collected and beta radiation was measured using a Perkin Elmer Liquid Scintillation Analyzer (TriCarb 2800 TR) and the QuantaSmart Software.Human beta globin mRNA carrying a poly (A) tail of 62 residues was 600 of 0.5 and 35S Methionine  was added to the culture. After 1 h, cells were lysed as described in buffer containing LiDS and 200 μl lysate was mixed with 100 μl oligo (T) beads. Flowthroughs after magnetic separation were mixed with 2.5 ml scintillation liquid (Perkin Elmer Ultimate Gold High Flash point KSC cocktail 6013327) and radioactive signals were measured.For protein labeling, 100 ml yeast cultures were grown to an OD2.2.4SDS-PAGE and Western blotting were performed according to standard protocols. Antibodies used: PUB1 and PAB1 , tubulin , histone H4 .2.2.5Total RNA from cells was isolated using the single step method 2.2.6Proteomic detection of the purified RBPs can be achieved by mass spectrometry. To determine the yeast RNA interactome, we first digested purified proteins (crosslinked samples as well as the non-crosslinked control) with Lys-C and trypsin. Then, sample and control peptides were differentially labeled using stable dimethyl isotopes. After mixing sample and control, peptides were split into 12 fractions according to their pH (ranging from pH 3–10) using a 3100 OFFGEL fractionator (Agilent) and the 12 fractions were finally analysed by LC-ESI-MS/MS using an Orbitrap Velos Pro (Thermo Fisher). Identification of peptides/proteins was performed using MaxQuant 2.3S. cerevisaeIf the interactome capture approach is not used to investigate individual RBPs (e.g. by western blotting) but to determine the RNA-bound proteome of yeast, the usage of mass spectrometry (see above) for unbiased detection of RBPs is necessary. However, after proteomics, a thorough statistical analysis is inevitable. I will discuss our approach to determine the RNA interactome capture of 2.3.1•For all proteins which were successfully identified by mass spectrometry, we collected the obtained fold-changes of proteins identified in crosslinked over non-crosslinked samples from all three repetitions. Our dimethyl labeling approach allowed us to directly obtain those ratios; if label-free proteomics techniques are used, ratios have to be generated from the quantified protein levels first see . Standar•Next, the means of the ratios from the individual biological repetitions were generated.2.3.2•t-test. This moderated t-test is advantageous over the normal t statistic as it is less influenced by under-estimated sample variances Inferring significance: We first tested differential enrichment using an empirical Bayes moderated •P-values alone are of limited use when interpreting high-dimensional data since many features could influence the identification and levels of recovered RBPs. Multiple testing was thus performed to estimate the false discovery rate (FDR) using the method of Benjamini and Hochberg. The resulting adjusted value was labeled ‘p adj’: all proteins within FDR of 0.01 were selected as RNA interacting proteins. We thus assume that 1% of the 678 RBPs are false positive hits.For the next steps, we used R/Bioconductor and the Limma package 2.3.3•We used scatter plots to compare enrichment of crosslinked proteins from individual repetitions and assess reproducibility inbetween biological replicates. We coloured dots of proteins which, after multiple testing of all replicates as described before, were within the 1% FDR range (p adj < 0.01).•A very useful approach to visualise all replicates in summary is to display the data in a volcano plot. Instead of the individual log-fold ratios, the mean log-fold ratio is plotted on the X-axis. On the Y-axis is the negative of the log of the adjusted p-value (−log2 p adj). This representation allows to quickly inspect how many proteins are enriched in the non-crosslinked control  and which are enriched as RBPs after crosslinking . We again coloured all proteins with an FDR of 0.01 or smaller.We mainly employ two types of plots to visualise outcome of the statistical analysis:2.4-We note that the ratio of lysed cells compared to the volume of oligo d(T) magnetic beads is crucial for a successful experiment. We routinely used 1 ml of beads per 1 L of yeast culture.-in vivo RNA labeling with 4tU using a dot blot approach Since our method relies on RNA labeling, it is important to test efficiency of RNA labeling during setup of the protocol. We tested -In some trials, white precipitates formed during the initial binding of poly (A) RNA with beads in the cold. These are most likely larger complexes formed by chromosomal DNA and proteins. Increasing the volume during the binding step with lysis buffer is then advisable to dissolve those precipitates.-During washes, we repeatedly noticed a brownish layer forming at the surface of the tubes. This is a result of beads sticking to the inner side of the tubes and can lead to significant loss of beads and purified RNA-protein complexes. Addition of 0.05% NP-40 to wash buffer 1 and wash buffer 2 helps to reduce the loss of beads. However, NP-40 may severely impair downstream applications such as some mass spectrometry protocols and we thus restrained from adding it to the low salt wash buffer or elution buffer.2.5-Day 1: Cell growth, RNA labeling & UV crosslinking-Day 2: 2 rounds of oligo d (T) binding and washing-Day 3: RNase digestion of the eluates, SDS-PAGE & visualisation of the RBPsYeast RNA interactome capture is a relatively straightforward protocol that does not require special equipment or devices that cannot be found in a standard molecular biology laboratory, maybe with the exception of the 365 nm UV bulbs and a magnet to separate beads in 50 ml volume. It takes 3 days to perform the complete protocol:However, the method requires a lot of hands-on time, esp. during day 2 in which 5 min wash steps alternate with magnetic separation (30–5 min).2.6bona fide RNA-binding capacity. Several methods exists from which I would like to highlight three:-Western blotting: This is maybe the most straightforward approach to validate a protein to bind RNA. Immunoblotting can be performed after oligo d(T) selection and RNA digestion as described above. However, since this approach is basically the interactome capture protocol again and is likewise based on UV crosslinking and oligo d(T) selection, this validation technique is best used to validate the mass spectrometry part of a high throughput screen for RBPs. Also, availability of antibodies might limit the usefulness of western blotting as validation technique. In summary, it is not advisable to solely rely on western blotting to validate novel candidate RBPs-in vivo UV crosslinking, individual candidate RBPs are immunoprecipitated with specific antibodies. After limited RNase digestion, covalently crosslinked shortened RNAs are 5′ phosphorylated with 32P-gamma ATP (radioactive isotope) using polynucleotide kinase (PNK). Next, the complex is analysed by SDS-PAGE. A radioactive signal detected at the molecular mass of the protein then indirectly proofs RNA-binding capacity. This assay has the advantage that the purification of the complex does not rely on oligo d(T) purification but on immunoprecipitation of the candidate RBP. Also, since all types RNA can be phosphorylated by PNK in principle, this validation approach does not distinguish which RNA targets are bound by the RBP.PNK assay: Following -CLIP-Seq: This could be seen as an extension of the PNK assay. Instead of radioactively labeled RNA as read-out, the crosslinked RNAs are identified by next generation sequencing. CLIP-Seq will yield much information on the RNA species of the RNP complex. However, this type of analysis also requires biological replicates and downstream bioinformatics analysis which could render it impossible to validate a larger number of candidate RBPs due to cost and time investment.After high throughput screens for potential novel RBPs, our described analysis Section  results In the study in which we determined the yeast interactome 33.1in vivo RNA labeled using 4tU and then UV-crosslinked by irradiation with light at 365 nm wavelength. Next, the cells are lysed with glass beads in a buffer containing 0.5 M salt, the anionic detergent lithium dodecylsulfate (LiDS) and a reducing agent . Such stringent conditions can be applied only as the initial UV irradiation resulted in a covalent crosslink inbetween RNA and interacting proteins. Then, poly (A) RNA was selected by binding to oligo d(T) magnetic beads and the captured (m)RNA  was washed in three different buffers prior to elution of the RNA from the beads.RNA interactome capture A consist32P-labeled spike-in RNA to ensure that buffer conditions do not impair the poly (A):oligo d(T) interaction; an in vitro-transcribed, body-labeled, human beta-globin mRNA carrying a poly (A)62 tail was incubated with whole cell lysate and levels of radioactive signal were determined from supernatants/flowthroughs of each intermediate step by scintillation counting. Since each wash step consists of two consecutive washes in the same buffer, we show pooled signals from both repetitions (ACT1) and GAPDH (TDH3) mRNA as well as 18S rRNA as non-polyadenylated, negative control  binding protein (PAB1) or poly U binding protein (PUB1). These results demonstrate that proteins recovered by interactome capture are enriched because of the interaction with RNA and that the crosslinking technique is indeed specific for proteins immobilised on RNA.For protein analysis, we digested RNA after elution from oligo d(T) beads using RNase A and RNase T1 as the covalently crosslinked nucleic acids add a huge mass to the denatured proteins, which would prevent size-dependent separation of the RBPs during gel electrophoresis. We routinely analysed samples +/− crosslinking using SDS-PAGE and silver staining B. When cDuring the setup of the protocol, we investigated the correlation of irradiation energies during UV crosslinking and RBP recovery. As expected, we detected higher yields of captured RNA-binders such as PUB1 when extending crosslinking times D. Howeve4bona fide RBP. Still, many of the so-identified proteins lack a role (yet) in RNA biology. However, not all of these novel RBPs must necessarily contribute to regulation of (m)RNA. We previously discussed that certain RNAs have evolved to bind and/or regulate proteins The RNA interactome capture approach has, in combination with proteomic analysis by mass spectrometry, been used for system-wide mapping of (m)RBPs in different cell lines and organisms. Hundreds of novel RBPs have been identified by this technique; many of them were unexpected to bind to RNA, contain no recognisable RNA-binding domain 4.14.1.1S. cerevisiae has no uptake mechanism for 4SU from the surrounding medium in vivo. In CV-1 monkey kidney cells, high concentrations of 4SU and UV irradiation have been shown to interfere in protein biosynthesis in vivo RNA labeling using >100 μM 4SU In interactome capture approaches in mammalian cell culture, 4-thiouridine (4SU) was used for RNA labeling instead of 4-thiouracil (4tU) After performing yeast interactome capture, we treated the significantly enriched recovered proteins as candidate RBPs, following the rationale that these proteins were crosslinked initially to RNA. Thus, it is of high importance to control for residual proteins and carry-over of abundant cellular proteins which are not related to RNA-binding. We already discussed the necessity to include a non-crosslinking control as this sample includes the background of proteins . In this respect, another advantage of UV crosslinking originates from the covalent bond which is formed upon irradiation in vivo crosslinking; corresponding to ∼7.2 J/cm2; others used energies up to 12 J/cm2Other interactome capture studies performed in mammalian cell culture benefitted from cell lines which grow adherent on the surface of the cell culture dish 4.1.2de novo transcription in eukaryotes; however animal cells harbour the mitochondrial poly (A) polymerase PAPD1 which was found to add a poly (A) tail of around 50 adenylates to RNA derived from mitochondrial genes S. cerevisiae, RBPs interacting with mitochondrial RNA species could not be selected in our approach in yeast, providing an explanation for the lower number of captured mitochondrial RBPs.Despite the fact that we sometimes refer to our method as “(m)RNA interactome capture”, we feel that it is important to remind the reader that we are actually determining a poly (A) RNA interactome since selection of mRNA is facilitated by usage of oligo d(T) beads during stringent wash steps. Although the majority of mature eukaryotic mRNA is poly-adenylated, some proteins such as histones are translated from poly (A) free mRNA 4.2In our efforts to understand the composition of yeast RBPs identified by mRNA interactome capture and the key factors that determine the outcome of efficient RBP recovery, we analysed newly found high confidence candidate RBPs along with the well established RNA-binders for various features. We found RBPs with various binding modes and with diverse specificities for RNA. RNA-binders with well-defined RNA target sites such as the poly (A) binding protein  stretches), as well as helicases which can be considered to move along RNA and thus do not bind a particular motif or site stably, to name only a few 4.2.12) When assessing the recovery of well-established RBPs in yeast such as PUB1, KHD1, PAB1  or in huTo rule out that highly abundant proteins are falsely selected as RBPs , we included staining for proteins such as tubulin and histones in our western blots, serving as negative controls since these proteins are not expected to bind to RNA C,D. AddiWe and others have previously used 254 nm UV light to crosslink RNA and proteins in an RNA-labeling independent manner (cCL) in vivo UV crosslinking experiment remains a black box for the time being: to date, we cannot predict which individual RBPs will crosslink to individual transcripts or at which efficiency. High-throughput studies such as the yeast RNA interactome determined via either cCL Taken together, none of the afforementioned features of RBPs such as abundance, localisation or domain composition of the purified RNA-binding proteins appear to dominate in the interactome capture process. Rather, UV crosslinking efficiency seems to be a main determinant for the outcome of the method which is also shown by the high dependance of individual proteins to be crosslinked and purified by one of the two irradiation methods (cCL or PAR-CL) exclusively. The technique of RNA-protein crosslinking utilising 4SU has been used from the 1980s on to study protein-binding to RNA and to map RNA:RNA interactions