The genome of viruses classified as picornaviruses consists of a single monocistronic positive strand RNA. The coding capacity of these RNA viruses is rather limited, and thus, they rely on the cellular machinery for their viral replication cycle. Upon the entry of the virus into susceptible cells, the viral RNA initially competes with cellular mRNAs for access to the protein synthesis machinery. Not surprisingly, picornaviruses have evolved specialized strategies that successfully allow the expression of viral gene products, which we outline in this review. The main feature of all picornavirus genomes is the presence of a heavily structured RNA element on the 5´UTR, referred to as an internal ribosome entry site (IRES) element, which directs viral protein synthesis as well and, consequently, triggers the subsequent steps required for viral replication. Here, we will summarize recent studies showing that picornavirus IRES elements consist of a modular structure, providing sites of interaction for ribosome subunits, eIFs, and a selective group of RNA‐binding proteins. cis‐ and trans‐regulatory elements coordinately determine picornavirus protein synthesis. Early in the infection, the cap‐dependent translation of host mRNAs is shut down, while viral RNA takes advantage of the IRES element, resulting in efficient synthesis of viral proteins. In this review, we discuss RNA structural motifs and host factors influencing viral RNA translation.Upon the entry of the viral RNA into susceptible cells,  A(n)poly‐A tractC(n)poly‐C tractCLclover leafCrecis‐replicative elementeIFseukaryotic initiation factorsEMCVencephalomyocarditis virusEV71enterovirus 71FMDVfoot‐and‐mouth disease virusHAVhepatitis A virusHCVhepatitis C virusIGRintergenic regionIRESinternal ribosome entry siteITAFsIRES trans‐acting factorsNtnucleotidePkspseudoknotsPRFprogrammed −1 frameshiftingPTVporcine teschovirusPVpoliovirusRBPsRNA‐binding proteinsSsmall fragment of aphthovirus RNASHAPEselective 2' ‐hydroxyl acylation analyzed by primer extensionuORFupstream ORFVPgviral protein genomePicornaviridae family includes a number of prominent human and animal pathogens, currently classified into 68 genera and 158 species [Picornaviruses are small, icosahedral, non‐enveloped RNA viruses, which belong to the vast group of positive‐strand RNA viruses. The  species . The EntPicornaviridae family consists of an infectious monocistronic single stranded positive RNA [The genome of all members of the tive RNA , with thtive RNA . The pictive RNA , 6, and tive RNA . The 3´Utive RNA .The picornavirus genome encodes a polyprotein, which is proteolytically processed by viral‐encoded proteases through a cascade of primary and secondary cleavages of the polyprotein precursors into 11‐15 mature proteins, depending on the genus . The strcis‐replicative element (cre), a stem‐loop of about 20 nt placed within the coding region [pro, 2B, 2Chel, 3A, 3BVPg, 3Cpro, and 3Dpol)  is characterized by the presence of a so‐called clover leaf (CL) structural motif (about 90 nt) at the most 5´end of the 5´UTR. The CL is involved in viral RNA replication  assistedg region . A shortg region . Viral‐eol) Fig. .cre element, and the IRES element  assisted by other viral proteins [Similar to most Picornaviruses, each Aphthovirus particle consists of a protein capsid comprised of 60 copies of the structural proteins VP4, VP2, VP3, and VP1 surrounding the genomic RNA . The genent Fig. . Both thproteins .pro) [A distinct feature of FMDV RNA is the presence of two in‐frame evolutionary conserved initiation codons, AUG1 and AUG2, which can be used as initiation sites for protein synthesis, generating the leader (L) proteins, termed Lab and Lb, respectively . Lb protpro) .pro, generating the mature viral proteins  [∗ from the Theiler´s murine encephalomyelitis virus (TMEV) is important for viral disruption of interferon signaling  , and as iewed in ).NPGP peptide, whereas similar to most Picornaviruses, it only encodes one 3BVPg protein.The genome organization of Teschovirus genus Fig.  differs In contrast to most Picornaviruses, the genome of members of the Dicipivirus genus is characterized by the presence of an intergenic region (IGR) separating the coding region of structural and nonstructural proteins  Fig. 1E1E.VPg polypeptide to the RNA replication sites. Viral RNA replication requires the covalent attachment to VPg of 5´‐terminal UMP (uracyl monophosphate) to the hydroxyl group of tyrosine, thereby yielding uridylylated VPg (VPgpU and VPgpUpU), an essential and differential step for Picornavirus RNA synthesis, which uses the first 2 adenines of the AAACA sequence of the cre element as a template [In all Picornavirus, the 3A protein delivers the 3Btemplate , 25. Thetemplate , are sentemplate . In thistemplate , consisttemplate .pro cleavage sites were identified by the sequence alignments of the identified processing sites on the Picornavirus polyprotein. In Enteroviruses, the cleavage site is a dipeptide (Q/G) [pro is more permissive and can accept E/G, E/S, Q/L, Q/I, and Q/T dipeptides  , whereasiewed in ). The 3C in [pro , it doespol). Similar to other RNA polymerases, its three‐dimensional structure folds into fingers, palm, and thumb subdomains, with the presence of an NH2‐terminal segment surrounding the active site [pol protein in the complex with the VPg‐UMP substrate has shown that two magnesium cations together with the catalytic aspartic acid residue participate in the uridylylation reaction. 3Dpol catalyzes the initiation of picornavirus RNA replication by the addition of two UMP residues to the Tyr‐3 residue of VPg [The picornavirus 3D protein is the RNA‐dependent RNA polymerase  tail. In FMDV, the deletion of the unique heterogeneous sequence that folds into two stem‐loops blocks infectivity . In contFlaviviridae family, as well as members of the families Dicistroviridae (CrPV) and Retroviridae (HIV‐1), reinforcing the fact that the use of IRES elements represents an optimized strategy for achieving successful viral infection [Picornaviruses are obligate intracellular parasites that rely on the host translation machinery to produce the proteins required for their replication and spread. Picornavirus viral particles, however, do not contain nonstructural proteins required for viral multiplication. Hence, the first phase of the picornavirus infection cycle requires the translation of the viral RNA, which is governed by the IRES element. This property is shared with other RNA viruses, such as hepatitis C (HCV), a member of the nfection , 38. A knfection .Similar to many other RNA viruses, picornaviruses co‐opt multiple host RNA‐binding proteins (RBPs) that assist with the recruitment of viral RNAs for viral protein synthesis and also with the assembly of the complexes regulating viral RNA synthesis . The picIn marked difference with the vast majority of cellular mRNAs, all picornaviruses contain regulatory elements in their genome that allow the recruitment of the translation machinery to the appropriate translation initiation codon using a cap‐independent mechanism, governed by the IRES element . IRES eli), eIF2, and GTP) and the small 40S ribosomal subunit, assisted by additional eIFs. The interaction of eIF4G with the poly(A)‐binding protein (PABP), in turn bound to the poly(A)‐tail of mRNAs, circularizes the 3´ and 5´ ends of the mRNA, stimulating translation. Under normal conditions, the 43S complex scans the 5´UTR of the mRNA until an initiator start codon (AUG) is located in optimum sequence context. AUG recognition triggers the joining of the 60S ribosomal subunit and eIF release, leading to the assembly of a translation elongation competent 80S ribosome [The cap structure present in the vast majority of cellular mRNAs allows the binding of the eIF4F trimeric complex . The recognition of the 5´cap by eIF4E allows the recruitment of the 43S complex  directs IRES‐driven protein synthesis [IRES‐mediated translation initiation is an important part of the strategy used by picornaviruses to recruit the translation machinery when cap‐dependent translation is inhibited by the action of viral proteases that induce the cleavage of eIFs and RBPs , and alsnslation , 45. On ynthesis , 47. Howynthesis , hamperiynthesis .In addition to eIFs, various ITAFs are targets of picornavirus proteases, such that the resulting cleavage products perform functions different from the uncleaved protein . In othe45 and PA2G4), or the cold‐shock domain containing E1 , among others [The replication and translation of the picornavirus genome takes place in the cytoplasm. Beyond the C‐terminal moiety of eIF4G, picornaviruses subvert a wide variety of nuclear and cytoplasmic RBPs to assist viral RNA translation using diverse strategies , 43. Theg others , 53, 54,g others , 55.Proteins recycled for picornavirus IRES activity in infected cells can act either as stimulators or repressors of viral RNA translation. The enhancer or repressor activity of the auxiliary host factors depends upon the mechanism of action supporting RNA‐protein or protein‐protein interactions. Indeed, different proteins can modulate IRES activity through changes in the local concentration of the protein, promoting unwinding of the IRES secondary structure, remodeling the RNA structure by stabilizing distant interactions between structural motifs and maintaining the active RNA conformation, inducing the proteolytic activity of viral proteases required to cleave repressor proteins or eIFs, enhancing RNA binding affinity, sequestering negative factors, and facilitating ribosome recruitment, among other possibilities Fig. . A few epro into N‐ and C‐terminal parts, so that the N‐terminal fragment retains the middle domain of eIF4G. However, since DAP5 has a lower affinity for the CVB3 IRES compared with authentic C‐terminal eIF4GI, the recruitment of other factors such as eIF4A is required for the full IRES function.Similar to other positive strand RNA viruses, picornaviruses have evolved strategies to target nucleo‐cytoplasmic trafficking of host proteins and RNA, and as a result, to increase the availability of factors required for their multiplication cycle. Among well‐known targets of picornavirus are the nucleoporins, the critical components of the nuclear pore. The disruption of the nuclear pore during early infection impairs active nucleo‐cytoplasmic transport in both directions . NuclearStudies carried out over the years have shown that picornavirus infection induces stress response, consequently leading to overall gene expression regulation . NuThere are various examples of RBPs interacting with picornavirus IRES elements, which are proteolyzed during infection, remodeling their activity on translation after cleavage. Among others, known examples include the PTB protein, the far‐upstream element binding protein 2 (FUBP2), FUBP1, the heterogeneous nuclear ribonucleoprotein (hnRNPK), PCBP2, the Ras GTPase SH3 domain binding protein 1 (G3BP1), and Gemin5 (reviewed in ). In thepro‐mediated eIF4G cleavage, favoring cap‐independent translation of the viral RNA. Second, the knockout of Hsp27 blocks hnRNP A1 translocation from the nucleus to the cytoplasm, abolishing the interaction of hnRNP A1 with the IRES element. Similarly, Hsc70 interacts with 2A protease to promote eIF4G cleavage, likely activating EV71 IRES activity [Instances of host factors promoting the activity of viral proteases on eIF4G have been recently reported, as shown by the heat shock proteins, HsP27 and Hsc70. Hsp27 is upregulated in response to EV71 infection . This practivity .pro cleavage in infected cells, such that the N‐terminal cleavage product retains inhibitory effects on IRES activity, whereas the C‐terminal cleavage product becomes a positive regulator of FMDV replication. The cleavage of G3BP1 by FMDV 3C protease renders two fragments that inhibit cap‐ and IRES‐dependent translation, but the Ct‐G3BP1 fragment shows a stronger effect on IRES‐dependent translation. The assembly of complexes with G3BP1 results in a significantly reduced local flexibility of the IRES element, consistent with the negative effect of this protein [Another strategy facilitating picornavirus RNA translation is exemplified by G3BP1 and hnRNP K proteins, which negatively regulate FMDV translation , 68. hnRIn addition to the strategies mentioned above, it has been reported that PV exploits specific ribosomal proteins, such as the ribosomal protein receptor for activated C kinase 1 (RACK1), to enhance ribosome recruitment, and therefore favor the translation of the viral proteins in infected cells . Anotherpol) in infected cells, it has been suggested that the inhibitory effect of hnRNP L on FMDV growth is presumably exerted through viral RNA synthesis.The permissive RNA‐binding capacity of ITAFs in conjunction with the multitasking activity of these factors, allows interaction with multiple targets, either RNA or proteins. The different steps of the picornavirus multiplication cycle are closely interconnected. Accordingly, there are examples of host factors interacting with IRES elements, which affect viral RNA replication, rather than RNA translation. For instance, the heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacts with the FMDV IRES to inhibit viral RNA replication . Given tThe 5´UTR of the picornavirus genome is characterized by the presence of many unused AUG codons, as shown in early studies of poliovirus RNA . In partAnother strategy proposed to modulate picornavirus translation relies on slowing down the translation kinetics of the viral RNA. The rationale behind this possibility relies on the observation that the rate of translation elongation can regulate cotranslational protein folding . Translacis‐acting RNA regions that promote internal initiation of translation using diverse cap‐independent mechanisms [Studies conducted since its discovery in 1988 have shown that IRES elements are chanisms , 81, 82.chanisms , 83. In chanisms , due to Despite performing the same function, the IRES elements present in the genome of different RNA virus families lack overall conserved features, and differ in the requirement of factors to assemble 48S initiation complexes , 82, 83.in vitro [Viral IRES elements have been classified into different types according to their mechanism of initiation. The simplest mechanism occurs by direct interaction of the IRES with the 40S ribosomal subunit . A more in vitro , 88. Howin vitro . In thisin vitro .Type IV IRES elements are eIF4G‐independent, but depend on eIF2 and eIF3. In fact, these group of IRES elements were designated HCV‐like because of the close similarity with the HCV IRES . The IREThe secondary structure of picornavirus IRES elements has been extensively analyzed over the years using different approaches , 94, 95.Previous studies reported that the AUG placed in domain VI was not utilized for initiation, and that the 6.5–9.0 kDa protein resulting from uORF translation was not detected in infected cells. However, recent data using selective 2'‐hydroxyl acylation analyzed by primer extension (SHAPE)  and baseThe relevance of the enterovirus IRES function for viral infection was highlighted earlier by the discovery of specific mutations within domain V of the PV IRES that inhibited viral RNA translation and also induced poliovirus attenuation properties in Sabin vaccine strains . As mentThe RNA structure of type II IRES elements is also arranged in modular domains, designated 2 to 5  , 104 Fi. Domain Sequence comparison of FMDV field isolates reveals shared conserved motifs exposed on loops and the junctions connecting stems, strongly supporting the hypothesis that the secondary structure is evolutionary constrained to deliver its function . Accordiin vitro using specific transcripts [in vivo using SHAPE methodology [A wide variety of RNA virus genomes fold into complex structures that include long‐range RNA‐RNA interactions relevant to control critical steps of the viral cycle . In the nscripts  and latehodology .The secondary structure of the HAV IRES element (a single member of type III) comprises six domains  Fig. 4C4C. This Type IV IRES elements are found in multiple picornavirus genera, including Teschovirus, Sapelovirus, Senecavirus, Tremovirus and Avihepatovirus , 84. ThiHost RBPs can modulate IRES function in multiple ways. For instance, they can act as RNA chaperones that promote specific conformations of the IRES structure, stabilize the interaction of the IRES with eIFs, mediate recruitment of the 40S ribosomal subunit, unwind RNA secondary structure near or at the start codon, and/or titrate IRES ligands Fig. .As already mentioned, the RNA architecture imposed by the presence of specific structural domains mediates IRES function (reviewed in ). One ofAnother example of a protein mediating IRES activity is PCBP2, a ubiquitously expressed RBP comprising three ribonucleoprotein K homology (KH) domains. The PCBP2 binding site in the enterovirus IRES elements is the ACCCC loop, a conserved motif located in domain IV in close proximity to the GNRA tetraloop , 109. ThA large diversity of RBPs involved in nuclear and cytoplasmic RNA‐dependent processes have been identified associated with viral IRES elements, as exemplified by Ebp1, the far upstream element‐binding protein 1 (FBP1) and FBP2, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), G3BP1, and Unr . HoAs picornaviruses have small genomes, their expression depends on outcompeting host cellular machinery to progress the different steps of the viral replication cycle. These strategies include the cleavage and/or post‐translation modification of eIFs and RBPs resulting in strong shutdown of the general cap‐dependent translation initiation. In contrast to the host mRNA, a key stage of the picornavirus cycle is translation of the incoming genomic RNA governed by IRES elements. Regarding IRES function, it is important to note that the functional features of RNA molecules are established in their three‐dimensional structure, but also in their ability to acquire distinct conformations in response to specific signals . RemarkaThe authors declare no conflict of interest.EMS conceived and wrote the manuscript with comments from all authors .


The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues. One of the basic cellular processes occurring in a eukaryotic cell is the post-transcriptional chemical modification of RNA bases. The data accumulated to date indicate that the methylation of cytosine residues at the C5 atoms in mammalian RNAs plays an important role in many aspects of RNA metabolism, including RNA export, RNA stability, RNA maturation, and mRNA translation ,2. This The biological functions of m5C residues in mRNA metabolism remain largely unclear. Only recently, data on cellular proteins that recognize this mRNA modification have appeared. One of these proteins is the mRNA export adaptor ALYREF, proposed as an m5C mRNA reader for the regulation of nuclear mRNA export . AnotherWe have previously demonstrated that NSUN2, along with YB-1, can be pulled down from a cytoplasmic extract of human embryonic kidney 293 (HEK293) cells using a short synthetic imperfect RNA hairpin containing the linear octanucleotide sequence CAGUGAGC (motif 2) . Similar4U). By analyzing samples of total mRNA and polysome-associated mRNAs from cells with or without NSUN2 knockdown using high-throughput RNA sequencing (RNA-seq), we identified differentially expressed genes (DEGs) at the transcriptional and translational levels, as well as genes with altered translational efficiencies (GATEs). Thus, mRNAs cross-linked to NSUN2 were defined, and the impact of NSUN2 deficiency on the cellular transcriptome and translatome was revealed.In connection with the foregoing, it was of interest to determine the specific features of NSUN2 binding regions in cellular mRNAs, substrates for NSUN2, and also find out whether the translation efficiencies of the mRNAs increase in response to a decrease in the enzyme level. To reveal NSUN2 target mRNAs, we applied the photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) method to HEK293T cells grown in the presence of 4-thiouridine , as well as histone genes   . Judgingnd H4C4) . At the tely 60% , implyintely 60% .We compared the set of NSUN2 target mRNAs found by PAR-CLIP in the current study with the set of 312 mRNAs that have previously been identified as contacting this protein using the methylation individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) method based on the spontaneous cross-linking of the NSUN2 C271A mutant to an RNA substrate . These sTo identify genes whose expression changes with a decrease in the efficiency of NSUN2-catalyzed RNA methylation, we knocked down this protein via RNA interference. To achieve this, HEK293T cells were treated with specific siRNAs designed against two regions in the CDS of NSUN2 mRNA , whereasp value (p.adj) was taken below 0.05 to sort out statistically significant genes between all replicates, and the minimal absolute shrunken Log2FoldChange (LFC) value was limited to 0.585 to only consider genes with more than 1.5-fold changes in expression levels  showed a high degree of clustering between biological replicates in both sequencing modes: RNA-seq A and poln levels B,D. Appln levels , as welln levels , respectn levels E.SRM, STMN3, TPM4, SRP54, NAMPT, and SEPTIN7, were selected. The values of changes in the expression of these genes at the transcriptional level and in their translation efficiencies, estimated using RT-qPCR, highly correlated with those obtained from analyzes with the NGS data  enrichment analysis for the up-regulated and down-regulated DEG sets. No enrichment for the up-regulated tDEGs and pDEGs was found in the biological process and molecular function categories. However, both sets exhibited weak enrichment in the cellular component category for the genes whose products are associated with lipid membranes and membrane-bound organelles, such as, in particular, the endoplasmic reticulum (ER), nuclear lumen, and centriole . In contNSUN2 itself appeared in the set of down-regulated GATEs; the level of its mRNA in the pool of polysome-associated mRNAs  in Bio-link ; the cells were not irradiated in the control experiment. After that, the cells were washed again with ice-cold PBS and pelleted by centrifugation at 1000× g , and then the cells (30 × 106 cells for each replicate) were lysed, triturated through a G30 needle, and centrifuged at 20,000× g . The lysate was treated with RNase T1 (0.5 U/μL) for 20 min at 25 °C and incubated overnight at 4 °C with the Protein G-conjugated magnetic beads  pre-bound to polyclonal antibodies to NSUN2 . After immunoprecipitation, the beads were incubated with RNase T1 (2 U/μL), washed, incubated with calf intestinal phosphatase (NEB), and then washed again. RNA fragments cross-linked to NSUN2 were 32P-labeled on the beads, washed, and eluted by heating in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The resulting eluate was resolved on 10% SDS-PAGE, after which the gel was blotted onto a nitrocellulose membrane, followed by exposure to a Phosphorimager screen . The radioactive signals were scanned using a Pharos FX Plus Molecular Imager with QuantityOne software (Bio-Rad). The RNA fragments were isolated as described in [PAR-CLIP was performed using HEK293T cells in 4 biological replicates according to  with somribed in .Raw data generated by 3 runs  and 2 on MGIseq 2000  for each replicate) were initially processed as described in . The preg . In control experiment, the set of non-targeting siRNAs was used. To confirm the knockdown of the mRNA of NSUN2, the content of the protein itself in the cells was determined by Western blotting using specific rabbit polyclonal antibodies against NSUN2 as described in [HEK293T cells were cultured in 10 cm dishes as described above. At 30% confluence, cells were transfected with specific siRNAs against the mRNA of NSUN2 using Lipofectamine 3000 according to the manufacturer’s protocol. After 48 h, the cells were passaged, transfected with NSUN2-specific siRNAs again, and harvested 48 h later at 80–90% confluence. To this end, the cells were placed on ice, washed with ice-cold PBS, kept for 10 min on ice with PBS and 0.1 mg/mL of cycloheximide, resuspended, and pelleted by centrifugation at 500× ribed in . Rabbit 2, 200 mM KCl, 1% Triton X100, and 0.1 mg/mL of cycloheximide. The lysates were clarified by centrifugation for 1 min at 14,000× g and layered on sucrose-density gradients (7−47%) in 50 mM Tris-HCl (pH 7.5) containing 100 mM KCl and 12 mM MgCl2 and centrifuged at 20,500× g in Beckman SW40 rotor . The gradients were then fractionated, and the A260 of the fractions was measured. Fractions corresponding to polysomes were supplemented with one volume of EtOH and MgCl2 (up to 20 mM) and centrifuged at 14,000× g . Polysome pellets were dissolved in the Trizol reagent. To evaluate the level of NSUN2 knockdown in the cells used, 1/100 of the lysate was analyzed by SDS-PAGE, and then the separated proteins were transferred from the gel onto a nitrocellulose membrane followed by immunoblotting as described above. To assess the ratio of 80S ribosomes in the polysome and monosome fractions (P/M), the integrated A260 values of the respective peaks were calculated in the MS Excel program. All other manipulations, including the isolation of RNA from the samples of cells and polysomes lysed with the Trizol reagent, along with the RNA integrity index assessment, RNA quantification, and RNA polyA enrichment, were carried out in accordance with the described protocol [For NGS, RNA samples in 4 biological replicates from one 10 cm plate of NSUN2 knockdown HEK293T cells were utilized. To isolate the samples for RNA-seq analysis, 1/5 cells from each replicate were lysed with the Trizol reagent. To isolate the samples for polysome profiling followed by RNA-seq analysis, 4/5 of cells from each replicate were lysed on ice for 10 min in 20 mM Tris-HCl (pH 7.5) containing 15 mM MgClprotocol .plotPCA function from the DESeq2 package based on rlog transformed read counts as described in the DESeq2 manual. DESeq2 with default parameters was utilized to perform DEA with the RNA-seq data and the polysome profiling followed by RNA-seq data separately, as well as when determining GATEs using the algorithm described in the systemPipeR package vignette. For the selection of DEGs, the p value adjusted (p.adj) cut-off was assigned to <0.05, the absolute shrunken LFC cut-off was assigned to >0.585, and the baseMean value was assigned to higher than 100, which excludes poorly covered genes, i.e., weakly expressed ones. Volcano plots were generated using the EnhancedVolcano package (v. 1.4.0). Calculations of translational efficiencies of genes were performed with the DESeq2 package. The sequences of mRNAs were extracted using the Biostrings package. The GO enrichment analysis of DEGs against the GO terms was carried out using the online-based resource www.geneontology.org (accessed on 15 May 2022) by selecting the Fisher’s Exact test type and False Discovery Rate (FDR) correction parameters. The motif discovery was carried out using MEME Suite. Secondary structure analysis was performed with the RNAFold tool from ViennaPackage (v.2.5.1). Motif analysis was carried out with the Streme tool from MEME Suite (v. 5.4.0), and the sequences of mRNA parts of non-DEGs were used as control.The quality-checking of RNA samples, DNA libraries preparation, their high-throughput sequencing with the use of the MGISEQ-2000 platform , and raw data processing were performed as described in . The respcr [For reverse-transcription, 2 g of total RNA from HEK293T cells with and without the knockdown of NSUN2 or from the respective polysome fractions was incubated with 100 pmol of random primer and 25 U of MMLV reverse-transcriptase according to the protocol . qPCR was performed on the LightCycler 96 (Roche) using the SYTO9 fluorescent dye (Thermo Fischer Scientific), HS-Taq polymerase , and gene-specific primers. The experiments were carried out for each of 3 biological replicates in 2–3 technical ones. The reaction conditions were as follows: 95 °C for 30 s; 45 cycles 95 °C for 10 s, 57 °C for 20 s, and 72 °C for 20 s (single acquisition). The relative quantification of gene expression levels was determined using R package pcr . The refThe effects of a deficiency of the RNA cytosine C5 methyltransferase NSUN2 on the composition of the transcriptome and translatome in mammalian cells enabled determining the dependence of gene expression on the methylation of mRNAs. A decrease in the NSUN2 level enhances the efficiency of translation of a large group of specific mRNAs, which includes shorter ones with a higher GC content, where motifs with C residues are located in GC-rich environments. The PAR-CLIP assay applied to endogenous NSUN2 revealed a group of its target mRNAs and its binding sites in them, showing the preferential location of these sites predominantly in the CDSs in GC-rich regions with a pronounced secondary structure. A fairly significant overlap between the sets of mRNAs that are targets of YB-1, the main packager of mRNAs that is most probably involved in their exosomal export, and of NSUN2 suggests that the latter may be implicated in the regulation of mRNA sorting into exosomes. The next research frontier in this area may be the discovery of a mechanism by which NSUN2 affects the cellular pathways related to the packaging of mRNAs, their sorting into exosomes, and extracellular export.


The brain estimates hand position using vision and position sense (proprioception). The relationship between visual and proprioceptive estimates is somewhat flexible: visual information about the index finger can be spatially displaced from proprioceptive information, resulting in cross-sensory recalibration of the visual and proprioceptive unimodal position estimates. According to the causal inference framework, recalibration occurs when the unimodal estimates are attributed to a common cause and integrated. If separate causes are perceived, then recalibration should be reduced. Here we assessed visuo-proprioceptive recalibration in response to a gradual visuo-proprioceptive mismatch at the left index fingertip. Experiment 1 asked how frequently a 70 mm mismatch is consciously perceived compared to when no mismatch is present, and whether awareness is linked to reduced visuo-proprioceptive recalibration, consistent with causal inference predictions. However, conscious offset awareness occurred rarely. Experiment 2 tested a larger displacement, 140 mm, and asked participants about their perception more frequently, including at 70 mm. Experiment 3 confirmed that participants were unbiased at estimating distances in the 2D virtual reality display. Results suggest that conscious awareness of the mismatch was indeed linked to reduced cross-sensory recalibration as predicted by the causal inference framework, but this was clear only at higher mismatch magnitudes (70–140 mm). At smaller offsets (up to 70 mm), conscious perception of an offset may not override unconscious belief in a common cause, perhaps because the perceived offset magnitude is in range of participants’ natural sensory biases. These findings highlight the interaction of conscious awareness with multisensory processes in hand perception. Where we perceive our hands in space has a substantial impact on how we carry out manual tasks. For example, when hammering a nail steadied by the thumb and index finger, misjudging the nail’s position could result in injured fingers. Through proprioception, the brain can estimate hand or finger position using signals from the muscles, joints, and skin even in the absence of vision . Visual When there is an externally-imposed spatial offset between available sensory cues, cross-sensory recalibration has been observed, where one or both unimodal estimates shifts toward the other . For exaWhile there is ample experimental evidence for cross-sensory recalibration, the principles by which it operates are unclear, and may depend on the task and context . The fraSome studies have suggested that perceiving a mismatch or conflict between cues affects cross-sensory recalibration in visual-auditory localization  as well One open question concerns the role of the magnitude of the cue mismatch. Prism exposure studies, in which the visual field is offset from the proprioceptive cue of hand position, have suggested that cross-sensory recalibration is affected by knowledge of the offset only if the offset is relatively large . This isIn the present study we build on this literature by assessing visuo-proprioceptive recalibration in response to a gradually imposed visuo-proprioceptive mismatch. Experiment 1 asked how frequently a gradually-imposed 70 mm visuo-proprioceptive mismatch  is conscA total of 96 healthy adults participated in this study, which consisted of three experiments. Experiment 1 participants each completed two sessions on different days. Experiments 2 and 3 comprised a single session each. 62  participated in Experiment 1. Twenty  participated in Experiment 2. Twenty  participated in Experiment 3. Six participants participated in both experiments 2 and 3. All participants reported being right-handed. All reported normal or corrected-to-normal vision, and no known neurological or musculoskeletal conditions. The study was approved by the Indiana University Institutional Review Board. All participants gave written informed consent before participating in the study.Participants completed two sessions each, on different days at least 5 days apart: a Mismatch session and a Veridical session. Session order was counterbalanced across participants. Time between sessions was 14.4 ± 13.8 days (mean ± SD).Participants sat at a 2D reflected rear projection apparatus composed of two touchscreen frames (PQ Labs) with a 3-mm-thick pane of glass in between. The touchscreens utilized infrared beams to detect touch with <0.5 mm resolution. Participants viewed the task display in a horizontal mirror positioned just below eye-level. This resulted in images that appeared to be in the plane of the touchscreens while also preventing the participants from seeing their hands . The totParticipants were asked to use their right index finger to indicate the perceived position of a series of three different target types related to the left (target) index finger: proprioceptive (P), visual (V), and visuo-proprioceptive (VP) targets. The V target was a projected image of a white box, and the P target was the participant’s target index finger placed on a tactile marker beneath the touchscreen glass . There wParticipants began each trial by placing their indicator finger in the starting position above the glass, as indicated by a 1 cm yellow box. To help the participant reach the start position, a 0.8 cm blue dot appeared when the indicator finger was in contact with the touchscreen glass and positioned near the yellow start box. The yellow start box could appear in any of five locations, arranged like a plus sign at the participant’s midline, about 15 cm in front of the chest. The blue dot disappeared as soon as the indicator finger left the start box, preventing participants from having online or endpoint feedback about indicator finger position.Once the indicator finger was correctly positioned, participants heard an audio cue instructing them to keep their eyes on a red cross that appeared at a random position within 10 cm of the target. However, eye movements were not recorded or enforced, and this was not intended to override subjects’ instinctive saccades to target position. The red cross was included in this paradigm  to discoNext, participants were instructed to place their target finger on one of the tactile markers (P or VP target), or to rest their target hand in their lap (V target), and the white box appeared in the display (V or VP target). Finally, participants heard a beep, cueing them to begin the trial. For VP trials, participants were told during task training that the white box would appear directly over their target fingertip and that they should place their indicator finger at that location.Participants were trained to lift their indicator finger off the glass from the starting position and place it down where they thought the target was positioned, without dragging their finger along the glass. Participants were notified that there were no speed requirements, and that adjustment was allowed. Once the participants had their indicator index finger on their estimated target position for 2 s, this position was recorded as the final estimate and the trial concluded.Certain aspects of the procedure were intended to prevent motor adaptation of the indicator hand, allowing us to assess changes in perception of the target hand. Multiple start and target positions were used and randomized to prevent memorization, and no performance feedback or knowledge of results was given. Thus, participants had no information about the accuracy of their indicator finger placements in relation to the target , to a maximum offset of 70 mm at the end of the single block of 84 trials. In the Veridical session, no offset was imposed, and the white square remained over the target finger throughout. At the end of each session, participants were asked to rate their attention level, quality of sleep the prior night, and fatigue caused by the experiment on a scale of 1–10.“Did it always feel like the white square was directly on top of your left finger, or did it feel off?” If participants replied with “it felt off,” they were then asked in what direction the white square felt offset from the left finger, and by how much at most. Participants were permitted to estimate this magnitude in either centimeters or inches. This approach was chosen to be consistent with previous studies using the visuo-proprioceptive recalibration paradigm . In the Mismatch session, visual and proprioceptive recalibration (ΔŷV and ΔŷP) were calculated as we have done in previous studies with this task structure  and those who did not (N = 52), in the Mismatch session. Recalibration was not normally distributed in these samples, so we used a non-parametric method .To test whether perceiving a forward offset was linked to reduced recalibration in the Mismatch session, we compared the magnitudes of visual and proprioceptive recalibration between participants who reported perceiving a forward offset , to a maximum offset of 140 mm at the end of Block 8.“Did it always feel like the white square was directly on top of your left finger, or did it feel off?” to screen out subjects who never noticed any offset. If participants replied with “it felt off,” they were asked in what direction the white square felt displaced from the left finger, and by how much at most. Both inches and centimeters were acceptable units. Only perceived offset magnitudes in the forward direction (true offset direction) were analyzed. All reported magnitudes were converted to centimeters. To prepare participants for this question being asked repeatedly, participants viewed task instructions in the form of a slideshow before beginning the experiment. This included the slides depicted in To test the participant’s awareness of the offset throughout the experiment, at the end of each block participants were first asked V(1) …ŷ. V(8)ŷ, P(1) …. yP(8)ŷ). These estimates were computed by taking the average of the y-coordinate of indicator finger endpoints on V trials and P trials, respectively . Visual and proprioceptive recalibration  were calculated as:Data consisted of the x,y coordinates of indicator finger endpoints and participants’ responses to the perceived offset question at the end of each trial block. Because the visuo-proprioceptive offset was imposed in the y  direction, we computed participants’ mean estimate of V and P target position in the y-dimension for each block (V△ŷ + P△ŷ) of 140 mm would indicate that 100% of the 140 mm offset was compensated for.Recalibration in the expected direction  comes out positive. Thus, a total recalibration .To test whether perceived forward offset was related to total realignment, we computed Pearson’s correlation between maximum perceived forward offset and total realignment at both Block 4 and Block 8. When this was significant, we also computed Pearson’s partial correlations between maximum perceived forward offset and each of visual and proprioceptive recalibration, with α of 0.05.Participants were shown a series of thick white lines on a black background with the same apparatus as in the two prior experiments . The lint tests were used for the rest. All hypothesis tests were performed two-sided, with α of 0.05.For each participant, we computed the mean estimated length of each of the eight line-orientation combinations and then converted it to a proportion by dividing estimated length by true length. At the group level, the Shapiro–Wilk test showed that the 3 and 6 cm estimated lengths were not normally distributed. To compare the proportion of each length perceived to 100%, the one sample Wilcoxon signed-rank test was used for the non-normally distributed lengths (3 and 6 cm), and one-sample n = 10) reported a forward offset (n = 7) reported a forward offset , which is inconsistent with the 70 mm forward offset in the Mismatch session being noticeable to subjects.In the Mismatch session, when a forward offset was imposed, 16% of 62 subjects (d offset i. Howeved offset ii. This N = 10) and those who did not (N = 52). These groups of participants did not differ significantly in visual recalibration , considered a large effect size , and vice versa . This suggests that participants who perceived the greatest max offset had reduced recalibration in both visual and proprioceptive modalities.In the first half of the experiment (Blocks 1–4), during which actual visuo-proprioceptive offset reached 70 mm, total recalibration was not significantly correlated with the maximum reported offset , vertical 6 cm line , or horizontal 6 cm line , with N = 20 in each case. Similarly, one-sample t-tests showed that line estimates did not differ from true length for the vertical 10 cm line , horizontal 10 cm line , vertical 16 cm line , or horizontal 16 cm line . For all except for the horizontal 3 cm line, there was no difference between perceived and actual length. For the horizontal 3 cm line, participants underestimated the line length .In Experiment 3, participants’ ability to judge line lengths was examined. Overall, for both horizontal and vertical lines, participants were able to judge the lengths fairly accurately . One-samHere we asked how frequently participants perceive a forward visuo-proprioceptive mismatch, both spontaneously and after being asked to attend to visuo-proprioceptive alignment, and whether such awareness is linked to reduced recalibration. The results suggest three main conclusions. First, at small offsets (<70 mm), awareness of the offset does not often occur spontaneously , but doeWe did not find evidence of participants spontaneously becoming aware of a gradual 70 mm visuo-proprioceptive offset in Experiment 1. These participants each completed two sessions on different days, in random order: One session with veridical visuo-proprioceptive calibration, and one with a gradual 70 mm forward offset. When questioned at the end of each session, 5% of participants reported perceiving a forward offset of any magnitude in the Mismatch compared to the Veridical session. However, the proportions of individuals who reported a forward offset in the two sessions did not differ statistically, suggesting that spontaneous awareness of this visuo-proprioceptive offset was uncommon. This result is not necessarily surprising. The gradual 70 mm offset was originally designed to be subtle enough that most individuals would not notice, while inducing a visuo-proprioceptive mismatch large enough that the brain would respond by recalibrating visual and proprioceptive estimates of hand position to compensate .Experiment 2 was intended to make visuo-proprioceptive offset easier to perceive. Participants were instructed in advance that they would be asked about their perceived visuo-proprioceptive calibration, and they were asked to report their perceived offset at frequent intervals instead of only at the end. With these changes, most participants correctly reported a forward offset at some point in the session. In contrast with Experiment 1, 70% (14/20) of the subjects in Experiment 2 had reported a forward offset by Block 4. Thus, conscious awareness of a 70 mm visuo-proprioceptive offset may require directing participants’ attention to the calibration of visual and proprioceptive stimuli. This finding is in line with a causal inference framework. Based on this framework, knowledge of a common cause acts as a Bayesian prior and instructions directing attention toward a common cause may influence its perception . As suchAn interesting finding is that when the offset was <70 mm, even though directing attention led to increased perception of an offset in Experiment 2, visuo-proprioceptive recalibration appears unaffected by this perception. Even with reported awareness of an offset, in the first half of Experiment 2 (0–70 mm offset), we did not detect a significant association between their max perceived offset magnitude and their total recalibration. Of course, we cannot rule out that there is a relationship , we found that when the offset is larger, awareness of the offset is clearly associated with reduced recalibration, consistent with the causal inference framework. In Experiment 2, max perceived offset was negatively correlated with total realignment by Block 8, when offset had reached 140 mm. The effect size of this correlation is considered large . HoweverThe causal inference literature makes clear predictions about cross-sensory recalibration in the context of offset awareness. In the case of visuo-proprioceptive recalibration, these predictions have been previously tested in experimental paradigms related to the rubber hand illusion (RHI) . The RHISimilar recalibration regardless of offset awareness suggests that explicit declaration of a separate cause may not override the intrinsic belief in a common cause at offsets of this magnitude. Indeed, others have suggested that unconscious belief in a common cause may continue even when subjects explicitly know about the offset . SpecifiOne possible explanation for the apparent boundary at 70 mm of offset is participants’ own biased visual and proprioceptive estimates even in veridical conditions; even in the absence of perturbation, visual and proprioceptive finger estimates do not agree perfectly . On averIn addition, while visuo-proprioceptive recalibration differs in many respects from visuomotor adaptation—a process requiring feedback about movement errors—the concept of error attribution may be a relevant parallel . It is pAttention is known to interact extensively with both sensory processing and behavioral performance. This includes regions known to be involved in multisensory integration, peripersonal space perception, and body ownership systems. Multisensory integration of visual and proprioceptive signals is largely associated with posterior parietal cortex (PPC). In monkeys, multimodal neurons responding to both “seen” and “felt” position of the limb exist in regions of PPC . NeuroimHuman neuroimaging work has revealed distinct frontoparietal networks for peripersonal space perception, which is often associated with sensorimotor tasks, and for the subjective sensation of body ownership, which is linked to attention and awareness tasks . FunctioAttention allows the completion of behavioral goals through the flexible selection and enhancement of a set of sensory inputs, thereby increasing the strength of the neuronal signals within that sensory area . This isWhen we found that participants consistently underestimated the magnitude of visuo-proprioceptive offset in Experiment 2, we wondered if this could be explained by participants being biased at estimating distances in general. For example, perhaps a participant actually perceives a 10 cm offset, but when asked to report that distance, estimates it to be only 4 cm. However, in Experiment 3, we found that participants were unbiased on average when asked to report the length of a series of white lines presented in the task display. This suggests that the under-reporting of perceived offset magnitude was not due to a systematic bias in estimating distances in general.did it always feel like the white square was directly on top of your left finger, or did it feel off?”. This question was asked of every participant the same way, but some needed clarification before they could answer it. When necessary, we clarified that we were not asking about V or P trials or their right hand, but rather about their perception of their left hand during VP trials. Importantly, this was simply the first question, intended to screen out subjects who never noticed any offset. For those who responded that they did feel an offset, we then asked them to estimate the direction and magnitude, which is what was analyzed. It may be advantageous for future studies to assess these parameters by alternative methods such as gaze tracking, given the importance of eye movements in attention.The conclusions of the present study are based on subjects’ verbal assessment of perceived offset, along with visuo-proprioceptive recalibration assessed by pointing with the other hand. One downside of relying on participants’ self-report of perceived offset is that participants may not be able to assess their perceptions accurately. Another could be a difference in the interpretation of questions, specifically the question “It should be noted that, although participants were asked to gaze at a red cross during each trial, the lack of eye tracking assessment in the present study means that we have no way to know where they were looking. Although participants heard a recording of this instruction at the beginning of every trial, we acknowledge it would not likely be sufficient to override a participant’s natural instinct to look toward the pointing target, at least initially. We must also offer the strong caveat that subjects may have employed different gaze strategies , which could explain substantial variations in offset detection or recalibration across participants.We chose this method of assessing perceived offset in order for the results to be comparable with our previous investigations using this task . There aAnother manipulation that might affect awareness of offset would be to make the visuo-proprioceptive mismatch occur abruptly. In the present study, visuo-proprioceptive offset increased gradually, 1.67 mm per VP trial. If the 70 or 140 mm full offset were reached in one or just a few trials, we might suppose that most participants would become aware of the perturbation. On the other hand, an abrupt shift may seem more natural and requiring of fewer cognitive resources; in real life, when we experience such an offset by viewing our hand under water, the visuo-proprioceptive offset occurs abruptly, not gradually.In a Bayesian causal inference framework , we woulHere we found that when a 70 mm mismatch is gradually imposed between visual and proprioceptive cues of hand position, individuals are unlikely to become aware of this spontaneously. When directed to attend to visuo-proprioceptive alignment by repeated questioning, conscious awareness of the mismatch was linked to reduced compensation only at higher mismatch magnitudes (70–140 mm). These results are consistent with causal inference predictions at larger offsets. At smaller offsets, conscious perception of an offset may not override unconscious belief in a common cause, perhaps because the perceived offset magnitude is in range of subjects’ natural sensory biases.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Indiana University Human Research Protection Program Board Affiliation: Indiana University. The patients/participants provided their written informed consent to participate in this study.AH: conceptualization, investigation, formal analysis, and writing—original draft. TL-M: formal analysis, software, visualization, and writing—review and editing. HB: conceptualization, methodology, software, writing—review and editing, funding acquisition, and supervision. All authors contributed to the article and approved the submitted version.


Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs.Previously, we have shown that apoplastic wash fluid (AWF) purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N The apoplast of Arabidopsis leaves contains diverse small and long-noncoding RNAs, including circular RNAs, that are bound to protein complexes and are located outside extracellular vesicles. IN A NUTSHELLBackground: To prevent infection by disease-causing microbes, plants secrete diverse antimicrobial compounds into their extracellular spaces. Included among these compounds are small RNA molecules 21-24 nucleotides in length (sRNAs) that can be taken up by microbes. These sRNAs are thought to cause the destruction of messenger RNAs having complementary sequences. The processes by which sRNAs are secreted, how they are protected from degradation, and how they are taken up by pathogenic microbes are all poorly understood.Questions: Although extracellular sRNAs have been shown to copurify with extracellular vesicles (EVs), their exact location had not been clearly established. Are sRNAs located inside or outside EVs? Also, do plants secrete RNAs longer than 24 nucleotides?Findings: We isolated EVs from the extracellular spaces of Arabidopsis leaves and then treated these preparations with RNase A to degrade naked RNA or with protease plus RNase A to degrade RNA protected by proteins. Our analyses revealed that sRNAs are associated with protein complexes that are located outside EVs. Significantly, we found that Arabidopsis secretes both sRNAs and much longer RNAs, ranging from 30 to over 500 nucleotides in length. These longer RNAs do not code for proteins, and many have a circular structure. Notably, both these long noncoding RNAs and the sRNAs were found to be highly enriched in a posttranscriptional modification known as N6 -methyladenine. We speculate that this modification might be required for secretion of RNA.Next steps: The discovery that plants secrete long noncoding RNAs, including circular RNAs, was unexpected and raises the question as to why. Do they play a role in cell-to-cell communication within the plant? Are they an important component of the immune system? How are these RNAs secreted and what are the roles of RNA-binding proteins and posttranscriptional modifications in this process?The apoplast is the extracellular space outside the plasma membrane of plant cells that comprises the cell wall, the xylem, and any space between cells . ApoplasIt is known that sRNAs from both plants and pathogens can hijack microbe or host RNA interference pathways to induce trans-kingdom gene silencing . HoweverArabidopsis thaliana) contains diverse species of sRNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and a previously overlooked class of tiny RNAs  with unknown functions  of Arabidopsis  are involved in immune responses to the fungal pathogen Magnaporthe oryzae. Several circRNAs in rice leaves were detected only upon infection with M. oryzae. Furthermore, this work showed that overexpression of one specific circRNA enhanced rice immunity to M. oryzae , including circular RNAs (circRNAs) . circRNATo understand the possible function of exRNAs in plants, we analyzed the sRNA and circRNA content of Arabidopsis apoplastic fluid both inside and outside EVs, as well as the RNA-binding proteins associated with these RNAs. Our data reveal that apoplastic fluid contains diverse RNA species, including sRNAs and lncRNAs (100 >500 nt), many of which appear to be circRNAs. The great majority of both sRNAs and lncRNAs were found to be located outside EVs. However, this extravesicular RNA is protected against degradation by RNases via association with RNA-binding proteins. The presence of abundant extravesicular sRNA- and circRNA–protein complexes in the apoplast suggests that these RNAs may play a central role in plant–microbe interactions and also contribute to host-induced gene silencing.g but that did not copurify with EVs in the density gradient.Our previous analyses of sRNAs associated with density gradient-purified EVs revealed that EVs contain relatively few RNAs in the 21, 22, and 24 nucleotide (nt) size range, and instead are highly enriched in shorter RNAs that are 10–17 nt in length, and have been termed tyRNAs . Those ag for 1 h . The P40 pellets contained a mixture of particles, including EVs. To distinguish between RNA located inside EVs from RNA located outside EVs, we treated P40 pellets with trypsin plus RNase A, which should eliminate RNA associated with proteins located outside EVs, while leaving RNAs located inside EVs intact. As controls, we treated pellets with just the buffer or with RNase A alone. The latter should degrade free RNA but not RNA bound to proteins or located inside EVs.To assess whether apoplastic fluid contains RNA-associated particles other than EVs, we generated sRNA libraries from pellets obtained after centrifuging AWF at 40,000 g and both were protected against digestion by trypsin, even after treating P40 pellets with RNase A first  and sequenced using an Illumina NextSeq platform. We observed that the distribution of read lengths was consistent between replicates, but substantially different between treatments . ControlTo further understand the nature of apoplastic sRNAs and tyRNAs, we analyzed their origin. We observed that most of the sRNA reads originated from rRNAs, mRNA, and products that were dependent on RNA polymerase IV (Pol IV) . We alsoWe further analyzed these sRNA-seq data by plotting the read-length distributions as a function of their origins . As expeAlthough we observed that, overall, apoplastic miRNAs were much more abundant outside EVs than inside EVs, this observation did not rule out the possibility that some miRNAs might be specifically loaded into EVs and thus could be enriched inside EVs relative to the general apoplastic miRNA population. To test this hypothesis, we compared the frequencies of individual miRNAs in each sample using a differential gene expression tool . To avoid false negatives due to low expression, we selected only miRNAs with more than one read per million (RPM) mapped reads in at least one sample. This filter reduced the data set from 427 mature miRNAs to 94. From these, 62 miRNAs displayed differential accumulation in at least one of the comparisons .Based on the differential accumulation pattern, we placed the miRNAs into one of six clades. Clade I comprised seven miRNAs that were highly accumulated in the trypsin plus RNase A-treated samples compared to control and RNase A alone-treated samples but were not differentially accumulated in RNase A alone treated versus control samples. This is the pattern expected for miRNAs located inside EVs because they should be protected against RNase A degradation regardless of trypsin treatment. Clade II comprised 10 miRNAs that were significantly more abundant in the RNase A alone-treated samples relative to the control samples and in the trypsin plus RNase A-treated samples relative to controls. Three of these also were significantly more abundant in the trypsin plus RNase A samples versus the RNase A-alone samples. This pattern is expected for miRNAs that are located both inside EVs and outside EVs, with the latter being protected against RNase digestion by proteins.Clades III and VI contained 24 miRNAs that exhibited low accumulation in trypsin plus RNase A-treated samples compared to control and RNase A alone-treated samples, but showed high accumulation in RNase A alone-treated samples compared to the controls. This pattern is expected for miRNAs that are located outside EVs and protected by RNA-binding proteins. The miRNAs found in Clades IV  and V  exhibited low abundance in RNase A alone-treated samples versus controls as well as trypsin plus RNase A alone-treated samples versus control samples. These are most likely miRNAs that are located outside EVs and are not protected by proteins. In summary, these data indicate that most plant miRNA species in the apoplast are located outside EVs, with only seven miRNAs apparently enriched inside EVs. Notably, of these seven miRNAs, six correspond to passenger strands of active miRNAs, and thus they might represent unneeded material that is being discarded from the cell.tasiRNAs are a subclass of sRNAs that have been proposed to mediate interkingdom RNA interference, possibly by transfer inside of plant EVs . The anaClade I (seven tasiRNAs) showed significantly higher relative abundance in RNase A alone-treated samples compared to control samples, suggesting these tasiRNAs are located outside EVs and are protected by proteins. Consistent with this conclusion, these seven tasiRNAs were relatively less abundant in trypsin plus RNase A-treated samples compared to RNase A alone-treated samples. Clade II tasiRNAs (four tasiRNAs) showed a very similar pattern to that of Clade I tasiRNAs, thus are also likely to be located outside EVs and protected by proteins.Clades III (three tasiRNAs) and IV (eight tasiRNAs) showed a relative abundance pattern opposite to that of Clades I and II—treatment with RNase A alone caused a decrease in relative abundance compared to control samples and treatment with trypsin plus RNase A caused an increase compared to RNase A alone-treated samples. This pattern suggests that tasiRNAs belonging to Clades III and IV are located outside EVs and are not protected by proteins. However, why trypsin plus RNase A treatment led to less efficient removal than RNase A alone is unclear. We speculate that residual trypsin activity in the former may have caused a slight reduction in RNase activity.Lastly, the five tasiRNAs included in Clade V showed a pattern more similar to RNAs in Clades I and II, suggesting that these are located outside EVs and are mostly protected by proteins. Notably, none of the tasiRNAs showed a pattern that would be consistent with protection inside EVs, as they should show a relative increase in abundance across all three comparisons. It has been previously reported that two tasiRNAs from Arabidopsis, Tas1c-siR483 (here named Tas1c_16_461) and Tas2-siR453 (here named as Tas2_0_566) are transferred into fungal cells via EVs . Howeverg. RNAs were isolated from total AWF, the P100 pellet, and from the supernatant of the P100 pellet and analyzed by denaturing polyacrylamide gel electrophoresis, followed by staining with SYBR Gold to detect nucleic acids. These analyses revealed that AWF contained abundant RNAs ranging in size from 14 nt to at least 500 nt , which promotes back-splicing, with the resulting circRNAs containing multiple m6A-modified sites  enrichment protocol, and Method 2 using an rRNA depletion protocol (see Methods). Analysis of the poly(A)-enriched library revealed that it contained very few products with inserts , indicatTo assess whether specific RNA species were associated with protein or were encapsulated inside EVs, we also made libraries from P40 pellets that were either treated with RNase A alone, which should eliminate RNA that is not protected by proteins or EVs, or treated with trypsin plus RNase A, which should leave mostly RNA encapsulated in EVs. Analysis of these libraries revealed that trypsin plus RNase A treatment reduced the relative proportion of most classes of exRNA , consistThe above analyses revealed that AWF contains abundant RNA species (including both sRNAs and long RNAs) that are protected from RNase degradation by proteins. This raised the question of what RNA-binding proteins are present in the apoplast. In our previous proteomic analyses of density-gradient purified EVs, we had identified the RNA-binding protein GLYCINE-RICH PROTEIN 7 (GRP7) as copurifying with EVs . GRP7 haTo confirm that GRP7 is secreted into the apoplast, we performed immunoblots on protein extracts isolated from the P40 and P100-P40 fractions of an Arabidopsis line expressing GRP7-GFP expressed under its native promoter . These aIn parallel to our analyses on GRP7, we also assessed whether the sRNA-binding protein ARGONAUTE2 (AGO2) was present in AWF. AGO2 was chosen from among the ten Arabidopsis AGO proteins because of (1) a known role in plant-pathogen interactions  and (2) To investigate the RNAs associated with GRP7 and AGO2 in the apoplast, we performed RNA immunoprecipitation on whole cell lysate (CL) and P100 fractions. RNAs were separated by size using polyacrylamide gels, followed by staining with SYBR Gold to detect nucleic acids. These analyses indicated that, in both whole CL and in the P100 fraction, Arabidopsis GRP7-GFP expressed under the native GRP7 promoter binds to RNAs of various sizes ranging from 50 nt to more than 500 nt . Similargrp7 and ago2 mutants following RNase R treatment. These analyses revealed a marked reduction in total circRNAs identified in each mutant  was obtained from the Arabidopsis Biological Resource Center at Ohio State University. The Arabidopsis ago2-1 mutant was obtained from James Carrington at the Donald Danforth Plant Science Center. The Arabidopsis HA-AGO2 transgenic line was also obtained from Dr Carrington. It expresses HA-AGO2 under the native AGO2 promoter in an ago2-1 mutant background , both of which were obtained from Hummert International . Seedlings were grown under a clear plastic dome for the first week following transfer.The ckground . The GRPckground . Seeds wckground . To induN-morpholino) ethanesulfonic acid, 2-mM CaCl2, and 0.01-M NaCl as described previously . The AWF was then filtered through a 0.22 µm membrane  and centrifuged at 10,000 g for 30 min to remove any remaining large particles. The supernatant was transferred into new centrifuge tubes and centrifuged at 40,000 g (P40) or 100,000 g (P100) for 1 h  to pellet EVs and other particles as noted in figure legends. The pellet was washed and pelleted again at 40,000 g or 100,000 g at 4°C using a TLA100.3 rotor, Optima TLX Ultracentrifuge (Beckman Coulter). The pellets were resuspended in 100 µL of cold and filtered VIB (0.22 µm) and either used immediately or stored at −80°C until further use.For a single biological replicate, AWF was isolated from 24 6-week-old Arabidopsis plants as described in eviously . After vg for 15 min. The aqueous phase was removed and mixed with one volume of cold isopropanol to precipitate the RNA. RNA pellets were washed using cold 80% ethanol. To isolate RNA from P40 and P100 pellets, 1 mL of TRIzol was added to 100 µL of resuspended pellet, followed by the same procedure as used for leaf RNA isolation. To isolate RNA either from the supernatant of P100 pellets, or from total AWF , the RNA was precipitated by mixing with 0.1 volume of 3-M sodium acetate (pH 5.2) and 1.0 volume of cold isopropanol, incubated at −20°C for 1 h and then centrifuged at 12,000 g at 4°C for 30 min. The resulting pellet was then resuspended in 1 mL of TRIzol followed by the same procedure as used for leaf RNA isolation. RNA pellets were resuspended in 10–12 µL of ultrapure DNase/RNase-free water  and stored at −80°C. The RNA quality and quantity was assessed using either a ThermoFisher NanoDrop One spectrophotometer or an Agilent 2200 Tape Station.Total leaf RNA was isolated from 100 mg of fresh or frozen leaf tissue using TRIzol Reagent . Briefly, to isolate RNA, leaf tissue was frozen in liquid nitrogen and ground into powder using a mortar and pestle. One milliliter of TRIzolReagent  was added to the ground leaf tissue and mixed vigorously by vortexing. The leaf and TRIzol mixture was then shaken at room temperature for 10 min, followed by the addition of 200 µL of chloroform. This mixture was then vortexed for 30 s and then centrifuged at 12,000 To assess whether RNAs were located inside or outside EVs, we performed RNase protection assays as follows. P40 pellets were treated with 1-µg/mL trypsin  in the presence or absence of 1% (v/v) Triton X-100  in 15-mM Tris–HCl . Samples were incubated at 37°C for 1 h followed by addition of 1.5 µg/mL trypsin inhibitor  to inactivate trypsin. For the RNase-treated samples, RNase A  was added to the mixture to a final concentration of 5 µg/mL  and the sample was incubated at room temperature for 30 min. Immediately after RNase A treatment, RNA was isolated using 1 mL of TRIzol as described above. To inhibit RNase A activity, a mixture of 10 µg/mL RNase Inhibitor, Murine (APExBIO) and 40 units/mL of RNase Out (Invitrogen) was added to the RNAs, which were stored at −80°C until library preparation.g for 10 min at 4°C to pellet cell debris. An aliquot of 30 µL of leaf lysate was combined with 10 µL of 4 × SDS loading buffer, and the mixture was heated at 95°C for 5  min. Then, 40 µL of P40 and 4 µL of leaf lysate were loaded onto gradient gels  and were separated at 150 V for 1 h in Laemmli electrophoresis running buffer  sodium dodecyl sulfate, 192 mM glycine, pH 8.3). After the proteins were transferred to a nitrocellulose membrane (GE Water & Process Technologies), membranes were washed with 1× Tris-buffered saline  containing 0.1% Tween-20 (TBST) and blocked with 10% Difco Skim Milk (BD) in TBST for 1.5 h at room temperature. Membranes were incubated overnight at 4°C with the following primary antibodies at the indicated dilutions: rabbit polyclonal anti-PEN1  sodium dodecyl sulfate, 40% (v/v) glycerol, 20% 2-mercaptoethanol and 0.004% (w/v) bromophenol blue) and were then heated at 95°C for 5 min. Leaf lysate samples were used as positive controls. The lysate was prepared by freezing 100 mg of leaf tissue in liquid nitrogen and grinding with a mortar and pestle. Ground leaf tissue was extracted in 800 µL of protein extraction buffer  Nonidet P-40, 1% (w/v) 2,2'-dipyridyldisulfide, and 1% plant protease inhibitor cocktail ) and centrifuged at 10,000 nti-PEN1 ; 1:1,000nti-PEN1 ; 1:1,000nti-PEN1 ; 1:5,000(RNA-IP). For leaves, we used 1 g of fresh or frozen leaf tissue, which was frozen under liquid nitrogen and ground with a mortar and pestle. Leaf powder was mixed with 5 mL of cold IP buffer , incubated on ice for 10 min, and then transferred to a 15-mL polypropylene screw-cap centrifuge tube. The tube was then centrifuged for 10 min at 12,000 g and the supernatant was filtered through a 0.45  µm membrane . The filtered supernatant was then incubated for one h at 4°C with 50 µL of anti-GFP agarose beads (Chromotek) to precipitate GRP7-GFP and with 50 µL of anti-HA agarose beads (Thermo Fisher) to precipitate AGO2-HA. Beads were then pelleted by centrifugation at 1,000 g for 2 min at 4°C and washed at least six times with 5 mL of cold IP buffer for 5 min at 4°C for each washing step. Finally, beads were washed two times with 1.5 mL of cold IP buffer followed by a final wash with 1 mL of ultrapure RNase-free/DNase-free water before pelleting by centrifugation at 538 g for 1 min. To immunoprecipitate GRP7-GFP and AGO2-HA from apoplastic fluid, P100 pellets were resuspended in 2 mL of cold IP buffer and proteins immunoprecipitated as described for whole leaf extracts.To isolate RNAs associated with GRP7-GFP and AGO2-HA in whole leaves and in apoplastic fluid of transgenic Arabidopsis plants, we performed RNA-immunoprecipitation g for 1 min, and RNA isolation was performed using the TRIzol reagent as described above.To isolate RNA, beads were incubated with proteinase K (Invitrogen) at a final concentration of 1.5 µg/µL in 100 µL of PK buffer  for 1 h at 55°C with intermittent shaking (every 3 min for 15 s). Beads were pelleted by centrifugation at 538 RNA samples were analyzed using denaturing polyacrylamide gel electrophoresis. Gels containing 15% polyacrylamide and 7 M urea were prepared using IBI InstaPAGE 40% acrylamide solution (37.5:1). RNA samples were denatured at 65°C in denaturing buffer (0.25 M EDTA (pH 8.0), 8-M urea, 0.2-mg/mL bromophenol blue, 0.02-mg/mL xylene cyanol) and were then separated on 0.5 × Tris-Boric Acid EDTA , 21 mM boric acid, 1.25-mM EDTA)-15% polyacrylamide urea gels. For size standards, we used New England Biolabs Low Range ssRNA Ladder  and Takara 14-30 ssRNA Ladder Marker . SYBR Gold Nucleic Acid Gel Stain (ThermoFisher) was used to stain gels for 30 min before UV transillumination. Gel images were acquired using a Bio-Rad ChemiDoc-MP imaging system.2 for 30 s. The membrane was then washed in clean RNase-free 1 ×  PBS buffer  and blocked in 5% nonfat milk in 1 × PBS containing 0.02% Tween-20 for 1 h at room temperature. The membrane was then incubated overnight with anti-m6A antibody  at a 1:250 dilution in 5% nonfat milk in 1 × PBS containing 0.02% Tween-20. The membrane was washed in 1 × PBS containing 0.02% Tween-20 three times and incubated with horseradish peroxidase-labeled goat anti-rabbit antibody  at a 1:5,000 dilution for 1 h. After a final wash in 1 ×  PBS contain 0.02% Tween-20, m6A modified RNAs were visualized using the Immune-Star Reagent (Bio-Rad) and imaged using X-ray film.RNA was isolated from leaf or apoplastic P40 and P100 fractions using TRIzol as described above, and the RNA concentrations were measured using a ThermoFisher NanoDrop One spectrophotometer. For all samples, equal amounts of RNA were prepared in equal volumes (6 µL) using UltraPure DNase/RNase-free distilled water (Invitrogen. RNA samples were denatured at 95°C for 3 min and placed on ice immediately to prevent the formation of secondary structures. RNA samples were applied directly to a piece of Hybond-N+ membrane (Amersham Pharmacia Biotech) using a micropipettor. To prevent the spread of RNA on the membrane, 2 µL of RNA solution was applied at a time, allowing the membrane to dry for three min before applying the next 2 µL drop to the same spot, until a total of 6 µL of RNA sample was applied. To crosslink the spotted RNAs to the membrane, an UVC-508 Ultraviolet Cross-linker (Ultra-Lum) was used to irradiate the membrane twice at 120,000 microjoules/cmTo determine the presence of circRNAs, RNA was isolated from the entire P100 pellet (resuspended in 100-µL VIB) obtained from 24 Arabidopsis plants using a PicoPure RNA isolation kit (Thermo Fisher). The RNA (1–3 µg) was then treated with 5 units of RNase R (Lucigen. RNR07250) for 1 h at 37°C. To visualize circRNAs, the RNA samples were separated on denaturing TBE-15% polyacrylamide urea gels and stained with SYBR Gold. To prepare RNA libraries for sequencing, it was necessary to remove RNase R from the RNA samples, and this was accomplished by repurifying the RNase R-treated RNA samples using a PicoPure RNA isolation column.sRNA libraries were constructed using the RealSeq-AC kit  following the manufacturer’s recommendations. To capture all types of sRNAs, we used 1 µg of RNA as starting material. Except for RNase-R treated samples, all RNA-seq libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina  using 500 ng of total RNA as the starting material. The rRNA was removed using the RiboMinus Plant Kit for RNA-Seq  and poly(A) RNA purification was attempted using the NEBNext Poly(A) magnetic isolation module . For sequencing of RNase R-treated samples, RNA-seq libraries were prepared using an Illumina TruSeq Stranded mRNA Library Prep kit  following the manufacture’s protocol except for skipping the poly(A) enrichment step. All libraries were sequenced on an Illumina NextSeq 550 instrument with paired-end 75-bp reads, except for the RNase R-treated samples, which were sequenced using paired-end 300-bp reads. Sequencing was performed at the Center for Genomics and Bioinformatics at Indiana University, Bloomington, IN, USA.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Clean reads were aligned to the Arabidopsis genome (TAIR version 10), and all subsequent analyses were performed using the software Bowtie2 (http://ibi.zju.edu.cn/plantcircbase/) (Sequences in the sRNA sequencing libraries were trimmed of adaptors using the software Cutadapt version 1.16  using a  Bowtie2 . For miR Bowtie2  was used Bowtie2  and sequ Bowtie2 . To idenrcbase/) . We onlyrcbase/) . In DESercbase/) . Graphicrcbase/)  in the Rhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183867) and GSE185133 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE185133). The accession numbers for Arabidopsis proteins discussed in this work are AT1G48410 (AGO1), AT1G31280 (AGO2), AT2G21660 (GRP7), AT1G72150 (PATL1), AT3G11820 (PEN1), AT1G59870 (PEN3), and AT3G25070 (RIN4).The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are accessible through GEO Series accession numbers GSE183867 (The following materials are available in the online version of this article. Apoplastic miRNAs and tasiRNAs are mostly located outside EVs and are protected by proteins. P40 RNA appears to lack polyadenylated RNA.koac043_supplementary_dataClick here for additional data file.


L being anti-apoptotic and Bcl-XS pro-apoptotic. In a number of cancers the Bcl-XL isoform is over-expressed, resulting in cancer cell survival and growth, so switching splicing to the Xs isoform could have therapeutic benefits. We have previously proposed that a putative G-quadruplex (G4) exists downstream of the XS 5′ss and shown that the ellipticine derivative GQC-05, a previously identified DNA G4-specific ligand, induces an increase in the XS/XL ratio both in vitro and in cells. Here, we demonstrate that this G4 forms in vitro and that the structure is stabilised in the presence of GQC-05. We also show that GQC-05 binds RNA non-specifically in buffer conditions, but selectively to the Bcl-x G4 in the presence of nuclear extract, highlighting the limitations of biophysical measurements taken outside of a functional environment. We also demonstrate that GQC-05 is able to shift the equilibrium between competing G4 and duplex structures towards the G4 conformation, leading to an increase in accessibility of the XS 5′ss, supporting our previous model on the mechanism of action of GQC-05.The BCL2L1 gene expresses two isoforms of Bcl-x protein via the use of either of two alternative 5′ splice sites (5′ss) in exon 2. These proteins have antagonistic actions, Bcl-X G4 struum (Cs+) . To dateum (Cs+) .RNA G4s have been implicated in a wide variety of biological processes, such as heterochromatin formation , mRNA trS) or an anti (XL) apoptotic protein variant , in which guanines are replaced by 7-deazaguanine . The use variant . In parttructure , but tha1 snRNPs . This mo1 snRNPs .s 5′ ss would be promoted by ligands that stabilise G4 structures. A small screen of known G4 ligands showed that the ellipticine derivative GQC-05, a previously-reported DNA G4-stabilising ligand  were purchased in their desalted and deprotected form from Horizon Discovery. RNAs were resuspended in 1 ml milliQ water and dialysed twice in 1 L of milliQ water for 12 h to remove any residual salts and impurities, using a 0.5–1 kDa Spectra-Por dialysis tubing . Samples were concentrated using a 3 kDa cut-off Amicon filter . The concentrations of RNAs were determined by measuring the absorbance at 260 nm at 95°C and using their respective molar extinction coefficients . RNAs wein vitro transcription following optimization of MgCl2, NTPs, DNA template and homemade T7 RNA polymerase concentrations. The transcription reactions were centrifuged to remove the pyrophosphate pellet and filtered before injection into a pre-equilibrated anion exchange preparative column (ThermoFisher DNAPac PA100 22 × 250 mm) on an UltiMate 3000 Max HPLC (ThermoFisher), using a 2 ml injection loop with a flow rate of 3 ml/min and a temperature of 80 C. The column was equilibrated with 12.5 mM Tris HCl, pH 7.4, and the RNA was eluted using a gradient of 1 M NaClO4 (from 10 to 50% over a period of 60 min) in 12.5 mM Tris HCl, pH 7.4. Relevant fractions were collected, pooled together and then concentrated to approximately 1 ml by centrifugation at 4,000 rpm for 60 min at 4 C using a 3 kDa cut-off Amicon filter . Samples were then dialysed against 2 L of milliQ water for >12 h using 0.5–1 KDa Spectra-Por dialysis tubing , followed by an additional 1 h against 2 L of fresh milliQ water. Aliquots of the samples were then analysed using an 8% denaturing polyacrylamide urea gel run at 180 V for 100 min and stained with a 0.1% toluidine blue solution (in 10% acetic acid) for 5 min to check for RNA purity. The RNA concentrations were determined via A260nm at 95°C.Long RNAs (>30 nucleotides) were prepared by large scale All circular dichroism (CD) experiments were conducted on a Chirascan CD spectropolarimeter (Applied Photophysics). RNA samples, with a concentration of 2 μM, were annealed as described in 1H NMR experiments were recorded on a 600 MHz Bruker AV III spectrometer at 283 K using a watergate scheme for suppression of the water signal. Spectra were acquired using 256 scans and an acquisition time of 200 ms with a relaxation delay of 2 s. A Fourier transform was applied using a cosine window function. For titrations with either KCl or GQC-05, a sample of 100 µM RNA was annealed in 10 mM Tris pH 7.0 in the absence of any salt, followed by the addition of D2O to a final concentration of 10% (v/v) post annealing. 1D NMR spectra were recorded following successive additions of small volumes of 4 M KCl to final concentrations of 10, 20 and 100 mM. In the case of GQC-05, small volumes of 10 mM GQC-05 (in 100% d2DMSO) were added to final concentrations of 100–300 µM.All 1D 2O (v/v) post annealing. An initial 1D spectrum was recorded and then the Q2 complementary strand was added to the Q2 RNA sample at an equimolar concentration, which defined the start of the experiment (t0). The acquisition time for each 1D spectrum was 160 s, and the total void time before acquisition of the first spectrum was 562 s. Spectra were recorded at 293 K over a period of 72 h as a pseudo-2D spectrum. This experiment was repeated in the presence of 4 equivalents of GQC-05, which was added to the sample post-annealing of the Q2 strand, followed by addition of the complementary strand. Data were analysed by peak integrals of the imino region (10–14 ppm) of the spectra using Dynamic Centre on Topspin. The kinetic curves of the peak integrals of the imino region versus time were plotted and fitted using first order kinetics with the equation available on GraphPad prism Version 9.0.100 µM of the Q2 RNA sample was annealed in 10 mM Tris pH 7.0, 20 mM KCl, followed by the addition of 10% DAll fluorescence spectra were obtained at 25°C on a Fluromax-4 fluorimeter using a 1 cm pathlength quartz cuvette designed for fluorescence measurements or a Hidex microplate reader on a 384 well plate (Corning) for the fluorescence titrations. For the N-methyl mesoporphyrin (NMM) experiments, 10 µM of the RNAs were annealed in the relevant salt concentrations in 10 mM Tris pH 7.0, followed by the addition of 1 µM NMM. Fluorescence emission spectra were recorded between 450 and 700 nm, with an excitation wavelength of 393 nm and a 5 nm excitation and emission slit width.For titration experiments using GQC-05 as the fluorophore, all fluorescence titrations were conducted on a Hidex microplate reader using 1 µM GQC-05, with an excitation wavelength of 320 nm and an excitation and emission slit width of 5 nm. 100 µM RNA samples were annealed in 10 mM Tris pH 7.0, 100 mM KCl, followed by the addition of 1 µM GQC-05 post annealing. Concentrations of the RNA were varied through serial dilutions in a buffer containing 10 mM Tris pH 7.0, 100 mM KCl and 1 µM GQC-05, in a total of 17 wells. The final fluorescence emission intensity of the RNA-GQC-05 complex was obtained after subtracting the fluorescence of the individual RNA and GQC-05 components. Data were analysed using GraphPad prism Version 9.0 and fitted to a single-site binding isotherm with the equation reported in .For fluorescence experiment in presence of nuclear extract, all experiments were conducted as described above, but with the addition of 10% HeLa nuclear extract (v/v) from Cilbiotech post annealing (approximately 2–2.5 mg/ml of nuclear proteins). Background fluorescence arising from interactions between GQC-05 and components of the nuclear extract were subtracted to obtain the final fluorescence signal.10 µM Q2 RNA samples were annealed in 10 mM Tris pH 7.0 with either 100mM KCl, NaCl or LiCl or increasing KCl concentrations (0–20 mM). Samples were loaded on a 12% native PAGE gel at 180 V for 100 min and stained with toluidine blue for 5 min and de-stained in water for >30 min. A mixture of poly(T) DNAs with sizes ranging from 20–120 nucleotides was used as a ladder.A fluorescent dye, AlexaFluor546, and a dark quencher, 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL), were conjugated to modified nucleotides on Q2 RNA located after the first and third G-tracts, respectively (with a 15 base separation). In addition, the 5’ end of Q2 RNA was modified to contain a biotin group. An RNA nucleotide was purchased from Eurogentec with modified thymine bases (X and Y) containing reactive amino and thiol groups, respectively, at the required fluorophore and quencher positions, 5′(Biotin) AAAAAGGGAXGGGGUAAACUGGGGYCGCAUUGUGG AAAAA3’. The A-tracts at the 5 and 3′ ends were included to avoid RNA degradation by nucleases in the nuclear extracts. The amino group on X was subsequently reacted with AlexaFluor546-succinimide (Thermofisher) and the thiol group with DABCYL-maleimide (Eurogentec), respectively. The custom-modified Q2 RNA oligonucleotide  was mixed with succinimide-AlexaFluor546  in a microcentrifuge tube, which was protected from light. The tube was placed in a rotary mixer at 2 r.p.m. for 6 h at room temperature. The standard buffer present in G25 columns, which had been stored at 4°C, was replaced with cold phosphate-buffered saline  and the sample containing dye-labelled Q2 RNA was added to the column. The column was spun for 2 min at 730 g and the eluent was collected, protected from light and stored in 20 μL aliquots at -20°C. The reducing agent tris(2-carboxyethyl)phosphine  was added under nitrogen to an aliquot of the dye-labelled Q2 RNA, which was placed in a rotary mixer (2 r.p.m.) at room temperature for 10 min. DABCYL-maleimide in DMSO  was subsequently added under nitrogen and mixed at room temperature using the rotary mixer. Again the Q2 RNA was added to a G25 column containing cold buffered saline, which was spun for 2 min at 730 g and the eluent collected, protected from light and stored in aliquots at -20°C.2, 20 mM HEPES-KOH (pH 7.5), 0.05% (v/v) NP40, 50 mM potassium glutamate, 20% nuclear extract (around 4–5 mg/ml of nuclear proteins), 1 mg/ml glucose oxidase, 0.04 mg/ml catalase, 1 mM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 0.8% (v/v) glucose and, where present, 0.1% (v/v) of 10 mM GQC-05 dissolved in DMSO. Experiments done in the absence of nuclear extract contained an additional 20 mM potassium glutamate to maintain the potassium ion concentration. Images of the emission from surface-tethered molecules of RNA were acquired in frames of 50 ms at ambient temperature.Time-resolved single-molecule fluorescence resonance energy transfer (FRET) was measured using total-internal reflection fluorescence (TIRF) imaging. The apparatus is custom-designed using an Olympus IX73P2F inverted microscope frame, with a TIRF objective (UAPON100XOTIRF). The AlexaFluor546 was excited by a 532 nm solid-state laser , and the emission collected by an electron-multiplied charge coupled device (Photometrics). The surfaces of microscope cover glasses were cleaned with piranha solution (3 parts concentrated sulphuric acid and 1 part 30% hydrogen peroxide), and subsequently functionalised by exposure to (3-aminopropyl)triethoxysilane (2% in acetone). Following washing in acetone and deionised water, the slides were assembled to form a fluidic chamber similar to the design previously described . MonolayS 38–206 RNA respectively was slow cooled in the absence or presence of 2 equivalents of GQC-05 in 10 mM Tris pH 7.0 and 100 mM KCl. To this, 6 µM of the U1 oligonucleotide (Horizon Discovery) was added post-annealing. The RNA was then serially diluted in an equivalent buffer containing the U1 oligo and with the presence or absence of GQC-05. Samples were loaded on a 16% native polyacrylamide gel, run at 150 V for 1 h and visualised on a Typhoon scanner using an excitation wavelength of 488 nm and detection at 550 nm.For the WT and optimised (OP) U1 oligo experiments, 150 and 50 µM HPLC-purified XS 5′ss (Q2)  that have the potential to fold into a G4: the G-tracts of Q1 which lie upstream of the 5′ss, and the G-tracts of Q2 which lies downstream of the 5′ss  containing parts of Bcl-X exons 2 and 3 with a shortened intron 2, we identified six candidate G4-forming sequences in Bcl-x-681 that had been labelled as Q1 to Q6  Figure . Previou′ss (Q2) . To confragments  in the pragments , Red, bu and KCl , Green, the 5′ss . Therefoith LiCl , suggestith LiCl , suggestS 38–206 and Q2 150–206 constructs even in the absence of NMM, as NMM may induce a G4 that would otherwise not be present. To do this we compared the CD spectra of the RNAs in the presence of KCl or LiCl in order to distinguish between G4 and duplex structures , consistent with the expected size of Q2 163–192. In 16 mM and 100 mM KCl we observed the formation of higher order structures migrating at positions around T-60 suggesting the possibility of a G4 dimer. This was also observed in 100 mM NaCl but not in 100 mM LiCl . We nextKnowing that a multimeric G4 element exists within the Q2, we next examined the specificity of GQC-05 for this structure, by determining its binding affinity and re-modelling capabilities.S 5′ss usage (D) for the interaction between GQC-05 and WT Q2 163–192 is 0.11 μM (95% CI: 0.026–0.26 µM) (D of 0.44 μM (95% CI: 0.21–0.78 µM) (D of 0.58 μM (95% CI: 0.34–0.93 µM)  . Surpris0.78 µM)  and Q2.20.93 µM) ; the KD ructures . This isructures .in vitro splicing assays is that fluorescence assays were done with RNA in buffer, while splicing assays are conducted in the presence of nuclear extract containing multiple RNA-binding proteins. We therefore conducted a structural characterisation of the Q2 RNA and investigated the binding of GQC-05 in the presence of HeLa nuclear extract. Even in the presence of nuclear extract, the CD spectra of Q2 163–192 display a greater amplitude as well as a higher melting temperature (ΔTm = 12°C) in 100 mM KCl than in LiCl   , consistTo confirm that the Q2 G4 could be stabilised by GQC-05 in the presence of nuclear extract, we used time-resolved single molecule FRET. We labelled the Q2 RNA after the first G-tract with AlexaFluor546 dye (the donor fluorophore) and after the third G-tract with DABCYL (the acceptor - a dark quencher of fluorescence). AlexaFluor546 and DABCYL will be adjacent in a folded G4 but separated by a greater distance, on average, in the unfolded state of Q2 RNA. This combination of donor/acceptor has a short Föster distance of 2.9 nm, which enables the folding status of Q2 RNA to be monitored sensitively. The unfolded state (non-G4) will exhibit a higher fluorescence photon count for AlexaFluor546, compared with folded RNA (G4) due to reduction in the fluorescence yield from a higher efficiency of resonance energy transfer to the DABCYL quencher.In the presence of nuclear extract, there are significant temporal fluctuations in the photon count from the Alexa546 dye in the absence of GQC-05  of duplex formation in the absence of GQC-05 is 1.4 h, whereas it is > 18 h in the presence of GQC-05 at 283 K. These data clearly indicate that GQC-05 can bind to and selectively stabilise the Q2 G-quadruplex structure over the duplex in the Bcl-x Q2 sequence.We then calculated the rates of duplex formation by integrating the peaks in the 13–15 ppm region using first order kinetics . The estS 5′ss. To examine this, we probed the binding of a small fluorescently labelled RNA oligonucleotide that would mimic the base pairing of U1 snRNP to 5′ss  construct is rather weak (KD > 150 µM) suggesting that the formation of the duplex indeed reduces the accessibility of the Xs 5’ss. However, the addition of two molar equivalents of GQC-05 significantly increases its affinity for Bcl-Xs (38–206)    indicating that the duplex is not stable enough to complete with its binding and as expected, addition of GQC-05 has no significant effect on the affinity   , in agrete usage . To inveU1 OP in . In this10.1 µM) . This suS 5′ss Bcl-x. GQC-05 was previously shown to selectively bind DNA G4 structures over their double stranded counterparts in vitro (in vitro, GQC-05 binds with similar affinities to both G4 RNA molecules and RNA molecules that are unable to fold into G4 structures, suggesting that GQC-05 does not display selectivity for G4 RNAs. However, surprisingly, we demonstrate that the specificity of GQC-05 towards G4 is achieved in the presence of HeLa nuclear extracts. To our knowledge, this is the first evidence that a ligand displays RNA G4 selectivity only in the presence of nuclear extract. This highlights the limitations of many experiments when determining binding specificities in highly purified in vitro systems, without taking into consideration the cellular environment, where the RNA is bound by multiple RNA-binding proteins and the true specificity of a G4 ligand is likely to be revealed. This could be true for many other G4-binding molecules that are known to display a high affinity but low specificity towards G4s in vitro conditions. The exact mechanism by which nuclear extract confers GQC-05 specificity to the Q2 G4 structure remains to be explored, though the potential for RNA-binding proteins to compete with non-G4 interactions is a likely possibility.We have previously suggested that the Bcl-x pre-mRNA contains two regulatory G4s, termed Q2 and Q5  and showin vitro . HoweverEven though GQC-05 lacks specificity for G4s in the absence of nuclear extract, our NMR data show that it induced and stabilised the formation of G4 structures over their duplex counterparts , 10. ThiS 5′ss to potential trans acting factors, such as the spliceosomal component U1 snRNP. Indeed our previous structural model of Bcl-x suggested that in the absence of Q2 G4, the Bcl-Xs portion of the RNA would adopt a long stem-loop that encompasses part of the Xs 5′ss, making it poorly accessible to the splicing machinery. By probing the binding capability of a U1 snRNA-mimicking oligo to a longer portion of RNA, that contained the sequences required to form the stem-loop, we show that GQC-05 can indeed increase the accessibility of the XS 5′ss, consistent with the observed enhancement in its usage in splicing assays, fully supporting our previously described model (S 5′ss binds strongly and the addition of GQC-05 shows no difference in accessibility, indicating that the stability this secondary structure would not be sufficient to regulate splicing if the 5′ss were a consensus sequence, perfectly complementary to the U1 snRNA. Thus, our model for regulation of this site by competition between the formation of secondary structure or a G4 would only hold in circumstances where, as with Bcl-Xs, the 5’ ss is intrinsically weak.In our previous work, we suggested that the competition between G4 and stem-loop formation could affect the accessibility of the Xed model . However