The dynamics of SARS-CoV-2 RNA structure and their functional relevance are largely unknown. Here we develop a simplified SPLASH assay and comprehensively map the in vivo RNA-RNA interactome of SARS-CoV-2 genome across viral life cycle. We report canonical and alternative structures including 5′-UTR and 3′-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in both cells and virions. We provide direct evidence of interactions between Transcription Regulating Sequences, which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE. More importantly, we find that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome domains remain stable but with strengthened demarcation of local domains and weakened global cyclization. Taken together, our analysis reveals the structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, the alternative conformations and the maintenance of global genome organization during the whole life cycle of SARS-CoV-2, which we anticipate will help develop better antiviral strategies. RNA secondary structure is important for viral replication, transcription and translation. Here the authors employ SPLASH method and map in vivo RNA interactions and dynamics of SARS-CoV-2 RNA genome in different viral life cycles. Although many efforts have been devoted to control the disease and vaccines are being approved for emergency use, the pandemic is far from being under control. Therefore, there is an urgent need to understand the basic molecular biology of SARS-CoV-2 coronavirus. SARS-CoV-2 belongs to the broad family of coronaviruses. It is a positive-sense, single-stranded RNA virus, with a single linear RNA segment of ~30,000 bases3. The transcription process is controlled by transcription-regulating sequences (TRSs) located at the 3′-end of the leader sequence (TRS-L) and preceding each viral gene (TRS-B), and requires base pairing between the core sequence of TRS-L and the nascent minus strand complementary to each CS-B (cCS-B), allowing for leader-body joining5. A three-step working model of coronavirus transcription was suggested6, which implies that long-distance RNA–RNA interactions are required prior to template switch. Long-distance interactions between B motif (B-M) and its complementary motif, and between proximal element and distal element, are important for forming high-order structures promoting discontinuous RNA synthesis during N sgmRNA transcription in the Transmissible Gastroenteritis Virus (TGEV) (see review5 for details). However, the motifs involved in these interactions are not conserved in β-coronaviruses and it is not known if similar interactions contribute to transcription of other sgmRNAs. Therefore, although it is widely assumed that TRS-L interacts with cCS-B, there has been no experimental evidence for direct interactions between TRS-L and TRS-Bs.Coronavirus RNA-dependent RNA synthesis includes two different processes as follows: continuous genome replication that yields multiple copies of genomic RNA (gRNA) and discontinuous transcription of a collection of subgenomic mRNAs (sgRNAs or sgmRNAs), which encode the viral structural and accessory proteins9. One unique feature of the coronavirus is frameshifting in ORF1ab, giving rise to RNA-dependent RNA polymerase (RdRP) and other proteins in ORF1b. The structure of the SARS-CoV frameshift element  was solved by nuclear magnetic resonance (NMR) to be a three-stem pseudoknot10 and was proposed to play key roles in translational control of ORF1b11. The global SARS-CoV-2 RNA structure in cells was also proposed by the genome-wide chemical probing strategies15. Recently, alternative conformations of the FSE were derived from in-cell SHAPE/DMS-MaP seq data15. Although these methods provided valuable insights into cis-acting RNA structures regulating important biological processes of virus life cycle, they could not elucidate long-distance interactions. In addition, short- and long-distance interactions within the SARS-CoV-2 RNA were described using the COMRADES and vRIC-seq methods. Importantly, Ziv et al.16 discovered networks of both gRNA and subgenomic RNA (sgRNA) interactions by applying specific probes to pull down each RNA species in cells, and Cao et al.17 reconstructed structures in virions. However, all of these experiments were performed in a specific stage of the virus life cycle. Thus, there is need to directly compare structures from different stages to investigate their dynamics and functional relevance during the whole life cycle.Functional studies have revealed the importance of RNA secondary structures for viral replication, transcription, and translation18 approach. We provide direct experimental evidence of comprehensive TRS-L interactions with TRS-B regions of sgRNAs, and identify and validate novel sgRNAs by analyzing additional TRS-L interaction peaks. We find multiple alternative interactions mediated by FSE, providing structural basis for ribosome stalling. In addition, we show that both proximal and distal genome RNA–RNA interactions are strengthened, whereas sgRNA-mediated interactions are significantly reduced in virions, suggesting thorough compaction of genome RNA in virions. Interestingly, although TRS-L-mediated interactions including genome cyclization are weakened, interactions between TRS-L and open reading frame ORF S are strengthened in later phase of infection cells and mature virions, which may contribute to rapid transcription of sgRNAs. Our data provides a global landscape of relationships between SARS-CoV-2 RNA structure and key processes of virus life cycle, such as replication, discontinuous transcription, and translation.In this study, to comprehensively map RNA–RNA interactions of SARS-CoV-2 RNA both in cells and in virions, we used a simplified sequencing of psoralen crosslinked, ligated, and selected hybrids (SPLASH)20, we also observed weak chimeric signals in non-ligated samples. Counts of chimeras in ligated and non-ligated samples were correlated, particularly in the C sample , in which cytopathic effect (CPE) was not observed. At a later stage, when 70% of cells underwent CPE, we collected the cell culture supernatant and harvested the mature virus particles (V), and at the same time used freeze–thaw methods to lyse the cells (L) to collect the cell and virus RNA Fig. . The maj 21 Fig. . Interes 21 Fig. . This is23. In our data, all five stems and recently identified stem loops 24 are supported by chimeras , possibly indicating that these stem loops form more alternative structures in cells .The 5′-untranslated regions (UTRs) of coronaviruses contain five evolutionarily conserved stem-loop structures (denoted SL1–SL5), which are essential for genome replication and discontinuous transcription29. All of these structures could be detected in our data  of 3′-UTR. We also evaluated base-pairing scores of this conformation and found BSL and P2 are supported, indicating bona fide alternative structure of a 3′-UTR , indicating that the pseudoknot is weakened in virions. It is possible that conformation of 3′-UTR is complicated and dynamic, with a dominant canonical conformation (conformation A in ref. 32), and alternative conformations, including conformation B in ref. 32 and alternative S2M.The 3′-UTR contains a bulged stem loop (BSL), hypervariable region comprising a conserved octonucleotide sequence and the stem-loop II-like motif (S2M), which are essential for sgRNA synthesis in mouse hepatitis virus (MHV)16. Interestingly, we found additional base pairing at genome cyclization sites  data set from SARS-CoV-2-infected cells3, which we assumed to represent sgRNAs rather than RNA–RNA interactions.Although RNA proximity ligation generates chimeric reads indicative of RNA–RNA interactions, cells also contain many spliced transcripts, or in the case of coronavirus-infected cells, sgRNAs, which resemble chimeras produced by proximity ligation, because they originate from disjoint regions of the genome. To correctly identify RNA–RNA interactions, it is therefore essential to filter chimeras that result from splicing or discontinuous transcription. In previous studies, filtering was performed by mapping reads to a database of known transcripts, and removing reads mapped to known splice junctions21); (2) the junctions between the arms of chimeras were precisely localized in sgRNAs, whereas ligation sites in proximity ligation were variable, due to the random nuclease digestion step used in proximity ligation36; and (3) sgRNA chimeras typically included regions of homology between TRS-L and TRS-B sides of the chimera37, whereas proximity ligation chimeras typically include no such regions36. Adjustment of the maximum gap/overlap setting in our analysis pipeline, hyb, allows detection  or removal  of most sgRNA chimeras in RNA-Seq data, whereas proximity ligation chimeras are detected with both settings (Supplementary Data We identified three characteristics that differentiated most sgRNA chimeras found in RNA-Seq from bona fide RNA–RNA interactions chimeras: 1) sgRNAs were ligated almost exclusively in the 5′–3′ orientation, whereas RNA proximity ligation chimeras can be ligated in both 5′–3′ and 3′–5′ orientation , the alternative arches are formed by FSE region and upstream ~620 nt (arch 2) and ~1.1 kb (arch 3) elements, respectively  < 0.05). These interactions are provided as Supplementary Data Contrary to the relative short-range interactions of most enriched interaction pairs in C samples, more interactions in V samples are long range , we found similar patterns of differential interactions in the comparisons of virions vs. cell (VvsC) and virion vs. lysate (VvsL) . These interactions were weakened in virions, compared to cells or lysates, both for TRS-L-sgRNA long-range interaction and local folding at the 5′-end of ORF1ab. However, the TRS-L-S interaction was stronger in the lysate than in cells  were identified by comparing SARS-CoV-2 to five related coronavirus genomes  RNase III was used to fragment RNA as in PARIS35 and COMRADES25, this treatment makes all the fragmented RNA ends compatible for T4 RNA ligase, and this is supposed to increase efficiency of ligation; (2) T4 Polynucleotide Kinase treatment is omitted; (3) considering low input of RNA amount in virions, we decided to omit all the enrichment steps; and (4) the purified ligation products were directly subjected to a commercial pico-input strand-specific RNA-Seq library construction kit. The major purpose of these modifications is to reduce the complexity of the whole protocol to increase the RNA yield adequate for library construction, so that the protocol is more suitable for low amount of virion RNAs. As a result, we obtained 28.9% chimeric reads in virions and more than 10% chimeras in cells and lysates. The higher chimeric rates in virions might result from highly compact genomeIn this study, we developed a simplified SPLASH protocol based on proximity ligation, to capture RNA–RNA interactions in the SARS-CoV-2 virus. RNA proximity ligation has previously been used to address many questions in models ranging from viruses to animal tissuesOur results provide the first direct evidence that TRS-L regions form long-distance interactions with TRS-B regions. Although it was widely believed that long-range interactions are required for discontinuous transcription, direct experimental evidence for such interactions was lacking. By comparing TRS-L : TRS-B chimeras ligated in 3′–5′ and 5′–3′ orientations, we distinguished two classes of reads, which represented (1) RNA–RNA interactions and (2) sgRNAs.We validated two of the putative sgRNAs by Sanger sequencing. Interestingly, although these novel sgRNAs do not have canonical cCS-B motif upstream of gene body, they both partially overlap with the canonical CS motif. This indicates that TRS-L and at least partial cCS-B base pairing at the negative strand are critical for template switching in discontinuous transcription of these sgRNAs. It would be interesting to further identify novel TRS-L-dependent sgRNAs and check if they play roles in SARS-CoV-2 biology.48. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal was proposed10 to regulate this process and was confirmed in SARS-CoV-238. This structure is supported, as all the stems in the pseudoknot are covered by chimeras  protein or other proteins involved in genome RNA packaging maintains its role as the regulator of the genome structure in infected cells, when the RNA is released. Interestingly, putative recombination breakpoints seem to locate closely to domain boundaries  VeroE6  were purchased from American Type Culture Collection (id: ATCC CRL-1586). VeroE6 cells were cultured in Dulbecco’s modified Eagles medium supplemented with 10% fetal bovine serum at 37 °C in a humidified CO2 incubator.g for 10 min to remove cell pellet. Then the clear supernatant was mixed with equal volume of saturated ammonium sulfate and incubate at 4 °C for 1 h. At the same time, the remaining unshed cells were washed three times by PBS and subjected to crosslinking.Infection experiments were performed under biosafety level 3 conditions. SARS-CoV-2 virus strain Wuhan-Hu-1 was kindly provided by (Wuhan Institute of Virology). Independent biological replicates were performed using 90–120 million cells each. VeroE6 cells were inoculated with SARS-CoV-2 strain Wuhan-Hu-1 at a multiplicity of infection = 0.01 pfu/cell for 24 h. Following inoculation, two flasks of cells were washed three times by phosphate-buffered saline (PBS) and then subjected to crosslinking. The remaining cells were cultured for another 48 h, when CPE was observed in about 70% cells, supernatant was collected and centrifuged at 4 °C 200 × For crosslinking, cells or virus pellet were incubated with 2 mM of EZ-Link Psoralen-PEG3- Biotin (ThermoFisher Scientific) at 37 °C for 10 min in PBS containing 0.01% digitonin. The cells were then spread onto a 10 cm plate and irradiated using 365 nm ultraviolet radiation for 20 min on ice. Cell and virion RNAs were extracted with RNeasy mini kit (Qiagen).The virus inoculation and crosslinking were performed on three independent replicates.Here, 500 ng of RNA was fragmented using RNase III (Ambion) in 20 μl mixture for 10 min at 37 °C and purified using 40 μl of MagicPure RNA Beads (TransGen). Each RNA sample was divided in two: one half was used for proximity ligation and then crosslink reversal . Proximity ligation was done under the following conditions: 200 ng fragmented RNA, 1 unit/μl RNA ligase 1 (New England Biolabs), 1× RNA ligase buffer, 50 mM ATP, 1 unit/μl Superase-in (Invitrogen), final volume: 200 μl. Reactions were incubated for 16 h at 16 °C and were terminated by cleaning with miRNeay kit (Qiagen). Crosslink reversal was done by irradiating the RNA on ice 254 nm UltraViolet C radiation for 5 min using a CL-1000 crosslinker (UVP). For the non-ligated controls, crosslink reversal was done directly after crosslinking, omitting proximity ligation .Sequencing libraries were prepared with 50 ng input RNA material using SMARTer Stranded Total RNA-Seq Kit v2—Pico Input Mammalian , according to the manufacturer’s instructions. The libraries were PCR amplified with 12 cycles using PCR primers in the kit and purified using 0.8× Ampure XP beads. The libraries were paired-end sequenced (PE150) using Illumina Nova seq platform.25. In brief, raw paired-end reads were trimmed for adaptors and checked for quality using cutadapt 50. Chimeric reads were identified and annotated to the respective genome using hyb51. SARS-CoV-2 samples were processed using SARS-CoV-2 sequence (NC_045512.2 [https://www.ncbi.nlm.nih.gov/genome/?term=NC_045512.2]).Sequencing data were collected by Illumina CASAVA 1.8. We obtained an average of about 70 M raw paired-end reads from each replicates of samples. Data preprocessing was performed according to ref. https://github.com/gkudla/hyb)51, using the command:Chimeric reads were called and annotated with the hyb package (default (stringent) parameters:hyb analyse in=sample_R2.fq db=SARS-CoV-2_no_polyA Format=fastq align=bowtie2 eval=0.001Relaxed pipeline:hyb analyse in= sample_R2.fq db=SARS-CoV-2_no_polyA Format=fastq align=bowtie2 eval=0.001 gmax=20The gmax setting represents the maximum gap/overlap between hybrid arms allowed when calling chimeras. Specifically, with the default setting (gmax = 4), hyb would only allow candidate chimeras where the two arms are either adjacent in the read (gap = 0), the mapped portions of the read are separated by a maximum of 4 nt (gap ≤ 4), or the mapped portions of the read overlap by a maximum of 4 nt (overlap ≤ 4). A stringent gmax setting (the default gmax = 4) reduces potential false-positive chimeras at the cost of increasing false-negative chimeras. In particular, we found that the default setting rejects chimeras that result from “spliced” TRS-L–TRS-B junctions, whereas the relaxed setting gmax = 20 allows the recovery of such chimeras.PCR duplicates were collapsed as part of the preprocessing step of the hyb pipeline.We also tested robustness of this pipeline by comparing read2 (sample_R2.fq) with pear merged PE reads:pear -e -j 32 –f sample_R2.fastq.gz -r sample _R1.fastq.gz –o sample.PEARthen detect chimeras using hyb:hyb analyse in= sample.PEAR.assembled.fastq db=SARS-CoV-2_no_polyA Format=fastq align=bowtie2 eval=0.001We found high similarity of contact matrices between the two pipelines (data are available upon request) and use R2 single reads (corresponding to RNA sense strand) hereafter, to make the analysis as simple as possible.25. We then randomly reassigned (shuffled) pairs of fragments found in chimeric reads and repeated the folding energy analysis. The folding energies of experimentally identified and shuffled chimeras were compared by Wilcoxon’s test.To evaluate the folding energy of chimeric reads, we used hybrid-min with default settings as in ref. 52, as previously described25, such that color intensity represents the coverage of chimeric reads at every pair of positions. The first read of each pair is plotted along the X axis and the second read along the Y axis. As a result, chimeras found in the 5′–3′ orientation are shown above the diagonal and chimeras in the 3′–5′ orientation are below the diagonal. Viewpoint histograms and arc plots were plotted with ggplot2 R package53.Virus interaction heatmaps were plotted using Java Treeviewz-scores for all the interaction pairs. The z-scores > 2.13  were considered as enriched interactions. Then, enriched TRS-L interaction were selected if either arm located in 1–100 nt.For TRS-L interaction peak calling, each chimeric read was split and mapped to two paired non-overlapping 10 nt bins; we first scored interactions by log2-transformed chimeric reads, then calculated 25, by comparing counts of chimeric reads in 10 nt × 10 nt bin pairs from ligated and control data sets.The statistical significance of RNA–RNA interactions was calculated using DESeq2 as previously reported in ref. https://github.com/gkudla/comrades).For short-range interactions, we assembled non-zero chimeric groups into uninterrupted stem structures and then fold RNA using COMRADES  command as: comradesMakeConstraints -i Data.hyb -f genome.fasta -b 1 -e 29870. This score is defined as chimeric read count for each base pair. Then the scores were divided by total mapped read counts and log2 transformed to make scores comparable between samples. For a given folded structure, normalized base-pairing scores of each base pair in each sample were compared to non-ligated controls by paired Wilcoxon’s rank-sum test. All the structures depicted in this study show significant (P < 0.05) enrichment of base pairing scores over non-ligated controls.Folded structures were visualized in VARNAv3-93. Paired bases are colored with normalized base-pairing scores as follows: scores were first calculated using COMRADES  square along each bin for calculating insulation score, A 50 nt (10× resolution) window was used for statistics of the delta vector and removed the weak boundaries, for which “boundary strength” < 1.Domain boundaries were identified by insulation scoreThe average insulation scores were normalized by around all domain and their nearby regions (±0.5 domain length). The heatmaps were binned at 10 nt resolution and an 800 nt window. Average insulation scores were plotted around boundaries from 1/2 domain upstream to 1/2 domain downstream.The size of the domain was homogenized to 400 nt, and the upstream and downstream extended 1/2 domain calculated the interaction frequency by the averaging all domain. The resulted matrices were plotted as heatmap by log2 average signals.56 software to model RNA genome in three dimensions. The final results obtained by Multidimensional scaling (MDS) algorithm were used for 3D visualization. For the spatial location of particular gene loci, we used 1 point/20 balls to calculate the position of specific genes in the whole 3D simulation and then modify the pymol results by a python script.We used pastis-0.1.057 and RDP58. Potential recombinant regions among analyzed sequences were identified by sliding a 200 nt window at a 10 nt step across the alignment using the Kimura two-parameter model. The positions of the analyzed sequence regions were based on those in the reference SARS-CoV-2 Wuhan-Hu-1 (MN908947). Regions between breakpoints were identified using a 99% confidence threshold.Potential recombination events and the location of putative breakpoints in coronavirus genomes were detected using Simplot39. A 100 nt bin interaction that displayed more than ±1 log2FC (FDR < 0.01) between C or L and V samples were considered as significantly differential interactions. Then, log2FC heatmaps were plotted in using Java Treeview. The full script to identify differential interactions can be found at github (see below)Differential gene expression analysis was performed using DESeq2RNA from infected non-crosslinked VERO cells  were extracted by miRNeasy Mini Kit (Qiagen). Then, 100 ng of RNA was subjected to retrotranscription using SuperScript VILO MasterMix (ThermoFisher) and cDNA was amplified with 2×Es Taq MasterMix (Cowin Biotech) by 0.4 μM of each primer: 3.9 K (5′-TGTTGTAACTTCTTCAACACAAGC-3′) or 12.3 K (5′-TGTTCAAGGGAACACAACCATC-3′) and TRS-L (5′-CCCAGGTAACAAACCAACCAAC-3′).Statistical analyses for differential interaction was conducted with the R Bioconductor package DESeq2 using three independent replicates as described above.Comparison of quantitative indicators, such as boundary strength, was performed with two-sided Wilcoxon’s rank-sum test. For a given structure, to test whether the base pairing is enriched in ligated samples, or to compare base-pairing dynamics, we performed paired Wilcoxon’s rank-sum test on normalized base-pairing scores.To test whether differential interaction spannings follow the same continuous distribution, two-sided Kolmogorov–Smirnov test was performed.Statistical significance of differences in odds ratios between two groups (Supplementary Fig. Correlation analysis of chimeric reads counts Fig.  and insuAll the statistics tests were performed with R package stats.Further information on research design is available in the Supplementary informationPeer Review FileSupplementary Data1Supplementary Data2Supplementary Data3Description of additional supplementary filesReporting Summary


Nucleotide addition cycle (NAC) is a fundamental process utilized by nucleic acid polymerases when carrying out nucleic acid biosynthesis. An induced-fit mechanism is usually taken by these polymerases upon NTP/dNTP substrate binding, leading to active site closure and formation of a phosphodiester bond. In viral RNA-dependent RNA polymerases, the post-chemistry translocation is stringently controlled by a structurally conserved motif, resulting in asymmetric movement of the template-product duplex. This perspective focuses on viral RdRP NAC and related mechanisms that have not been structurally clarified to date. Firstly, RdRP movement along the template strand in the absence of catalytic events may be relevant to catalytic complex dissociation or proofreading. Secondly, pyrophosphate or non-cognate NTP-mediated cleavage of the product strand 3′-nucleotide can also play a role in reactivating paused or arrested catalytic complexes. Furthermore, non-cognate NTP substrates, including NTP analog inhibitors, can not only alter NAC when being misincorporated, but also impact on subsequent NACs. Complications and challenges related to these topics are also discussed. RNA viruses are a large collection of diverse, rapidly evolving viruses with a wide host range covering bacteria and eukaryotes . In rece1-S4) are usually used to help depict these microsteps (2 to S3) and translocation (S4 to S1) (Thermus aquaticus (Taq) DNA polymerase close the active site through a large-scale conformational change of their fingers domain , and translocation, while four structural reference states (S4 to S1) . In vira4 to S1) . By conts domain . S3 and reaction . Two typreaction . Similarreaction , suggestAlthough numerous NAC-state-related structures from various viral RdRPs have provided a relatively complete structural view of the cognate NTP-driven NAC, several aspects directly or indirectly related to NAC have rarely been addressed structurally in RdRPs including nucleotide addition-free translocation, intrinsic product cleavage activities, and perturbation of NAC by non-cognate NTPs and NTP analogs. As discussed below, these events are possibly related to important events including but not limited to RdRP catalytic complex dissociation, proofreading, reactivation, fidelity control, and effective intervention. In the following sections, a synoptic introduction of these events and structural challenges to approach them are discussed.1 and S4 represent the post- and pre-translocation states, respectively. In a classic NAC, a post-translocation S1 complex directs the binding of the incoming NTP, while the polymerase in the pre-translocation S4 complex needs to translocate to the next register and reopen its active site for the next NAC . In DdRPs, there is a channel to accommodate the single-stranded 3′-portion of the product RNA and transcription factors such as bacterial GreA/GreB and eukaryotic TFIIS can facilitate intrinsic endonuclease activity of DdRP to reactivate transcription . ProofreExcept for recently reported backtracked SARS-CoV-2 RdRP structures obtained using RNA scaffolds with designed mismatches at the 3′-portion of the product RNA , structui) is the byproduct of the NTP/dNTP-driven phosphoryl transfer reaction in NAC. Under certain circumstances , PPi can reverse the reaction through pyrophosphorolysis  reverse transcriptase (RT), a non-cognate NTP can induce the cleavage of the product strand 3′-nucleotide, forming a dinucleotide with a 5–5′ poly-phosphate (tetra- or tri-phosphate) linkage when the incoming cognate dNTP is not available . Similari- and NTP-mediated cleavage activities by RdRP itself may play key roles in maintaining viral genome stability and keeping the virus away from the error-catastrophe threshold , a high-fidelity DNA polymerase  at enzymology and structural levels is a key not only to identify repurposed NA drugs, but also to design new NA drugs (The successful usage  of nucleNA drugs . As an eNA drugs . A delayNA drugs , demonstNA drugs . The nonNA drugs . For exaNA drugs . Unlike To date, structures of RdRP-RNA complexes containing non-cognate NTP-derived mismatches are rarely reported, while related structures depicting intervention mechanisms of representative NAs have been emerging with the fast growing of RNA virus research . NAs witRepresentative NAC-related RdRP-RNA complex structures have been reported in picornaviruses, HCV, influenza viruses, bacteriophage ϕ6, and more recently in bunyaviruses and CoVs . However


Although many of these maturation processes also affect other RNA species, the Tlc1 RNA exploits them in a new combination and, therefore, ultimately follows its own and unique pathway. In this review, we highlight recent new insights in maturation and subcellular shuttling of the budding yeast telomerase RNA and discuss how these events may be fine-tuned by the biochemical characteristics of the varying processing and transport factors as well as the final telomerase components. Finally, we indicate outstanding questions that we feel are important to be addressed for a complete understanding of the telomerase RNA lifecycle and that could have implications for the human telomerase as well.As the limiting component of the budding yeast telomerase, the Tlc1 RNA must undergo multiple consecutive modifications and rigorous quality checks throughout its lifecycle. These steps will ensure that only correctly processed and matured molecules are assembled into telomerase complexes that subsequently act at telomeres. The complex pathway of Tlc1 RNA maturation, involving 5'- and 3'-end processing, stabilisation and assembly with the protein subunits, requires at least one nucleo-cytoplasmic passage. Furthermore, it appears that the pathway is tightly coordinated with the association of various and changing proteins, including the export factor Xpo1, the Mex67/Mtr2 complex, the Kap122 importin, the Sm RiboNucleoProtein (RNP) complex that is a specialised reverse transcriptase. Telomere extension by telomerase involves addition of telomeric repeats onto the 3' end of the G-rich strand , small nucleolar RNAs (snoRNAs) and messenger RNAs (mRNAs). However, ultimately, the Tlc1 RNA follows a unique maturation pathway, and thus, constitutes a separate RNA niche. The core and, in many species, the highly conserved function of the telomerase RNP is to provide the RNA template for reverse transcription. However, different species utilise a wide variety of RNA stabilising mechanisms and maturation pathways   cap at its 5'-end  that in yeast is comprised of Sto1 and Cbc2  complex, comprised of THO subunits , Yra1 and Sub2, associates with mRNAs in a CBC-dependent manner  connects Xpo1(CRM1) with telomerase RNA and other RNA types  were not sensitive to a loss of Mex67/Mtr2 function anymore , would lead to subsequent degradation  , in which after a temperature shift virtually no new Tlc1 molecules are generated, the nuclear vs cytoplasmic distribution of the stable pre-existing Tlc1 molecules remains normal. Therefore, these molecules appear to continuously shuttle in and out of the nucleus even in the absence of a functional Mex67 protein  molecules require Mex67/Mtr2, Xpo1 (CRM1), Los1 and Msn5 for the first round of nuclear export, while only requiring Mex67/Mtr2 and Xpo1 (CRM1), but neither Los1 or Msn5 for a subsequent second re-export , just distal to the Est1 binding site Fig. . These pThe yKu heterodimer, comprised of yKu70 and yKu80, originally was discovered as the yeast homologue of human proteins associated with DNA end-binding in preparation for non-homologous end joining, a form of DNA double-strand break repair  units are required for nuclear import  likely causes a requirement for one or more NLSs for it to pass through the nuclear pore. These NLS sequences could be located on Est, yKu and/or Pop proteins, which, thus, would help to drive nuclear re-entry of the telomerase RNP . This would cause a major change in the biochemical properties of the RNP and exposed NLSs could strongly favour nuclear re-entry of the RNP (Fig. Taken together, we speculate that the changing telomerase RNP composition could be a major reason for its biophysical behaviour in vivo. On the freshly made RNA, the assembled protein complexes may strongly favour its export to the cytoplasm, presumably mostly driven by the CBC, the TREX-1 and the Mex67/Mtr2 proteins (Fig. The budding yeast telomerase RNA, Tlc1, is a large non-coding RNA that shares certain maturation and transport processes with snRNAs and mRNAs, but ultimately follows its own unique pathway. Despite the currently available information on Tlc1 transcription, modification and shuttling, many questions remain in regard to the full telomerase RNP lifecycle. What is the precise role of Mex67/Mtr2 on Tlc1 RNA after transcription? One possibility is that this complex acts as a chaperone and export adaptor, associating with newly transcribed Tlc1 and channelling it for Xpo1-mediated export, thereby preventing its degradation by the nuclear exosome (Vasianovich et al.


O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C’/D’ motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2’-O-methylation enzymes.Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2’-OH of RNA. The central ‘guide RNA’ has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C’/D’ motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C’/D’ motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2’- Ribosomal RNA (rRNA) is methylated at the 2’-OH position (2’-O-methylation) in all kingdoms of life7; this modification ensures correct rRNA folding, structural and thermodynamic stability, translational efficiency and proof-reading11. In humans, deregulation of 2’-O-methylation is involved in several diseases, including cancer and neuropathologies13.Chemical modifications of RNA base or ribose diversify the structures and functions of RNAO-Me modifications are well defined in rRNAs and those located in functional regions of the ribosome are highly conserved from bacteria to eukaryotes17. Ribonucleoprotein (RNP) complexes called Box C/D RNPs methylate 2’-OH groups during ribosome biogenesis21. These RNPs assemble around a small nucleolar (sno) ‘guide’ RNA that recognizes individual substrates by base complementarity.The positions of the 2’-22. The k-turn structure takes its name from the characteristic 50° kink of the RNA backbone ; it is formed by a box C sequence motif (5’-RUGAUGA) at the 5’ end of the guide RNA and a box D sequence motif (5’-CUGA) at the 3’ end  or a loop27. These structures provide a second binding site for L7Ae, the archaeal orthologue of Snu13. In eukaryotes, however, the predicted box C’/D’ motifs diverge from the consensus sequence and often lack one of the two sheared G•A base-pairs. They are thought to fold in a k-turn structure, as in archaea; however, the evidence for this is not conclusive and their ability to recruit a second copy of Snu13 to snoRNA is controversial29.Guide RNAs form a kink-turn (k-turn) three-dimensional structure that binds the Snu13  protein28, the C-terminal domain of Nop58 was found to bind to the composite surface formed by the RNA box C/D motif and Snu13 , whereas the C-terminal domain of Nop56 was found near the predicted box C’/D’ motif. The N-terminal domains of the eukaryotic Nop56–Nop58 heterodimer bind the methyltransferase Nop1 in yeast (fibrillarin in humans) and recruit it to the RNP .The methyltransferase Nop1 (or fibrillarin) is brought to the Snu13–snoRNA complex by the two scaffolding proteins Nop56 and Nop58, which form a heterodimer through their coiled-coil domains. In cross-linking studies34. In archaea, the homodimer Nop52 substitutes Nop56–Nop58; each Nop5 monomer recognizes one k-turn or k-loop L7Ae–RNA site, with the coiled-coil dimerization domains running roughly along the axis of the guide RNA . The two fibrillarin copies, recruited by the Nop5 N-terminal domains, transfer a methyl group to the ribose 2’-OH of two substrate RNAs, each base-paired with one of the two guide-RNA sequences upstream of box D and of box D’. The methyl group transfer is selective and occurs to the fifth nucleotide upstream of both box D and box D’. The substrate-loaded archaeal Box C/D RNP may adopt a monomeric or dimeric form, depending on the guide RNA sequence 35. In both cases, the restraint imposed on the RNA geometry by the length of the coiled-coil domains, which run between the L7Ae-bound box C/D and box C’/D’ motifs, specifies the methylation position on the substrate RNAs.Unfortunately, all attempts to reconstitute a methylation-competent and homogeneous eukaryotic Box C/D RNP in vitro have failed, precluding structural studies. Instead, there are numerous structures of archaeal Box C/D RNPs36. In this structure, the U3 snoRNP is symmetric and features two identical protein assemblies at the box C’/D (corresponding to box C/D) and box B/C  sites. However, the U3 snoRNP does not function as a methyltransferase, but acts as a chaperone to aid rRNA folding38.Because of the lack of a three-dimensional structure of a eukaryotic, methylation-competent Box C/D enzyme, the archaeal monomeric Box C/D RNP is generally used as a proxy of the eukaryotic enzyme. The assumption that eukaryotic and archaeal methylation machineries have similar architectures is reinforced by the electron microscopy structure of the eukaryotic U3 snoRNP bound to the pre-ribosomal 90S subunitPyrococcus furiosus Nop5 and yeast Nop56/Nop58 C-terminal domains), and because the interaction surfaces between the Nop56/Nop58 C-terminal domains and both the RNA kink-turn and Snu13 in the U3 snoRNP structure are similar to those of archaeal Box C/D complexes, eukaryotic Nop56/Nop58 C-terminal domains should also be unable to bind box C/D motifs in the absence of Snu1339. Thus, if eukaryotic box C’/D’ motifs do not bind Snu13, it remains unclear how the C-terminal domain of Nop56 recognizes the RNA at this site and whether the eukaryotic methylation machinery adopts the symmetric architecture seen in archaeal Box C/D complexes and in the eukaryotic U3 snoRNP.In archaea, the C-terminal domain of Nop5 is incapable of binding box C/D motifs in the absence of the primary binding protein L7Ae. Because of the high sequence similarity between archaeal and eukaryotic Nop5 and Nop56/58 C-terminal domains  snoRNAs, snR41, snR51 and snR54, and tested their ability to bind the L7Ae protein of archaea and the Snu13 protein of eukaryotes, which specifically recognize k-turn structures. We annotated the box C’ and D’ sequences of these three snoRNAs manually, as described in Methods. The box D’ sequences corresponded to those annotated in the yeast snoRNA database UMass-Amherst41. The box C’ sequences of snR51 and snR54 corresponded to those annotated in26. The box C’ sequence annotated for snR41 (UACAUGU), differed by three nucleotides from that annotated in26 (AUGUGCA), because our choice maximized the number of base-pairs in stem II matching those of a canonical box C’/D’ motif. We used the sequences from snR51, snR41 and snR54 predicted to form box C’/D’ motifs and their flanking structural elements to generate three RNAs: snR51-kl1, snR41-kl1, and snR54-kl1 47 using 5’-Cy5 fluorescently labeled RNAs.Of the 43 known snoRNAs in yeast, only two have box C’/D’ motifs matching the canonical sequencesD of the complex is at least one order of magnitude smaller than the total RNA concentration, corresponding to a KD ≤ 100–200 nM). According to its annotation, the snR51-kl1 box C’/D’ motif contains the 1n−1b A•G base-pair of the consensus sequence, as well as base-pairs –1 and –2 of stem I. By contrast, the predicted snR41-kl1 box C’/D’ motif lacks the 1n−1b A•G base-pair, and snR54-kl1 lacks both base-pair –1 of stem I and the 2n−2b G•A base-pair of stem II. The positive control sR26-kl has the consensus box C/D sequence but lacks stem I.Of the three putative box C’/D’ motifs tested, only snR51-kl1 bound to L7Ae  of the complex, leading to smearing of the gel bands. On the other hand, snR51-kl4, with the 2n−2b G•A base-pair but without the −1 base-pair, bound to L7Ae similarly to snR51-kl1.The results of Fig. D at least one order of magnitude higher than the total RNA concentration (corresponding to a KD ≥  ~ 10 μM). Weak binding with band smearing was seen for the snR51-kl2 mutant containing the 2n−2b G•A base-pair  or do so with a very weak affinity (KD ~ 100 μM). As a control Snu13 bound canonical k-turn sequences at a ratio of the total concentrations of RNA and protein of 1:1 .Finally, we repeated a subset of EMSAs using a 50 times higher RNA concentration of non-fluorescently labelled RNA  and visualized the RNA by staining with ethidium bromide. Also in this case, snR51-kl2 was the only RNA that showed binding to Snu13, confirming that all other RNAs either do not bind Snu13 . In our structure the stretch 1GUAC4 of one RNA forms base-pairs with the same stretch of the neighboring RNA molecule.The 2n−2b G•A base-pair of stem II, whose absence abolishes binding to Snu13 but not to L7Ae, is conserved in archaeal guide RNA. Thus, to understand whether non-canonical box C’/D’ motifs lacking the 2n−2b G•A base-pair adopt a k-turn-like structure in the presence of L7Ae, we set out to solve the structure of the L7Ae–snR51-kl1 complex. We obtained crystals for L7Ae in complex with a snR51-kl1 mutant, termed snR51-kl1-S, in which stem I was shortened to eight base-pairs and determined the X-ray crystallographic structure at a resolution of 1.9 Å  and the N6 of the A originally annotated as 5b stacked on top of A(1n) . In general, binding to RNAs without a stable stem I was promoted by an increase in the hydrophobicity of loop 9. By contrast, introducing a negative charge, as in Snu13 S94E, lowered the binding affinity  . A second peak related to a dimeric RNP . This result demonstrates that the presence of Nop52–Fib2 does not promote binding of a second copy of Snu13 to the non-canonical box C’/D’ motif of snR51.We then analyzed the particles assembled with snR51 and sR26 by size-exclusion chromatography and multiple angle light scattering (MALS). The RNP assembled with snR51 Fig. C resulte2–Fib2 and Snu13  rather than only one copy of Snu13 . This data suggests that the presence of Nop52–Fib2 promotes binding of Snu13 to canonical k-loop structures, as that formed by sR26.A similar elution profile was obtained for the complex assembled with the archaeal sR26 RNA, Nop5u13 Fig. B, with t29; on the other hand, it is unable to bind the non-canonical box C’/D’ motif of Xenopus U25 snoRNA28.These findings may explain conflicting literature data on the ability of Snu13 to bind snoRNA box C’/D’ motifs: on the one hand, Snu13 associates with the box C’/D’ motif of human U24 because in this RNA the box C’/D’ motif forms a canonical k-loopThe archaeal Box C/D RNPs are often used as a proxy for eukaryotic Box C/D RNPs, but the similarities and differences between them remain unclear. One major question is whether the predicted, internal, non-canonical box C’/D’ motifs in eukaryotic snoRNAs bind the box C/D motif-binding protein Snu13, leading to a similar architecture of the box C/D and box C’/D’ protein-assembly sites. In this study, we revisited the question of the existence of box C’/D’ motifs in yeast snoRNAs by defining a functional box C’/D’ motif as two juxtaposed sequences capable of base-pairing  and the remaining 17 lack one of the two sheared base-pairs . According to our systematic mutational analysis, Snu13 binding to the box C’/D’ motif requires the presence of both an intact stem I and the tandem A•G sheared base-pairs. Thus, we predict that only snR60 and snR70, and possibly snR58, snR65, snR66, snR69, snR71 and U24, recruit Snu13 to their putative box C’/D’ motif. In humans none of the 32 snoRNAs homologous to yeast sRNAs contains a canonical box C’/D’ and stem I seems to be absent in most cases. Thus, functional box C’/D’ motifs are by no means universal in eukaryotic snoRNAs50.Among the 43 yeast snoRNAs, only two (snR60 and snR70) contain box C’/D’ motifs that match the consensus sequence; six others  contain both the A•G and G•A sheared base-pairs of stem II and the first base-pair of stem IO-methylation complexes depend on this bipartite, symmetrical architecture. We show here that a similar bipartite architecture is unlikely to exist in the 2’-O-methylation complexes of eukaryotes, which have a number of asymmetric features. It follows that archaeal Box C/D RNPs are not a satisfactory proxy for eukaryotic Box C/D RNPs in all their functional and regulation aspects.In archaea, the Nop5 dimer directs the methyltransferase, fibrillarin, to the methylation sites by anchoring its C-terminal domains to the box C/D and C’/D’ motifs of the guide RNA, thereby orienting the RNA guide sequences along its coiled-coil domains . The activity and specificity of archaeal 2’-The prediction that no more than 8 of the 43 yeast methylation guide RNAs contain a functional box C’/D’ motif calls into question their role in binding Nop56 and indicates that a RNA three-dimensional structure different from both the k-turn and the k-loop might recognize this protein. Based on the observation that box C’/D’ motifs are not universal in eukaryotic snoRNAs, we propose that the interaction of Nop56 with the guide RNA does not depend on a box C’/D’ motif but on a yet-unknown RNA motif or structure, which has evolved exclusively in eukaryotes from the box D’ sequence and may partially overlap with it. In this scenario, the box C/D sequence would have a leading role in initiating complex assembly by the recruitment of Snu13 and Nop58, followed by a chaperone-aided dimerization of Nop58 with Nop56. Only at this point Nop56, readily recruited to the complex, would be able to recognize the RNA next to the box D’ site in a yet-unknown manner. It is also possible that the C-terminal domain of Nop56 is not involved in RNA binding and that the guide-substrate RNA duplex is recognized in the right register by the Nop56-N-terminal-domain-fibrillarin complex with the help of transiently-associated chaperones.29, mutation or depletion of the box C/D motif abolished methylation at both sites upstream of box D and box D’. By contrast, depletion of box D’ (or box C’) affected methylation only upstream of box D’29, indicating that the box C’/D’ motif is required neither for the assembly of a functional complex nor for methylation specificity at the site upstream of box D. The sequence of the engineered snoRNA used in this study was derived from human U24, which has a canonical box C’/D’ k-loop sequence and was shown to recruit two copies of Snu13. The fact that even in this case the conserved box C’/D’ element is unable to nucleate the assembly of the methylation complex indicates that in vivo the snoRNP does not adopt a symmetric bipartite structure like the archaea Box C/D RNPs. These findings and our own data together strongly suggest that the architecture of Box C/D snoRNPs at the box C/D and (putative) box C’/D’ sites differ from each other. This conclusion is not in the disagreement with the symmetric architecture of the U3 snoRNP bound to the pre-ribosomal 90S subunit36, as the U3 snoRNP does not function as a methyltransferase and the U3 RNA has two canonical box C/D sequences, unlike most of the methylation competent snoRNAs. The apparent existence of different assembly modes for Snu13, Nop56 and Nop58 on Box C/D snoRNAs demonstrates the versatility of the eukaryotic snoRNP machinery: the sequence of the snoRNA determines the assembly mode at the Nop56 site to support different functions.The role of the box C/D element in initiating the assembly of snoRNPs is supported by functional data in vivo. In a study of the effect of mutation or depletion of various secondary structure elements in an engineered guide snoRNA on rRNA methylation in yeast51. In eukaryotes, the picture is less clear cut: methylation of the substrate upstream of box D is (moderately) sensitive to alterations of the spacer/guide length between box C’ and box D, whereas methylation of the substrate upstream of box D’ is sensitive to alterations of both spacer/guide sequence lengths29.The idea that the symmetric bipartite architecture seen in archaeal Box C/D RNPs does not exist in methylation-competent eukaryotic snoRNPs is further supported by the biochemical and in vivo evidence of the importance of the spacer/guide sequences between box C and box D’ or box C’ and box D in archaea and eukaryotes. In archaea, the optimal spacer/guide length is 12 nucleotides; alteration of the length of one of the two spacer/guides impacts the methylation only of the corresponding substrate, in agreement with similar architectures at the box C/D and box C’/D’ sitesIn summary, we show that a functional box C’/D’ element does not exist in most yeast guide RNAs, leading us to conclude that this RNA element is not the specific recognition motif for Nop56. We propose that eukaryotic, methylation-competent RNPs have a non-symmetric architecture with different protein–RNA contacts and assembly geometries at the box C/D and putative box C’/D’ sites.The asymmetric nature of the eukaryotic complex may result into different mechanisms for the regulation of methylation levels at the substrates upstream of box D and box D’ and thus into a higher flexibility for the coupling of site-specific methylation to other cellular processes.P. furiosus  were obtained by PCR from genomic P. furiosus (Pf) DNA. The genes were cloned into expression vector pET-M11 containing a TEV (tobacco etch virus) protease-cleavable N-terminal His6-Tag using BamHI and NcoI restriction sites31. The full-length SNU13 gene from S. cerevisiae (UniProtKB accession code P39990) was ordered from Invitrogen with codon-usage optimized for E. coli translation. The gene was amplified via PCR and cleaved with NcoI and XhoI restriction enzymes . Cleaved PCR products were ligated into the cleaved pET-M11 expression vector. The final Snu13 construct contained an N-terminal His6-Tag cleavable with TEV-protease.Genes encoding full-length L7Ae, Nop5, and fibrillarin in E. coli OmniMax cells. Positive mutants were verified by sequencing (Eurofins).Snu13 point mutations were accomplished using the Pfu Plus! DNA Polymerase (Roboklon) according to the protocol provided by the manufacturer. PCR products were cleared from the starting material by DpnI (NEB) digest; the enzyme was heat-inactivated before transformation of the cleared PCR products into S. cerevisiae (Sc) guide RNAs snR51 (Gene-ID: 9.164.983), snR41 (Gene-ID: 9.164.986), and snR54 (Gene-ID: 9.164.960) were ordered as synthetic genes in cloning vector pUC57 from GENEWIZ (Sigma-Aldrich). All templates contained a 3’ PstI cleavage site for DNA linearization. For all other RNA constructs, the template DNA was ordered as single-stranded DNA with EcoRI (5’ GAATTC) and HindIII (5’ AAGCTT) cleavage sites at the 5’- and 3’-end, respectively, as well as a PstI cleavage site upstream of the HindII site. Complementary single-stranded DNA molecules were annealed, cleaved with EcoRI-HF, and HindII-HF (NEB), and purified with the QIAquick PCR purification kit (Qiagen). The inserts were ligated into a cleaved pUC19 cloning vector using T4 DNA ligase (NEB). Correct insertion was verified by sequencing (Eurofins).Full-length DNA templates for 6-tagged L7Ae, Nop5, and fibrillarin (Fib) were expressed in E. coli BL21(DE3). Cells were grown in LB Medium at 37 °C until an OD600 of 0.6–0.8, and expression was induced at 20 °C with 1 mM final concentration of IPTG (Carl Roth). Cells were harvested 18–20 h after induction by centrifugation at 4500 rpm and 4 °C.His6-Tag was removed by overnight cleavage with TEV-protease (produced in-house) at room temperature. The reaction mixture was loaded on a 5 ml HisTrap FF column, which retained the TEV-protease and the cleaved His6-Tag, while the cleaved protein was collected with the flow-through.The cell pellet was resuspended in lysis buffer A . The transformed cells were grown in LB medium at 37 °C until an OD600 of 0.6–0.8, and expression was induced at 16 °C with 1 mM final concentration of IPTG. Cells were harvested 18–20 h after induction by centrifugation at 4500 rpm and 4 °C. The cells were then resuspended in lysis buffer D .41. All snoRNAs considered here have experimentally verified methylation sites52. Box D and D’ motifs were manually annotated using the consensus motif 5’-CUGA and had to start five nucleotides downstream of the canonical base-pair formed between the snoRNA and the target nucleotide in the rRNA. Our annotation was identical to that of the yeast snoRNA database UMass-Amherst41. Box C was annotated using the consensus sequence 5’-RUGAUGA and had to start within 5 nucleotides upstream of the 5’ end. Box C’ was annotated manually using the consensus sequence 5’-RUGAUGA and had to be positioned between the box D’ motif and the guide sequence upstream of the box D motif. Our annotation corresponded to that reported in26 for all snoRNAs but snR41. For snR41 we chose the sequence 5’-UACAUGU instead of 5’-AUGUGCA as in26, because it yielded a stem II that was more similar to that of a genuine box C’/D’ motif than the sequence chosen in26.Yeast snoRNA sequences were obtained from the yeast snoRNA database UMass-AmherstE. coli Top10; transformed cells were grown in LB medium overnight at 37 °C and harvested by centrifugation at 4500 rpm and 4 °C. Plasmids were extracted using the Qiagen Plasmid Mega Kit (Qiagen) and cleaved with PstI-HF (NEB). Linearized plasmid DNA was purified by phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol (Carl Roth) extraction and concentrated by precipitation with pure ethanol and NaCl.All RNAs used for crystallization and the guide RNAs used for activity assays were produced by in vitro transcription, using T7 RNA polymerase produced in-house. Plasmids containing DNA templates were transformed into 2 and T7 polymerase were optimized to maximize the yield. Large-scale transcription reactions were run for five hours at 37 °C. All RNAs were purified using preparative, denaturing polyacrylamide gels containing 8 M urea. Purity was verified using analytical denaturing polyacrylamide gels.For the transcription of each RNA construct, the concentrations of DNA, nucleoside triphosphates , MgClAll RNAs used for the electrophoretic mobility shift assays (EMSA) were purchased from Integrated DNA Technologies (IDT) with a 5’-Cy5 label for fluorescence detection. Three nucleotides were added at the 5’ end as a spacer between the Cy5 label and the desired RNA sequence (Supplementary Table 1). To ensure that the spacer nucleotides did not interfere with the RNA structure, a subset of binding assays were repeated with non-labelled RNAs lacking the spacer nucleotides and analyzed using ethidium bromide. The results were equivalent in all cases.53.In the fluorescence-detected binding assays, 10 pmol of RNA were mixed with sterile LC–MS grade water (Merck) and annealing buffer  in a total volume of 5 µl and annealed by heating to 80 °C and slow cooling to 4 °C in a T100 Thermocycler (Bio-Rad). After annealing, 0, 2.5, 5, 10 and 20 pmol of protein were added and incubated for up to 30 min at 4 °C. Afterwards, 5 × native loading dye  was added to each sample. All samples were analyzed on 10% native polyacrylamide gel at 4 °C. Each gel was pre-run for 0.5–1 h before sample loading. After sample loading, gels were run overnight at 4 °C and 10 mA. Gels were analyzed using a Typhoon Trio system  with a 670 nm-bandpass (670 BP 30) emission filter for Cy5 detection. Intensities were extracted and analyzed using FijiA subset of EMSAs  was repeated with non-labeled RNAs, lacking the spacer nucleotides between the RNA and the dye at the 5' end. In these assays, 0.5 nmol of non-labeled RNA were mixed with pure and sterile LC–MS grade water (Merck) and annealing buffer  in a total volume of 5 μl and annealed by heating to 80 °C for 1 min and slow cooling to 4 °C in a T100 Thermo Cycler (Bio-Rad). After annealing, 0, 0.125, 0.25, 0.5 and 1 nmol of protein (L7Ae or Snu13) were added, and the mixture was incubated for 30 min at 4 °C. All samples were further were analyzed on 10% native polyacrylamide gel at 4 °C to prevent diffusion and degradation of the protein and the RNA. Gels were stained with ethidium bromide, and the RNA was visualized using a Gel Doc XR + gel documentation system (Bio-Rad).Purified archaeal L7Ae and snR51-kl1-S RNA were mixed in a 1:1 ratio, incubated for 15 min at 80 °C and slowly cooled down to room temperature. The protein–RNA complex was purified in crystallization buffer  using a HiLoad 16/600 Superdex 75 pg column (Cytiva) on an Äkta Pure system. The purified complex was concentrated using Amicon Ultra-15 3 K centrifugal filters (Merck).2, 0.1 M sodium acetate, 30% 2-Methyl-2,4-pentandiol (MPD) (G12 from Qiagen JCSG Core I Suite). Cryo-protection was achieved by the addition of 10% -2,3-butanediol before flash-freezing.A concentrated solution of ~ 10 mg/ml of L7Ae–snR51-kl1-S was used for crystallization by sitting drop vapor diffusion. Initial crystallization screens were set up with a Crystal Phoenix crystallization robot (Art Robbins Instruments) using NeXtal DWBlock Suites (Qiagen); JCSG Core I Suite, JCSG Core II Suite, JCSG Core II Suite, JCSG Core IV Suite, Nucleix Suite, PEG Suite, and PEG II Suite. The drop solution was equilibrated against 200 µl of reservoir solution at 18 °C. Crystals appeared after one week in several conditions across all initial screens. The best crystal was obtained in 0.02 M CaCl54. The dataset yielding the structure was recorded at 100 K and a wavelength of 1 Å and processed using the AutoPROC toolbox 55 executing XDS56, Pointless57, Aimless58 from the CCP4 program suite59. The high-resolution cut off was determined using a signal/noise ratio (I/σ(I)) of 2.0.Data were collected at beamline P11 of the PETRA III storage ring, DESY 60 from the Phenix software package61. L7Ae from P. furiosus  was identified as a suitable search model for molecular replacement by executing Balbes62 from the CCP4 program suite59. The molecular replacement solution containing only the protein component was improved by executing the AutoBuild tool63 from the Phenix software package61. The missing RNA component was then built by the AutoBuild tool around the fixed model of the improved protein component. The crystal structure of the protein-RNA complex was further improved and finalized by iterative cycles of model building in Coot64 and refinement in Phenix.refine65. Data collection and refinement statistics are summarized in Table The number of molecules in the asymmetric unit was determined using Xtriage31. The Nop52–Fib2 complex was purified by size-exclusion chromatography. The guide RNA (Supplementary Table 2) was annealed in annealing buffer  at 90 °C for 2 min followed by snap-cooling. Nop52–Fib2, Snu13, and the guide RNA were mixed in a 1:2:1 ratio and incubated for 15 min before purification via size-exclusion chromatography.All RNP complexes were assembled in complex buffer  using a Superdex S200 Increase 10/300 GL (Cytiva) on an Äkta Pure system. Nop5 and fibrillarin were mixed in a 1:1.1 ratio, incubated for 15 min at 80 °C and cooled to room temperatureMulti-angle-light scattering (MALS) data were collected using an on-line SEC-MALS set-up, consisting of a Superdex S200 Increase 10/300 GL column on an Åkta pure system coupled to a MALS miniDawn TREOS system and Optilab T-rex refractive index detector (Wyatt Technologies). Data were analyzed using the ASTRA 7.0 software package (Wyatt Technologies).Supplementary Information.


Chromosome instability is a hallmark of cancer and is caused by inaccurate segregation of chromosomes. One cellular structure used to avoid this fate is the kinetochore, which binds to the centromere on the chromosome. Human centromeres are poorly understood, since sequencing and analyzing repeated alpha-satellite DNA regions, which can span a few megabases at the centromere, are particularly difficult. However, recent analyses revealed that these regions are actively transcribed and that transcription levels are tightly regulated, unveiling a possible role of RNA at the centromere. In this short review, we focus on the recent discovery of the function of human centromeric RNA in the regulation and structure of the centromere, and discuss the consequences of dysregulation of centromeric RNA in cancer. S. cerevisiae) nucleosomes interspersed with classical Histone 3 nucleosomes  allowing the activation of Aurora B . The train vitro with ∝ satellite RNAs through an RNA binding motif  . TogetheIn pancreatic and other epithelial human cancers, overexpression of α-satellite transcripts (cenRNA)  is correThus, detection of cenRNA might be a good predictor of cancer prognosis. In support of this concept, detection of pericentromeric SatII RNA, satellite RNAs present at specific heterochromatin located at the edges of the centromere, in biopsies is more tightly correlated with detection than the standard method . SensingCentromeres are essential for chromosome segregation, and while this function is preserved across species, their structure and mechanisms underlying presents large differences. However, one common particularity is that the centromere DNA sequence is actively transcribed. The resulting RNA is then processed to result in RNAi in yeast and in mammals or cenRNA in mammals. These cenRNAs have been shown to be an indispensable part of the centromere by interacting with CENP-A and CENP-C, as well as actively recruiting other parts of the kinetochore. The dysregulation of the cenRNA expression leads to chromosome instability, which can result in some cases in metastasis of a cancer. One possible treatment to counter this effect is the injection of a synthetic CDN that activates the cGas-STING pathway, able to detect the presence of cytosolic DNA resulting from CIN and activating an immune-response. One point to further investigate is with which mechanism higher eukaryotes can finely tune the expression of cenRNAs, and if a drug can avoid the overexpression of cenRNA in cancer cells.SL wrote the manuscript. KK directed how to write the manuscript and edited it. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diagnosis of peritoneal metastasis in gastric cancer (GC) is essential for determining appropriate therapeutic strategies and avoiding non-essential laparotomy or gastrectomy. Recently, a variety of activatable fluorescence probes that can detect enzyme activities have been developed for cancer imaging. The aim of this study was to identify the key enzyme involved in peritoneal metastasis in GC. The enzymatic activity of gamma-glutamyl transpeptidase, dipeptidyl peptidase IV, and β-galactosidase  was assessed in lysates prepared from preserved human GC (n = 89) and normal peritoneal  samples. β-Gal activity was significantly higher in the human GC samples than in NP samples, whereas no differences were observed in the activities of the other enzymes. Therefore, we used SPiDER-βGal, a fluorescent probe that can be activated by β-Gal, for imaging GC cell lines, peritoneal metastasis in a mouse model, and fresh human resected GC samples (n = 13). All cell lines showed fluorescence after applying SPiDER-βGal, and metastatic nodules in the mice gradually developed high fluorescence that could be visualized with SPiDER-βGal. The human GC samples showed significantly higher fluorescence than NP samples. β-Gal is a useful target enzyme for fluorescence imaging of peritoneal metastasis in GC. Peritoneal metastasis is considered to be non-curable2, and gastrectomy for advanced GC patients with peritoneal metastasis did not show a survival benefit in phase III randomized controlled trials3. Thus, an accurate diagnosis of peritoneal metastasis is essential for determining the appropriate therapeutic strategies and avoiding laparotomy or gastrectomy whenever possible.Gastric cancer (GC) is one of the most common types of cancer worldwide6. However, the probability of obtaining false-negative results with staging laparoscopy is approximately 10.6–17.2%9. Although false-negative cases are clinically important, cases with tiny nodules, nodules inside of the bursa, or nodules where the shape of the metastasis is not formed may be missed completely.Staging laparoscopy is more useful for the diagnosis of peritoneal metastasis compared to computed tomography (CT) with a sensitivity of 64–94% and 33%, respectively12. However, both 5-ALA and ICG need to be administered prior to surgery. Moreover, a variety of activatable fluorescence probes targeting cancer-associated enzymes have been developed15. These probes are originally nonfluorescent; however, after an enzymatic reaction, they become highly fluorescent within a few minutes. Thus, malignancy can be rapidly detected in cancer samples simply by topically spraying the activatable probes targeting the cancer-associated enzymes. For example, gGlu-HMRG, a gamma-glutamyl transpeptidase (GGT)-activatable fluorescent probe, can detect breast cancer16, lung cancer17, hepatocellular carcinoma18, and lymph node metastasis of colorectal cancer19. Furthermore, a dipeptidyl peptidase IV (DPPIV)-activatable fluorescence probe can detect esophageal cancer14. Moreover, SPiDER-βGal, which is activated by β-galactosidase , has been tested on ovarian cancer cell lines21. However, it is important to select specific target enzymes for each cancer type according to its upregulated expression in the cancer tissue compared with that in the surrounding normal tissue to achieve specific fluorescence imaging.In recent years, various fluorescence techniques have been developed to improve the diagnostic results for peritoneal metastasis. For example, photodynamic diagnosis using 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) is useful in detecting peritoneal metastasisSince there is no known target enzyme, fluorescence imaging of peritoneal metastasis in GC by activatable fluorescence probes has not been reported to date. Thus, the aim of the present study was to find a suitable target enzyme and a compatible enzyme-activatable fluorescence probe to detect peritoneal metastasis in GC.p < 0.001) in pH 5.0  and NP (n = 20) samples Fig. a. The enThere was no significant difference in the β-Gal activity according to various clinicopathological characteristics of the patients Table .Table 1βNext, we planned a fluorescence imaging study using GC cell lines and SPiDER-βGal. SPiDER-βGal is an activatable fluorescence probe that is originally nonfluorescent. However, it emits high fluorescence after cleavage by β-Gal.First, we prepared lysates of six GC cell lines, MKN28, MKN45, MKN74, Kato3, HGC27, and NUGC4, and mouse NP prior to conducting live cell imaging and imaging of a peritoneal metastasis mouse model. We assessed the β-Gal activity of these GC cell lines, and NP was used as a control  are shown in Fig.  and 7 arWe evaluated a total of 13 cases (including the above 2 cases). The patient characteristics of these 13 cases are listed in Table Here, we revealed that the β-Gal activity in GC is significantly higher than that of the NP, suggesting the potential of β-Gal to serve as a target enzyme for the development of an activatable fluorescence probe for imaging peritoneal metastasis in GC. We confirmed that SPiDER-βGal is useful for sample imaging of GC using fluorescence imaging of GC cell lines, a mouse model of peritoneal metastasis, and fresh human GC samples.21. However, there are no reports on the use of such probes for the detection of peritoneal metastasis in GC. To apply an activatable fluorescence probe for cancer imaging, a target enzyme that is specifically overexpressed in each cancer needs to be identified. In this study, we investigated potential candidate enzymes for detecting peritoneal metastasis by assaying the enzymatic activity of lysates prepared from preserved clinical samples.Activatable fluorescence probes are useful for detecting various cancers24; however, there are few reports in GC. In this study, the theoretical reason why β-Gal activity in GC is significantly higher than that of NP was not determined. β-Gal is known as a senescence marker25, and SPiDER-βGal is also used for detecting senescence-associated β-Gal 26. Recently, the association between cellular senescence and cancer has been reported27. Hence, we suggest that SA-β-Gal might be over-expressed in GC cells which enables their detection by SPiDER-βGal.β-Gal activity has been reported to be upregulated in some malignancies15. However, since the resected samples did not have a blood supply, the pH of the cells and their organelles in these samples was unclear. In the lysate assay, we assessed the β-Gal activity at not only pH 5.0 but also at pH 7.4. In both acidic and neutral environments, the β-Gal activity of GC was significantly higher than that of NP. Thus, the difference between the tumor and NP was significant regardless of the pH of the samples. This was further validated in the imaging of clinical samples using SPiDER-βGal.β-Gal is a lysosomal enzyme; thus, the assessment of the β-Gal activity of the lysate was considered to be appropriate at pH 5.0We imaged 12 clinical samples of the primary tumors and visceral peritoneum, demonstrating higher autofluorescence in the peritoneum than in the tumors before spraying SPiDER-βGal in cases 4, 8, 9, 10, 11, and 12. However, the peritoneum showed little increase in fluorescence over time in all cases. Thus, the fluorescence increase from the 0-min time point may be a more effective marker for diagnosis than the fluorescence intensity itself.19, similar enzymatic activity cannot be guaranteed in metastases and primary tumors. In this study, the imaging of peritoneal metastatic nodules was carried out in only one case (Case 7). However, the peritoneum in all other cases showed little increase in fluorescence; case 7 showed a greater increase in fluorescence. Thus, we speculate that β-Gal is a promising target enzyme for detecting peritoneal metastasis in GC. To gather a sufficiently large number of cases with peritoneal metastasis from a single institution would take a long time; however, we plan to continue imaging peritoneal metastasis with SPiDER-βGal in the future to strengthen our observations.A major limitation of this study is that we used the comparison of primary tumor vs. peritoneum as a proxy for assessing peritoneal metastasis. As we have previously reported11. In addition, ICG was shown to detect peritoneal metastasis of colorectal cancer12. Activatable fluorescence probes are novel tools in the field of fluorescence imaging, with advantages of convenient and non-invasive use, requiring simple topical spraying without preoperative preparation to obtain conclusive results. For the intraoperative investigation of the peritoneal cavity by laparoscopy or laparotomy, activatable fluorescence probes may be useful options for improving the diagnostic results of peritoneal metastasis.Previously, 5-ALA was reported to be useful for the fluorescence imaging of peritoneal metastasis of GCThus, we conclude that β-Gal is a promising target enzyme of an activatable fluorescence probe for imaging peritoneal metastasis in GC. Further, SPiDER-βGal can be applied to clinical samples. We believe that the results of this study significantly contribute to improving the clinical diagnostic results in GC patients.2.The following six human gastric cancer cell lines were used in this study: MKN28, MKN45, MKN74, Kato3, HGC27, and NUGC4. All cell lines were purchased from RIKEN cell bank . HGC27 cells were cultured in minimum essential medium , and the other cell lines were cultured in RPMI1640 . Both culture media contained 10% fetal bovine serum , penicillin (100 U/mL), and streptomycin (100 μg/mL); all cells were cultured at 37 °C under 5% COA total of 89 primary GC and 20 normal peritoneal (NP) samples were analyzed in this study. The GC samples were obtained from a total of 98 consecutive patients with GC who underwent surgery between January 2014 and December 2017 and whose samples could be collected. Nine patients were excluded because either the sample volume was too low to be used in experiments (n = 7) or they were diagnosed with neuroendocrine tumors (n = 1) or lymphoma (n = 1). We collected NP samples from 20 patients who underwent surgery for various benign or malignant diseases between May and June of 2018. All patients underwent surgery at the University Hospital of Kyoto Prefectural University of Medicine (KPUM), and written informed consent was obtained from each participant. Human samples were collected and preserved at − 80 °C. All investigations conformed with the tenets of the Declaration of Helsinki and the study was approved by the Institutional Review Board (IRB) of KPUM (ERB-C-67 for sample collection and ERB-C-1288 for assessing enzymatic activity).g at 4 °C. The supernatant was collected, and the protein concentration was quantified using the BCA method .To evaluate the enzymatic activity of β-Gal, we prepared cell and tissue lysates. For the preparation of the cell lysates, cultured cells were washed with phosphate-buffered saline (PBS), CelLytic M (Sigma) was added, and the cells were homogenized using an ultrasonic homogenizer . Similarly, for the preparation of tissue lysates, CelLytic M was added, and the tissues were cut with scissors and then homogenized using an ultrasonic homogenizer. Both cell and tissue homogenates were centrifuged for 10 min at 14,000 × For measuring GGT and DPPIV activity, we used gGlu-HMRG and EP-HMRG, which are GGT and DPPIV-activatable fluorescence probes, respectively. The GGT and DPPIV activity was measured in 384-well black plates with 5 μL/well lysate (1 mg/mL protein concentration) and 15 μL/well fluorescence probe solution . HMRG was used as the positive-control fluorophore. An EnVision multilabel plate reader   was used to measure the fluorescence intensity for GGT activity. A GloMax Discover System (excitation/emission = 475 nm/ 500–550 nm)  was used for measuring DPPIV activity. The activity was calculated as follows: Activity = (Fluorescence increase rate)/(Fluorescence intensity of HMRG in lysate—Fluorescence intensity of gGlu-HMRG or EP-HMRG just after the addition of lysate)/(protein concentration).d-galactopyranoside solution . The plate was incubated at 37 °C for 30 min. After incubation, a stop buffer  was added when the measurement was performed in a pH 5.0 acetate buffer; however, no stop buffer was added when the measurement was performed in pH 7.4 PBS. The fluorescence intensity (excitation/emission = 390 nm/460 nm) was measured using a microplate reader . The β-Gal activity was calculated as the ratio of the fluorescence intensity of each sample to that of the positive control ; 0.1 mM 7-hydroxycoumarin-3-carboxylic acid was used as the positive-control fluorophore.For measuring β-Gal activity, we used the FluoReporter lacZ/Galactosidase Quantitation Kit . The β-Gal activity was measured in 96-well black plates using 10 μL/well lysate (1 mg/mL protein concentration) and 100 μL/well 3-carboxy-umbelliferyl β-5 cells and incubated at 37 °C under 5% CO2 for 2 days. The cells were washed using Hanks’ balanced salt solution  twice, and 1 μM SPiDER-βGal in HBSS was added to the cells and incubated for 15 min. Fluorescence images were captured using a fluorescence microscope . The TRITC filter set  was used for fluorescence images; bright-field images were also acquired.Cells from each cell line were plated on a cover glass-bottomed culture well at a density of 1 × 106 MKN45 and NUGC4 cells, suspended in PBS, were injected into the peritoneal cavity. Fluorescence imaging was performed 2–4 weeks after the injection of tumor cells. The study was carried out in compliance with the ARRIVE guidelines.All procedures with animals were carried out in compliance with the Manual for Animal Experiments and approved by the Kyoto Prefectural University of Medicine. BALB/c Ajcl-nu/nu 5-week-old female mice were purchased from SHIMIZU Laboratory Supplies Co., Ltd., Japan. A total of 1 × 10To evaluate the fluorescence intensity of SPiDER-βGal, we performed fluorescence imaging of the mouse model as described above. Mice were sacrificed using deep anesthesia, and the mesentery and bowel were excised. SPiDER-βGal  was sprayed on the samples. Fluorescence images were obtained using an in vivo imaging system (IVIS Lumina Series III). Filter settings of excitation 520 nm and emission 570 nm were used for the detection of SPiDER after the enzymatic reaction. Fluorescence images were captured every 2 min using a sequence setup.Thirteen GC patients who underwent various surgeries, including distal gastrectomy, proximal gastrectomy, total gastrectomy, and metastatic nodules resection, consecutively at the university hospital of KPUM and who provided written informed consent for this study were included for imaging analysis. Twelve primary tumor samples and one peritoneal metastatic sample were obtained. The resected samples were carried to the IVIS immediately, and fluorescence imaging was performed with SPiDER-βGal. We substituted the primary tumors vs. visceral peritoneum for imaging peritoneal metastasis because it would take a long time limited to the peritoneal metastasis samples. Both mucosa and peritoneum sides were imaged. Fluorescence intensity of the tumor and peritoneum was evaluated in the region of interest (ROI) with Living Image version 4.4 . This study was approved by the IRB of KPUM .U test was used to compare the β-Gal activity and fluorescence intensity in vitro and ex vivo. JMP 13.0 software  was used for comparing the β-Gal activity for each clinicopathological factor with the Wilcoxon test and for drawing the receiver operating characteristic (ROC) curves. Results with p < 0.05 were considered to be statistically significant.Statistical analysis was performed using ystat 2008 software. The two-tailed Mann–Whitney Supplementary Information 1.