The hepatitis C virus (HCV) co-opts numerous cellular elements, including proteins, lipids, and microRNAs, to complete its viral life cycle. The cellular RNA-binding protein, poly(rC)-binding protein 1 (PCBP1), was previously reported to bind to the 5′ untranslated region (UTR) of the HCV genome; however, its importance in the viral life cycle has remained unclear. Herein, we sought to clarify the role of PCBP1 in the HCV life cycle. Using the HCV cell culture (HCVcc) system, we found that knockdown of endogenous PCBP1 resulted in an overall decrease in viral RNA accumulation, yet resulted in an increase in extracellular viral titers. To dissect PCBP1’s specific role in the HCV life cycle, we carried out assays for viral entry, translation, genome stability, RNA replication, as well as virion assembly and secretion. We found that PCBP1 knockdown did not directly affect viral entry, translation, RNA stability, or RNA replication, but resulted in an overall increase in infectious particle secretion. This increase in virion secretion was evident even when viral RNA synthesis was inhibited, and blocking virus secretion could partially restore the viral RNA accumulation decreased by PCBP1 knockdown. We therefore propose a model where endogenous PCBP1 normally limits virion assembly and secretion, which increases viral RNA accumulation in infected cells by preventing the departure of viral genomes packaged into virions. Overall, our findings improve our understanding of how cellular RNA-binding proteins influence viral genomic RNA utilization during the HCV life cycle. Flaviviridae family (genus: hepacivirus) that typically causes a persistent liver infection ,18T straectively B–D. Quannockdown E. Surpriectively F. OveralJ6/JFH-1 . To veriJ6/JFH-1 . AlthougTo clarify PCBP1’s precise role(s) in the HCV life cycle, we used assays to specifically examine different steps of the viral life cycle . FirstlyTo examine PCBP1’s impact on viral translation, we used a full-length J6/JFH-1 RLuc reporter RNA containing an inactivating mutation in the NS5B polymerase gene (GNN). The RLuc activity thus served as a direct measure of HCV IRES-mediated translation, and over time, this signal also served as a proxy measure for viral RNA stability. We found that the PCBP1 knockdown and control conditions had similar RLuc activity at all timepoints B. MoreovTo assess the impact of PCBP1 knockdown on HCV RNA replication, we made use of a J6/JFH-1 subgenomic replicon RLuc reporter RNA, with a deletion of the structural proteins (Δcore-p7), which allowed us to assess viral RNA replication in the absence of virion assembly C. We obsT and, three days post-infection, treated cells with 2′CMA or vehicle (DMSO), and monitored viral RNA accumulation as well as intracellular and extracellular virion production over the next 12 h , to block viral RNA synthesis by the HCV NS5B RNA-dependent RNA polymerase and monitored virion production over time  23]. Si. Si23]. ext 12 h A. In agrDuring virion secretion, viral genomes that have been packaged into new infectious particles are removed from the cell. Since our previous findings suggested that PCBP1 knockdown increases virion secretion, we wondered if this increased egress of assembled viral particles  might explain our initial observation that PCBP1 knockdown led to an overall reduction in viral RNA accumulation C,D. To dHerein, we investigated the role of PCBP1 in the HCV life cycle. Previous studies had found that PCBP1 interacts with the HCV 5′ UTR in vitro ,15, and T, a cell culture-adapted strain that produces greater yields of infectious virions in cell culture than the parental JFH-1 strain [T strain itself, we validated our findings with J6/JFH-1, a chimeric virus that is widely used in HCVcc studies, commonly referred to as Jc1 [T and J6/JFH-1 protein expression, RNA accumulation, and extracellular viral titers; however, we found that while JFH-1T intracellular titers were not significantly different from control condition, J6/JFH-1 intracellular titers were significantly decreased by approximately 2.6-fold  writers, and the YTHDF m6A-binding proteins during HCV infection [6A modification of the HCV genome inhibit virion assembly as components of a common pathway, or through distinct and/or additive mechanisms. Interestingly, high-throughput affinity capture and proximity ligation studies have found that PCBP1 interacts with hnRNP K, IGF2BP2, YBX-1, METTL3 and METTL14, although this has been demonstrated only in non-hepatic cell lines to date [Finally, PCBP1′s closely related paralogs, hnRNP K and PCBP2, have been more extensively studied during HCV infection. While the hnRNP K protein was shown to restrict infectious virion production, the PCBP2 protein has been suggested to modulate viral translation and RNA replication ,14. Sincnfection ,38,39. A to date ,41,42. S6A modification of the HCV genome offer promising leads for future investigations.Our results support a model whereby endogenous PCBP1 limits HCV infectious particle production. By preventing virion assembly and secretion, PCBP1 indirectly enhances viral RNA accumulation. The model presented herein helps to inform our understanding of how cellular RNA-binding proteins modulate HCV genomic RNA utilization during the viral life cycle, specifically as it pertains to virion assembly. While the precise molecular mechanism(s) employed by PCBP1 to inhibit HCV assembly and egress remain to be characterized, similar phenotypes reported for hnRNP K, IGF2BP2/YBX-1 and m


Our technique is an alternative to the classical isotope labelling approach for the measurement of 2′-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2′-O-MTase.The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2′-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m Coronaviridae family that has already produced at least two other deadly human viruses during the last two decades. The severe acute respiratory syndrome (SARS) virus was identified as the virus causing atypical pneumonia in the Guangdong Province of China in 2002 [The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current COVID-19 pandemic [ in 2002 .pro) and 3C-like main proteases (3CLpro); the nsp13 helicase; the nsp15 endonuclease; and two methyltransferases [pro were recently described [Coronaviruses are now recognized as a major threat to global human health . Their gsferases . Therefosferases , althougsferases ,9,10,11.escribed . In addiescribed ,15,16, aescribed ,18,19.Innate immunity is a crucial part of the human immune system, and viruses have evolved abilities to evade it . The 5′-7Gp3N cap, RNA can be capped by cofactors, such as nicotinamide adenine dinucleotide [nNs) and are incorporated into RNA co-transcriptionally by the RNA polymerase [nNs are also present in eukaryotic cells, we hypothesize that they might be incorporated into RNA as non-canonical initiating nucleotides where they can represent an additional layer of information. Moreover, NAD or flavin adenine dinucleotide (FAD) capped RNA was detected in viral particles of the Dengue 2 virus [nNs, have not been studied as substrates for any viral encoded enzyme.The chemical variations in RNA caps and their physiological implications are not fully understood. Recently, it has been shown that, beside the common canonical mcleotide ,26 or cocleotide ,28. Whercleotide , its funcleotide , albeit cleotide . These clymerase . Dinuclelymerase . They arlymerase . As NpnN 2 virus , suggest7Gp3A, Gp3A, m7Gp3G, Gp3G, and Gp4A. We discovered that the m7Gp3A-RNA was the best substrate for nsp10-nsp16 in accordance with studies on MTases from other coronaviruses [50 value of 138 ± 30 nM.Here, we aimed to characterize the SARS-CoV-2 nsp10-nsp16 2′-O-MTase. We prepared a recombinant nsp10-nsp16 complex and analyzed its substrate specificity using LC-MS. First, we tested whether nsp10-nsp16 is capable of methylation of free caps or short hexamer RNA capped with canonical and non-canonical RNA nucleotides. As we did not observe any methylation of the free caps and the methylation of the short hexamer RNA was only partial, we used a longer RNA (35mer). Usually, the methylation of RNA at the 2′-O of ribose is studied by radioactive labelling . We deveaviruses ,36,37. Waviruses  and obta7GpppA was synthesized in house according to Baranowski et al. [All chemicals were either purchased from Merck or Jena Biosciences and used without further purification. Oligonucleotides were purchased from Generi Biotech. mi et al.  as detaiE. coli BL21 cells, and the cells were grown at 37 °C in LB media supplemented with 25 µM ZnSO4 until the OD600 nm reached 0.5. Subsequently, the expression was induced by IPTG , and the temperature was lowered to 18 °C overnight. Cells were harvested, resuspended, and lysed by sonication in lysis buffer . Proteins were purified by affinity chromatography using the NiNTA agarose (Machery-Nagel), dialyzed against lysis buffer, and digested with Ulp1 protease at 4 °C overnight. The last purification step was size exclusion chromatography at the HiLoad 16/600 Superdex 200 gel filtration column  in the SEC buffer . Purified proteins were concentrated to 7 mg/mL and stored in −80 °C until needed.The plasmid encoding for nsp10 and nsp16 proteins was described previously, as was the purification protocol . BrieflynNs, 5% DMSO, 0.12% triton X-100, 12 mM DTT, 4.8 mM MgCl2, and 1x reaction buffer for T7 RNAP and 62.5 units of T7 RNAP . The mixture was incubated for 2 h at 37 °C. After incubation, the samples were injected, without any further purification, in the HPLC, and only the hexamer RNA was collected. The purified RNA was dried on a Speedvac system three times to remove excess triethylammonium acetate (TEAA).In vitro transcription was performed in a 50 μL mixture containing 80 ng/μL of template DNA (6A), 1 mM NTPs (only those necessary for the RNA production), 1.6 mM NpnNs (or ATP or GTP for the control experiments), 5% DMSO, 0.12% triton X-100, 12 mM DTT, 4.8 mM MgCl2, and 1x reaction buffer for T7 RNAP and 62.5 units of T7 RNAP . The mixture was incubated for 2 h at 37 °C.In vitro transcription was performed in a 50 μL or 75 μL mixture containing 80 ng/μL of template DNA (35A or 35G) , 1 mM NT2, 0.5 mM CaCl2, pH 7.6 at 25 °C, supplied with the enzyme), and 4 units of DNAse I (NEB) were incubated at 37 °C for 60 min. The enzyme was thermally deactivated at 75 °C for 10 min followed by immediate cooling on ice. All samples were purified with RNA Clean and ConcentratorTM from ZYMO research and eluted in 25 μL of water for further use.After the transcription, the DNA template was digested by DNAse I to obtain pure RNA. The transcription mixture (50 μL), 6 μL of 10× the reaction buffer for DNAse I  in the solution of 1× buffer in a total volume of 30 μL for 1 h at 37 °C. All samples were purified with RNA Clean and ConcentratorTM and eluted in 15 μL of water for further use.The RNA was treated with 1 unit of Terminator ™ 5′-phosphate-dependent exonuclease (Epicenter) in the solution of 1× buffer A in a total volume of 30 μL, and the mixture was incubated at 30 °C for 1 h. All samples were purified with RNA Clean and ConcentratorTo test the methyltransferase activity, the cap or the capped RNA samples were divided into two parts. The positive control contained ~3.5 µM (~40 µM for mixture of capped RNA and ppp-RNA) of the RNA, 80 µM (1 mM for mixture of capped RNA and ppp-RNA) of SAM, and 1.5 μM of nsp10-nsp16 in the reaction buffer . Nsp10-nsp16 was replaced by water for the negative control. The reactions were performed in 50 µL volume. The mixture was incubated at 30 °C for 1 h (2 h for mixture of capped RNA and ppp-RNA). The enzyme was heat-deactivated at 75 °C for 10 min followed by immediate cooling on ice. The reaction with free caps was analyzed without further purification by HPLC, and capped RNA was digested before analysis by LC-MS.® XB-C18 column . Mobile phase A was 100 mM TEAA pH 7, and mobile phase B was 100% acetonitrile. The flowrate was kept at 1 mL/min, and the mobile phase composition gradient was as follows: linear decrease from 0% to 12% B  over 20 min, linear decrease to 100% B over 7 min, maintaining 100% B for 3 min, returning linearly to 0% B over 10 min. Waters Fraction Collector III was used for collection of the hexamer RNA.HPLC was performed using a Waters Acquity HPLC e2695 instrument with PDA detector and with a Kinetex The capped RNA after nsp10-nsp16 reaction was digested using 3 U of Nuclease P1 (Merck) in 50 mM ammonium acetate buffer (pH 4.5) at 37 °C for 1 h. The digested RNA was purified using Amicon-Millipore filters 10 kDa (Merck) to eliminate the Nuclease P1. The flowthrough was dried on a Speedvac system and dissolved in 10 μL of a mixture of acetonitrile (10%) and ammonium acetate .LC-MS was performed using a Waters Acquity UPLC SYNAPT G2 instrument with an Acquity UPLC BEH Amide column . The mobile phase A consisted of 10 mM ammonium acetate, pH 9, and the mobile phase B of 100% acetonitrile. The flowrate was kept at 0.25 mL/min, and the mobile phase composition gradient was as follows: 80% B for 2 min, linear decrease to 50% B over 4 min (Method Y) or 14 min (Method Z), linear decrease to 5% B over 1 min, maintaining 5% B for 2 min, returning linearly to 80% B over 2 min. For the analysis, electrospray ionization (ESI) was used with a capillary voltage of 1.80 kV, a sampling cone voltage of 20.0 V, and an extraction cone voltage of 4.0 V. The source temperature was 120 °C, the desolvation temperature was 550 °C, the cone gas flowrate was 50 L/h, and the desolvation gas flowrate 250 L/h. The detector was operated in negative ion mode. In total, 8 μL of the dissolved material was injected and analyzed.MassLynx software was used for the data analysis and the quantification of the relative abundance of all caps. The Area Under the Curve (AUC) for all caps in the positive and negative samples was calculated and normalized for the area of GMP of each negative. The decreasing AUC of the starting material (unmethylated cap) in the nsp10-nsp16-treated sample was compared with the AUC of the starting material (unmethylated cap) in the untreated sample and expressed as a percentage.7Gp3A-RNA, 3.6 μM of SAM, 500 nM of nsp10-nsp16, and 5 nM–3 μM of Sinefungine were added to the reaction buffer . The mixtures were incubated at 30 °C for 1 h. The enzyme was heat-deactivated at 75 °C for 10 min followed by immediate cooling on ice. The m7Gp3A-RNA was digested by Nuclease P1 and analyzed by LC-MS.For each reaction, ~0.7 µM of pure m7Gp3Am RNA cap. LC-MS was performed using a Waters Acquity UPLC SYNAPT G2 instrument with an Acquity UPLC BEH Amide column . The mobile phase A consisted of 10 mM ammonium acetate, pH 9, and the mobile phase B of 100% acetonitrile. The flowrate was kept at 0.25 mL/min, and the mobile phase composition gradient was as follows: 80% B for 2 min, linear decrease to 50% B over 4 min, linear decrease to 5% B over 1 min, maintaining 5% B for 2 min, returning linearly to 80% B over 2 min. For the analysis, electrospray ionization (ESI) was used with a capillary voltage of 2.7 kV, a sampling cone voltage of 30.0 V, and an extraction cone voltage of 3.0 V. The source temperature was 120 °C, the desolvation temperature was 500 °C, the cone gas flowrate was 70 L/h, and the desolvation gas flowrate 600 L/h. The detector was operated in positive ion mode. In total, 8 μL of the dissolved material was injected and analyzed.The LC-MS conditions were optimized for the highest signal to noise ratio of the mnNs) [nNs. We let m7Gp3A, Gp3A, Ap3A, m7Gp3G, Gp3G, and Np4N  react with nsp10-nsp16 complex in the presence of SAM for 2 h at 30 °C or 37 °C. The reaction mixture was analyzed by HPLC. We did not observe any 2′-O-methylated products. This finding was in an agreement with previously observed SARS-CoV nsp10-nsp16 activity [In the light of our recent discovery of a new class RNA caps based on dinucleoside polyphosphates (NpnNs) , we testactivity  , only m7Gp3A-RNA was methylated in approximately 20% yield  capped with various dinucleoside polyphosphates can be methylated by this complex. The hexameric RNA was prepared by in vitro transcription with T7 RNA polymerase and free caps. As in vitro transcription with T7 RNA polymerase allows only the preparation of a bigger amount of RNA with sequences typical for T7 bacteriophage, we could not prepare the 5′-terminal sequence (AUUA--) of the SARS-CoV-2 genome. Therefore, we used the template with the T7 promotor φ 2.5, leading to RNA with a sequence of AGGGAA as the model RNA. After HPLC purification, RNA was treated by nsp10-nsp16 complex with SAM for 2 h at 30 °C. The samples were then digested by the nuclease P1 to release 5′-mononucleotides and intact RNA caps and analyzed by HPLC. From all the tested substrates  and observed the formation of 2′-O-methylated m7Gp3A (m7Gp3Am). The conversion of the reaction was calculated from the difference of the unreacted cap before and after the reaction and confirmed by the formation of the methylated cap. After 2 h, all of the m7Gp3A cap was converted to m7Gp3Am  as substrates for the SARS-CoV-2 nsp10-nsp16 MTase complex. The RNA was prepared as a 35mer by in vitro transcription and treated by 5′ polyphosphatase and terminator exonuclease to degrade the uncapped RNA. Afterward, pure capped RNA was treated by the nsp10-nsp16 complex in the presence of SAM at 30 °C for 1 h. Subsequently, the samples were digested by nuclease P1, and the disappearance of the unreacted cap and formation of the methylated cap were observed , three other capped RNAs  were methylated at the 2′-O position of the +1 nucleotide. All of them were approximately methylated from 70% to 20% (7Gp3A-RNA. The yield of the reaction with m7Gp3G-RNA (30%) may have been negatively influenced by the presence of the side product Gp3m7G-RNA, which was created by a reverse incorporation of m7Gp3G during the in vitro transcription. This side product could not be removed from m7Gp3G-RNA by enzymatic treatment or chromatographic separation. As we encountered the loss of materials during the purification of the capped RNA from the ppp-RNA, the amount of Gp4A-RNA may have decreased under the limit of detection. When Ap3G was incorporated into RNA in the opposite manner [nNs-RNA, which has not been detected in eukaryotic cells so far, we also tested the recently discovered eukaryotic NAD- [7Gp3A cap, we did not observe any methylated products. Ap3-5A-, m6Ap3A-, Gp5A-, Gp4G(A)-, m7Gp4G-, and CoA-RNA were also not accepted as substrates. Nevertheless, when the methylation reaction of nsp10-nsp16 was performed with a crude transcription mixture (in the presence of ppp-RNA), we also observed the partial methylation of Gp4A-RNA  RNAs that can be methylated only when m7Gp3A capped [3A, Gp3G, m7Gp3G, and Gp4A) which are also accepted as substrates for the nsp16 MTase could also play a regulatory role in the stability of viral RNAs. Besides the ~30 kb genomic RNA (serves as mRNA for nsp1-16 proteins), coronaviruses produce up to 10 subgenomic mRNAs that encode structural and accessory proteins [nN-RNAs.Here, we report the development of an LC-MS-based method for the analysis of RNA methylation. Our method is non-radioactive, which is the current trend for safety reasons and is also advantageous for high-throughput screening ,42. We aGp3A-RNA . HoweverA capped . We obseed short mer RNAs proteins . Howeverproteins . To dateThe SARS-CoV-2 nsp16 MTase is an important drug target. Often, drug-like candidate molecules are found using high-throughput screening (HTS)  and subs3A has important implications for the viral life cycle because it reveals that nsp16 can, in principle, act before the nsp14 N7 MTase.Recently, an alternative MS method was published for the analysis of methylation of 25 mer canonically capped RNA by nsp16 MTase without Nuclease P1 digestion . This me


O-methyl groups that are introduced sequence specifically by box C/D small nucleolar RNAs (snoRNAs) in ribonucleoprotein particles. Most data on this chemical modification and its impact on RNA folding and stability are derived from organisms of the Opisthokonta supergroup. Using bioinformatics and RNA-seq data, we identify 30 novel box C/D snoRNAs in Dictyostelium discoideum, many of which are differentially expressed during the multicellular development of the amoeba. By applying RiboMeth-seq, we find 49 positions in the 17S and 26S rRNA 2′-O-methylated. Several of these nucleotides are substoichiometrically modified, with one displaying dynamic modification levels during development. Using homology-based models for the D. discoideum rRNA secondary structures, we localize many modified nucleotides in the vicinity of the ribosomal A, P and E sites. For most modified positions, a guiding box C/D snoRNA could be identified, allowing to determine idiosyncratic features of the snoRNA/rRNA interactions in the amoeba. Our data from D. discoideum represents the first evidence for ribosome heterogeneity in the Amoebozoa supergroup, allowing to suggest that it is a common feature of all eukaryotes.A hallmark of ribosomal RNA (rRNA) are 2′- This first indication for rRNA as the catalytic entity in protein biosynthesis, rather than proteins, was subsequently confirmed by ground-breaking and highly decorated crystallographic work4. Maturation of ribosomes is amongst the most complex cellular processes and requires about 200 facilitating proteins, as reviewed recently5. Amongst many other processes, the introduction of post-transcriptional, covalent modifications in rRNA is of utmost importance for ribosome biogenesis and function, as summarized in Ref.6. The most prominent nucleotide modifications in rRNA are 2′-O-ribose methylation (2′-O-Me) and pseudouridylation (Ψ) that are introduced site-specifically. These modifications are thought to be important for RNA folding, ribosome stability and translational fidelity9. In recent years, a specialization of ribosomes in response to environmental changes and/or developmental processes has been suggested, with substoichiometric chemical modifications being implicated as a major source of ribosome heterogeneity10. As such, examples for fractional rRNA modifications are found in various species, including Saccharomyces cerevisiae, where 18 positions are modified in less than 85% of the ribosomal population11, and also approximately a third of the 2′-O-Me positions in rRNA of Homo sapiens are found hypomodified12. Recently, altered 2′-O-Me levels were also discovered during the development of Danio rerio13. Functionally, ribosome heterogeneity has been proposed to constitute a fine-tuning mechanism for translational activity of an unknown subset of mRNAs15.Early on, the peptidyltransferase reaction of the ribosome was shown to be resistant to protein degradative treatment16. They come in two flavors: H/ACA snoRNPs catalyze the conversion of uridine to Ψ, while box C/D snoRNPs introduce methyl groups at the 2′-hydroxyl of ribose residues18. For each class of snoRNPs, a conserved and distinct set of four proteins form the catalytic complex, of which dyskerin in the H/ACA snoRNPs isomerizes uridine19, while fibrillarin in box C/D snoRNPs acts as the methyltransferase on 2′-hydroxyl groups20. The rRNA target positions are defined by the individual snoRNA components of the RNPs. For both classes, specific base pairing patterns define the nucleotide to be modified. In the following, we briefly summarize the interaction of box C/D snoRNAs with rRNA and refer for H/ACA snoRNAs to two recent excellent reviews10. Box C/D snoRNAs possess conserved box C (5′-RUGAUGA-3′) and box D (5′-CUGA-3′) motifs that are essential for their structure, function and biogenesis -dependent reaction26. Since both, the D and D′ box can potentially guide 2′-O-Me with their antisense elements, these snoRNAs can guide in principle two sites in rRNA. Furthermore, a reorientation of the box C/D snoRNA inside the snoRNP complex might lead to alternative base pairing, increasing the target number of a given snoRNA even further28.Ribose methylations and pseudouridylations in eukaryotes are introduced in rRNA site-specifically by small nucleolar ribonucleoprotein particles (snoRNPs), as summarized recently30, information for the Amoebozoa supergroup remains scarce. Dictyostelium discoideum is a well-established model organism and arguably the best studied organism of the Amoebozoa31. A wide spectrum of experimental tools has been established for the amoeba32, and these are frequently used to study mechanisms governing cell motility, autophagy, social evolution (reviewed in Ref.33), mobile genetic elements34, and their domestication by the RNA interference (RNAi) machinery37. D. discoideum single cells usually propagate by mitotic division; upon starvation, however, a complex developmental process is initiated, in which about 100,000 cells aggregate to form a multicellular mobile slug after 16 h, resulting in a fruiting body within 24 h38. This allows to study fundamental developmental processes in the amoeba.While a substantial amount of data on rRNA modifications by snoRNPs is available from organisms of the evolutionary supergroups of Opisthokonta and ArchaeplastidaD. discoideum, however, they are not encoded in chromosomes but localized on extrachromosomal elements40. Each nucleus contains about 100 copies of these elements of 88 kb, that each feature two rRNA transcription units organized as palindromes41. A first model for the processing of rRNAs from the primary 37S transcript in the amoeba has been proposed, and sequences of the mature rRNAs in D. discoideum were determined experimentally42. Earlier work has identified several box C/D snoRNAs in D. discoideum, and verified a function in rRNA 2′-O-methylation43. The study employed a shotgun cloning approach to identify novel non-coding RNAs (ncRNAs) in D. discoideum. This work led also to the discovery of the functionally important Class I RNAs, which recently were shown to be involved in the evolution of multicellularity in Dictyostelia44. Next to these, sequencing of cloned fragments yielded 17 box C/D and one box H/ACA snoRNA(s) in D. discoideum, besides other ncRNAs.In most metazoans, the genes for the rRNAs are organized in rDNA clusters, an arrangement that is thought to facilitate efficient rRNA transcription. Such rDNA clusters exist also in O-Me pattern(s) in the amoeba. Employing RiboMeth-seq (RMS)11, we created a comprehensive map of the 2′-O-Me sites in Dictyostelium’s 17S and 26S rRNAs. We thereby positioned methylated residues in functional important parts of the rRNAs, for which we have determined sequence homology-based models of their secondary structures. Further, we also have identified bioinformatically and validated experimentally additional box C/D snoRNAs with which we can at large explain methylated rRNA positions in the amoeba.Owing to these observations, we set out here to elucidate the global 2′-D. discoideum strains AX245 and ∆drnB46 were cultivated in HL5 medium containing 50 μg/mL ampicillin, 250 ng/mL amphotericin, 500 U/mL penicillin/streptomycin at 22 °C in shaking suspension.The 8 of axenically grown D. discoideum cells pelleted for 5 min at 500×g and washed three times with Sørensen buffer . The pellet was resuspended in Sørensen buffer and transferred in a 6-cm dish containing two layers of Whatman® paper topped off with a nitrocellulose membrane. After 16 h, the slugs were harvested by washing the nitrocellulose membrane with Sørensen buffer and spun down by centrifugation at 500×g for 5 min. RNA was isolated from the resulting pellet.Filter development was performed using 5 × 10∆drnB in axenic growth and slug stage of development were acquired from the sequence read archive (https://www.ncbi.nlm.nih.gov/sra) and used for RNA-seq validation of box C/D snoRNA candidates and expression analysis. Accession numbers of the utilized data sets can be found in Supplementary Table 47.RNA-seq datasets of AX2 and www.dictybase.org) and the sequences of the 17S and 26S rRNA42 were retrieved from GenBank  disabled. Candidates with a combined box C and box D score higher than 9 and a box C-D distance between 50 and 100 nt were selected for RNA-seq validation. Sequencing reads from the axenic AX2 dataset were aligned to the genomic coordinates ± 150 bp using bowtie v. 1.2.349 allowing for one mismatch. Box C/D snoRNAs were considered validated, if reads specifically matched the predicted loci and read coverage calculated with BEDTools coverage v. 2.29.250 indicated a distinct 5′ end, yielding an expression score of 15. Box C/D snoRNA candidates lacking expression or a distinct 5′ end received a penalty of − 15. All scores were combined into a classifier score containing C/D box scores, terminal stem score, Box C–D distance score, and the expression score  method. Principal component analysis was performed on DESeq2-normalized reads using R-stats v. 4.0.0 and visualized with R-ggplot2 v. 3.3.2. The heatmap of log2 fold-change of box C/D snoRNAs was generated using ComplexHeatmap v. 2.5.353.Reads were aligned using bowtie v. 1.2.32, 5 mM DTT, 100 µM spermidine, and 0.37 MBq [γ-32P]-ATP. The reaction was stopped at 80 °C for 5 min, the radiolabeled oligonucleotides were phenol/chloroform-extracted and purified using a Sephadex G50  column.DNA oligonucleotides were purchased from Merck and are listed in Supplementary Table 7 axenically grown D. discoideum cells washed with pre-cooled Sørensen buffer . Cells were pelleted and resuspended in TRIzol reagent (Invitrogen) containing 10 mM EDTA (pH 8.0). RNA was extracted according to the manufacturer’s instructions. RNA concentration was determined spectrophotometrically.RNA was isolated from 2 × 10For primer extension, a box C/D snoRNA-specific 5′-radiolabeled oligonucleotide was annealed to 4 µg RNA at 65 °C for 5 min and cooled for at least 1 min on ice. Upon annealing, 1× SuperScript IV buffer , 1 mM dNTP mix, 5 µM DTT, 40 U RiboLock RNase Inhibitor  and 50 U SuperScript IV Reverse Transcriptase  were added. The reaction was incubated at 55 °C for 30 min and stopped at 85 °C for 5 min. Products were phenol/chloroform-extracted, recovered by ethanol precipitation and separated on a polyacrylamide gel  for 3 h at 25 mA.TM-NX) by electroblotting for 30 min at 20 V. Blotted RNA was crosslinked by 0.5 J/cm2 UV illumination. Blots were probed overnight with 5′-radiolabelled DNA oligonucleotides in Church buffer  SDS, 1% (w/v) BSA in 0.5 M Pi buffer, pH 7.2). Probed Blots were washed two times for 20 min with each 2×, 1×, and 0.5× SSC buffer . Hybridization with an oligonucleotide complementary to tRNAUUC was used as a loading control.For the detection of snoRNAs, 20 μg of total RNA was separated by gel electrophoresis on a 12% polyacrylamide gel . The RNA was transferred to a nylon membrane  using Bowtie255 and scored for read-end counts. RMS scores representing “fraction methylated” were calculated as described previously (“score C”) in Ref.11 and barcode correction was applied when necessary56. The commercial RNA oligonucleotides used as 3′adaptors were found to be slightly heterogeneous in length, which can cause a fractional shift in the 3′-read-end count, if the 3′-library fragment nucleotide is identical to the expected 5′-end of the oligonucleotide. As the experiments were made in triplicate with barcodes carrying different 5′-ends, such errors were easily detected, and a manual correction was made at a few sites to counter the effect by excluding the 3′-read-end counts from the analysis.The RiboMeth-seq analysis was performed in triplicates with barcoded adapters according to previously described protocolsO-Me sites in the mature rRNA, we predicted the secondary structure by comparative analysis with the LSU and SSU rRNAs of A. thaliana, C. elegans, H. sapiens, and D. melanogaster. For that purpose, we retrieved the corresponding SSU and LSU rRNA sequences for these organisms from GenBank  2′-O-Me site is located at the 5th base paired nucleotide upstream of a D or D′ box and (II) a box C/D snoRNA/rRNA duplex length of minimum 7 bp with (III) a maximum of 1 mismatch. Conservation of box C and box D motifs was visualized using WebLogo v. 3.760. Calculation of the predicted duplex’ minimum free energy (MFE) in kcal/mol was performed using RNAduplex v. 2.4.1561. Box C/D snoRNAs that were not mapped to any predicted 2′-O-Me sites but were validated by RNA-seq, were classified as orphans.Mapping of box C/D snoRNAs to the predicted 2′-D. discoideum prior to this study is relatively small for normally-sized rRNA sequences42 compared to orthologous RNAs found in other species62. Therefore, we set out here to search for additional box C/D snoRNAs in the amoeba. To this end, we employed an in silico-approach for the identification of novel box C/D snoRNAs by using the probabilistic model-dependent search tool snoScan48, which we combined with RNA-seq analyses. The sizes of previously described box C/D snoRNAs of D. discoideum range between 66 and 113 nt, with box C-D distances between 50 and 97 nt43. We searched accordingly first with snoScan in the genome of D. discoideum (available at www.dictybase.org) for sequences containing box C and box D motifs with a box C-D distance between 50 to 100 nt. Since inverted repeats at the 5′ and 3′ ends were not observed before43, we did not pre-require the presence of a terminal stem structure for a classification as a bona fide box C/D snoRNA. Using these settings, we identified 577 box C/D snoRNA candidates in the genome of D. discoideum (data not shown), including the set described before43. To refine our search, we next addressed the expression of these candidates in publicly available RNA-seq data of the axenic AX2 wild type strain, deposited in duplicate47 at the sequence read archive (https://www.ncbi.nlm.nih.gov/sra). Specifically, we mapped reads to the genomic loci of the candidates and selected only those sequences that exceeded a read count of 100 and were not part of a longer transcript, as indicated by a distinct 5′ end. Both, the lack of specific RNA-seq reads or of a distinct 5′ end, were penalized  was missing in the 17S reference sequence42, and its presence was independently confirmed by sequencing of a PCR product on total DNA. Using the criteria outlined above, we determined in total 17 and 32 positions with a 2′-O-Me moiety on the 17S rRNA and the 26S rRNA, respectively  rRNA sequences from A. thaliana, Caenorhabditis elegans, Drosophila melanogaster, and H. sapiens (Supplementary Table S3). The inferred secondary structure models of the 17S and 26S (with the 5.8S) rRNAs of D. discoideum are shown in Figs. As methylated rRNA positions are required for folding and structural stabilization of rRNAs, thereby contributing to ribosome functioneplastida1. In bri72, which often harbor species-specific sequences. This is exactly what the models for the amoebal rRNA structures display ay Figs.  and 4. TO-Me patterns in wild type D. discoideum to those in S. cerevisiae, H. sapiens, and A. thaliana, we found 28 of the 2′-O-Me sites conserved in at least one of these organisms, and therefore, the other 21 sites are specific to D. discoideum 73.About half of the 2′-O methylated positions were found in the vicinity of nucleotides residing in the A, P and E sites, and the other half in other regions of the rRNAs Figs.  and 4. TO-Me sites with at least one, occasionally two box C/D snoRNA guides  of − 13.9 kcal/mol  of box C/D snoRNA expression on two biological replicates for each time point per strain. The analysis revealed global changes of box C/D snoRNA abundance in the development of the AX2 and ∆drnB strains  fully modified in differentiated tissues70. These aforementioned studies also all used RMS, as the preferred high-throughput analysis method of 2′-O-Me patterns, allowing for single nucleotide analysis in a quantitative manner, unlike alternative approaches. The advantages of RMS were also highlighted in a comparative study on rRNA from Trypanosoma brucei that further revealed 2′-O-Me patterns, which depended on the living conditions of the parasite77. Similar methodological advantages to RMS are also realized by the recently introduced and validated RiboMethSeq tool79 and the methylated positions reported here for the AX2 strain were at large confirmed independently using this method .A single 2′-O-methylated position, 26S-A1463, displayed altered RMS scores in the development of the amoeba and between the investigated strains Fig. . Such ch43, and we have added here additional 21 novel box C/D snoRNAs with a target in rRNAs, plus nine without. Previously, small non-coding RNAs in the amoeba were all called DdR-x , for Dictyostelium discoideum RNA43. With a functional association, we now have decided to rename the box C/D snoRNAs with an rRNA target to CDx , and those without to ORx RNA (for orphan).For the majority of 2′-O-methylated rRNA positions, we have bioinformatically identified suitable CD RNAs Fig. , Table 131. In the rRNA models for the Amoebozoan D. discoideum, about half of the 2′-O-methylated nucleotides are found close to the A, P and E sites of the ribosome. The remainder localize either in formally single stranded regions or at the very beginning of helical stems where they presumably fulfil a stabilizing function or support rRNA folding. Our models of the D. discoideum rRNAs are greatly supported by the previously introduced Cryo-EM structure of the nascent 60S subunit of Dictyostelium71, that features parts of the proposed structural elements of the 26S rRNA  in the A-site via base pairing with C75 in their CCA-tail80. This suggests that Gm3255 might fulfill the same function in Dictyostelium. U3254 is likely modified by the CD25 RNP , however, a guide for Gm3255 is missing , and by analogy we hypothesize that its gene product might fulfil the same function as Spb1 in yeast. We can, however, not exclude that the CD25 RNP might also introduce that methylation by using its + 6 position, in analogy to two D. rerio snoRNPs that guide neighbouring positions in the rRNAs13.In D. discoideum are encoded in intergenic regions or as part of introns of protein-coding genes, and in either set-up, they can be generated from mono- or poly-cistronic transcriptional units62. The selected set of 38 CD RNAs and their encoding genes display overall features similar to those seen in the original 17 sequences43. We found all box C/D snoRNAs in intergenic regions except for CD38, which is encoded in an intron of DDB_G0283293  and before the separation of the Opisthokonta encompassing as diverse organisms as D. melanogaster, H. sapiens, S. cerevisiae, or S. pombe31. This current situation might be explained by snoRNA numbers and their intronization having evolved after the split of the individual supergroups to meet the needs of the individual organism.Not only in 82. However, only 10 base pairs actually fit in the substrate binding channel, as observed for an archaeal box C/D snoRNP24. Overall, the interactions that we are proposing for the CD RNA/rRNA pairs adhere to these rules  that are predicted in individual interaction pairs. G*U base pairs have been observed also in analogous pairs of other species13, and they can be isosteric to Watson–Crick base pairs74. However, their occurrence appears more frequent in the amoeba, and in the extreme case of the CD12/26S-U2580 interaction . Therefore, we speculate that these sequences might be present in presumed precursor molecules.The mature box C/D snoRNAs in NAs Fig. . A stablNAs Fig. . Such stDictyostelium discoideum CD RNAs are predicted to use the antisense elements associated with the weakly conserved D′ box sequences more frequently than those with the highly conserved D boxes 90. Positions targeted by CD1, CD7 and CD19  must have taken place, as it requires the accessibility of the target sequences. We also cannot exclude that CD13 binds its targets after they reach proximity . Also, some box C/D snoRNAs are involved in the processing of precursor rRNA molecules in a variety of organisms (summarized in Ref.87). While 2′-O-Me in tRNA is usually introduced by specialized stand-alone methyltransferases, e.g. Ref.93, certain positions are also guided by specific box C/D snoRNAs (reviewed in Ref.16), either alone or together with a dedicated box C/D scaRNA, like in the case of the wobble cytidine 34 of human tRNAMet94. Further functions that are conceivable also for D. discoideum box C/D snoRNAs encompass rRNA acetylation95, regulation of 3′ pre-mRNA processing97 or even the generation of small, sno-derived RNAs that might have regulatory functions, as described for other organisms100. Future work will show whether these possible functions are realized in D. discoideum by any of the OR RNAs or those CD RNAs, in which one antisense sequence lacks an identified rRNA target.We noted that a substantial set of 22 box C/D snoRNAs are differentially accumulated in the development of the amoeba compared to axenic growth, however, without manifesting in altered 2′-ons Fig. . This inSupplementary Information.


Coronaviral methyltransferases (MTases), nsp10/16 and nsp14, catalyze the last two steps of viral RNA-cap creation that takes place in cytoplasm. This cap is essential for the stability of viral RNA and, most importantly, for the evasion of innate immune system. Non-capped RNA is recognized by innate immunity which leads to its degradation and the activation of antiviral immunity. As a result, both coronaviral MTases are in the center of scientific scrutiny. Recently, X-ray and cryo-EM structures of both enzymes were solved even in complex with other parts of the viral replication complex. High-throughput screening as well as structure-guided inhibitor design have led to the discovery of their potent inhibitors. Here, we critically summarize the tremendous advancement of the coronaviral MTase field since the beginning of COVID pandemic. Coronavirinae of the family Coronaviridae, order Nidovirales, which belong to the realm Riboviria. We can distinguish four genera of CoVs which are named according to the Greek alphabet: Alpha-CoV, Beta-CoV, Gamma-CoV and Delta-CoV. Whereas Alpha-CoVs and Beta-CoVs contain a large number of mammal and human pathogens, their Gamma and Delta siblings are mostly avian, although some of them can be found in cetaceans and pigs, respectively (Coronaviruses (CoVs) are important human and animal pathogens that belong to positive-sense single-stranded RNA (+RNA) viruses. CoVs refer to members of the subfamily ectively .Currently, only seven coronaviruses are known to infect humans, causing a wide range of disease severity. Human coronaviruses (HCoVs) HCoV-229E and HCoV-NL63 belong to Alpha-CoVs, and they are mostly responsible for milder forms of upper respiratory illnesses. However, both of these viruses have been implicated in the pathogenesis of rather severe diseases such as pneumonia, usually in immunocompromised patients  that are essential for the installation of the viral RNA cap, with a special focus on their potential as molecular targets for future therapeutic intervention. Therefore, a significant portion of this review article is devoted to structural studies of these enzymes and to already known inhibitors that may serve as starting points for the preparation and optimization of therapeutic compounds.Eukaryotic mRNA possesses a special chemical structure, called a cap, on its 5′ end. This RNA cap is important for at least four reasons: (i) mRNA-splicing, (ii) mRNA-stability, protecting its 5′ end from premature degradation by exonucleases, (iii) mRNA transport from nucleus and (iv) efficient mRNA translation ,5.7Gppp-pre-mRNA. This structure is referred to as cap-0 and it is the final product in lower eukaryotes such as the budding yeast. However, in humans, the cap-0 is further methylated by the cap-specific mRNA MTase 1 (CMTR1) at the 2′-O-ribose position of the first nucleotide giving rise to the cap-1 which can be further methylated by CMTR2 at the 2′-O-ribose position of the second nucleotide yielding a cap-2  RNA  and by the nsp12 NiRAN (Nidovirus RdRp associated nucleotidyl transferase) domain (GTP transfer) (O-MTase (nsp10/nsp16 complex) carries out the next methylation of the 2′ OH ribose group of the first nucleotide resulting in cap-1  RNA  and lateMHV) RNA . The mosMHV) RNA . Until rMHV) RNA , howeverMHV) RNA . Yan et in cap-1 . This prnsp10/nsp complex ficiency ,18. Howeficiency ,18 suggeab initio mechanism when a DNA-dependent RNA-polymerase uses them as a substrate  and IFIT (interferon-induced protein with tetratricopeptide repeats) . mRNAs bO-MTase nsp16   and the induction of an anti-viral state. It is perhaps not surprising that viruses have evolved various intriguing mechanisms to protect and/or hide their RNAs . Installtein 16) ,15,31–35O-MTase is the nsp16 protein. However, it would be more accurate to describe the 2′-O-MTase as a heterodimeric protein complex composed of the catalytic subunit nsp16 and the activating subunit nsp10 because the nsp16 is inactive unless bound to this small activating protein  binding site  bound. This study provided a nice confirmation of previous studies based on a single frozen crystal. The structures obtained at room temperature were almost identical, and revealed the movement of the two gate loops (AAs 28–35 and 131–146). This conformational change leads to the formation of a hydrogen bond between Tyr30 and Lys137 which is necessary to create the cap-0 binding site  and requires a divalent ion, Mgated RNA . The actO-MTase is well conserved among coronaviruses, the similarity of the overall fold with other 2′-O-MTases from +RNA viruses from different orders such as the Zika virus  is rather small  activity. Each activity is associated with one of its two domains: the N-terminal domain bears the ExoN and the C-terminal domain bears the MTase activity. These two domains are connected with a small hinge region which is actually located within the MTase domain  Figure . Small-aof nsp14 . This coof nsp14 . Fortunaof nsp14 ,50.Both enzymatic activities of nsp14 are being considered as targets for antivirals. In addition, due to high conservation of nsp14 between individual coronaviral species , nsp14 iThe ExoN activity of nsp14 is responsible for proofreading, an unusual feature in the realm of +RNA viruses . Due to The N7-MTase domain of nsp14 is located at its C terminus and, interestingly, the N-terminal ExoN domain is important for its enzymatic activity . Howeverin silico docking studies are emerging. One postulated model based on in silico docking studies proposed by Perry et al. suggests that the RTC could be formed by a large protein assembly (2), the capping and ExoN enzymes (nsp14/nsp16/(nsp10)2), the helicase (nsp13), nsp9 and the nucleocapsid (N) protein . However, given the rather large distance between the NiRAN and MTase active sites, it is quite possible that another co-transcriptional capping complex is involved in cap-0 synthesis (trans mechanism). The ExoN catalytic site is located ∼80 Å away from the polymerase active site and 120 Å away from the dsRNA exit site, implicating the trans mechanism of proofreading . These compounds are usually derived from SAM or SAH by a slight modification of their chemical structure. The second group is composed of several derivatives of an RNA cap that can inhibit either nsp14 or nsp16. Next, a small group of compounds acts as inhibitors of protein-protein interaction between nsp16 and its crucial cofactor nsp10. Finally, there are a number of compounds that have been identified in high-throughput screens against either nsp14 or nsp16 where the mechanism of action is not entirely clear.Although the importance of these enzymes has been known for almost two decades ,74, seleThe use of SAM as a methylation agent is not specific to viruses; SAM is also used by many cellular enzymes. Therefore, there is a need to develop SAM analogues specific for viral MTases. Although achieving high selectivity is always difficult, the binding site for SAM has been already exploited for the design of novel human MTase inhibitors and it has resulted in several promising selective drug candidates . From a There are two naturally occurring compound that highly resemble a SAM molecule that have been used as a standard control for most of the MTase inhibitory assays: SAH and sinefungin. Both of these compounds can be regarded as amino acid residue modifications.Streptomyces griseolus and presented as an antifungal agent (50 = 16 μM) and nsp10/nsp16 (IC50 = 12 μM) in comparison to sinefungin . Aouadi et al. showed that these two compounds can also inhibit nsp10/nsp16 from MERS-CoV  even at submicromolar concentrations. The authors were able to show that these inhibitors do not inhibit human N7-MTase (RNMT), however, selectivity towards other human MTases has not been studied , whereas non-methylated analogues were inactive ,79. Alth2 , SARS-CoV and MERS-CoV together with P29 resulted in a significant inhibition of MTase activity by >50%. For P29 to be effective in cells, it was necessary to fuse P29 with the Tat protein from HIV (TP29), as the Tat allowed the peptide to cross the cytoplasmic membrane. The cell culture was infected with MHV strain A59 (MHV-A59) and TP29 was added one hour after infection. Analysis after 20 hours showed an 80% lower viral titer compared to the control. The authors think that the peptide inhibits the formation of the nsp10/nsp16 protein complex, leading to inhibition of genome replication and deficiency of caps on RNA. Consequently, without the cap, proper translation does not occur, and the cytosolic receptors of cap-0 mRNA trigger a cellular response in the form of interferon production. The advantage of using TP29 is that it targets a structure that is specific to coronavirus nsp16 and therefore the treatment should result in lower incidence of side effects.Wang and colleagues confirmed this using a peptide (P29) that consisted of a segment of nsp10 (amino acids 68–96) from the murine hepatitis virus (MHV) . In vitrSeveral high-throughput screening (HTS) campaigns against both nsp10/nsp16 and nsp14 leading to a number of structurally diverse hit compounds has been reported recently.10, 11 and 12 , ExoN activity and the helicase (nsp13) were reported recently ,90–99. Pgkab1279_Supplemental_FileClick here for additional data file.


Mycobacterium smegmatis in apoprotein and cofactor-bound forms. Based on structural analysis and mutagenesis, we identified several catalytically critical, positively charged residues at a putative RNA-binding site. We found that mutation of any of these sites is sufficient to abolish methylation activity, whereas the corresponding RNA-binding affinity of Erm38 remains unchanged. The methylation reaction thus appears to require a precise ensemble of amino acids to accurately position the RNA substrate, such that the target nucleotide can be methylated. In addition, we computationally constructed a model of Erm38 in complex with a 32-mer RNA substrate. This model shows the RNA substrate stably bound to Erm38 by a patch of positively charged residues. Furthermore, a π-π stacking interaction between a key aromatic residue of Erm38 and a target adenine of the RNA substrate forms a critical interaction needed for methylation. Taken together, these data provide valuable insights into Erm–RNA interactions, which will aid subsequent structure-based drug design efforts.Erythromycin resistance methyltransferases (Erms) confer resistance to macrolide, lincosamide, and streptogramin antibiotics in Gram-positive bacteria and mycobacteria. Although structural information for ErmAM, ErmC, and ErmE exists from Gram-positive bacteria, little is known about the Erms in mycobacteria, as there are limited biochemical data and no structures available. Here, we present crystal structures of Erm38 from  One major mechanism of AMR in Gram-positive and mycobacterial pathogens affects three broad classes of front-line antibiotics — macrolides, lincosamides, and streptogramins (MLS) — and is conferred by a family of horizontally and vertically transmitted genes that encode the so-called erythromycin resistance methyltransferases (Erms)  in virtually every clinically important bacterial pathogen represents a true crisis with major societal and economic impact  (https:/s (Erms) , 4, 5, 6s (Erms) . Horizonbscessus , 9, 10. B, and C , which germ genes, such as erm37 in M. tuberculosis, erm38 in Mycobacterium smegmatis, and erm41 in M. abscessus, as well as ermE of Saccharopolyspora erythraea, are genomically encoded, whereas the erm genes of Gram-positive bacteria, including ermA and ermC of methicillin-resistant S. aureus and ermB of vancomycin-resistant enterococci, are found on highly mobile plasmids involved in horizontal gene transfer. In terms of size, Erms A, B, and C share similar molecular weights (∼30 kDa), whereas Erm37 and Erm41 are smaller (∼20 kDa) because of the lack of a C-terminal domain  (1QAO) (6NVM)  , ErmC (P) (1QAO) , 16, and) (6NVM) . However) (6NVM) , and Ermfections . Here, wEscherichia coli resulted in a heterogenous population of partially degraded proteins with molecular weights ranging from 30 to 45 kDa, in spite of length variations , with analysis using the Database of Disordered Protein Predictions (riations . The comriations . The higriations  that difA). Generally, Erm38 shares highly a similar structural fold with all published Erm structures and KsgA, a 16S rRNA methyltransferase (B) reveals that both structures are highly similar with RMSD of 2.3 Å for 237 superimposed alpha carbon atoms, despite sharing only 22% sequence identity. Compared to ErmC, the N-terminal region of Erm38 has a longer α1 helix and longer loops connecting α3–β4 and α5–α6. In the C-terminal helical domain, Erm38 possesses a longer α8 helix but shorter α9 and α10 helices, along with an extra 310 helix-labeled η5.Erm38 features at its N-terminal catalytic domain the classic Rossmann fold, an α/β sandwich that contains the SAM cofactor-binding site and a helical C-terminal domain A. Generansferase . A compansferase B revealsB). This was expected given the high chemical homology between SAM and sinefungin. The SAM-binding pocket of Erm38 was also compared with ErmC (A and C). Despite sharing only 22% sequence identity, residues at the SAM-binding pocket are highly conserved between Erm38 and ErmC proteins with minor differences found at V62/I60, A41/S39, and F93/I85, whereas residues F15, G40, G42, E61, D92, N108, and P110 are identical for the two enzymes. Particularly, G40, G42, E61, D92, and P110 of Erm38 are strictly conserved among Erms A, B, C, E, 37, 38, and 41  to cause full MLS . As these solvent-exposed basic residues are likely to be involved in binding the RNA substrate, we produced an alanine mutant for each site and compared the enzymatic activities of the mutant and WT enzymes. R31A was selected as a negative control based on the fact that it is distant from the SAM- and RNA-binding sites, and we concluded that it does not participate in any intramolecular interaction needed to maintain the fold of the protein. E61K is known to reduce methylation activity by disrupting the SAM-binding site . Surprisingly, the RNA-binding profiles for WT and mutants were similar, with an interaction model was best represented by 2:1 heterogeneous binding and two dissociation constants (KD)  . The micE. coli 23S rRNA) lying in the vicinity of F111 of Erm38, an aromatic residue known to interact with the adenine to facilitate methylation  simulation to further refine the binding interface. 3FTF). The Erm38–RNA complex is stable in the 10-ns simulation, whereas this alternate model lacks the π-π stacking interaction and it does not interact with R140 of Erm38, which was shown to be important according to our mutagenesis data  that resulted in complete loss of erythromycin resistance, whereas mutations R112A and K133A in ErmC  reduced erythromycin resistance. These three amino acid positions are highly conserved across all seven Erms compared in this study . This rules out X-ray crystallography, cryo-EM, and solution-state NMR techniques for structure determination. Solid-state NMR offers a solution to analyze the precipitate, whereas the 40 kDa size of the Erm38–RNA complex poses a problem for NMR resolution.et al.  was produced by the NTU Protein Production Platform (Singapore). The truncated Erm38 is referred to throughout as Erm38. Site-directed mutagenesis was performed on Erm38 to introduce the single mutations R31A, R119A, R140A, R141A, R142A, and K166A, using primers listed in E. coli BL21(DE3)-T1R Rosetta strain, Erm38 proteins were expressed by growing in LB broth containing 34 μg/ml chloramphenicol and 50 μg/ml kanamycin at 37 °C, with expression induced with 0.2 mM IPTG for 18 h at 16 °C. Cell pellets were lysed by sonication in 50 mM Na Hepes pH7.5, 500 mM NaCl, 10% glycerol, 0.5 mM DTT, and 10 mM Imidazole. After removal of cell debris by centrifugation at 16,000g, the supernatant was sterile filtered and subjected to a series of purification steps. For WT Erm38, high-purity protein was critical for crystallization studies, which necessitated a three-step purification involving immobilized metal affinity chromatography (IMAC), cation-exchange chromatography, and size-exclusion chromatography  on Intelli-plates 96-3 (Art Robbins Instruments) , 27, 28.3FTF and 3FUV as search models. Several rounds of manual model building were then performed using program COOT  were determined by plotting the SAH concentration as a function of RNA concentration tested and the data were fitted by nonlinear regression with GraphPad Prism version 9 .All assays were performed in 10 μl total volume in a 384-well plate . Erm reaction kinetics were quantified using the MTase-Glo methyltransferase assay kit (Promega), which detects the SAH product of SAM demethylation in a coupled luminescence reaction , 40 that1 mM DTT , 42. To d values for each Erm38 protein, a reference sensor with loaded RNA but no protein was subtracted from the data before fitting. Curve fitting using a 2:1 interaction model was used to measure the dissociation constant (KD) for WT and mutants of Erm38.The binding affinities of WT, R119A, and R130A Erm38 proteins with the 32-mer RNA substrate were measured by biolayer interferometry using an Octet RED96e (ForteBio), with binding data acquired (kinetics mode) and analyzed using built in software. Biosensors were hydrated in phosphate-buffered saline with Tween (PBST) buffer  for 20 min at ambient temperature preceding data acquisition, and experiments were performed at 25 °C. Erm38 proteins were diluted in PBST buffer. A biotinylated 32-mer RNA substrate was synthesized (Sigma-Aldrich) with biotin appended at the 3′-end of the oligo. The biotinylated RNA was diluted to 100 nM and immobilized on a streptavidin-coated biosensor with a signal threshold of 0.2 nm. A baseline level was established for 60 s before Erm38 proteins were exposed to RNA-loaded biosensors for 120 s and dissociated in PBST buffer for 120 s. To determine KE. coli numbering) pointing outwards, which makes this RNA model primed to interact with the methylation site of Erm. Docking of Erm38 and 32-mer RNA was performed using HDOCK 2, where k is 1 kcal mol−1 Å−2 and xref is the initial atom coordinates. Finally, 20 ns production simulations were performed without constraints. The simulation was performed under the NPT ensemble assuming the CHARMM36 force field , 7F8B (Erm38 complexed with SAM), and 7F8C (Erm38 complexed with SFG).This article contains The authors declare that they have no conflicts of interest with the contents of this article.


CCND1/IGH), usually resulting in cyclin D1 overexpression. In some cases, particularly at low levels of disease, it can be morphologically challenging to distinguish between these entities in the bone marrow (BM) since PCN with t are often CD20‐positive with lymphoplasmacytic cytology, while MCL can rarely have plasmacytic differentiation. We compared the difference in CCND1/IGH by fluorescence in situ hybridization (FISH) in PCN and MCL to evaluate for possible differentiating characteristics. We identified 326 cases of MCL with t and 279 cases of PCN with t from either formalin‐fixed, paraffin‐embedded tissue or fresh BM specimens. The “typical,” balanced CCND1/IGH FISH signal pattern was defined as three total CCND1 signals, three total IGH signals, and two total fusion signals. Any deviation from the “typical” pattern was defined as an “atypical” pattern, which was further stratified into “gain of fusion” vs “complex” patterns. There was a significantly higher proportion of cases that showed an atypical FISH pattern in PCN compared with MCL . There was also a significantly higher proportion of cases that showed a complex FISH pattern in PCN compared with MCL . We confirmed these findings using mate‐pair sequencing of 25 PCN and MCL samples. PCN more often have a complex CCND1/IGH FISH pattern compared with MCL, suggesting possible differences in the genomic mechanisms underlying these rearrangements in plasma cells compared with B cells.Plasma cell neoplasms (PCN) and mantle cell lymphoma (MCL) can both harbor t ( Data including the final diagnosis, number of individual CCND1 and IGH signals, and fusion signals were collected. Thirty‐four consult cases in which the final diagnosis was unavailable were reviewed by a hematopathologist (JCD) to confirm the pathologic diagnosis.This study was approved by the Mayo Clinic Institutional Review Board. The Mayo Clinic Genomics laboratory database from 2007 to 2018 was retrospectively reviewed to identify all cases of PCN and MCL investigated by FISH. Cases positive for 2.2CCND1/IGH, a break‐apart probe set for MYC (8q24.1) rearrangement, and an enumeration probe for TP53 deletion or monosomy 17 (centromere 17/TP53) were utilized . The IGH probe is labeled with Spectrum Green (Abbott Molecular) and the CCND1 probe is labeled with Spectrum Orange (Abbott Molecular); the imaging of the Orange fluorophore is herein referred to as Red. A total of 100 or 200 interphase nuclei were evaluated in each case, with 50 or 100 nuclei evaluated independently by two qualified clinical cytogenetic technologists and interpreted by a board‐certified  clinical cytogeneticist. The “typical,” balanced CCND1/IGH FISH signal pattern was defined as three total CCND1 signals (red), three total IGH signals (green), and two total fusion signals (yellow) or 1R1G2F  or fresh BM specimens using standard FISH pretreatment, hybridization, and fluorescence microscopy protocols.F Figure . Any devF Figure . The per2.3Mate‐pair sequencing (MPseq) was performed on a subset of 16 PCN and 9 MCL cases. DNA extraction and mate‐pair library preparation methods have been previously described.3CCND1/IGH FISH complexity between MCL and PCM, we analyzed the CCND1/IGH FISH patterns for 326 cases of MCL with CCND1/IGH  and 279 cases of PCN with CCND1/IGH . A variety of FISH patterns were observed, including balanced translocations, the gain of fusion, loss of fusion, and a combination of abnormalities    Table . These dCCND1/IGH FISH complexity was associated with an increased incidence of TP53 deletion, MYC rearrangement, or tetraploidy.TP53 deletion is associated with increased genomic complexity and poorer outcome in both PCN and MCL, including an association with the more highly proliferative blastoid and pleomorphic variants of MCL.TP53 deletion was identified in six cases (32%) of MCL and eight cases (3.2%) of PCN  was not considered significant (P = .4182). MYC rearrangements can also occur as a secondary cytogenetic abnormality and contribute to progression in PCNMYC rearrangement was identified in 3 cases of MCL (11%) and 19 cases of PCN (7.8%)  was also not considered significant (P = .6571). A tetraploid clone, reported to be more common in pleomorphic and blastoid variants of MCL,CCND1/IGH FISH subtypes  (P = 1)  Table . Similar1) Table .CCND1 breakpoint locations revealed that the breakpoints of the t translocation in MCL were located in a region closer to the CCND1 gene  compared with PCN  suggests differences in the genomic mechanisms underlying these rearrangements in PCs compared with B cells. The breakpoint in CCND1 typically occurs at the 5′ end (centromeric) in both PCN and MCL, with an increased frequency of breakpoints clustered near the major translocation cluster in MCL. However, the IGH breakpoint has been shown to be different in MCL vs PCN.IGH and frequently involves the IGHV3‐23 and IGHV4‐59 genes.IGH switch region.IGH segments, duplications and losses of the IGH gene on the normal non‐translocated chromosome 14 as well as IGH/CCND1 fusion on der (14) and CCND1/IGH fusions on der (11).We show that PCNs have a significantly higher propensity to have atypical and complex IGH rearrangements in MCL appeared to be due to aberrant VDJ recombination (RAG1/2 mediated), while in PCN IGH rearrangements appeared to be due to aberrant class switch recombination (AID mediated).CCND1 and IGH observed in PCN cases could be described as templated insertions, a previously reported complex event found in about 20% of plasma cell myeloma cases.Previous studies showed that cyclin D1 leading to the 870G > A polymorphism has been reported to be associated with a risk of t PCM and AL amyloidosis.CCND1/IGH positive cases were more likely to have TP53 deletions or MYC rearrangements; however, our sample size was small and, our findings were not considered significant. We also investigated the presence of tetraploid clones; however, there was also no significant difference in the association of tetraploidy with the complex CCND1/IGH positive cases. In our cohort, only one PCN case with t amplification was identified, while no MCL cases with amplification were observed. It is noted that previous studies have shown that amplification can also be seen in MCL,TP53 deletions and MYC rearrangements and the absence of clinical outcome for all cases, limiting evaluation of the prognostic significance of FISH complexity in PCN and MCL. Similar to our FISH results, our MPseq data also show that PCN demonstrates a more complex t pattern in comparison to MCL. However, the sample size tested by MPseq was also small. At the time of this study, we did not have the ability to perform MPseq on FFPE samples and required fresh tissue for testing, thus limiting MCL case selection for MPseq.The single nucleotide polymorphism rs603965  occurring at the splice site of CCND1/IGH FISH patterns compared with MCL. While we initially sought to determine if this could potentially be used to aid in the diagnosis of challenging cases, despite the significant difference we observed, there remains significant overlap with a subset of MCL cases associated with a complex FISH pattern, thus limiting the application of these findings for diagnostic purposes. Future studies to evaluate whether there is any association of genomic complexity to clinical outcome may be useful to determine if this observation may be of prognostic significance.Our studies indicate that PCNs have a significantly increased frequency of atypical and complex Shaji Kumar served as a consultant for Celgene, Takeda, Amgen, Janssen, and Bristol‐Myers Squibb and received research funding from Celgene, Takeda, Novartis, Amgen, AbbVie, Janssen, and Bristol‐Myers Squibb. The remaining authors declare no competing financial interests.SUPPLEMENTARY FIGURE 1 Reconstruction of complex t translocation in PCN. Partial reconstruction of 8 samples of PCN that had a gain near CCND1 resulting in gain of the IGH/CCND1 fusion. P1 in A, P2 in B, P4 in C, P6 in D, P7 in E, P9 in F, P10 in G and P14 in H. Due to the complexity and existence of subclonal variation in the rearrangement in patient 2, what is depicted is one possible solution to the rearrangement. The actual rearrangement is more complex and varied in structure. In patient 4, both sides of the t balanced junction have evidence of complexity. What is depicted is the portion of the complex rearrangement which includes two copies of the CCND1 gene and two copies of the IGH/CCND1 fusion. Due to the complexity and existence of subclonal variation in the rearrangement in patient 6, what is depicted is one possible solution to the rearrangement. The actual rearrangement is more complex and varied in structure. In patient 9, due to the telomeric nature of the chr2 templated insertion, the connection between chr2 and chr22 is extrapolated from copy number changes. For this reason, this is a plausible rearrangement, however possibly a simplified version of the actual rearrangement. It is possible that the actual rearrangement is more complex than depicted. The complexity in patient 10 is subclonal H. Like the other complexities this rearrangement in patient 14 results in a copy of the t. However, unlike the others, this does not result in a copy of CCND1. Instead the complexity jumps to the MYC region of chr8 and would bring IGH in proximity to both MYC and CCND1 in the course of the complex rearrangement. Zoomed in regions of the chromosomes show IGH and CCND1 in relation to the rearrangement. Other genes in the regions are unlabeled and depicted as blue and red boxes if they are on the forward or reverse strand of the chromosomes, respectively. Green lines show the path of reconstruction. Dashed lines denote junctions that connect discordant regions of the genome. Double lines denote regions or junctions that are passed through twice in the reconstruction and thus indicate regions/junctions that have been gained and are depicted twice in the derivative chromosomes. If no lines overlap a region, this indicates an area of loss and these regions are not depicted in the derivative chromosomesClick here for additional data file.


The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome. In almost all eukaryotic cells, mitochondria are essential organelles that are mainly known for their role in ATP production via oxidative phosphorylation (OXPHOS). OXPHOS is carried out by the electron transport chain (ETC), which consists of a series of inner membrane-embedded electron transporters forming complexes I to IV and by the ATP synthase, or complex V, which generates ATP from ADP and inorganic phosphate . In mammAll these complex processes do not occur in membrane limited submitochondrial compartments. Instead, mitochondria have developed specialised nucleic acid-protein rich domains that are key sites in the organization of mitochondrial gene expression . The mosIn 2004 Iborra et al.  reportedUntil now, the precise identity of the transcripts present in the MRGs has not been revealed, and it would be interesting to know whether polycistronic, immature transcripts occupy the same or a different location as the mature transcripts ready for translation. A recent study by Zorkau et al.  has suggIn addition, recently, using the J2 antibody, which specifically recognizes double-stranded RNA (dsRNA) , Dhir etAs expected, a large number of proteins were found to localize within MRGs. Although previously characterized as a cytoplasmic poly(A)+ mRNA binding protein that is preferentially bound to RNA with a G-rich element , GRSF1 iIn addition to FASTK family members, several enzymes involved in mtRNA processing and maturation, as well as components of the mitoribosomes, were found to localize, at least in part, in MRGs ,32,33,34As mentioned above, the proteome of MRGs suggests that these structures display complex functions, which are likely to be distributed in specialized subcompartments to ensure efficacy. How MRGs are compartmentalised is a key question that can be addressed using super-resolution microscopy. Using such an approach, the diameter of MRGs, when stained for its proteins GRSF1 or FASTKD2, was found to measure ~150 nm, and when assessed using BrU staining, their size was found to be ~100 nm. The core of the MRGs would consist of compacted RNA, enveloped by RNA binding proteins (RBPs) . The preIn mammalian cells, granules composed of RNA complexed with RBPs are found in homeostatic and stress-induced conditions. Processing bodies  and streRey et al.  have recRNA has been found to be essential for the formation of MRGs A. IndeedRecently, Rey et al.  observedInhibition of mitochondrial fission using the overexpression of a dominant-negative mutant of the fission factor dynamic related protein 1 (Drp1_K38A) led to the formation of ‘mitobulbs’ which are enlarged domains ballooning out of an elongated network. Using super-resolved stimulated emission depletion (STED) microscopy and correlative fluorescent and electron microscopy, both nucleoids and MRGs were found to form clusters of several MRGs, similar to a bunch of grapes A. HoweveIn another recent study, Kleele et al.  studied In 2018, Dhir et al. identified dsRNA molecules forming discrete foci along the mitochondrial matrix, with the use of the J2 antibody, which recognizes dsRNA of lengths > 40 nucleotides, irrespective of its sequence . ImportaDrosophila melanogaster (Dm) models silenced for the Dm orthologs of SUV3, PNPase, LRPPRC and mtPAP. Their results indicated that the absence of DmPNPase, DmSUV3 and, surprisingly, DmmtPAP caused an accumulation of mt-dsRNA foci in the cytosol of dissected brains from Dm larvae. As yet, LRPPRC, SLIRP and mtPAP in mammalian models and their effects of mt-dsRNA as visualized by the J2 antibody have not been studied and are interesting research questions.The binding of RNA binding proteins, during, or just after, transcription could also potentially prevent the formation of long dsRNA transcripts. Other possible RBP candidates for the regulation of mt-dsRNA are leucine-rich PPR motif-containing protein (LRPPRC), stemloop interacting RNA-binding protein (SLIRP) and mitochondrial poly(A) polymerase (MTPAP). Mutations in LRPPRC gene are responsible for the neurodegenerative disease Leigh Syndrome ,61. LRPPEscherichia coli, the degradosome machinery of mt-RNA in Saccharomyces cerevisiae termed mtEXO is the most well studied in eukaryotes. The mtEXO of S. cerevisiae is made up of the ATP-dependent RNA helicase, SUV3 and the exoribonuclease, DSS1 [The degradosome is another specialised domain within the mitochondrial matrix, which has been suggested to be the site of mt-RNA degradation. The existence of the degradosome is highly conserved from bacteria to humans and is crucial in RNA surveillance and degradation . Althougse, DSS1 .The human degradosome similarly consists of the evolutionarily conserved human orthologs: SUV3 and PNPase. SUV3 and PNPase form a complex that was visualized as foci within the mitochondrial network ,45. ThisAs mentioned above, transcription of the cytosine-rich L strand of the mitochondrial genome results in RNAs that are guanine-rich (G-rich). These transcripts are mostly non-coding except for MT-ND6, which is the most G-rich sequence of all the mitochondrial coding transcripts . G-rich Most recently, REXO2, a 3′ to 5′ exonuclease, has been reported to localise to mitochondria , and itsThe degradosome has also been shown to participate in the proper functioning of mt-DNA by preventing the accumulation of mitochondrial R loops . R loopsAs mentioned above, the dsRNA content of mitochondria is regulated by the activity of the mitochondrial degradosome, consisting of the SUV3-PNPase protein complex . The losAs mentioned previously, REXO2 is considered to act downstream of the SUV3-PNPase complex to degrade nanoRNA sequences that are smaller than 4 nucleotides. Szewczyk et al.  found thIn this review, we have highlighted the different membraneless RNA enriched granules of the mitochondria that delineate the states of processing and degradation of mt-RNA. We have also detailed how the overall mitochondrial state including the proper functioning of the nucleoids, the various mitochondrial proteins and the network dynamics have an impact on the maintenance of the RNA granules relative to their number, size and arrangement within the mitochondrial matrix. The field of mt-RNA granules is relatively young, and as of now, the majority of its study is based on in vitro experiments. Furthermore, many of the results have been obtained using cancer cell lines cell lines, and it has been reported that these structures are barely detectable in primary fibroblasts. This of course raises the question of the MRG status in non-cancer cells, such as neurons or cardiomyocytes, which are highly depend on mitochondria for their metabolism. The study of MRGs in these cells would be of key interest. Furthermore, as MRGs are sites of a high number of critical reactions involving essential enzymes, it would also be interesting to study whether pathologies associated with mutations in enzymes involved in RNA processing, degradation or in ribosome assembly lead to perturbations of the physical and structural properties of MRGs.


The progress of the cell cycle is directly regulated by modulation of cyclins and cyclin-dependent kinases. However, many proteins that control DNA replication, RNA transcription and the synthesis and degradation of proteins can manage the activity or levels of master cell cycle regulators. Among them, RNA helicases are key participants in RNA metabolism involved in the global or specific tuning of cell cycle regulators at the level of transcription and translation. Several RNA helicases have been recently evaluated as promising therapeutic targets, including eIF4A, DDX3 and DDX5. However, targeting RNA helicases can result in side effects due to the influence on the cell cycle. In this review, we discuss direct and indirect participation of RNA helicases in the regulation of the cell cycle in order to draw attention to downstream events that may occur after suppression or inhibition of RNA helicases. Cell division can be formally presented as a series of coordinated events that form the cell cycle. The transition of the cell cycle through all phases is determined by the interaction of cyclin-dependent kinases (CDK) and CDK-bound proteins with cyclins . The intDisturbances in the cell cycle can result in the development or enhancement of many pathophysiological conditions—cancer, ischemia/reperfusion injury, atherosclerosis, inflammation and neurodegeneration . VariousThere are numerous reports indicating that RNA helicases are involved in the regulation of transcription, translation, splicing and RNA transport. Thus, the participation of RNA helicases in the main processes of RNA metabolism makes them important indirect regulators of the cell cycle . RNA helThe cell cycle is a sequential one-way process that guides the cell through initial growth (G1 phase), DNA replication (S phase), another growth period (G2 phase) and finally segregation of the chromosomes into two new nuclei (M phase), followed by cell division resulting in the birth of daughter cells. These major cell cycle events are triggered by CDKs controlled by cyclin binding and/or phosphorylation. Each stage of the cell cycle is controlled by a set of specific CDKs and cyclins A. CDK1 fHowever, the cell cycle is not just a mechanical automated cycle—before moving to the next phase, the cell must go through checkpoints to ensure that each stage is fully completed without any defects. In general, checkpoints include a sensor that monitors defects at the stage of the cell cycle, signal transducers and an effector that inhibits the transition of the cell cycle if something goes wrong ,4,22. DuRNA helicases unwind duplexes and stem-loops in RNA, thereby introducing structural changes into the RNA and RNA–protein complexes and switching their activity using NTP as an energy source ,35. RNA RNA helicases belong to the main superfamilies SF1 and SF2, which are also subdivided into families based on their motif composition. The catalytic cores of SF1 and SF2 helicases share almost identical folds and significant structural similarity. The presence or absence of a specific motif determines the functions of RNA helicases. The central structural element of SF2 superfamily is formed by two RecA-like domains carrying conservative motifs important for these RNA helicases. Motifs I, II, V and VI are required for binding and hydrolysis of a nucleoside triphosphate; motifs Ia, Ib and IV are involved in RNA binding; and the motif III participates in coupling of ATPase and unwinding activities B. In theThe main role of DDX3 helicase from the DExD protein family is the regulation of translation. DDX3 represses cap-dependent translation by trapping eIF4E into a translationally inactive complex ,48 or biHelicase DDX46 is involved in pre-mRNA splicing  and innaDDX6 interacts with several protein complexes and regulates the mRNA life cycle and translation rate. DDX6 can interact with decapping  and translational machinery, such as eIF4E; translational repression factors ; and other RNA-binding proteins (RBPs), such as YBX1, IGF2BP2, FXR1, polyA-binding and ribosomal proteins. The association of DDX6 with EDC3 reduces stability of KLF4 mRNA , an esseHelicase DDX21 is involved in the processing of ribosomal RNA through interaction with 45S and 32S precursors and regulates 18S and 28S rRNA levels in the cell ,71. DDX2DDX51 binds to pre-60S subunit complexes and facilitates the displacement of U8 snoRNA from pre-rRNA, which is necessary for the removal of the 3′ external transcribed spacer from 28S rRNA and productive downstream processing . DDX51 iSnail1 gene by displacing histone deacetylase from the Snail1 promoter [Cdkn1a (p21). DDX5 also binds to a number of cyclin mRNA transcripts and influences its nuclear export and stability [WTIP [CDKN1, and, conversely, cyclin D1 in a different context [DDX5 locus. In addition, this data supports the idea that DDX5 is highly context sensitive presumably due to post-translational modifications or specific rules. Thus, DDX5 may not be an effective universal therapeutic target. Furthermore, simultaneous delivery of inhibitors to healthy cell can induce toxic or off-target effects.DDX5  is one of the prototypic members of the DEAD box family of RNA helicases. DDX5 and related DDX17 (p72) are involved in a variety of cellular processes, including transcription, pre-mRNA and rRNA processing, alternative splicing and miRNA processing, and they are also deregulated in a range of cancers ,81,82,83promoter , which itability . DDX5 inty [WTIP . On the ty [WTIP . DDX5 coty [WTIP ,97. Howe context , suggest context . Thus, DMMP9, MMP14 and PLAU genes involved in the regulation of cancer cell invasion and migration [cyclin A2, cyclin B2, cyclin E2, MCM4, MCM7, cdc6, cdc20 and E2F1. Expression of DHX33 is necessary for continuous cell proliferation as it promotes cell cycle progression at the G1/S, G2/M and metaphase–anaphase transitions [MCM2, MCM4, CDC26, CCNB2, CCNE1, CCNE2 and CCND3 [cdc6, cyclin D1 and Ki-67. Thus, DHX33 contributes to the regulation of various aspects of cell proliferation and migration during cancer development. Hence, the exact molecular mechanism remains to be revealed [DHX33 interacts with the architectural protein UBF and indirectly regulates RNA polymerase I-mediated transcription and rRNA synthesis ; promoteigration ; and parigration . In addinsitions . DHX33 insitions . In addind CCND3 . DHX33 prevealed .DDX41 acts as an intracellular DNA sensor in myeloid dendritic cells via the STING-TBK1-IRF3 pathway . BesidesHelicase eIF4A is a canonical protein of the translation initiation process that unwinds long and complex secondary structures in the 5′-UTR, which are common for many eukaryotic mRNAs . In addiDHX9 mediates binding of the epidermal growth factor receptor (EGFR) with the cyclin D1 promoter, which stimulates its transcription activation . DHX9 alWEE1, a G2–M cell cycle checkpoint. Hence, the molecular mechanism of WEE1 abnormal splicing of regulation by DDX56 remains unknown [RNA helicase DDX56 is overexpressed in different cancer types  and leads to a poor prognosis. DDX56 can promote cell proliferation by inducing oncogenic splicing alteration in a cell cycle checkpoint gene,  unknown . Inhibit unknown . RNA helicase UAP56/DDX39B participates in the resolution of the nonscheduled R loops, representing a major source of DNA damage and replication stress in human cells. UAP56 depletion causes replication fork stalling and leads to a significant increase in the percentage of damaged cells in all phases of the cell cycle but has a major impact in G1 cells .We also want to mention a set of helicases that can indirectly influence the cell cycle. Among them is helicase SUV 3, which is capable of unwinding the secondary structures of RNA and DNA. This helicase is localized in the mitochondria and is part of the mitochondrial degradosome. A decrease in the amount of SUV 3 leads to the accumulation of nondegraded mitochondrial RNA and, as a consequence, to changes in the cell cycle . In embrRNA helicases are highly conserved enzymes crucial for RNA metabolism. Most of the RNA helicases discussed here have become  or may bHowever, RNA helicases are involved not only in the regulation of RNA metabolism. They also participate in various aspects of cell cycle regulation . In someWEE1 [Thus, inhibition or downregulation of RNA helicases, discussed above as antiviral and anticancer targets, will also lead to a significant effect on the cell cycle. Inhibition of DDX3 causes a decrease in the amount of cyclin E1, A1 and CDK2 and stops the progress of cell cycle ,53,147. WEE1 . All of WEE1 ) are invWEE1 ,150. DowWEE1 ,152 can WEE1  and, finWEE1 . Multiple functions, sometimes with the opposite direction depending on the type of cells or conditions, make some RNA helicases potential biomarkers and selective targets for therapy. Moreover, their contribution to various processes may depend on the level of the protein in the cell and/or on the inhibited protein domain responsible either for ATP- or RNA-binding. Here, we reviewed the effects of RNA helicases on cell cycle and cell proliferation. These follow-up events may be crucial for development of therapeutics due to potential off-target effects. Problems with pharmacological intervention can also be resolved by targeted delivery of inhibitors to cancer or infected cells . On the