Vertebrate early embryogenesis is initially directed by a set of maternal RNAs and proteins, yet the mechanisms controlling this program remain largely unknown. Recent transcriptome-wide studies on RNA structure have revealed its pervasive and crucial roles in RNA processing and functions, but whether and how RNA structure regulates the fate of the maternal transcriptome have yet to be determined.cis-regulatory elements important for maternal-to-zygotic transition (MZT). We find that the RNA-binding protein Elavl1a stabilizes maternal mRNAs by binding to the cis-elements. Conversely, RNA structure formation suppresses Elavl1a’s binding leading to the decay of its maternal targets.Here we establish the global map of four nucleotide-based mRNA structures by icSHAPE during zebrafish early embryogenesis. Strikingly, we observe that RNA structurally variable regions are enriched in the 3′ UTR and contain cis-regulatory elements during zebrafish early embryogenesis. We reveal that Elavl1a regulates maternal RNA stability in an RNA structure-dependent fashion. Overall, our findings reveal a broad and fundamental role of RNA structure-based regulation in vertebrate early embryogenesis.Our study finds that RNA structurally variable regions are enriched in mRNA 3′ UTRs and contain Supplementary information accompanies this paper at 10.1186/s13059-020-02022-2. Embryogenesis in vertebrates is initially directed by maternal mRNAs deposited in the egg –4. Befortability , 13. BeyRNA’s folding into intricate secondary structures is crucial for its function in vivo, and its misfolding is known to be associated with genetic disorders , 15. Recdgcr8 [P value = 2.5 × 10− 9). The receiver operating characteristic (ROC) curve was then plotted to evaluate the concordance between icSHAPE reactivities and the conserved secondary structures of dgcr8. A high agreement between the icSHAPE reactivity with the conserved secondary structure was observed, and the area under curve (AUC) was positively associated with the agreement .We found that the RNA structuromes are highly dynamic from 0 h.p.f. (fertilized egg) to 6 h.p.f. (shield), with a structural opening immediately after fertilization and a gradual close until 2 h.p.f. (64-cell) followed by a reopening Fig. d. To cheThe previous study by Beaudoin et al. showed that translation drives ORF structural opening . The strP value less than 0.05 (two-sided paired Student’s t test) was also included for selecting the significant structural change for a 10-nt sliding window. Briefly, we identified structurally variable nucleotides that show a significant structural change at the cutoff of Δreactivity score > 0.2. Overall, about 18–28% of the nucleotides were defined as structurally variable across different developmental stages , which is consistent with previous observations that adenosine and uracil are structurally more flexible nucleotides when comparing RNA structures across different cellular compartments [reactivity score > 0.05 and P value < 0.05 (two-sided paired Student’s t test) between two developmental stages as structurally variable sites , especially for the shared structurally variable regions  that are functionally posttranscriptional regulators Fig. , includicis-regulatory sequence-and-structure elements that could possibly regulate maternal RNA degradation. We performed a motif search in the structurally variable regions during 4 to 6 h.p.f. since the majority of maternal transcripts are degraded rapidly during this period [Based on the above results, we speculated that RNA structure might regulate maternal RNA degradation. We therefore repeated the computational screening for s period . The scrs period , 39, in elavl1 gene orthologs in zebrafish, elavl1a and elavl1b, with the former one being more similar to human ELAVL1 in genomic structure. To further explore the function of Elavl1 in early embryogenesis, we examined the expression of homologous genes in zebrafish embryos. The result showed that the expression level of elavl1a was much higher than that of elavl1b, suggesting that elavl1a is likely the dominant homologous gene during zebrafish early development  assay was used to analyze the in vivo targets and structural preference of Elavl1a in zebrafish embryos  injection for translation inhibition  and Elavl1a knockdown samples at stages of 4 h.p.f. and 6 h.p.f.. Elavl1a knockdown was carried out by To further examine whether Elavl1a prefers to bind single-strand RNA (ssRNA) or double-strand RNA (dsRNA), biotin-labeled RNA pulldown assays were carried out by six probes containing endogenous Elavl1a binding sites Fig. e. The reTo test whether RNA structure sufficiently influences Elavl1a’s binding ability, we repeated the above analyses using different structural conformations of biotin-labeled probes containing an endogenous Elavl1a binding site, including P1-wild-type with partially base paired Elavl1a binding site, P1-mutant with disrupted base pairing at the binding site, and P1-rescue with restored base pairing at the binding site Fig. h. The fiP value = 6.9 × 10− 15, two-sided Wilcoxon test), suggesting that potency of Elavl1a in stabilizing RNAs is substantially influenced by the structural shift in its binding regions, and moreover, Elavl1a stabilizes its target transcripts when its binding sites within single-stranded regions are easily accessible. Through comparing the gene expression levels between 2 and 6 h.p.f., we categorized the genes into three groups: (i) genes with RPKM > 1 at 2 h.p.f., log2(FC) > log2(1.2), and FDR < 0.05 were defined as maternal decay genes; (ii) genes with RPKM > 1 at 2 h.p.f., log2(FC) < log2(1.2), and FDR < 0.05 were defined as maternal stable genes; and (iii) genes with RPKM < 1 at 2 h.p.f. and RPKM > 1 at 6 h.p.f. were defined as zygotic genes  of group I transcripts belong to the maternal decay genes  could reach the 1k-cell stage through completing cell cycle 10 at 3 h.p.f.. However, the transition from the 1k-cell stage to sphere was moderately postponed, and the subsequent gastrulation was markedly delayed in elavl1a morphants  by cross-mating heterozygous mutants (elavl1a+/−) , suggesting a protective role of Elavl1a during MZT. Consistently, Elavl1a deficiency accelerated the degradation of maternal stable RNAs to a greater extent than the maternal decay ones . We also found that zygotic gene expression is also downregulated upon Elavl1a deficiency, which is probably owing to the delayed ZGA. Further GO analysis showed that downregulated transcripts are enriched in the processes of protein metabolism, cell homeostasis, transmembrane transport, and energy metabolism  containing Elavl1a binding site with different structural contexts, (i) the wild-type in which part of the Elavl1a binding site is base paired, (ii) a mutation that disrupts base pairing of the binding site, and (iii) a rescue that restores the base pairing  reporter mRNAs, which are derived from partial endogenous 3′ UTR pardgb contaiThese observations clearly indicate that the RNA structure regulates the mRNA stability and consequent protein synthesis in zebrafish early embryogenesis through affecting Elavl1a’s accessibility to its binding motif. In conclusion, we proposed a model depicting the overall role of structurally variable changes of Elavl1a’s binding regions in regulating RNA stability through affecting Elavl1a’s binding ability to its mRNA targets Fig. e.Vertebrate early embryogenesis is characterized by the conversion of the maternal oocyte state to the embryonic totipotency via the involvement of a series of programmed genetic and epigenetic events for genome remodeling, such as DNA demethylation, chromatin remodeling, and spatial genome reorganization . RNA strHere we investigated the RNA structure-based regulation of transcriptomic fate and function during zebrafish early embryogenesis. We generated the global maps of RNA structures at various stages during early embryogenesis in zebrafish, covering all four nucleotides of several thousands of transcripts. The results revealed the dynamic nature of RNA structuromes during zebrafish early development.cis-regulatory elements corresponding to specific RBPs. The previous studies [cis-regulatory elements were enriched in structural changes region in 3′ UTR by comparative analysis, but the intrinsic bias of DMS to A and C leads to the uncomplete identification of the structure-based cis-elements. With the all-4-base structural maps at six early developmental stages in zebrafish, we were therefore able to reconstruct large-scale structure-based posttranscriptional regulomes for zebrafish in different developmental contexts of early embryogenesis. Three factors are required for the regulome construction and the following desirable analyses: (1) the RNA structures of multiple developmental stages along the full zebrafish early embryogenesis were probed, which allow the identification of structurally variable regions along the development process; (2) by using icSHAPE, we obtained RNA structure maps of all four nucleotides enabling the motif search in those structurally variable regions; and (3) about 4000 transcripts were probed, which is also critical for the construction of a large-scale regulome.Comparative analysis of RNA structures at different time points revealed many hot regions of structural changes, which are enriched for  studies , 30 alsoDrosphila, Pumilio participates in deadenylation, translation repression, and maternal mRNA clearance, and Smaug can initiate poly(A) tail shortening followed by mRNA elimination [From the analysis of the large-scale structure-based regulomes, we predicted and finally validated that the RBP Elavl1a regulates zebrafish maternal RNA stability in a structure-switch fashion, i.e., Elavl1a stabilizes its bound maternal mRNAs, whereas the secondary structure formation in maternal decay RNAs during 4–6 h.p.f. period blocks Elavl1a’s binding and consequently leads to maternal RNA degradation. It is known that association of RNA molecules with RNA-binding proteins (RBPs) is crucial for RNA metabolism, such as splicing and degradation in eukaryotes. Several RBPs have been demonstrated to be involved in maternal gene posttranscriptional regulation in different organisms. In mination . Howevermination . This ismination . As ElavOverall, our study reveals the extensive role of RNA structures in regulating early embryogenesis in vertebrates. By generating the RNA structure landscape and structure-based regulomes, we can predict the on-and-off binding of RBPs on the transcriptome in early embryogenesis. Further investigations with additional complementary approaches, including experiments to perturb RNA structures in vivo, are warranted to fully dissect the complex posttranscriptional regulatory network and to facilitate functional studies on early embryogenesis in vertebrates.Zebrafish wild-type strain AB was raised in system water at 28.5 °C under standard conditions. The zebrafish embryos were acquired by natural spawning.2, 37 °C cell culture incubator.293T cells were originally purchased from ATCC, authenticated by short tandem repeat (STR) analysis, and routinely confirmed to be free of mycoplasma. 293T cells were maintained in standard DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1 × penicillin/streptomycin (Invitrogen) in standard humidified 5% COelavl1a atgMO: 5′-TGTGGTCTTCGTAACCGTTCGACAT-3′, Control MO: 5′-CCTCTTACCTCAGTTACAATTTATA-3′. MOs were purchased from GeneTools. MOs  were injected into one-cell stage embryos. For elavl1a overexpression experiments, myc-tagged or flag-tagged elavl1a was cloned into pCS2+ vector, mRNAs were generated using mMACHINE™ (Invitrogen) and injected into one-cell stage embryos. For mRNA structure reporter experiments, GFP reporters and Control mCherry were subcloned into pCS2+ vectors, capped and polyadenylated mRNAs were generated using mMACHINE™ and Poly(A) tailing kit (Invitrogen) according to the manufacturer’s protocol. A total of 20 pg of either GFP reporter mRNA and 20 pg of mCherry mRNA were injected into one-cell stage embryos.Morpholino oligonucleotides (MOs) of genes include the following: elavl1a mutant was generated using CRISPR/Cas9, and the method for Cas9 mRNA and guide RNA synthesis was described previously [elavl1a gRNA, target sequence: GGCCAAGCTCTGACTCCATCAAGG, was generated with in vitro transcription by T7 RNA polymerase . Mutant identification was carried out by DNA sequencing analysis.The eviously . pXT7-CaBright-field and fluorescent images of embryos were observed with a Nikon SMZ1500 fluorescent microscope and captured with a Nikon digital camera. The relative mean fluorescence intensities were analyzed using ImageJ.elavl1a. For probe synthesis, PCR products of elavl1a F:5′-ATGCCAGGCCAAGCTCTGACTC-3′, elavl1a R:5′-TGCAGGCAAACCCTGCTTTCTAC-3′ were cloned into pGEM-T vector (Promega), and the RNA probe was transcribed with T7 RNA polymerase (Promega). After hybridization, RNA probes were detected by AP-conjugated anti-DIG antibody (Roche), and color reactions were carried out using BM purple (Roche) as substrate.Whole-mount in situ hybridization was carried out using a ZF-A4 in situ hybridization machine  with digoxigenin-uridine-5′-triphosphate (Roche) labeled single-stranded RNA probes for Western blotting was performed as previously reported  using th2, 333 mM NaCl) was added and the RNA was allowed to equilibrate at 37 °C for 10 min. Then, injecting the folded RNA probe to each stage  of zebrafish embryos (10 ng/embryo). After injection, NAI-N3 modification or vehicle control DMSO (unmodified group) was performed immediately. Total RNA was isolated by using TRIzol® Reagent . Cy3-labeled DNA primer (5′-GAACCGGACCGAAGCCCG-3′) was annealed to 20 μg of total RNA by incubating at 95 °C for 2 min followed by a step-down cooling (2 °C per s) to 4 °C. To the reaction first-strand buffer, dithiothreitol (DTT) and dNTPs were added. The reaction was pre-incubated at 52 °C for 1 min, then SuperscriptIII  was added. Extensions were performed for 10 min. To the reaction, 1 μl of 4 M NaOH was added and allowed to react for 5 min at 95 °C. Ten microliters of Gel Loading Buffer II (Ambion) was then added, and cDNA extensions were resolved on 8% denaturing (7 M urea) polyacrylamide gels . cDNA extensions were scanned on a Typhoon 9400  imager.The manual SHAPE analysis assay was performed as a previously reported method . RNA pro3, 0.9 mM CaCl2). In total, 500 μl of 2 M NAI-N3 in DMSO (modified group) or vehicle control DMSO (unmodified group) was added drop-wise, immediately mixed by inversion, and incubated at 28.5 °C on end-over-end rotation for 5 min. Total RNA was isolated by using TRIzol® Reagent . mRNA was extracted with using Dynabeads® mRNA Purification Kit .In vivo SHAPE modification of zebrafish RNAs was performed as described previously  with som2, 5 mM DTT, 4 U/μl RiboLock, 0.1 U/μl FastAP (Life Technology), 2 U/μl T4 PNK (NEB)) before 3′ adapter ligation with addition of 10 μl ligation mix , 0.66 U/μl T4 RNA ligase , 15% PEG8000, 1X RNA ligase buffer) and incubation at 25 °C for extra 3 h. Then the excess adaptor was removed as described below. The purified RNA was incubated in the mix of 1 μl FastAP, 0.2 μl SSB (Promega), 0.8 μl Ribolock, and 1 μl 5′ Deadenylase (NEB) in 1× NEB buffer 2 (NEB) at 30 °C for 90 min. And then 1 μl RecJf (NEB) was added with another incubation at 37 °C for 1 h. The following procedures, including reverse-transcription, biotin-streptavadin enrichment, size selection of cDNA, circularization, and PCR amplification, were the same as described in the standard protocol.The purified mRNA was subjected to icSHAPE library preparation as previously described with some modifications . The in flag-elavl1a mRNA-injected embryos were collected at 4 h.p.f. and 6 h.p.f.. A total of 400 zebrafish embryos were irradiated twice with 0.8 J/cm2 , lysed, and subjected to mild RNA fragmentation. Crosslinked RNA protein complexes were immunopurified using Anti-FLAG M2 Magnetic Beads  for 4 h.p.f. and 6 h.p.f. at 4 °C. RNA was extracted and subjected to library construction using Smarter smRNA-Seq kit (Clontech Laboratories Inc).iCLIP was carried out as previously described . flag-elTotal RNA was isolated from zebrafish embryos at different time points with TRIzol reagent and mRNA was further purified using Dynabeads mRNA purification kit . Fragmented mRNA was used for library construction using the KAPA Stranded mRNA-Seq Kit  according to the manufacturer’s protocol.myc-elavl1a mRNA-injected embryos were collected at 4 h.p.f. and 6 h.p.f., further lysed in NETN lysis buffer . The Elavl1a RIP was carried out with a modified procedure [− 1 proteinase K  in 200 μl PK buffer  for 20 min at 37 °C, followed by incubation with 200 μl PK-urea buffer  for 20 min at 37 °C. After washing, RNA was collected by EtOH precipitation and then used for RT-qPCR.rocedure . In brie2 UV (254 nm) and snap frozen in liquid nitrogen (UV+), while nonirradiated embryos were served as negative controls (UV−). The embryos were subsequently lysed in lysis/binding buffer , 5 mM DTT, Protease Inhibitor Cocktail) and subjected to two rounds of 1 h mRNA capture at room temperature using 200 μl Dynabeads™ Oligo (dT)25 (Invitrogen). After three times of 10 min washes with Lysis/binding buffer, beads were washed three times with NP40 wash buffer . mRNPs were heat eluted from the beads in 20 μl low-salt buffer  at 70 °C. Proteins were released from mRNPs by 1 h RNase I (Ambion) treatment at 37 °C in low salt buffer and separated on a 4–12% NuPAGE NOVEX gradient gel in NuPAGE buffer (Thermo). The protein-containing gel was analyzed by mass spectrometry in the Institute of Biophysics, Chinese Academy of Science. The data files have been uploaded to http://www.peptideatlas.org with the access number: PASS01264.The zebrafish mRBP capture was performed as previously described . Briefly293T cells were transiently transfected with pCS2-Flag-Elavl1a plasmids using PEI transfection reagent. Forty-eight hours later, cells were lysed with lysis buffer  and then sonicated  for 1 min by a Sonic Dismembrator (Thermo Fisher). After removing the cell debris through centrifugation at 13,300 rpm for 20 min, the lysates were incubated with anti-Flag M2 Affinity Gel (Sigma-Aldrich) for 4 h at 4 °C. After washing with lysis buffer for five times and TBS buffer  for twice, the bead-bound proteins were eluted with 1 mg/ml 3× Flag peptide (Sigma-Aldrich) for 1 h at 4 °C. The elute containing purified protein was condensed using VIVASPIN 500 (Sartorius Stedim Biotech) and quantified by coomassie brilliant blue staining and western blotting.2, 333 mM NaCl) was added, and the RNA was allowed to equilibrate at 37 °C for 10 min. Zebrafish embryo extracts were precleared for 1 h at 4 °C by incubation with streptavidin-conjugated magnetic beads (NEB) in binding buffer  supplemented with 0.4 U/μl RNasin (Promega). Biotin-labeled RNA oligonucleotides were incubated with precleared nuclear extracts for 2 h at 4 °C under gentle rotation together with streptavidin-conjugated magnetic beads which were precleared by incubation with 0.2 mg/ml tRNA (Sigma) and 0.2 mg/ml BSA (Amresco) for 1 h at 4 °C under gentle rotation. Beads were washed three times with wash buffer ). For western blotting analysis, samples were separated on SDS-PAGE and transferred onto PVDF membrane. After blocking with 5% non-fat milk in TBST for 1 h, the membrane was then incubated for 1 h at 4 °C with Monoclonal anti-Hur  diluted at 1:1000 in 5% milk. Protein levels were visualized using ECL Western Blotting Detection Kit .The biotin-labeled RNA probes were synthesized by the GenScript Biotech Corp. In vivo RNA pulldown assays were carried out using zebrafish embryo extracts as previously described  with som2, 333 mM NaCl) was added and the RNA was allowed to equilibrate at 37 °C for 10 min. Then, 10 pmol of purified Flag-Elavl1a protein and 10 pmol of biotin-labeled probes were incubated with 15 μl streptavidin-conjugated magnetic beads (NEB) in binding buffer  for 1 h at 4 °C. After washing with binding buffer for three times, the bead-bound proteins were heated in NuPAGE™ LDS Sample Buffer (4×) (Invitrogen) and then separated on the NuPAGE™ 4–12% Bis-Tris Gel (Invitrogen), and subjected to western blotting analysis with anti-Flag antibody .In vitro RNA pulldown assay was performed according to the previously reported method  with somPurified Flag-tagged Elavl1a proteins were diluted to a series of concentrations of 0.2 μM, 0.5 μM, 1 μM, and 2 μM in binding buffer . One microliter synthesized Cy3-labeled RNA probes  and 1 μl purified protein  were mixed and incubated at room temperature for 30 min. Then, 1 μl glutaraldehyde  was added into the mixture which was incubated at room temperature for 15 min. The entire 11 μl RNA protein mixture was mixed with 5 μl 5× Hi-Density TBE Sample buffer and separated on 6% TBE gel on ice for 30 min at 80 V. The gel was scanned on a Typhoon 9400  imager. Quantification of each band was carried out using ImageJ. The RNA binding ratio at each protein concentration was determined by (RNA protein)/((free RNA) + (RNA protein)).P < 0.05; **P < 0.01; ***P < 0.001. The primers used for RT-qPCR in this study are listed in Additional file Quantitative reverse-transcription PCR (RT-qPCR) was carried out to examine the relative abundance of target RNA. 0.1 μg RNA were used for cDNA synthesis using RevertAidTM First Strand cDNA Synthesis Kit (Thermo). Experiments were performed with Takara SYBR Premix Ex Taq (Takara) according to the manufacturer’s instructions and examined by a CFX96 Real-Time PCR System (Bio-Rad). *Raw sequence reads were first trimmed to remove 3′ adaptors by cutadapt (V 1.16), filtered out low-quality bases with Trimmomatic  (V 0.33)icSHAPE reactivity was calculated as previously described , 53 withFor structurally variable nucleotides, we chose the same cutoff 0.2 used in our previous study  to distit-test. P value < 0.05 was considered as a significant structural change. In sum, the average Δreactivity score > 0.05 and P value < 0.05 (two-sided paired Student’s t test) were used to assess the significant structural change for a 10-nt sliding window. The whole process is very similar as the differential gene expression analysis  were marked as less structural windows; < − 0.05 and P value < 0.05 were marked as more structural windows; and others were marked as stable ones. The less structural and more structural windows were used to define structurally variable regions. Next, we extended these windows with 10-nt flanking regions and then merged the overlapped extended windows and divided them into 30 nt regions to remove redundancy. These re-bounded windows were finally defined as variable structure regions with the same annotation as that of their origin.For structurally variable regions, we used the similar strategy to choose the cutoff by ranking all windows according to the average Δreactivity score. About 95% windows have the average Δreactivity score lower than 0.05. For every window, we also calculated the significance of the difference by using the two-sided paired Student’s For the definition of “hot” structurally variable sites, the structurally variable regions that are shared by two comparisons, e.g., comparison of 0 h.p.f. and 0.4 h.p.f. stage and comparison of 1 h.p.f. and 2 h.p.f., are defined as two-way “hot” structurally variable regions. Those shared by three comparisons are termed three-way “hot” structurally variable regions and so on.To assess the enrichment of the structurally variable windows in 5′ UTR, CDS, and 3′ UTR, we assigned each window to one of the three nonoverlapping transcript segments: 5′ untranslated region (UTR), coding sequence (CDS), and 3′ untranslated region (UTR) by applying BEDTools  , and ELAVL1 from ATtRACT database [https://attract.cnic.es/).To explore the sequence feature of structurally variable windows at 3′ UTR, we used MEME suite Dreme , a tool database  , and low-quality bases were trimmed and filtered by cutadapt (V 1.16) and Trimmomatic [The quality of raw sequencing reads was screened using FastQC (mmomatic  (V 0.33)mmomatic  (V 2.2.9mmomatic  (V1.0), 2 (FC) > log2 (1.2), and FDR < 0.05 were defined as maternal decay genes (N = 6589); (ii) genes with RPKM > 1 at 2 h.p.f., log2 (FC) < log2 (1.2), and FDR < 0.05 were defined as maternal stable genes (N = 2479); (iii) genes with RPKM < 1 at 2 h.p.f. and RPKM > 1 at 6 h.p.f. were defined as zygotic genes (N = 1225).The fold change of expression level of transcripts between samples were calculated using DEGseq  . Firstly, the reads were trimmed off adapter with cutadapt followed by collapsing duplicates with CTK tool (fastq2collapse.pl). After stripping the 5′ degenerated barcode, we mapped the clean reads to the transcriptome using bwa (v0.7.17). For each time point (4 h.p.f. and 6 h.p.f.), two replicates were merged before subsequent peak calling. We used CTK tool CITS mode to call the peaks. The reads were clustered using tag2cluster.pl with parameters -big -s -maxgap “-1” and the peaks are identified using tag2peak.pl with parameters -big -ss -v --prefix “CITS” -gap 25 -p 0.05.The processing of iCLIP data is similar to pipeline as described in CTK tool  and −UV (negative) sample are obtained from the detector with replicates . GO classification for biological process was performed using the genes with RPKM > 1 for respective zebrafish embryo developmental stage as background.Gene ontology (GO) analysis was performed using the DAVID  website The dataset of RBP binding sites of 23 iCLIPs in zebrafish embryos at 4 h.p.f. (sphere) stage was collected . The bint test using t.test function in stats R package. Statistical significance was set to P < 0.05.Statistical comparison of structurally variable regions between stages was carried out with two-sided paired Student’s P values in Additional file All the correlations and P values were less than 2.23 × 10− 308 ; *, P < 0.05; **, P < 0.01; ***, P < 0.001 ; *, P < 0.05; **, P < 0.01; ***, P < 0.001 ; *, P < 0.05; **, P < 0.01; ***, P < 0.001 ; *, P < 0.05; **, P < 0.01; ***, P < 0. 001  using functions: scipy.stats.mannwhitneyu, scipy.stats.ttest_ind, scipy.stats.ks_2samp, scipy.stats.fisher_exact.All the statistical tests listed above, if not specified, were carried out with scipy Python package (Additional file 1: Supplementary Figures S1-S7.Additional file 2:Table S1. Summary and statistics of icSHAPE, RNA-seq and iCLIP.Additional file 3:Table S2. LFQ intensity of proteins in UV+ and UV- samples at 0, 0.4 and 4 h.p.f.Additional file 4:Table S3. Structurally variable regions between neighboring stages and hot structurally variable regions.Additional file 5:Table S4. Results of motif enrichment analysis in 3′ UTR structurally variable regions.Additional file 6:Table S5. Summary of DLE element in structurally variable regions during early development and its associated biological function.Additional file 7:Table S6. Elavl1a binding sites at 4 h.p.f. Elavl1a binding sites identified by Flag-Elavl1a iCLIP at 4 h.p.f. and 6 h.p.f.Additional file 8:Table S7. Maternal and zygotic gene sets categorized by gene expression and SNP.Additional file 9:Table S8. GO term enrichment analysis of down-regulated genes upon elavl1a knockdown at 6 h.p.f.Additional file 10:Table S9. List of oligos used for this Study.Additional file 11: Review history.


Coronaviruses are known as enveloped viruses with a positive-sense single-stranded RNA genome that their helical symmetry nucleocapsid is about 26–32 kilobases in size, making it the largest investigated genome among RNA viruses. The disease caused by 2019 new coronavirus (2019-nCoV) was named coronavirus disease-19 (COVID-19) by the World Health Organization in February 2020. The 2019-nCoV is phylogenetically related to severe acute respiratory syndrome-coronavirus (SARS-CoV) .Our information on the probability and effect of 2019-nCoV on the peripheral and central nervous system is still scarce and therefore unreliable. There have been various studies evaluating coronavirus’s effects on the central nervous system. These studies suggest that the human central nervous system (CNS) may be susceptible to coronavirus infection . Routes Numerous reports of loss of sense of smell and taste have been received from Iranian people as one of the most heavily involved countries with COVID-19 during the outbreak of the disease , 9. SignAlthough there is not sufficient evidence to make a definitive judgment and need more comprehensive investigations, two scenarios are more likely to be suggested as the cause of such an incident. The appearance of dysosmia and dysgeusia whether can be attributed to olfactory nerve and trigeminal nerve damage caused by the 2019-nCoV infection or excessive exposure to chemicals and disinfectants that are more commonly used by people due to the viral epidemic. Designing a study to assess the validity of such a hypothesis is important in that it can be considered a relatively acceptable diagnostic criterion for both the individual and the physicians. Since the existence of such a relationship is likely, it also seems likely that during the COVID-2019 outbreak, those who experience complications such as dysosmia and dysgeusia should be considered potential carriers of the virus and that in addition to necessary hygiene measurements, they must quarantine themselves.


Aspergillus nidulans. In this review, we present evidence that shows that transporter traffic to the PM takes place through Golgi bypass and transporter endocytosis operates via a mechanism that is distinct from that of recycling membrane cargoes essential for fungal growth. We discuss these findings in relation to adaptation to challenges imposed by cell polarity in fungi as well as in other eukaryotes and provide a rationale of why transporters and possibly other housekeeping membrane proteins ‘avoid’ routes of polar trafficking. Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental, or stress signals. Sorting of transporters from their site of synthesis, the endoplasmic reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway. Endocytosis of specific eukaryotic transporters has been studied more systematically and shown to involve ubiquitination, internalization, and sorting to early endosomes, followed by turnover in the multivesicular bodies (MVB)/lysosomes/vacuole system. In specific cases, internalized transporters have been shown to recycle back to the PM. However, the mechanisms of transporter forward trafficking and turnover have been overturned recently through systematic work in the model fungus  Aspergillus nidulans has been used, which eventually emerged as a unique organism for studying eukaryotic transporters [A. nidulans is changing our views on transporter biogenesis and turnover. The pioneer biochemical work of Ronald Kaback on the LacY permease before the era of crystallographic analysis of transporters <2003) had a huge impact on all of us who decided to devote our research to understanding how transporters work. His ingenious experimental inventions for studying a prokaryotic transporter, which led to the establishment of the generally accepted ‘alternating access mechanism of transport’ ,2, coupl003 had asporters ,14,15,16sporters ,18,19,20The first step in the biogenesis of nascent eukaryotic membrane proteins, including transporters and other polytopic transmembrane proteins, is their co-translational translocation from ribosomes to the membrane of the endoplasmic reticulum (ER) ,22,23,24Saccharomyces cerevisiae Shr3 protein, which mediates COPII–cargo interactions required specifically for the packaging of amino acid transporters into vesicles [cis-Golgi has been defined and called the ER-intermediate compartment (ERGIC) [cis-Golgi and then ‘reach’ the trans-Golgi network (TGN) via Golgi maturation [Rab11, package in AP-1/clathrin-coated vesicles, which translocate to the PM either directly or indirectly via highly motile endosomes [Membrane cargo–Sec24 interaction and packaging into COPII vesicles is often assisted by specific ER-resident chaperones and/or cargo receptors. One of the best characterized ER-exit membrane chaperone is the vesicles . Shr3 havesicles . Additiovesicles ,32,33. Ovesicles ,35,36,37vesicles ,29,38. I (ERGIC) . After sturation ,40,41,42ndosomes ,44. AP-1ndosomes ,43,45. Tndosomes . In filandosomes ,48,49. Noticeably, however, the aforementioned brief account on membrane cargo forward trafficking towards the PM is heavily based on cargoes that are not transporters or other polytopic transmembrane proteins . In fact, very few studies have addressed directly and systematically how transporters reach the PM. The dogma seems to be that, being transmembrane proteins, transporters, channels, and receptors use the conventional Golgi- and post-Golgi-dependent vesicular secretion route, described above. However, some lines of evidence supported that specific transporters might not follow known conventional secretion routes. For instance, the insulin-regulated glucose transporter GLUT4 accumulated at the PM, rather than being sequestered in the Golgi or other intracellular compartments, after the deletion of proteins involved in TGN-dependent membrane cargo sorting , suggesting the presence of alternative routes out of the TGN . In lineS. cerevisiae, several transporters specific for amino acids  or uracil (Fur4) transporters are preferentially located in PM microdomains of 200-300 nm called MCC (membrane compartments of Can1), which do not overlap with other microdomains, called MCP or MCL, defined by distinct transporters, such as the H+-ATPase Pma1 or sterol transporters Ltc3/4, respectively [A. nidulans, several nucleobase transporters of the NCS1 family also do not define microdomains, at least within the limits of conventional epifluorescence microscopy [A. nidulans, where >30 different transporters specific for purines, pyrimidines, amino acids, sugars, carboxylic acids, or drugs have been studied at the level of subcellular localization via epifluorescent microscopy. All studied fungal transporters involved in the uptake or efflux of solutes or ions are known to localize in a rather homogeneous manner all along the PM. Some appear in distinct foci, whereas other evenly mark the entire PM. For example, in ectively . Still, croscopy ,60,61,62croscopy ,63. In gAspergillus transporters highly contrasts the polar localization of several other membrane cargoes involved in continuous plasma membrane or cell wall synthesis at the apex of growing hyphae, such as, for example, chitin synthase ChsB, lipid flippases DfnA and DfnB, synaptobrevin SynASnc1, or components of the exocyst complex, needed for regulation of apical recycling and repositioning of specific cargoes necessary for growth [The non-polar localization of r growth ,65,66,67r growth ,69. In aA. nidulans transporters, we noticed that transporters are localized principally in two compartments: The PM (including septa) and in vacuoles, the latter reflecting the fraction of transporters undergoing constitutive or signal-elicited degradation after endocytosis . In the first approach, we blocked the synthesis of proteins involved in conventional secretion via the tight repression of a regulatable promoter. In particular, we followed transporter localization in strains that did not express Sec24 or Sec13 (COPII generation), SedVSed5 or GeaAGea1 (early-Golgi functioning), HypBSec7 (TGN functioning), RabERab11, AP-1σ or clathrin ClaHChc1 (post-Golgi secretion), RabA/BRab5 (early and recycling endosomes), or SsoASso1 (major PM tethering t-SNARE). Notice that although persistent transcriptional repression  of secretion eventually leads to A. nidulans cell death, given that in our system full repression needed 10–12 h to occur, this provided a period of time for conidiospore germination and the development of germlings, and also sufficient time, after full repression of secretion, for inducing and studying the trafficking of transporters [To identify the trafficking pathways of nutrient transporters in sporters . Besidessporters . Using these approaches, we showed that trafficking of de novo made transporters initiates by COPII-packaging  and subsequently requires clathrin heavy chain and the PM t-SNARE SsoA but is not dependent on Golgi functioning  or key effectors for post-Golgi conventional secretion (RabE and AP-1), microtubule polymerization (benomyl-insensitive), or early and recycling endosome formation . Actin polymerization was shown to be required for transporter trafficking , surprisingly due to a previously non-described essential role in COPII formation. Co-localization studies strongly supported the presence of nascent transporters in COPII structures and the absence of transporters from the late Golgi/TGN. These findings contrasted the dynamic trafficking of model apical cargoes , followed in parallel by analogous approaches, which showed a clear dependence on early and late-Golgi/TGN and post-Golgi secretory routes . The essential role of clathrin heavy chain  on tranThe above findings, and in particular the common dependence of transporters and apical cargoes on Sec24 and Sec13, led to a simple but major assumption of considering the existence of distinct subpopulations of COPII vesicles as elements of different trafficking routes. Thus, the main question to address is to identify the particular features of distinct subpopulations of COPII and understand what makes these vesicles traffic through different routes and ending at different sections of the fungal membrane. PM transmembrane proteins can undergo regulated endocytosis in response to physiological or developmental signals or stress. This a ubiquitous biological phenomenon that serves adaptation to changes of the cell environment , signaling , and renewal of membrane components, especially under chemical and biological stress, senescence, or aging. Endocytosis of membrane proteins is followed by sorting to early endosomes, which in most cases mature to late endosomes and multivesicular bodies (MVBs) that eventually fuse with the lysosome  or vacuole (plants/fungi), where membrane protein degradation takes place. Notably, several membrane cargoes can recycle back the PM instead of being degraded ,75,76. RS. cerevisiae and A. nidulans, have led to important findings concerning the molecular mechanism underlying endocytosis, turnover, or recycling of nutrient transporters. In most cases, the primary molecular signal preceding endocytosis is ubiquitylation of cytoplasmically exposed terminal regions of transporters [Studies in fungi, mostly in sporters ,84. Transporters ,85,86. Hsporters ,88. Signsporters ,90,91,92sporters ,94,95. Fsporters ,97,98,99S. cerevisiae, a detailed temporal scheme of arrival and departure of over 60 proteins at sites of endocytosis has been established [Following transporter ubiquitination, several proteins are involved in subsequent steps necessary for fission of endocytic vesicles from the PM and sorting into early or recycling endosomes ,101. Theablished . Endocytablished ,105. Wheablished . Deubiquablished .A. nidulans transporters, which showed that internalization from the PM is AP-2 independent, despite being clathrin dependent, as probably expected [Aspergillus polarly localized  membrane proteins, such as chitin synthase, lipid flippases, or synaptobrevin A, proved to be AP-2 dependent but clathrin independent [A. nidulans showed formally that clathrin-dependent and clathrin-independent mechanisms of endocytosis do exist in lower eukaryotes, similar to metazoa. Furthermore, these findings very probably concern all higher fungi, since the σ subunit of the AP-2 complex lacks the entire C-terminal domain containing the putative clathrin-binding box in all cases [Figure 3.As far as it concerns fungi, the above brief account on endocytosis has been based on the studies concerning the internalization of selected transporters and polar markers. However, a surprising result came from studies with expected . A furthependent . Thus, sll cases ,19. OverA. nidulans transporters revealed that transporters homogenously localized all along the PM and polarly localized apical cargoes follow distinct forward trafficking and endocytic routes. In endocytosis, the distinct mechanisms followed seem to depend on the involvement of AP-2  vs. clathrin (transporters). What makes a cargo follow different biosynthetic trafficking routes , implicating distinct COPII subpopulations, remains less clear. What is, however, apparent is that trafficking and endocytosis are both highly dependent on intrinsic information contained in membrane cargoes. Specific sequence or structural motifs/domains, the number and length of transmembrane segments, interactions with membrane specific chaperones, receptors or lipids, oligomerization, and partitioning in specialized membrane microdomains might all play roles in determining the route and mechanism of trafficking followed. Work with In mammalian neurons, most cargoes involved in neurotransmission are polarly localized in the synaptic region by conventional Golgi-dependent secretion, but specific cargoes serving dendrite, soma homeostasis, or the initial segment (AIS), such as glutamate receptor GluA1, neuroligin, or the potassium channel Kv2.1, are sorted via mechanisms bypassing the Golgi ,108,109.Given the plethora of cellular roles cargoes have, the assumption that trafficking is cargocentric has a strong physiological rationale. For example, most transporters serve the cell nutrient supply so that there is no obvious physiological need to drive transporters to a specialized domain of the PM. This is well reflected in fungi and other organisms, where transporters are localized non-polarly and rather homogenously all over the PM of hyphae. In contrast to transporters, membrane cargoes that serve membrane or cell wall synthesis, polarity maintenance, and apical growth in fungi need to be polarly localized at the hyphal tips, and thus face the challenge of long-distance sorting, coupled with recycling, which apparently need the involvement of conventional vesicular secretion via the Golgi/endosome and microtubules. Similarly, endocytosis of transporters occurs all along the PM and most often leads to vacuolar degradation, whereas apical cargo endocytosis needs to be restricted at the fungal apical region to serve rapid recycling at the apical tip necessary for growth. In the case of transporters, by4passing the Golgi might also serve the need to avoid polar localization at the tip, and thus avoid competition for PM translocating with apical cargoes needed for polar growth.


Genetic analyses of patients with amyotrophic lateral sclerosis (ALS) have identified disease-causing mutations and accelerated the unveiling of complex molecular pathogenic mechanisms, which may be important for understanding the disease and developing therapeutic strategies. Many disease-related genes encode RNA-binding proteins, and most of the disease-causing RNA or proteins encoded by these genes form aggregates and disrupt cellular function related to RNA metabolism. Disease-related RNA or proteins interact or sequester other RNA-binding proteins. Eventually, many disease-causing mutations lead to the dysregulation of nucleocytoplasmic shuttling, the dysfunction of stress granules, and the altered dynamic function of the nucleolus as well as other membrane-less organelles. As RNA-binding proteins are usually components of several RNA-binding protein complexes that have other roles, the dysregulation of RNA-binding proteins tends to cause diverse forms of cellular dysfunction. Therefore, understanding the role of RNA-binding proteins will help elucidate the complex pathophysiology of ALS. Here, we summarize the current knowledge regarding the function of disease-associated RNA-binding proteins and their role in the dysfunction of membrane-less organelles. SOD1) as an ALS causative gene in 1993 [TARDBP), FUS RNA-binding protein (FUS), TATA-box binding protein associated factor 15 (TAF15), EWS RNA-binding protein 1 (EWSR1), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), HNRNPA2B1, Ataxin 2 (ATXN2), and TIA1 cytotoxic granule associated RNA-binding protein (TIA1), have been shown to cause or influence the disease risk for ALS and/or FTD [FUS and TARDBP encode RNA-binding proteins and lead to disrupted RNA metabolism [FUS or TARDBP show abnormal stress granule formation with defects in translation, the formation of pathogenic RNA foci, the dysregulation of nucleocytoplasmic shuttling, as well as other forms of disrupted RNA metabolism [C9orf72) gene [C9orf72 gene [C9orf72 repeat expansion’s toxicity increases with age, repeat length, and expression level [C9orf72 gene can form RNA foci enriched with RNA-binding proteins in induced pluripotent stem cell (iPSC)-derived neurons from ALS and FTD patients, as well as in motor neurons of C9orf72-ALS patients [C9orf72 mutations [C9orf72 mutations, the production and accumulation of DPR is considered to be one of the main contributors to its pathogenesis. Studies demonstrating the mechanisms of toxicity of DPRs in vitro and in Drosophila revealed that poly-GR and poly-PR DPRs interact with RNA-binding proteins or proteins that contain a low complexity sequence domain (LCD), also called the prion-like domain [In 1939, the major league baseball player Lou Gehrig was diagnosed with amyotrophic lateral sclerosis (ALS), which is often called Lou Gehrig’s disease. The first descriptions of the disease in 1869 connected the symptoms and underlying neurological problems, and Charcot introduced the term ALS in 1874 . The pro in 1993 ,16,17,18 in 1993 . Althoug in 1993 ,22. Inted/or FTD ,29,30,31tabolism ,33. The 72) gene ,35. ALS f72 gene , and theon level . Repeat-patients ,38,39,40utations ,41, RNA utations . Dipeptiutations ,44,45,46e domain ,48. LCD e domain ,47. Indee domain . RNA-bine domain ,51,52,53Here, we focus on the cellular and functional features of ALS/FTD-related RNA-binding proteins, and discuss the complex pathophysiology of ALS by highlighting the role of RNA-binding proteins .FUS also impair nucleocytoplasmic transport [FUS impede its nuclear localization, and the increase in the altered localization pattern of mutant FUS in the cytoplasm correlates with rapid disease progression and a lower age of disease onset [SFPQ mRNA [C9orf72 hexanucleotide repeat mutations and defects in nucleocytoplasmic shuttling [C9orf72 hexanucleotide repeat expansion has been shown to cause nuclear import deficits in Drosophila and iPSC neurons, including an abnormal cytoplasmic accumulation of TARDBP [C9orf72 model system [C9orf72 mutation. Heaxanucleotide repeat expansion of C9orf72 sequestered and perturbed the subcellular distribution of MATR3 in C9orf72-ALS patient-derived motor neurons [A defining characteristic feature of ALS and FTD is the loss of specific RNA-binding proteins from the nucleus and their mislocalization into cytoplasmic aggregates ,55. Emerransport ,63,64. Aransport . ALS-assse onset ,66. It hFPQ mRNA . MislocaFPQ mRNA . ALS-assFPQ mRNA . StudiesFPQ mRNA ,69. SeveFPQ mRNA ,71. Sevehuttling ,72. Thesf TARDBP . Severall system . The ALS neurons .C9orf72-derived DPR poly-GR and poly-PR have been shown to interact with several mediators of nucleocytoplasmic shuttling, including IPO5, IPO7, KPNA2, KPNB1, NUP205, XPO1, and TNPO1, as well as with RNA-binding proteins consisting of nuclear pore complexes, such as PARP1, YBX1, and LBR [C9orf72-patient-iPSC-derived motor neurons have shown the mislocalization of U2 snRNP in the cytoplasm, and poly-GR as well as poly-PR are both associated with U2 snRNP [C9orf72 hexanucleotide repeats leads to an accumulation of pathological dipeptide repeat protein-containing inclusions [HNRNPA1 and HNRNPA2B1, especially in the prion-like domains of genes, promote cytoplasmic inclusion formation [PFN1), an actin-binding protein involved in actin cytoskeleton dynamics, have been identified in ALS [C9orf72 repeat expansion or mutant PFN1 [ and LBR . C9orf72U2 snRNP . The DPRU2 snRNP . Poly-GAU2 snRNP . The nucclusions . ALS-cauormation ,77. Seved in ALS ,79, and d in ALS . The modant PFN1 .TIA-1 [ATXN2 [TIA-1 have been identified [FUS and HNRNPA2B1 [FUS mutation causes FUS to colocalize with stress granules that are larger and more numerous than wild-type FUS-mediated stress granules [HNRNPA1, especially the Gly-rich low-complexity domain of HNRNPA1, have shown increased incorporation into stress granules [C9orf72 gene impair the formation, dynamics, and function of stress granules [C9orf72 mutation can cause reduced stress granule internal dynamics, with HNRNPA1 and TIA1 phase separation [PFN1 cause not only defects in nucleocytoplasmic shuttling but also impairments in stress granule formation or clearance [SOD1 ALS mutations cause G3BP1-positive SOD1 aggregation in spinal cord motor neurons [FUS that cause ALS [Stress granules are a specific type of RNA granule and are composed of RNA and RNA-binding proteins, including repressed translational complexes. Stress granules are cytoplasmic members of the RNA granule family and are formed during cellular stress, particularly in neurodegenerative diseases and myopathies, resulting in dynamic membrane-less compartments . DefectsTIA-1  or ATXN21 [ATXN2 , which a1 [ATXN2 ,87, have1 [ATXN2 ,25,88. Sentified ,89,90, aentified ,91. MutaNRNPA2B1  have alsgranules ,93. ALS-granules ,94. Hexagranules ,95, whicgranules . Poly-GRparation  leading paration ,97. Polyparation ,102,103.learance ,104. SOD neurons ,105. Mut neurons ,107. Strause ALS .C9orf72 mutations preferentially accumulate in the nucleoli and disrupt the transport of ribosomal proteins, rRNA processing, and ribosome assembly, leading to cell death [C9orf72 transcripts [C9orf72 mutation and shows an altered distribution pattern, eventually inducing nucleolar stress [C9orf72 also interact with NCL RNA-binding protein and disrupt its molecular dynamics in the nucleolus [The nucleolus is the largest nonmembrane-bound nuclear compartment and an important region for rRNA synthesis and the assembly of the eukaryotic ribosome . Althougll death ,116,117.ll death ,118,119.nscripts , and NPMnscripts . NPM1 nunscripts ,109,118.nscripts . Anotherr stress ,122,123.r stress . In addiucleolus .The nuclear bodies termed Cajal bodies are regions within the nucleus that are enriched in proteins and RNAs involved in mRNA processing. They are the main sites for the assembly of snRNPs. These Cajal bodies show frequent close proximity to the nucleolus but are structurally distinct from the nucleolus, although it can be difficult to clearly delimit the border between the two structures .FUS mutations induce the cytoplasmic accumulation of snRNAs and associated Sm proteins, leading to a decrease in the nuclear distribution of snRNPs in Cajal bodies [Toxic DPR poly-GR and poly-PR interact with Cajal body components such as RNA-binding proteins SRRM2 and XPO, which do not bind mRNA directly; however, other adaptor proteins act as a bridge to the interaction between XPO and mRNAs . In livel bodies ,69. The l bodies .Nuclear speckles, also known as interchromatin granule clusters, are membrane-less nuclear compartments enriched in pre-mRNA splicing and transcription factors located in the interchromatin regions of the nucleoplasm of cells . NuclearC9orf72 mutations, have also been shown to accumulate in nuclear speckles and disrupt their dynamics [C9orf72, has been shown to co-localize with C9orf72 antisense RNA foci in C9orf72 ALS patients [TARDBP levels also impairs splicing events and especially disturbs the splicing of disease-associated transcripts [TARDBP decreases the expression of the nuclear speckle-specific noncoding RNA, MALAT1 [FUS are expected to disrupt the structure and function of nuclear speckles [The self-organizing structure of nuclear speckles has dynamic liquid-like properties ,127. DPRdynamics ,97. Polydynamics . The altdynamics . SRSF2, patients ,133. SRSpatients . In ALS,patients ,135. Thenscripts . The dep, MALAT1 . FUS is NEAT1, which acts as a seed for recruiting RNA-binding proteins and building a paraspeckle as well as more than 60 paraspeckle proteins [NEAT1 RNA [TARDBP accelerates paraspeckle assembly [TARDBP diminished NEAT1 non-coding RNA-mediated TARDBP liquid-liquid phase separation and resulted in a specific defect in the nuclear body and paraspeckle [FUS, especially in the low complexity domain, lead to the redistribution of FUS to the cytoplasm [NEAT1 non-coding RNA in cells [C9orf72 mutations are predominantly associated with paraspeckle proteins SFPQ, NONO, RBM14, FUS, and HNRNPH in cells and brain tissue in FTD [NEAT1 non-coding RNA [NEAT1 expression and suppresses the function of HNRNPF and HNRNPH1, leading to increased paraspeckle formation and neuronal toxicity [Paraspeckles are mammalian-specific nuclear bodies that contain a long non-coding RNA, proteins ,139. Parproteins . A numbeproteins ,143,144.EAT1 RNA . Spinal assembly . ALS-assaspeckle . FUS is aspeckle , and ALSytoplasm . Mutatioin cells ,149. GGGe in FTD . ALS-assding RNA ,151. Poltoxicity .Although a limitation of this study includes the fact that we were not able to include all identified studies in the manuscript due to space restrictions, we have reviewed RNA-binding proteins in the context of ALS and highlighted how they are involved in many diverse membrane-less organelles. Most components of membrane-less organelles and ALS-related genes also have common features. As mentioned in this paper, many RNA-binding proteins have been directly or indirectly implicated in ALS and FTD.Several membrane-less organelles have distinct function, composition, and localization but appear to be closely connected. It has been shown that the activation of the integrated stress response induces the accumulation of several factors into stress granules that are crucial mediators of nucleocytoplasmic transport, including import and export factors such as importin-β1, -β2, -α1, and Exportin-1, as well as nucleoporins, the core subunits of the nuclear pore complex ,69. ConsTARDBP, C9orf72, and FUS [C9orf72 hexanucleotide repeat expansion have been associated with HNRNPH1, HNRNPH3, HNRNPF, HNRNPA1, HNRNPA3, and other nuclear speckle related RNA-binding proteins [HNRNPA1 pre-mRNA splicing and eventually leads to HNRNPA1B cytoplasmic accumulation [With regard to the components of membrane-less organelles, one example, TARDBP, is one of the protein components of paraspeckles but is also found in stress granules, Cajal bodies, and in the nucleoplasm. Therefore, many RNA-binding proteins are components of several membrane-less organelles simultaneously. The interactome of ALS-associated genes shares somewhat similar RNA-binding proteins. Each interactome of the hnRNP family or key pathological genes is associated with ALS, including  and FUS . In the  and FUS ,152. Man and FUS . RNA focproteins ,153. Themulation . We haveGiven this connectivity, a precise and systematic understanding of RNA binding might be important. Despite structural heterogeneity in RNA-binding proteins, many RNA-binding proteins have common functional roles. RNA-binding proteins can form dynamic and cooperative complexes with other RNA-binding proteins to fulfill their role in membrane-less organelles and in diverse RNA regulatory steps. Therefore, it is not at all surprising that so many RNA-binding proteins have been linked directly or indirectly to the pathogenesis of ALS. The molecular pathogenesis of ALS appears to be driven by various and complex inter-connected RNA metabolic processes that cannot be solely attributed to any one dysfunctional event. The dysregulation of one RNA-binding protein might disrupt the structure or function of a specific complex of RNA-binding proteins. This might disrupt the dynamics and function of membrane-less organelles that harbor a dysfunctional complex of RNA-binding proteins. An abnormality in one RNA-binding protein will also lead to an unusual RNA-binding protein complex that harbors an abnormal RNA-binding protein; this will eventually lead to the dysfunction of other membrane-less organelles as well as the malfunction of RNA metabolism, known as the domino or snowball effect. Given the diverse functional and structural role of RNA-binding proteins, the dysregulation of specific RNA-binding proteins might lead to diverse functional abnormalities and complex pathophysiology. The complex and various pathophysiologies of ALS might be linked directly or indirectly to each other because of RNA-binding proteins in some but not all cases.For a better understanding of the complex and linked pathophysiology of ALS, the functional role of RNA-binding proteins could be important. In terms of the functional role of RNA-binding proteins, they generally bind to miscellaneous RNA transcripts and exhibit a widespread distribution in cells. The relationship between target mRNA and RNA-binding protein should also be considered in order to understand the exact function and importance of RNA-binding proteins in ALS. Knockdown or overexpression of specific RNA-binding proteins often shows exactly the opposite effect according to target mRNAs ,151. ForSporadic ALS cases are still more common than familial ALS cases, although some sporadic ALS cases have been identified as being caused by pathogenic mutations. It is thought that a complex and various interaction between genetic and environmental factors might be involved in sporadic ALS. Although RNA-binding protein abnormalities cannot account for all sporadic ALS cases, RNA binding proteins such as TARDBP, FUS, TAF15, EWSR1, HNRNPA1, HNRNPA2B1, MATR3, and TIA1 are strongly linked with sporadic ALS ,161,162.In the field of ALS, the reason why there is a selective affection of particular types of neurons in ALS is one of the more perplexing conundrums. Accumulating evidence demonstrates that neuronal stimulation and signal transmission require the trafficking and local translation of mRNA for proper neural function ,166. TheThis information indicates that the context-dependent complex of RNA-binding proteins may warrant further study; it may be the key to understanding the complex, linked, and entangled pathophysiology of ALS. The elucidated map of RNA-binding proteins in ALS will shed light on strategies for the development of therapeutics for ALS.


Fecal water syndrome (FWS) is long‐standing and common in horses, particularly in central Europe. No large epidemiological data sets exist, and the cause remains elusive. Dysbiosis could play a role in pathogenesis.To evaluate whether dysbiosis is present in horses with FWS when compared to stable‐matched control horses in spring and autumn.Fecal samples were collected from horses with FWS  and controls .The bacterial microbiome of samples collected in spring and autumn of 2016 was analyzed using high‐throughput sequencing. Differences in relative abundance of bacterial taxa, alpha diversity, and beta diversity indices were assessed between horses with FWS and controls based on season.Differences in microbial community composition based on time point and health status were not observed on any taxonomic level. Limited differences were seen on linear discriminant analysis effect size analysis. No difference in alpha diversity indices was observed including richness, diversity based on health status, or time point. No effect of health status on microbial community membership structure was observed.Limited differences were found in the bacterial microbiota of horses with and without FWS, regardless of season. Further research is needed to elucidate the role of microbiota in the development of FWS. AMOVAanalysis of molecular varianceANOSIManalysis of similaritiesbpbase pairsEGUSequine gastric ulcer syndromeFDRfalse discovery rateLEfSelinear discriminant analysis effect sizeOTUoperational taxonomic unitPCoAprincipal coordinate analysisRDPribosomal database project1Fecal water syndrome (FWS) in horses is a common problem, but so far it is reported in the literature only in central Europe.The bacterial microbiota plays a key role in human and animal health. Recent studies indicate that imbalances in the microbial communities and their function (dysbiosis) can be associated with diseases in the gastrointestinal tract and beyond.Our objective was to analyze differences in the fecal bacterial microbiota between horses with FWS and stable‐matched healthy controls at 2 time points.2This study was conducted with the approval of the Swiss Cantonal Veterinary Office (BE100/13).2.1A prospective case‐control study was performed. Referring veterinarians of the equine clinic of the University of Bern were asked to provide contact information of owners of horses suffering from FWS. Horse owners then were called by a veterinary student in her final year (LC) and asked to take part in a telephone survey. The questionnaire contained in‐depth information about the horses' health status in general, use, and the individual history of the FWS  and in autumn (August‐September 2014) of the same year. The autumn samples were collected by the owners after providing a fecal container at the spring visit. The packages were shipped overnight to the laboratory. The time points were chosen because FWS in this population was more frequent during spring than during autumn based on results of the telephone questionnaires. The samples were stored at 4°C for a maximum of 2 weeks before being transported to the laboratory where all were frozen at −80°C until DNA extraction. Some horses also had gastroscopy, measurement of saliva cortisol concentration after ACTH stimulation,2.3A sample of DNA was extracted from 1 g feces using a commercial kit  according to manufacturer's recommendations. Adequate DNA quality and quantity were assessed by spectrophotometry .Amplification of the V4 region of the 16S rRNA gene, purification, and sequencing were performed as previously described.2.4Distribution of age, sex, breed, equine gastric ulcer syndrome (EGUS) grade, fecal egg counts, and salivary cortisol concentrations were compared between horses with and without FWS using Fisher's exact or chi square tests.https://www.mothur.org/wiki/MiSeq_SOP).http://rdp.cme.msu.edu/index.jsp). Subsampling to the level of the sample with the smallest number of sequences  was performed to normalize sequence numbers for further comparison. Subsampling therefore consisted of random selection of 134 583 sequences from each sample. Completeness of sampling effort was assessed using Good's coverage. The sequences were uploaded to the University's data verse server (https://doi.org/10.5683/SP2/A5NZZP).Sequence processing was performed using Mothur 1.42.3 as outlined in the Mothur MiSeq SOP  adjustments were performed using the Benjamin Hochberg procedure for comparisons of relative abundance of taxa.P‐value of <.05 was considered significant for all comparisons. A commercial program was used for all statistical analyses .Community overlap and structure were compared between groups by parsimony test, analysis of molecular variance (AMOVA), and analysis of similarities (ANOSIM) applied to the Jaccard (community membership) and Yue and Clayton (community structure) indices, respectively. Dissimilarity was visualized using principal coordinate analysis (PCoA). Linear discriminant analysis (LDA) effect size (LEfSe) analysis, involving Wilcoxon and Kruskal‐Wallis tests was performed to identify differentially abundant OTUs with 97% sequence similarity between groups.33.1P = .16, .57, and .15, respectively). The main breeds represented in the FWS group in our study were breeds used for pleasure riding including Freiberger, Frisian, and Frisian mix breeds, Fjord ponies, PRE and Haflinger (Supplementary Information SP = .6) and McMaster egg count (P = .8). Salivary cortisol concentration after ACTH stimulation was normal in all tested horses and therefore comparison could not be performed.Thirty‐one horses were included, 16 horses with FWS and 15 stable‐matched controls. Age, sex, breed, and farm distribution are presented in Supplemental Information. Nine of 16 affected horses were female (56%) and middle‐aged . No statistical difference was found among age, breed, and sex between control horses and horses with fecal water . Median sequence numbers were 44 300  in the control group and 48 067 (range 134 584‐375 450) in the FWS group.3.3Thirty different phyla, 80 classes, and 137 orders were identified. Seven phyla, 11 classes, and 12 orders had a mean relative abundance >1%  were not visually evident on Jaccard and Yue and Clayton PCoA Figure  at eithe4Fecal water syndrome is common in horses in Europe. Dysbiosis has been suggested to play role in FWS in horses, but this possibility has not been studied to date. We found only minor differences in microbiota composition between horses with FWS and stable‐matched controls. Alpha and beta diversity did not differ between affected horses and stable‐matched controls.4.1No difference was seen in alpha diversity between horses affected with FWS and stable‐matched controls. In humans with functional gastrointestinal disorders, particularly irritable bowel syndrome, the diversity of microbial populations is decreased.Fecal water syndrome likely is a multifactorial disease. Stress associated with low hierarchical status in the herd has been suggested because it is a factor for the development of FWS.Selenomonas).The microbiota produces a substantical proportion of the gases present in the gastrointestinal tract, including hydrogen sulfide. Hydrogen sulfide recently has been recognized as a gaseous neuromodulator or neurotransmitter capable of modulating intestinal inflammation.Differences based on health status and season were evident on LEfSe analysis. Statistical tests dependent on taxonomic categories such as phyla and order often fail to detect community‐level differences because they mainly take into account relative abundances.In healthy horses, temporal variation is limited and differences usually are based on individual composition of the microbiota and husbandry, particularly feeding practices.There is individual animal variation in the composition of the microbiota in horses, but there is also a substantial impact of husbandry. Different feeding and management conditions have been shown to cause significant shifts in the microbiota.5The main limitation of our study was the selection of cases and controls. Because different diseases have been suggested as causes for chronic diarrhea and also for individual FWS cases, a standardized approach to diagnose FWS does not exist. Therefore, we chose to include horses for which the veterinarian in charge of the case perceived the horse to be suffering from FWS and for which the history was consistent with this syndrome. Additional tests for the patients depended on the cost to the owners and therefore could not be regarded as inclusion criteria. Controls were selected based on normal manure production, normal clinical examination, and similar age and breed. The abnormal findings for some of the diagnostic tests  were not considered as the cause of FWS, but this possibility could not be ruled out completely.Storage of fecal samples at 4°C for 14 days is not ideal because data from human medicine indicate stability for 24 hours at 4°C, and current recommendations are to store samples for a maximum of 24 hours at 4°C.Fecal water syndrome is likely a multifactorial disease. Minor changes in the gut microbiota were present between affected horses and healthy horses, indicating that the microbiota is a potential factor in the development of clinical signs. Additional research is needed to further elucidate the role of the microbiota in FWS and to try and exploit this knowledge for new therapeutic strategies.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.This study was conducted with the approval of the Swiss cantonal veterinary office (BE100/13).Authors declare human ethics approval was not needed for this study.AppendixS1. Supplemental Table 1. Information to dysbiosis not present in horses with fecal water syndrome when compared to controls in spring and autumn: Population data on horses studiedClick here for additional data file.AppendixS2. Supporting Information.Click here for additional data file.AppendixS3. Supplementary Table 2. LEFSE resultsClick here for additional data file.AppendixS4. Supporting Information.Click here for additional data file.


A distinguishing feature of the Mason-Pfizer monkey virus (MPMV) packaging signal RNA secondary structure is a single-stranded purine-rich sequence (ssPurines) in close vicinity to a palindromic stem loop  that functions as MPMV dimerization initiation site (DIS). However, unlike other retroviruses, MPMV contains a partially base-paired repeat sequence of ssPurines (bpPurines) in the adjacent region. Both purine-rich sequences have earlier been proposed to act as potentially redundant Gag binding sites to initiate the process of MPMV genomic RNA (gRNA) packaging. The objective of this study was to investigate the biological significance of ssPurines and bpPurines in MPMV gRNA packaging by systematic mutational and biochemical probing analyses. Deletion of either ssPurines or bpPurines individually had no significant effect on MPMV gRNA packaging, but it was severely compromised when both sequences were deleted simultaneously. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) analysis of the mutant RNAs revealed only mild effects on structure by deletion of either ssPurines or bpPurines, while the structure was dramatically affected by the two simultaneous deletions. This suggests that ssPurines and bpPurines play a redundant role in MPMV gRNA packaging, probably as Gag binding sites to facilitate gRNA capture and encapsidation. Interestingly, the deletion of bpPurines revealed an additional severe defect on RNA propagation that was independent of the presence or absence of ssPurines or the gRNA structure of the region. These findings further suggest that the bpPurines play an additional role in the early steps of MPMV replication cycle that is yet to be identified. Retroviruses are versatile mobile genetic elements that have historically been used to study the regulation of eukaryotic gene expression . Retrovipsi-ψ) assume higher-order structures and are confined to the first ∼100 to ∼400 nucleotides (nt) of the gRNA that invariably extend into the gag gene  containing sequences of the molecular clone MPMV/6A. The nucleotide numbers refer to the GenBank accession number M12349 .psi region (from R in the 5′ LTR to 282 nt of gag) of the MPMV sub-genomic vector, SJ2  that helps to monitor the effect of mutations introduced into this transfer vector upon propagation of the packaged viral RNA (XhoI site at the 5′ end) and an outer anti-sense (AS) primer, OTR788 (containing a BamHI restriction site at the 3′ end) along with internal overlapping primers containing the desired mutations, as previously described (psi fragments containing appropriate flanking cloning restriction sites (XhoI at the 5′ end and BamHI at the 3′ end), which were used to clone the fragments containing the mutations into SJ2, resulting in clones LA-I to VII. Details of the oligonucleotides and the templates used for making the mutations are listed in Site-directed mutations were introduced into the tor, SJ2 . SJ2 expiral RNA . Splice iral RNA  was emplescribed , 2019. Tin vitro dimerization and SHAPE assays, the psi region harboring the wild-type and the abovementioned mutations was also cloned into a pUC-based cloning vector, pIC19R  and OTR1005 . The PCR products were cleaved with HindIII and XmaI and ligated to pIC19R that had been previously digested with the same restriction enzymes, resulting in the wild-type  supplemented with 10% (v/v) heat-inactivated fetal bovine serum. To monitor the propagation efficiency of the transfer vector RNAs, the human cervical cancer cell line HeLa T4 was used and maintained at 37°C in the presence of 5% CO2 in DMEM supplemented with 7% (v/v) heat-inactivated fetal calf serum.Human embryonic kidney cells 293 (HEK 293T) were used for virus production. These cells were maintained at 37°C in the presence of 5% COin vivo to observe the effect of the introduced mutations on gRNA packaging and propagation. Toward this, the MPMV sub-genomic wild-type and mutant transfer vectors that contained the cis-acting sequences needed for genome replication, including transcription, polyadenylation, encapsidation, reverse transcription, and integration  sucrose cushion to concentrate the virus particles, as described previously . The relative quantitation (RQ) values for MPMV expression in the mutants were compared to the wild type to get the relative expression of each mutant vector RNA, both of which were normalized to the β-actin expression levels in each sample. As previously reported, the viral particles were observed to have an equivalent amount of β-actin mRNA irrespective of the amount of viral vector RNA that was packaged, thus providing a good proxy for the amount of virions produced  as described earlier  of the resulting RNA. After phenol/chloroform extraction, the RNAs were further purified on a TSK gel column  by Fast Protein Liquid Chromatography (FPLC)  in the presence of a buffer containing 200 mM sodium acetate (pH 6.5) and 1% (v/v) methanol. RNA fractions corresponding to the relevant peaks were pooled and ethanol-precipitated. The purified RNA pellets were resuspended in Milli-Q water, quantified by nanodrop (Thermo Fischer Scientific), and 800 ng of each RNA sample was electrophoresed on an 8% acrylamide/8M urea gel to check for its quality.The wild-type (RCR001) as well as mutant plasmids created for  earlier . A portiin vitro transcribed RNAs were subjected to SHAPE analysis, as described before  or monomer  buffer for 30 min at 37°C. This was followed by electrophoresis in native 1% agarose TBM  gel at 4°C. The gels were stained with ethidium bromide and visualized for dimeric or monomeric bands using ultraviolet (UV) transillumination. Band intensities were quantified using ImageLab software , and the percentage of dimerization was calculated for each RNA employing the following formula: (Intensity of dimer band – Intensity of background)/[(Intensity of monomer band – Intensity of background) + (Intensity of dimer band – Intensity of background)]. Results for each mutant RNA were presented relative to the wild type to determine the effect of mutations on their dimerizing ability. RCR001;  and FN s RCR001;  accordin RCR001; . BrieflyTo investigate the role of the ssPurines in MPMV gRNA packaging and propagation, we constructed two deletion mutants, LA-I and LA-II along with a substitution mutant LA-III . LA-I coin vivo packaging and propagation assay which involved co-transfecting the wild-type or mutant transfer vectors mentioned above with the packaging constructs TR301 and MDG  on the cDNAs prepared from the cytoplasmic RNA fractions of the samples ; LA-II (P = 0.48); LA-III (P = 0.04); Estimation of the relative packaging efficiency (RPE) of the transfer vector RNAs revealed that despite partial or complete deletion of ssPurines, RNA packaging of the mutant vectors  was not significantly reduced when compared to the wild-type  RNA [LA-I (P < 0.001) compared to the wild-type SJ2 vector RNA  and 0.74 (P = 0.12), respectively, P < 0.001; To investigate the significance of the redundancy of the purine-rich sequences during MPMV gRNA packaging and propagation processes, two double-deletion mutants involving both the ssPurines and bpPurines were created  relative to the WT; Finally, mutant LA-VII was designed to study the relevance of maintaining the base pairing in the repeat sequence (bpPurines) at the base of SL3 , 2A. Thiin vitro transcribed mutant RNAs was performed employing SHAPE, and their structure–function relationship was established to better understand the results obtained using biological assays.The predicted Mfold RNA secondary structures for the ssPurines mutants LA-I to III did not show any noticeable structural disruptions due to deletion or substitution when compared to the wild type . To expeThe SHAPE validated RNA secondary structure for mutant LA-I harboring five-nucleotide deletion in the former part of ssPurines  is depicThe SHAPE-validated structure of the LA-II mutant revealed that deletion of the ssPurines, while inducing local remodeling of the RNA secondary structure, did not affect the major structural motifs that have been shown to be important for MPMV gRNA dimerization and packaging . Brieflyin vitro transcribed RNAs of these mutants to identify any structural changes that may have implications for the observed biological results. In contrast with the Mfold prediction, the SHAPE-validated structure of the double deletion mutant LA-IV  revealed a complete abrogation of RNA propagation , indicatThe SHAPE-validated RNA structure of mutant LA-V, in which the duplicated sequence (GAAAGUAA) in ssPurines and bpPurines had been deleted and maintained the overall structural motifs critical for gRNA packaging. Noticeably, among the RNA conformers for this mutant, two differently base-paired structures for the primer binding site (PBS) were consistent with the SHAPE data . While tThe SHAPE-validated structure for mutant LA-VI (deletion of only the bpPurines) revealed that while SL1, SL2, GagSL1, GagSL2, LRI-I, LRI-II, and PBS structures were maintained, Pal SL and most of SL3 structure were remodeled . Thus, cThe SHAPE-validated structure of LA-VII containing deletion of the sequence (5′ ACUCUC 3′) complementary to the base paired region in bpPurines was not very different from the wild-type structure , the onlin vitro transcribed wild-type (RCR001) and mutant RNAs were incubated in a monomer or dimer buffer and analyzed for the percentage of dimerization after running them on agarose gel in TBM buffer at 4°C. The dimers formed in the TBM buffer at 4°C  on MPMV gRNA with purified full-length Pr78Gag polyprotein.The current study was undertaken to establish the role of ssPurines and its base-paired partial repeat, bpPurines, in MPMV gRNA packaging and propagation. The presence of a stretch of purines in the packaging sequences on retroviral gRNA has been proposed to facilitate RNA packaging by functioning as a potential Gag binding site . In supptrans-complementation assays with the ssPurine mutants (LA-I, LA-II, and LA-III, Experiments performed employing genetic Earlier structure prediction analyses of a mutant harboring a deletion of the ssPurines suggested that the stretch of duplicated bpPurines may become single stranded, restoring RNA packaging to almost wild-type levels . HoweverOn the other hand, mutants LA-V (deletion of only the duplicated sequence in ssPurines and bpPurines; in vivo or in virio probing experiments, as has been done for HIV-1 (in virio SHAPE data show minimal differences with in vitro data, supporting the usefulness of the in vitro data.In summary, this study suggests that both the ssPurine- and bpPurine-rich sequences play a redundant role in MPMV life cycle perhaps by acting as potential Gag binding sites, while maintaining the overall structure and stability of the MPMV packaging signal RNA. Additionally, our results also suggest a potential role of the bpPurines in the early steps of the MPMV life cycle that affect propagation of the packaged RNA . Furtheror HIV-1 . NeverthAll datasets presented in this study are included in the article/TR and RM conceived the concept. TR, FM, and RM supervised the project. LA, FP, VV-B, AC, AJ, and VP performed the experiments. LA, FP, AC, FM, RM, and TR contributed toward data analysis. LA and FP wrote the original manuscript. TR, FM, RM, LA, FP, and AC contributed toward reviewing and editing the manuscript. All authors discussed the results and commented on the manuscript.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.


However, it is also found increasingly in a plethora of RNA species, predominantly transfer RNAs, but increasingly found in cytoplasmic and mitochondrial ribosomal RNAs, enhancer RNAs, and a number of long noncoding RNAs. Moreover, this modification can also be found in messenger RNAs and has led to an increasing appreciation that RNA methylation can functionally regulate gene expression and cellular activities. In mammalian cells, the addition of m5C to RNA cytosines is carried out by enzymes of the NOL1/NOP2/SUN domain (NSUN) family as well as the DNA methyltransferase homologue DNMT2. In this regard, NSUN2 is a critical RNA methyltransferase for adding m5C to mRNA. In this review, using non-small cell lung cancer and other cancers as primary examples, we discuss the recent developments in the known functions of this RNA methyltransferase and its potential critical role in cancer. The first intimation that RNA could be modified came from studies in the 1960s which demonstrated that pseudouridine  could be incorporated into transfer RNA (tRNA) and ribosomal RNA (rRNA) . Since tThe most commonly found RNA modification in mRNA is called N6-methyladenosine (m6A) and was first described in 1974 by Desrosiers et al. . However5-methylcytosine (m5C) has frequently been associated with tRNA  but was Early methodologies to identify m5C utilized liquid chromatography mass spectrometry (LC-MS) but can be considered currently unsuitable for analysis of m5C for technical reasons . Since tOne of the most established is RNA-bisulfite sequencing. Bisulfite sequencing is currently the most accurate and reliable method to detect m5C marks at nucleotide resolution in both DNA and RNA and basically involves the chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion, allowing users to determine m5C methylation at the singe nucleotide level by sequencing . With thArabidopsis [Other methodologies based on NGS have also been developed. One, methylated RNA-immunoprecipitation sequencing (meRIP-Seq) was initially developed to study changes in m6A . This tebidopsis  and in bbidopsis ,18 and, bidopsis . Two othbidopsis , althougbidopsis . Methylabidopsis . When cobidopsis . By usinbidopsis . Using tbidopsis ,24,25,26At present, the known mammalian m5C RNA methyltransferases (RNMTs) belong to either the DNA Methyltransferase family   or belonBoth of these families utilize S-adenosyl-L-methioinine (SAM) as a donor for the transfer of methyl groups to RNA, and the mechanisms by which these proteins catalyze this transfer have been recently well reviewed ,32. WhilA large body of evidence has now shown that NSUN2-mediated RNA methylation plays many cellular functions and are summarized in NSUN2 is a predominantly nucleolar protein and plays important roles in tissue homeostasis, spindle stability, and early embryogenesis . During Various indirect functions for NSUN2 in cellular processes have been described and include cellular differentiation, testis differentiation, and early embryogenesis ,43,44,45A significant body of evidence now links m6A RNA methyltransferases with both memory and neural development ,68,69,70Aside from neurological disorders, some emerging evidence now links the activities of both m6A and m5C RNMTs in the vascular setting. Various m6A RNMTs have now been shown to have roles in endothelial cells, vascular calcification, and abdominal aortic aneurysm ,85,86,87A significant body of work has now shown that RNMTs are aberrantly expressed and play important roles in the development and pathogenesis of cancer ,91. In tn = 89 tumors), but that this upregulation showed no correlation with the expression of Ki67, Estrogen Receptor (ER) status, or MYC amplification [Several studies have now shown that NSUN2 is overexpressed in breast cancer. In their original report on NSUN2, Frye and Watt observed that NSUN2 was overexpressed in 7/7 human breast carcinomas . Using afication . The uprfication . DNA hypfication . A more fication . While tfication , in contfication . These sIn addition to their original description of NSUN2 as an RNMT, Frye and Watt also observed high expression of NSUN2 in 3/4 colon cancers , an obseCircular RNAs (circRNAs) are a class of noncoding RNA (ncRNA) that, unlike linear RNAs, form covalently closed continuous loops and act as gene regulators in mammals . IntriguThe current available published evidence for altered expression of m5C RNMTs in lung cancer is sparse. Due to its original identification as a marker of proliferation ,104, higDuring early embryogenesis, NSUN2 is constitutively expressed , but it Most recently, a pan-cancer analysis of RNMTs has shown that alterations to NSUN2 are common in lung cancer , althougn = 566) and LUASC (n = 487) samples. Mutations, putative copy number alterations, and mRNA expression were selected for analysis using default settings. The results are presented as OncoPrints, a compact means of visualizing distinct genomic alterations, including somatic mutations, copy number alterations, and mRNA expression changes across a set of cases. Given the large number of alterations observed in the cBioPortal analysis, we subsequently conducted additional analyses for NSUN2 using Lung Cancer Explorer (LCE) [cBioPortal analysis ,112 was er (LCE) . For tumor-normal gene expression difference, Lung Cancer Explorer generates searchable tables for LUAD and LUSC that contain the summary statistics from meta-analysis of standardized mean difference  using Hedges’ G as an effect size metric. Studies included for meta-analysis must have at least 10 samples in each group, and meta-analysis was only performed for genes with data available from at least three qualifying studies . For surMeta-analysis of all available datasets on LCE for gene expression of tumor vs. normal and survival are also presented graphically in Given the significant alterations observed in the meta-analysis for tumor versus normal gene expression changes, we reanalyzed the TCGA datasets using UALCAN . In agreAs a strong association was observed for NSUN2 with overall survival in LUAD, we also examined the prognostic value of NSUN2 mRNA using KM-Plot  Figure A. OveralThe data presented here clearly demonstrate that NSUN2 is significantly overexpressed/altered in NSCLC, and while evidence for a functional role for this protein in lung cancer is currently lacking, it would suggest that this protein may play important roles in lung cancer pathogenesis and suggests that its expression may also have utility as a prognostic marker.The roles for m5C RNMTs have now been identified in several other cancers. In one early study, elevated protein expression of NSUN2 was confirmed in various cancers including esophageal, stomach, liver, pancreas, uterine cervix, prostate, kidney, bladder, thyroid, and breast cancer . In skinKip2 in an m5C-dependent manner [In bladder cancer, Y-Box Binding Protein 1 (YBX1) has been identified as a an m5C “reader” and was shown to promote carcinogenesis by stabilizing Hepatoma-Derived Growth Factor (HDGF) through an interaction with Elav-Like RNA-Binding Protein 1 (ELAVL1). Importantly, high co-expression of NUSN2, YBX1, and HDGF was found to predict the poorest survival in urothelial carcinoma of the bladder (UCB) . High ext manner .Sphingosine kinases have been shown to be potential targets for therapy in breast cancer ,122. In Vaults are ribonucleoprotein complexes widely associated with the induction of chemoresistance and long-term chemotherapy failure  and for Molecularly targeting m5C RNMTs using drugs previously developed to target DNMTs is a potential possibility as demonstrated from using Aza-IP  and prelFinally, the identification of NSUN2 expression as a potential biomarker for stratifying patients for immune-checkpoint inhibitors at least in HNSCC  is a finSeveral companies have emerged to target RNA Epigenetics. At the present moment in time, many of these are focused on targeting m6A RNMTs , but givWhilst still a relatively new field, the recent discoveries of the role of the epitranscriptome in cancer clearly links RNMTs as a candidate therapeutic target moving forwards. While m6A RNMTs are currently the focus of several targeting strategies in the pharmaceutical setting, emerging evidence now begins to link m5C RNMTs to cancer.Targeting the proteins that add m5C modifications to DNA (particularly DNA methyltransferases) has been an area of active research in cancer. Agents such as 5-azacytidine (vidaza) or 5-aza-2-deoxycytidine (decitabine) originally developed to target these proteins have been approved by the U.S. Food & Drug Administration (FDA) ,142 for