B6/D2 hybrid and Dnmt2−/−C57BL/6) resist and do not change their phenotype while their sperm RNAs after microinjection into fertilized mouse eggs transfer the newly acquired phenotypes in a susceptible inbred line . Unexpectedly, in the same line of experiments, sperm RNA from animals raised on a normal diet when mixed with the sperm RNA from animals raised on a diet high in fat or synthetic miR-19b (inducer of obesity) affects or prevents the development of obesity and diabetes. However, it remains unclear what happens to ncRNA signaling under diet. With a comprehensive new analysis of the transcripts maintained as an RNA/DNA hybrid in sperm, we suggest that a fraction of the RNAs are stably attached to the genome. Thus, we propose that changes in the dynamics of ncRNA retention on DNA by factors such as transcriptional variations or lack of adequate methylation could serve as molecular markers to trace these epigenetics events.A wide range of diseases result from environmental effects, and the levels of many native transcripts are altered. The alteration of non-coding RNAs (ncRNAs) and transmission of the variation to the next generation is increasingly recognized as a marker of disease. However, the determining signals and mechanisms of RNA-induced heritability remain unclear. We performed functional tests with four different genotypes of mice maintained on a high-fat diet to trace the transfer of the obesity/diabetes phenotype to the next generation in order to detect common signals. Two founders of four mouse lines ( C57BL/6 , and a mutant Dnmt2−/−C57BL/6  maintaiSeveral studies now show changes in diet induce RNA variations also in the sperm cells (see recent review ). The RNIn addition, Chen et al.  identifiAspGUC occurs by the Dnmt2 protein. Subsequently, a group of RNA modifying enzymes has also been reported . In addormation . In factnscripts . In prepnscripts . Accordid by HFD . Thus, id by HFD .Dnmt2−/−C57BL/6 inbred background are found at varying levels from male to male regardless of diet. These varying levels of ncRNAs in Dnmt2−/−C57BL/6 mice are suspected to be produced by uncertain changes in RNA stability in the absence of Dnmt2. In addition to Dnmt2−/−C57BL/6, we reveal here another line B6/D2 hybrid mice (wild type) resistant to HFD. Organisms with hybrid genetics are known for their variation in the levels of transcripts  in the same male . Our resTogether, these results highlight the combinatorial effects of ncRNAs in the field of RNA-mediated epigenetic hereditary variation and its consequence on cross-generations.C57BL/6 and Balb/c with B6/D2 hybrids (F1 cross between the C57BL/6 and DBA2 lines) and the Dnmt2−/−C57BL/6 mutants. To minimize the effect from the environment, all experiments were conducted using animals born during the same week under the same housing and feeding conditions. From founder males (F0) raised in HFD, we derived two generations by sexual mating (F1 and F2). All mice other than F0 males were kept on normal diet (ND) and were followed by monitoring their body weight and health for up to 20 weeks. During this time, lineage-dependent differences of mice in test results for weight gain, glucose and insulin tolerance were observed. A summary of the experiments with four lines  is presented in the Supplementary Figures: founder (F0) To answer the question of susceptibility to HFD (21% butter) among different genotypes and its transgenerational maintenance, we maintained two inbred lines, Additionally, all glucose tolerance test (GTT) and insuline tolerance test (ITT) plots with area under the curve (AUC) were analyzed before and after normalization to baseline glucose levels (see the guidelines provided in PMID: 34117483). Analyses of AUC for GTTs and ITTs involve first normalizing the data . This is thought to be important, because a GTT and an ITT the response to a glucose and insulin boost, respectively. The calculated AUC, after normalizing data to baseline glucose levels, is proposed to reveal a much more subtle effect than usually reported without normalization. However, there are conflicting opinions on the application of AUC in chronically treated animals [see references ]. BasicaC57BL/6) for body weight gain, glucose, and insulin tolerance were noticeable in the F0 generation  diet as previously described using a larger number of animals, 10 males and eight females; neither sex gained weight . We observed that lineage-dependent phenotypic variations in the disease spectrum  were transferable by microinjection of total RNA from spermatozoa of C57BL/6, B6/D2, Balb/c and Dnmt2−/−C57BL/6 mice in fertilized eggs from naïve Balb/c mice. A higher effect is observed on female mice born after microinjection with sRNAs.Another generation (G1) was obtained in Dnmt2−/−C57BL/6 and B6/D2 hybrids, also contained RNAs that could recapitulate the metabolic phenotypes after microinjection into fertilized eggs of a susceptible line . We assume that unmethylated sRNAs from Dnmt2−/−C57BL/6 are methylated by wild-type Dnmt2 protein present in fertilized Balb/c eggs. The dynamism of methylation remains to be assessed. These results again confirm the role of RNA methylation mediated by Dnmt2 protein in the transfer of hereditary acquired epigenetic traits. In addition, these results reveal signals from as yet unexplained sRNAs from the B6/D2 hybrid line which required further investigation.Thus, the total spermatic F0 RNAs of HFD-resistant founders, in particular the In addition, we microinjected RNase enzymes with sRNAs to initially abolish RNA inducing signals. The results of the RNase enzyme microinjection are however contradictory with the expected results. Under these conditions we believe that the RNase treatment removes more than the sRNAs at the time of microinjection into the fertilized eggs. We report this negative result to signal in future experiments that the RNase enzyme should be removed from the aliquot prior to the microinjection step.Finally, co-injection with sperm RNAs from ND could compromise obesity . Previouin vivo except under extreme conditions. Under natural conditions this scenario does not occur for a given miRNA of spermatozoa from animals reared in HFD. It is unlikely that a small amount of changes in individual RNAs is immediately responsible for the complete phenotype. On the other hand, several ncRNAs  are also increased during HFD; as such, we expect a cumulative role of ncRNAs in the inheritance of diet-induced metabolic phenotypes. To answer the question of the specificity of miR-19b-5p, we used the same amount of miR-19b-5p  as that used previously, now in combination with total sperm RNAs (sRNA) as carrier from animals raised in an ND. As shown in In studies reported from our laboratory, we have shown that microinjection of miR-19b-5p is sufficient to recapitulate HFD-mediated phenotypes (HFD-MPs) . Indeed,Under these conditions sRNA-HFD mixed with sRNA-ND always maintains a detectable level of activity. RNA-mediated transfer of phenotype is confirmed by variation in the phenotype expression of weight gain and GTT and ITT tests . Microinjection of a synthetic miR-19b is completely abolished by sRNA-ND; on the other hand, under the same conditions, sRNA-HFD maintains detectable levels of activity. We predict that synthetic miR-19b-5p is less stable than endogenous sRNA-ND or sRNA-HFD. The stability of RNA molecules would secure their activity.C57BL/6 and Balb/c), while that in the B6/D2 F1 hybrid miR-19b-3p is increased. In contrast, in Dnmt2−/−C57BL/6 males, none of the mir-19b was increased under HFD conditions. Dnmt2−/−C57BL/6 mice from male to male, regardless of food. To exclude effects of litter in the expression levels of miR-19 in sperm of Dnmt2−/−C57BL/6 under ND, we tested five males from two separate litters and the results are shown from male to male in Dnmt2−/−C57BL/6 mice in ND. This means that in Dnmt2−/−C57BL/6 mice, an alteration of miRNA-19b already exists in males raised on ND, and this marker suggests sporadic changes in the proportion of miR-19b-5p and -3p in its sperm RNA. We also confirm, see below, by RNA-seq analysis with four different Dnmt2−/−C57BL/6 RNA samples from sperm, the presence of other transcripts variations.From a previous study, we found a short list of deregulated miRNAs in the sperm of males raised on HFD. By quantitative RT-PCR analysis, we confirmed the deregulation of miR-182, miR-19a, miR-19b, miR-29a, and miR-340 in testis and sperm RNA of the HFD males compared to ND males. Therefore, we focused our study here on the analysis of one miRNA (miR-19b) given its HFD-MPs inducing activity. Dnase. The RNAs associated with the DNA and the free RNA fraction from the sperm are recovered in equivalent quantities, with the hypothesis that the comparison between the proportion of strongly maintained RNA with the DNA fraction on free RNA of the spermatozoa could reveal a change of the hereditary epigenetic marks.To get an overall view of sperm RNA profiles, we addressed the issue of examining different sperm RNA fractions. In particular, the RNA fraction associated with the genome in hybrid DNA/RNA structures. Recently, we reported that with a conventional sperm RNA extraction protocol, two sperm RNA fractions could be obtained . Unlike C57BL/6 wild-type and Dnmt2−/−C57BL/6): free (F) and fraction associated with DNA (D) [for more details see Methods and references . This analysis aimed to efficiently assign peaks in terms of important genomic characteristics. Regions of the genome were annotated by sperm RNA fractions of DNA-bound sperm RNAs versus free RNA. The analysis was performed to obtain information if a peak is found in the promoter TSS (transcription start site), 5′UTR, exon (coding), intron, 3′UTR, TTS (transcription termination site), and intergenic and non-coding regions. In normal mice, there was no difference between the number of counts in the samples of ND/HFD and the results are homogenous between the replicates of each sample group −/−C57BL/6 > 60% than in the samples with wild-type around 50%. In contrast, transcripts from intron regions are somewhat lower in Dnmt2−/−C57BL/6 samples (35%) compared to normal animals (38%) regardless of diet conditions  has been reported to be associated with reduced rates of protein synthesis  and genowith HFD . In otheMus musculus ERF, Erf1), Gm42418, and Gm26917 . The transcripts are visualized using the UCSC genome browser. The expression signal is very high and comparable between samples according to the scaling of the data view on the left , transcript variant 1 on chromosome 9 of Dnmt2−/−C57BL/6 to wild-type C57BL/6 in HFD/ND.Here, we focus on the set of abundant mouse transcripts presented in the left , and ove samples . We reveDnmt2−/−C57BL/6 samples exhibits a unique profile and shows variation in transcripts among the four samples. The unpredictable state of transcript levels from Dnmt2−/−C57BL/6 animals would suggest altered RNA stability as a cause of variation in Dnmt2−/−C57BL/6 sperm transcripts. Our working hypothesis is that the loss of the Dnmt2 protein triggers random changes in the overall stability of the transcripts and thus influences the half-life dynamics of DNA/RNA hybrids, which may be one of the causes of loss of acquired inheritance in Dnmt2−/−C57BL/6 line.Unlike the wild type (four samples), each biological replicate of the Dnmt2−/−C57BL/6 in normal diet (ND) and 21% butter (HFD) through generations. The transcript set comprising the mouse gene AY036118 (transcription factor linked to the ETS of Mus musculus ERF (Erf1), Gm42418, and Gm26917 (A 45S pre-rRNA transcripts) serves as a precursor for 28, 18, and 5.8S rRNA on chromosome 17, overlapping elements repeated by Repeat Masker on the mouse assembly (GRCm38/mm10).The snapshot of the genome browser of pre-rRNA transcripts and leucyl-tRNA synthetase, mitochondrial (lars 2) of normalized BigWig files of two types of sperm RNA fractions (DNA bound RNA and free RNA), shows a normalized expression RNA-seq signal in two mouse lines, wild-type and The complexity of the genetic contribution to a common phenotype of weight gain with a diet high in sugar and fat poses many challenges. One question raised here is whether the RNA alterations produced in the sperm of laboratory mouse lines reared on the same HFD (high-fat diet) vary from line to line. In other words, what are the common signals (RNAs) produced for the transmission of high-fat diet-mediated phenotypes (HFD-MPs) between different genotypes? Here, we show that RNA variations are produced in male mice maintained on HFD and are transported by sperm regardless of the genetic background, at least in the four mouse lines tested.C57BL/6, B6/D2, Balb/c and Dnmt2−/−C57BL/6) nevertheless under the same conditions respond differently in the expression of HFD-MPs and that all founders even with the wild-type genome could be resistant, like the B6/D2 hybrid, and yet pass the changes on to their offspring. Two of the four founder mouse lines do not develop HFD-MPs as indicated previously for the Dnmt2−/−C57BL/6 mice, and here the hybrid B6/D2. C57BL/6 is a commonly used inbred mouse line that responds effectively to HFD by increasing fat and body weight, being insulin-resistant (Type 2 diabetes-like), and transmitting all metabolic alterations to at least two generations as previously indicated  is another laboratory inbred line which, in HFD condition that does not increase body weight but develops Type 2 diabetes-like phenotypes and passes on HFD-MPs to the F1 and F2 generations. Thus, with C57BL/6 and Balb/c two inbred lines maintained in the same homing, the same food and the same conditions respond differently to HFD. B6/D2 is a hybrid between C57BL/6 and DBA2; under the same feeding and environmental conditions as the above inbred lines, founder males appears to be resistant to HFD, but in their offspring certain aspects of the phenotype are found as predicted by genetic segregation. Transmission of the phenotype from B6/D2-resistant male founders in backcrossing is reminiscent of genetic anticipation , the Type 2 diabetes-like phenotype is detectable, and in the F2 generation with different genotypic segregation, obesity is displayed. This means that the body weight and Type 2 diabetes-like characters are separately segregating. Finally, a Dnmt2 null mutation in the C57BL/6 background abolished the sensitivity to HFD (21–33% butter), and the animals do not gain weight and do not exhibit Type 2 diabetes-like traits, but unlike B6/D2 the transmission of obesity and diabetes in the offspring is also well abolished. Likewise, using a higher concentration of butter (33% fat), the Dnmt2−/−C57BL/6 founder mice maintained the same weight, but without symptoms, profiles in both conditions glucose and insulin tolerance tests are changed , it is expected that both fractions are active in microinjection tests in fertilized mouse eggs. We suggest that the activities of RNAs could be dynamic and dependent on the half-life of each RNA as DNA-bound or circulating molecules. The robustness and mechanisms underlying these hereditary changes will require further investigation.The free RNA fraction of sperm from four lines induces HFD phenotypes described here. The DNA-bound RNA fraction is not yet tested https://arriveguidelines.org). Mice were maintained in building with accord to European and international guidelines and policies in accordance with the animal welfare laws in Turkey in an animal facility at Genkok Institute, Kayseri, Turkey. The experiments were approved by the Turkish ethics committee (see file number below). 1) Institutional and/or licensing committee Erciyes University, Kayseri, Turkey; 2) we confirm all experiments were performed in accordance with relevant guidelines and regulations with permission from Erciyes University committee in an animal facility at Genkok Institute, Kayseri, Turkey. 3) All mice were maintained according to European and international guidelines and policies according to the animal welfare laws in Turkey in an animal facility at Genkok Institute, Kayseri, Turkey.All animals (mice) and experiments were carried out in compliance with the ARRIVE guidelines .The mice were maintained in a facility under controlled conditions . The animals were cared for and treated according to the Principles of Laboratory Animal Care (European rules). All tests were performed between 10:00 and 16:00 in isolated rooms.C57BL/6, B6/D2, Balb/c and Dnmt2−/−C57BL/6 . After weaning, 4-week-old males were divided into two groups. One group was maintained on a normal diet (ND), a standard chow diet , energy 3.5 kcal/g) and the other was fed a Western diet containing, in addition, 17% casein, 0.3% DL-methionine, 34% sucrose, 14.5% cornstarch, 0.2% cholesterol, 5% cellulose, 7% CM 205B, 1% vit200, and 21% or 35% butter .Four lines of mice were maintained: Overnight fasted mice were injected with glucose or insulin in saline buffer. The baseline glucose measurements were analyzed from tail blood before intraperitoneal (ip) glucose injection (2 mg/g body weight) using the OneTouch Vita  system.®, Novo Nordisk, Bagsværd, Denmark) diluted in 0.08 mU/μl saline was injected to a final delivery of 0.8 mU/g body weight.Insulin  was prepared in a filtered microinjection buffer  at a concentration of 3-5 × 10 (RNA from purified sperm (centrifugation) was extracted using TRIzol reagent . All total RNA preparation from mouse sperm were treated with proteinase K 400 microg and Dnase 10–100 ng. The quality of the RNA preparations was verified by spectrometry on an Agilent Bioanalyzer 2,100 apparatus  . Microin3-5 × 10  moleculemiRNA quantitation was performed using miScript Primer Assays , following the manufacturer-recommended protocols. Real-time PCR was performed with a Light Cycler Instrument  using the miScript SYBR Green PCR kit .Dnase enzyme treatment. Here, we have released sperm cells from epididymis and washed and separated from somatic cells by centrifugation and overnight incubation at 56°C in Tris buffer 20 mM pH8, EDTA 50 mM, with 0.5% SDS, 20 µM dithiothreitol, and 400 μg/ml Proteinase K. Total nucleic acids after enzymatic removal of the proteins was followed by fractionation of RNA and DNA-bound RNA. Extracts were fractionated by the ZR-Duet™ DNA/RNA MiniPrep Plus protocol according to the specifications of the manufacturer (www.zymoresearch.com). After digestion by DNase, 10–100 ng of RNA was prepared and sent to Eurofins for high-throughput sequencing on Illumina Hiseq 2,500 or Illumina MiSeq. Libraries of all samples corresponding to DNA-associated RNA and free-RNA molecules were generated. Small RNA high-throughput sequencing was performed by Eurofins (Europe). Number of mapped reads are summarized in Dnmt2−/−C57BL/6 samples is lowered than HFD/ND samples.A classic TRIzol protocol produces two fractions of RNAs: one in aqueous phase (free fraction) and the other at the junction of the phenol-chloroform DNA fraction. The DNA fraction consists of DNA/RNA hybrid molecules and RNAs which are tightly associated with the genome . The RNADnmt2−/−C57BL/6 samples.FastQC version 0.11.5 was used to do some quality control checks on the RAW sequence data. RNA-seq reads adapters removed by cutadapt v1.16. Based on the FastQC results, trimming of bad-quality reads was performed using Trimmomatic version 0.36  . Transcrhttp://genome.ucsc.edu)  Hisat2 was used . Quality of alignment was assessed using stats and plot-bamstats utilities of samtools . Bam files were converted into BigWig format using deep tools bamCoverage with the following parameters: ‐of = bigwig - 50- normalizeUsing RPKM. Visualization of normalized BigWig files were performed using the UCSC genome browser (csc.edu) .HOMER version 4.9 was used for peak finding {Heinz, 2010 #10325}. Statistically significant peaks of expression were identified using HOMER  rate threshold (cut off) < 0.001). After obtaining sets of peaks, peak annotation was performed using Homer “annotate_peaks” to relate each peak to nearby genes and genomic features. Homer (annotate_peaks) is widely used to assign peaks to positions annotated for gene, exon, intron, promoter, 5′ untranslated region (UTR), 3′ UTR, and other genomic features. The promoter-TSS, exons, introns and other features were based on the mm10 transcripts.Statistical analysis was performed by two-tailed, one-way analysis of variance (ANOVA), uncorrected Fisher’s least significant difference (LSD). Data are expressed as the mean with sem. p < 0.05 was considered to be statistically significant.


LMNA, and two additional variants, one each in SETX (g.intron10-13delCTT) and FUS (p.Gly167_Gly168del). These latter genes have been linked to ALS. All family members were genotyped and each variant, and each combination of variants detected, were functionally evaluated in vitro regarding effects on cell survival, expression patterns and cellular phenotype. Muscle biopsy retrieved from the individual with ALS showed leakage of chromatin from the nucleus, a phenotype that was recapitulated in vitro with expression of all three variants simultaneously. Individually expressed variants gave cellular phenotypes there were unremarkable. Interestingly the FUS variant appears to be protective against the effects of the SETX and the LMNA variants on cell viability and may indicate loss of interaction of FUS with SETX and/or R-loops. We conclude that these findings support genetic modifications as an explanation of the clinical heterogeneity observed in human disease.Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), can be clinically heterogeneous which may be explained by the co-inheritance of multiple genetic variants that modify the clinical course. In this study we examine variants in three genes in a family with one individual presenting with ALS and lipodystrophy. Sequencing revealed a p.Gly602Ser variant in The online version of this article (10.1007/s11010-021-04103-7) contains supplementary material, which is available to authorized users. SETX, and fused in sarcoma, FUS), and one variant in a gene  which exhibits profound allelic heterogeneity. However, only the individual with all three variants has presented to date with ALS.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that targets motor neurons and commonly results in death within 2–5 years of diagnosis. Mutations in several genes have been detected and linked to ALS. However, the disease process is quite heterogeneous, and patients with the same mutation can exhibit varying severity or progression of the disease , 2. ThisTDP-43) , F, FTDP-4343) . Lysates were clarified by a 30 s spin at maximum RPM, and protein concentration determined with the DC protein assay kit (BioRad) or the Pierce protein assay kit (ThermoFisher). For whole cell lysates 50 µg of total protein was diluted in Laemmli sample buffer, denatured at 90 °C for 15 min and separated using 7.5% or 6% SDS containing polyacrylamide gels (SDS.PAGE). Proteins were transferred to nitrocellulose membrane and blocked for 1 h at RT in blocking buffer [10% skim milk powder in PBS containing 0.1% Tween-20 (PBST)]. Membranes were probed with antibodies as listed in Supplemental Table 1 in blocking buffer, then washed twice for 20 min each with PBST, then incubated with appropriate secondary antibodies  for 1 h at RT in blocking buffer. Membranes were washed twice in PBST for 20 min each, then incubated with ECL reagent , and visualized using a BioRad gel documentation center and ImageJ software.For immunoprecipitations, 1 mg of total protein was subjected to immunoprecipitation (IP) using pansorbin  as a carrier and 1 μg of antibody for 1 h at RT on a rotating platform. Samples were then spun at 21.1 × g, supernatant removed, and the pellet washed in lysis buffer. Washes were repeated 3 times with lysis buffer wash, and then followed by 2 washes in PBST. After the final spin pellets were fully resuspended in Laemmli PAGE sample buffer and denatured at 90 °C for 5 min. Samples were then spun to pellet the pansorbin and the supernatant run as per the western blotting protocol (below). Negative controls consisted of IP with pansorbin and rabbit IgG or mouse IgG as appropriate.. [Immunoprecipitation using S9.6 antibodies methodology was modified from Cristini et al. . Cells w. . After wLMNA from the proband was indicated due to the presence of lipodystrophy and cardiac conduction abnormalities. We detected g.156108384G>A within exon 11 in LMNA, producing a glycine  to serine  substitution at amino acid residue 602 (p.Gly602Ser). Sanger sequencing revealed the proband was heterozygous for the LMNA variant  in the protein or contribute to alternate splicing through creation or abolition of a splicing donor or acceptor site. Due to these results suggesting at best a modest effect of LMNA, we used ONDRISeq, a neurodegeneration gene panel, to search for variation within ALS genes to explain the ALS phenotype in the proband. Two heterozygous variants were identified: a rare intronic deletion in SETX (g.intron10-12delCTT) and an in-frame deletion in FUS p.Gly167_Gly168del  and was predicted to be deleterious (score −114.84). In contrast, the FUS deletion removes two glycine residues at the N-terminal region of the glycine rich domain and was predicted by Provean to be pathologically neutral. SETX g.intron10-12delCTT was observed in one individual in gnomAD with an allele frequency of 0.000004. Interestingly, this variant is not observed when subsetting to the non-neurological subset of gnomAD suggesting the carrier of this variant in gnomAD may have a diagnosis of ALS, Alzheimer’s disease, schizophrenia, or another allied neurological condition. Importantly, due to a lack of information on this individual’s phenotype or genomic information there is not enough evidence to speculate on the impact of this variant on the individual’s neurological disease diagnosis. This SETX variant was not observed in the ALS Knowledge Portal or Project MinE data browser, the two largest ALS exome and genome databases. While the FUS variant was not observed in gnomAD, other deletions of glycine residues within this region were observed at an elevated frequency in individuals free of neurodegeneration in keeping with a benign variant classification. Sequencing of the proband’s father revealed that he was a heterozygous carrier of only the FUS deletion, and while the maternal genotype could not be determined experimentally, it can be deduced that she is an obligate carrier of the LMNA and SETX variants as the proband’s siblings are also carriers.Sequencing of The deltoid muscle biopsy from the proband demonstrated both acute and chronic neurogenic atrophy with fiber type grouping. No abnormalities were noted on Gomori trichrome or PAS staining (not shown). In normal muscle tissue, both lamin and emerin immunoreactivity delineate rod-shaped nuclei in a continuous manner with no evidence of nuclear membrane interruption in normal muscle cells and chromatin is easily seen with Hoechst staining within the nuclei Fig. a. In conTo determine the effect of the co-expression of these mutations, constructs were transfected to HEK293T cells Figs. . The expSETX, LMNA and FUS protein was examined in HEK293T cells to determine the normal expression of these proteins  strongly suggesting that tightly controlled expression levels of SETX are also likely required in normal cellular homeostasis. Interestingly, the co-expression of MT FUS with MT SETX resulted in rescue of viability back to control levels, while MT FUS expressed with MT LMNA resulted in a partial rescue (p < 0.01) when compared to control transfections  strongly suggestive that MT SETX is driving the toxicity. The co-expression of all three mutant proteins was also significantly detrimental to cell viability (p < 0.001) compared to control transfections, and the inclusion of MT FUS appears to have no effect on MT SETX toxicity, suggesting that MT SETX is the protein likely driving the reduced viability in this model. An alternative explanation is that the combination of MT SETX and MT LMNA expression is too detrimental for the expression of MT FUS to have any effect on viability.Cells were transfected with constructs for expressing WT or MT proteins alone or in combination and viability assessed using the MTT assay. Over three replicates, and using ANOVA, the significance was determined against the EGFP control transfection Fig. . UntransSeveral of the ALS associated proteins are known to be involved in DNA/RNA binding including R-loop interaction/resolution or double stranded break repair, including FUS  and SETXp < 0.0008; Student’s t-test compared to non-etoposide treatment), while MT SETX amounts detected were not significantly different (p = 0.77) upon treatment, suggesting the MT SETX is likely associated only at extremely low levels, if at all, with R-loops , anrg337His , 87). ThFUS variant (p.Gly167_Gly168del) was predicted to be non-detrimental and expression of this mutation in culture appears to be consistent with this prediction, it may have some physiological function since MT FUS appears to partially rescue MT SETX and MT LMNA phenotypes in culture and only the presence of all three variants causes the dramatic abnormal nuclear phenotype. The SETX variant  was predicted to result in the loss of exon 11 (causing a deletion of aa 1792–1849). The function of exon 11 is unclear. However, this variant appears to affect cellular localization of SETX, its ability to associate with R-loops, and possibly its interaction with FUS.While the SETX variant, but only one met the El Escorial criteria for diagnosis of ALS. The examination of viability confirms that the SETX variant is deleterious, and confocal data shows MT SETX expression is also localized to the peri-nuclear region. The reason for this is unclear and deserves further study. Of interest, the expression of MT FUS with either MT SETX or MT LMNA proteins appeared to be able to partially rescue the toxicity, and the sibling positive for FUS and SETX variants was not affected by ALS. This prompted the examination of the expression of, and interactions between, these proteins. The expression pattern of MT FUS was unremarkable, and the expression of MT LMNA paralleled other reports of lamin A variants in which cells expressing LMNA mutations exhibit nuclear blebbing and reduced ability to withstand shearing forces [LMNA variants [In this family, two individuals were heterozygous for the g forces –93. Howevariants . In the We hypothesize that changes in interactions seen between FUS and SETX proteins when mutations are involved is likely contributing to this phenotype, while MT LMNA is weakening the nuclear membrane to shear forces such that the combination of both these is leading to the disease state. It will be of interest to examine the interaction between FUS and SETX proteins further to determine if both of these proteins are involved in common functions concurrently, potentially in R-loop regulation as would be suggested by their colocalization and immunoprecipitation with S9.6 antibodies. SETX is known to have a role in the resolution of R loops, and while the function of R-loops is still the subject of considerable debate, they appear to be involved in the modulation of methylation of gene promoters , 30. VarSETX variant eventually becomes affected by ALS, since heterozygous SETX variants have been linked strongly to the ALS phenotype. However, to date the sibling carrying this variant remains asymptomatic. This study suggests that the combinatory effect of several variants may be important in the development of pathology in cases where single disease-predicting mutations are not present. While we cannot unequivocally determine that the combination of variants expressed in this family is the cause of ALS, and while our targeted panel approach captures most of the genes known to be involved in ALS, an exome-wide approach may reveal additional genes contributing to the development of ALS beyond the three variants reported herein. However, our in vitro data recapitulates the pathological nuclear phenotype detected in the proband, showing that expression of variants are important to the development of the nuclear pathology detected in this affected individual, and supports the current view that combinations of variants are likely of importance for clinical manifestation of disease.It will also be important to follow family members to determine whether a heterozygote for the (DOCX 14 kb)Below is the link to the electronic supplementary material.


C. elegans MEC-4 and UNC-8 belong to the DEG/ENaC family of voltage-independent Na+ channels and have been implicated in mechanosensation and synaptic remodeling. MEC-4 and UNC-8 hyperactive mutants, designated (d) mutants, conduct enhanced currents and cause cell death due to uncontrolled influx of cations. We show here that MEC-4(d) but not UNC-8(d) currents are further potentiated by treatment with the protease plasmin and that this effect is dependent upon co-expression with the chaperon protein MEC-6. Mammalian DEG/ENaC channels are cleaved by plasmin in the channel finger domain and both MEC-4 and UNC-8 have a predicted plasmin cleavage site in this domain. We previously showed that MEC-4(d), but not UNC-8(d), currents are increased by co-expression with MEC-6, which interacts with the channel via the finger domain. We suggest that interaction of the channel subunit with MEC-6 may render the plasmin cleavage site more accessible. Given that C. elegans expresses a homolog of plasmin, these effects might be relevant in vivo. While MEC-4 is expressed in body touch neurons where it mediates the transduction of gentle touch sensation , UNC-8 is primarily expressed in motoneurons where it is involved in synaptic remodeling during development . Both MEC-4 and UNC-8 can be hyperactivated by genetic mutations that hinder channel closing, called (d) mutations . C. elegans neurons and Xenopus oocytes expressing these hyperactive variants of MEC-4 and UNC-8 undergo cell death due to uncontrolled flux of ions into the cell. Cell death in Xenopus oocytes and in cultured C. elegans neurons can be prevented by incubation with the DEG/ENaC channel blocker amiloride .MEC-4 and UNC-8 are subunits of the DEG/ENaC family of voltage-independent Naet al. 2003, Hughey, Bruns et al. 2004) and is completed once the channel arrives at the cell surface by transmembrane and extracellular proteases such as plasmin . While the effect of proteases on mammalian DEG/ENaCs has been extensively studied, little is known about whether nematode DEG/ENaC subunits are sensitive to these enzymes.Potentiation of the mammalian α, γ, and δ subunits of the DEG/ENaC ENaC subfamily can be also achieved via proteolytic cleavage of the extracellular domain (Kleyman and Eaton 2020). This cleavage results in the release of inhibitory peptides and probably the conformational change of the channel that leads to larger whole-cell currents. DEG/ENaC proteolytic processing begins in the Golgi by the Golgi resident protease furin  for 30 minutes with currents recorded in mock-treated cells (ND96). We found that MEC-4(d) currents were nearly doubled in plasmin-treated oocytes  . However, plasmin did not increase UNC-8(d) current amplitude  . Mammalian ENaCs are cleaved by plasmin in the channel finger domain where this protease has at least one recognition site . We thus used the SitePrediction website (https://www.dmbr.ugent.be/prx/bioit2-public/SitePrediction/) to predict potential plasmin cleavage sites in the finger domains of MEC-4 and UNC-8 . We found that both proteins contain at least one potential plasmin cleavage site in the finger domain . This suggests that lack of effect of plasmin on UNC-8(d) is not due to lack of potential recognition sites for plasmin in UNC-8.We coexpressed MEC-4(d) with stomatin-like protein MEC-2 and paraoxonase-like protein MEC-6, and, in a separate experiment, UNC-8(d) with MEC-2 and MEC-6 in et al. 2002) with chaperone function  that increases the number of functional MEC-4 channels at the cell surface . We recently showed that MEC-6 mediates MEC-4(d) regulation via interaction with the channel finger domain . We also showed that MEC-6 does not enhance UNC-8(d) currents in oocytes. We thus postulated that the effect of plasmin on MEC-4(d) might depend on MEC-6 and that the lack of effect on UNC-8 might be related to the fact that MEC-6 does not change UNC-8 currents in Xenopus oocytes. To test this hypothesis, we repeated the experiments in oocytes injected with only MEC-4(d) and MEC-2 or with only UNC-8(d) and MEC-2, but without MEC-6. As previously reported, MEC-4(d) currents are much smaller in the absence of MEC-6 . More importantly, we found that plasmin does not increase MEC-4(d) currents in oocytes that do not express MEC-6  . UNC-8(d) currents remain unaffected by plasmin treatment  . Taken together, these results support the idea that chaperon MEC-6 is needed for enhancement of MEC-4(d) current amplitude by protease plasmin. Interestingly, Brown and colleagues showed that activation of MEC-4(d) by extracellular chymotrypsin is independent of both MEC-2 and MEC-6 . Collectively, these data suggest that C. elegans DEG/ENaC channels might be sensitive to different extracellular proteases based, not only on channel primary sequence but also on its quaternary structure. These results also suggest that chaperon MEC-6 may help expose potential cleavage sites. Given that C. elegans expresses svh-1, the homolog of mammalian plasmin precursor plasminogen, we suggest that these effects might be relevant in vivo both in physiological conditions and when DEG/ENaC channels are hyperactivated .MEC-6 is a paraoxonase-like protein . The MEC-6 and UNC-8(d) cDNA were cloned in a PGEM vector and were published previously . cRNAs were synthesized using T7 mMESSAGE mMACHINE kit (Invitrogen) following manufacturer’s protocols. cRNAs were purified, run on denaturing agarose gels for size and integrity verification, and quantified spectroscopically.Xenopus oocytes expression and electrophysiologyStage V-VI defolliculated oocytes from Xenopus laevis were purchased from Ecocyte Bioscience US LLC  and injected with 10 ng/oocyte of each cRNAs except MEC-6 cRNA, which was injected at 2 ng/oocyte. Injected oocytes were incubated in ND96 medium consisting of 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.5, supplemented with penicillin and streptomycin (0.1 mg/ml) and 2 mM Na-pyruvate at 20°C. The next day the medium was changed to ND96 plus 500 µM amiloride to prevent cell death. Whole-cell currents were measured three days after the injection using a two-electrode voltage-clamp amplifier  at room temperature. Electrodes with resistance between 0.3–0.7 MΩ were filled with 3 M KCl. To measure MEC-4(d) currents, oocytes were perfused with a physiological NaCl solution containing (mM): 100 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, and 10 HEPES, pH 7.2 (NaOH). Due to the strong block by divalent cations , to measure UNC-8(d) currents a divalent cation–free NaCl solution containing (mM): 110 NaCl, 2 KCl, 1 EGTA, and 10 HEPES, pH 7.2 (NaOH) was used. To elicit the whole-cell currents, the oocyte membrane was clamped at −30 mV and stepped from −160 to +100 mV in 20 mV increments. Current amplitude was measured at -100 mV and 300 ms. We used the pCLAMP suite of programs (Axon Instruments) for data acquisition and analysis. Currents were filtered at 200 Hz and sampled at 1 kHz. Prism was used to plot data and for statistical analysis.Plasmids encoding MEC-4(d) and MEC-2 were a gift from the Chalfie lab .


The click azide = alkyne 1,3-dipolar cycloaddition (click chemistry) has become the approach of choice for bioconjugations in medicinal chemistry, providing facile reaction conditions amenable to both small and biological molecules. Many nucleoside analogs are known for their marked impact in cancer therapy and for the treatment of virus diseases and new targeted oligonucleotides have been developed for different purposes. The click chemistry allowing the tolerated union between units with a wide diversity of functional groups represents a robust means of designing new hybrid compounds with an extraordinary diversity of applications. This review provides an overview of the most recent works related to the use of click chemistry methodology in the field of nucleosides, nucleotides and nucleic acids for pharmacological applications. Tw. TwTLDNATLMO) oligonucleotides , the morpholino incorporation in the triazole-linked sequence improved the binding (Tm TLMO:DNA = 56.1 °C) compared to the TLDNA (Tm TLDNA:DNA = 55.1 °C). The stabilizing effect of the morpholino moiety was more pronounced in the case of duplexes with RNA, where the ΔTm between TLMO:RNA (Tm = 56.6 °C) and the control DNA:RNA (Tm = 58.5 °C) was −1.9 °C. Instead, a −4.4 °C difference was determined between the control and TLDNA:RNA (Tm = 54.1 °C) duplex. Nevertheless, for future therapeutic applications, further structural studies were required. Aimed at developing therapeutic nucleic acids, Hayes et al. reported on the synthesis of triazole-linked morpholino (leotides  60]. Th. ThTLMO)78A–C, 81, 84, Conformationally restricted locked nucleic acid (LNA) is a bicyclic RNA analog with interesting antisense properties ,62. At t85 bearing an alkyne and a fixed Cas9-binding chemically modified 79-mer 86 bearing an azide. Click ligation of the two components, the second step of the synthesis, generated sgRNAs 87 containing an artificial triazole backbone that is well tolerated in functionally critical regions of the sgRNA enabling effective Cas9-mediated DNA cleavage in vitro and in cells  . Th. ThN-ace100. The chosen modifiers were those targeting sugar GalNAc for the click ligation and the lipophilic pentylamine for the amidation. Complete incorporation was achieved when amidation was performed first, followed by CuAAC reaction with the complex CuBr·SMe2 in a DMSO:H2O mixture. Interestingly, after lipid-mediated in vitro transfection, modified siRNA retained the ability to reduce ApoB (apolipoprotein B) mRNA levels. Moreover, when GalNAc was present in a clustered manner on the sense strand, binding of siRNA to ASGPR  was improved, proving that heavy modification in the 2′ ribose position of the sense strand is possible in the siRNA therapeutic approach.The orthogonality of the CuAAC was exemplified in the work of Zewge and co-workers, where a sense RNA, employed for siRNA approach, was internally modified by subsequent amidation and cycloaddition reactions to provide a platform for rapid structure–activity relationship analysis of variously substituted oligonucleotides . An opti12 enabled the uptake of oligonucleotides in Escherichia coli and Salmonella enterica [12-2′OMeRNA conjugate 103a targeting the bacterial mRNA encoding RFP (red fluorescent protein) and its scrambled sequence 103b were prepared by optimized CuAAC reaction in solution phase  after 48 h, thus representing a possible candidate for future development of oligonucleotide-based antibiotics.Specific antisense oligonucleotides were designed and synthesized as promising candidates as antibacterial drugs. The main obstacle in the development of new oligonucleotide antibiotics comes from the fact that bacterial cells do not spontaneously uptake oligonucleotides from their environment . Howeverenterica . The staon phase . Conjuga105a modified at the nucleobase was found to be lower than that of unmodified oligonucleotide. However, oligonucleotides 107a–c showed silencing levels promising for potential therapeutic applications (83–95% decrease in EGFR expression). The same approach was fruitfully employed for the synthesis of more heavily modified, boron cluster-bearing antisense oligonucleotides [110 demonstrated increased lipophilicity and low cytotoxicity (MTT assay on HeLa cells). However, the silencing activity of 110 was found to be lower than that of the control-unmodified oligonucleotide. This could be attributed to a less favorable recognition of the complex RNA/DNA by RNase H due to the boron cluster presence on the antisense strand.Boron clusters have been used in medicinal chemistry predominantly against cancer via boron neutron capture therapy . Recentlleotides . Up to f113a,b in which Dmd and Acvr2b targeting PMO were conjugated via alkyne-azide click chemistry as shown in Several synthetic splice-switching oligonucleotides (SSOs) targeting nuclear pre-mRNA molecules have been recognized by FDA as drugs, and others are in clinical trials . HoweverIn a striking example of click reaction use, Murthy and co-workers  employed114  as a Cu (I)-stabilizing agent. The full restoration of the normal levels of L-type calcium channel, which can recover myocardial function in pathological heart conditions, was achieved.Nucleic acid aptamers are short single-stranded DNA or RNA (ssDNA or ssRNA) molecules and are commonly produced by systematic evolution of ligands by exponential enrichment (SELEX). They can selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, etc., with recognized therapeutic applications . In a re114 A in whicEX cycle B, after EX cycle . The conIn conclusion, this review highlights the extraordinary diversity of pharmacological applications related to the use of one of the most important click reactions, the 1,3-dipolar cycloaddition of alkyne and azide to form 1,2,3-triazole moiety, in the field of nucleosides, nucleotides and nucleic acids. Nucleoside, nucleotides and nucleic acids are biomolecules with a crucial role in all biological cellular life functions; thus, the development of analogs for diverse biological targets represents a research topic of great interest. Considering the well-established role of 1,2,3-triazoles as biocompatible pharmacophores and bioisosteric linkers, click reactions are a very attractive tool for the construction of modified and/or conjugated nucleosides, nucleotides and nucleic acids including oligonucleotides. In addition to the high efficiency of these reactions, azide and alkyne moieties can be easily incorporated into oligonucleotides without affecting biophysical and biochemical properties. Azide-alkyne cycloadditions can be carried out in solid-phase conditions and can give rise to a plethora of novel, tailored modified nucleic acids and modified nucleic acid libraries.Furthermore, the present review indicates that, in recent years, many studies have been dedicated to oligonucleotide modifications using click azide-alkyne cycloaddition. Studies in vitro and in vivo show that several pharmacokinetic and pharmacodynamic properties of modified compounds result improved, suggesting that the 1,2,3-triazole does not act as a passive linker but, on the contrary, is an important moiety in drug design and delivery. Finally, although many nucleosides and oligonucleotides possessing the 1,2,3-triazole moiety have been reported in the last years, further studies are required to assess the in vivo efficacy and low toxicity of lead compounds. In the future, we believe that more elucidations about 1,2,3-triazole chemistry, biochemistry and biocompatibility would facilitate the development of triazole-based nucleosides, nucleotides and nucleic acids for clinical applications.


S-adenosyl-L-methionine to 6N amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from  N6-methyladenosine (m6A) is considered a major covalent modification of the adenosine (A) base in coding and noncoding (nc) RNAs  and METTL14 methyltransferases (S-adenosyl methionine (SAM)-dependent methyltransferases (N6-MTases) (6N-deoxyadenosine DNA methyltransferases (m6dA MTases), especially those from Type III restriction-modification (R-M) systems in bacteria  (nc) RNAs . It is lnc) RNAs , stem cenc) RNAs , oncogennc) RNAs , and DNAnc) RNAs . In humasferases , both of-MTases)  that alsEcoP15I) . The firEcoP15I) . We analEcoP15I) .6dA in bacterial DNA is deposited in a strictly sequence-dependent manner in double-stranded DNA (dsDNA)  , promoter-associated RNA, enhancer RNA, and repeat RNA elements  (XIST (NEAT2 (MALAT1)  , m6A in = A/C/U) . Such rean cells . Of noteelements . Consist3K36me3)  or the t3K36me3) . Certain3) (XIST  and NEAT(MALAT1) , also hauggested . In thosted RNAs . METTL3 ted RNAs .6A in nucleic acid metabolism thus seem to converge to a common point: the occurrence of m6A at chromatin, a predominantly DNA- and RNA-rich environment inside the nucleus. A recent study showed that METTL3-METTL14 can convert dA to m6dA within equivalent DRACH  motif in a single-stranded DNA (ssDNA) with a 2.5-fold preference over the equivalent RNA in vitro  and thought to originate from RNA catabolism rather than from SAM-dependent MTase reactions  methylation in mammalian genomes.The increasingly appreciated roles of min vitro . Since meactions , fundameB) of EcoP15I , we chose ModB and METTL14 for this comparison. While the MTase domain of METTL14 is enzymatically inactive, it harbors features that contribute to substrate binding (-RGG) from insect cells  (6A activity of the METTL3-METTL14 enzyme complex (We compared the MTase cores of (Mod binding . These osection) . The RGGsection) . In huma complex . The seq complex .6A signature motif (RRACH), including an RNA oligo  in which two RRACH sites were arranged in a palindromic fashion  with at least one canonical m fashion . We incl fashion  without  fashion .6A mark in mammalian RNAs is widespread and expands beyond internal adenines in mRNAs to lncRNAs (XISTNEAT2 lncRNA can relocate a large subset of transcription units across cellular compartments from polycomb bodies to interchromatin granules (NEAT2 (nt 4917–4966) facilitates the recruitment of a mega transcription complex (NEAT2 fragment harbors one RRACH motif (AAACA) in its loop region and fulfils the criteria of m6A peak signature (RRACH ACH ACA rNEAT2-30) of NEAT2-49 in which both the m6A signature motif and bulged stem regions were well preserved  at the end of the short palindromic sequence (5’-CCG•CGG-3’). We thus hypothesized that d6T , respectively, were replaced by two thymidines to disrupt base pairing and palindrome formation without affecting the core RRACH motif required for m6dA installation (We observed higher enzymatic activity of METTL3-METTL14 on the d6T DNA oligo compared with its RNA counterpart (r6T) . By closallation . We reasallation . Consistallation , METTL3-rNEAT2 , rC-flip wherein the RRACH motif resided at the 3’-end. The other three RNA oligos, FoxM1-p1 , c-Myc-p5 (8-nt), and c-Myc-p6 , were derived from the FOXM1 and MYC genes because of their involvement in m6A-mediated processes in glioblastoma and acute myeloid leukemia  methyltransferase activity on rNEAT2 . We thenrNEAT2 . We alsoleukemia . We covaWe used a radiometric assay and fluorescence polarization (FP) to determine methylation activity and RNA binding, respectively. As shown in ACA-3’) changed to an ideal m6A target sequence, that is, 5’-GGACU-3’  that higher affinity substrates tend to show lower methylation levels, possibly due to slow off rates of m6A RNA.If METTL3-METTL14 were to methylate ssDNA in vivo, and the evidence to date does not support its direct enzymatic origin , then whGGACU-3’ . AlthougGGACU-3’ . MoreoveGGACU-3’ . ImportaGGACU-3’ . These r50 = 216 nM) showing 4-fold stronger inhibition than rTCE23 (IC50 = 887 nM) (ACH motif). These results are consistent with those obtained by oligonucleotide intact mass analysis. Altogether, the methyltransferase and binding assays confirm that the binding to structured RNAs, especially those lacking the GGACU sequence, almost completely abolishes the methyltransferase activity METTL3-METTL14.To further investigate RNA-mediated inhibition, we showed that the two structured RNAs (rTCE23 and rNEAT2) inhibited the DNA methylation activity of METTL3-METTL14 in a dose-dependent manner, with rNEAT2 (IC 887 nM) . Next, w 887 nM) . We conf-RGG  localization of METTL3 to sites of dsDNA breaks, and its role in DNA/RNA hybrid accumulation , (b) reccriptome . Future N6-deoxyadenosine methylation activity on ssDNA, which is regulated by binding of METTL3-METTL14 to shape-dependent structured regions of ncRNAs and chromatin-associated nascent pre-mRNAs (CA-RNA)  . The strcription , DNA reccription , damage cription , and R-lcription .6dA is a predominant modification mark in bacterial DNA, whereas the m5dC is more prevalent in eukaryotic genomes. How is the eukaryotic genome then protected from m6dA deposition despite having an efficient enzyme machinery  to accomplish such task? A comparative sequence/structural analysis of the bacterial EcoP15I and human METTL3 and METTL14 MTases provides some hints  in human cells.A major difference in prokaryotic and eukaryotic genomes is the contrasting occurrence of the base methylation. The mme hints –3a. Of nme hints , whereasme hints . It is lme hints  to ensurme hints  of m6dA Interestingly, a recent study showed that a comprehensive network of RNA-RNA and RNA-DNA interactions mediated by ncRNAs (including MALAT1) creates distinct nuclear compartments and facilitates recruitment of proteins for RNA processing, heterochromatin assembly, and gene regulation . Thus, thttp://n2t.net/addgene:101892; RRID:Addgene_101892). The coding sequence of full-length human METTL14 (NCBI reference number GI: 172045930) was cloned into the pFastBac1 vector between BamHI and NotI restriction enzyme sites. For expression of recombinant proteins, we used ExpiSf Expression System (Thermo Fisher). First, the viral DNA bacmids for each gene were prepared from individual plasmids transformed into MAX Efficiency DH10Bac competent cells (Thermo Fisher). We confirmed the identity of the inserted genes by PCR amplification and DNA sequencing. The recombinant P0 virus for each gene were generated in ExpiSf9 insect cells following the manufacturer’s recommendations (Thermo Fisher). The amount of each virus needed for infection has been optimized for maximal production of the two proteins. Infected cells were grown in ExpiSf CD medium (Thermo Fisher) at 27°C on an orbital shaker (speed 125 rpm) in a controlled  environment. Cells were harvested at 72 hr post-infection by spinning at 300× g for 5 min, washed with phosphate buffered saline (PBS) twice, and resuspended in a buffer A ] glycerol) supplemented with 0.5% (v/v) Igepal, two mini-protease inhibitor cocktail tablets (Roche), DNase I, and lysozyme. Cells were lysed using a microfluidizer  and the soluble fraction was separated by centrifuging the lysate at 40,000 rpm for 40 min. The supernatant was passed through a 0.22 µm filter.The coding sequence of full-length human METTL3 (NCBI reference sequence GI: 33301371) was used in this study. The gene was cloned into a plasmid suitable for expression of proteins in insect cells with an N-terminal poly histidine tag followed by a tobacco etch virus (TEV) protease site. This plasmid (5TEY (METTL3)) was a kind gift from Dr Cheryl Arrowsmith  of METTL14. It was expressed and purified using same method as the full-length METTL3-METTL14 complex.The clarified soluble fraction was loaded on to a Nuvia IMAC column (Bio-Rad) pre-equilibrated in buffer A. The proteins were eluted by increasing the concentration of imidazole. The 6XHis tag was then proteolytically removed using TEV protease. Any uncleaved fractions and remaining protein impurities were removed by second passage through an IMAC column. The METTL3-METTL14 complex was further purified by successive passage through HiTrap heparin and HiLoad Superdex 200 columns . The protein complex was eluted as a single homogenous species in a final buffer containing 0.02 M Tris-HCl pH 8.0, 0.15 M NaCl, 0.1 mM TCEP. The purified protein complex was concentrated to 3–5 mg/mL and used immediately, or flash-frozen in liquid nitrogen and stored at –80°C. The METTL3-METT14All RNA and DNA oligonucleotides used in this study were synthesized, purified by HPLC, and received in deprotected (for RNAs) and desalted form from Dharmacon and Integrated DNA Technologies, respectively. Oligos were dissolved in 1× buffer containing 0.01 M Tris-HCl pH 8.0, 0.05 M NaCl. To prepare double-stranded oligos, the two strands were mixed in roughly equimolar amounts. Annealing was carried out by heating the mixture to 95°C for 2 min followed by gradual cooling to 25°C over 45 min in a thermocycler (Eppendorf).3H] SAM (PerkinElmer), 10 µM substrate RNA/DNA probe, and 1 µM co-purified full-length METTL3-METTL14 enzyme complex. The reactions were incubated at 37°C for 1 hr and 3 µL of each reaction was quenched by blotting on Hybond-N+ membrane (Amersham). RNA/DNA probes were crosslinked to membrane by exposure to ultraviolet light (254 nm) for 2 min. The membranes were successively washed three times by 1× PBS followed by three ethanol washes for 5 min each. The membranes were air-dried in the hood for 15 min and the counts per minute (c.p.m.) of the RNA/DNA probes were measured using a scintillation counter (Beckman LS6500). We also performed this assay in a reaction buffer containing higher salt (50.0 mM NaCl) and observed consistent results  as presented in Each reaction was carried out in a 5 µL mixture containing 50 mM HEPES pH 7.5, 5 mM NaCl, 1 mM dithiothreitol (DTT), 5 µM The reviewer believes that the study will benefit immensely if the structural details are expanded to explain substrate specificity in their discussion. The authors use structural models in their figures but do not use these models to their advantage in explaining their results. This should be included.As shown in the revised Figures 1a-e and supplementary figure. 1, we have made a detailed comparison of the methyltransferase domains of EcoP15I, METTL3, and METTL14. We have added a description of these models to explain our results on pages 3 (lines 50-54) and 5 (lines 89-97).Reviewer #2:[…]1. The enzymatic reactions performed in this work are at non-physiological conditions in 5mM NaCl. Hence, it is difficult to comment on the biological processes for the activity observed under such conditions.We followed the same protocol in a previous study that reported, for the first time, the m6dA methyltransferase activity of METTL3-METTL14 . In this study they used a 14-mer DNA substrate (d6T). Since we wanted to measure the methyltransferase activity of METTL3-METTL14 on different RNA/DNA substrates and compare it to its activity on the original DNA substrate (d6T) used in the study by Woodcock et al., we used their identical experimental conditions to minimize experimental bias. Moreover, an independent research laboratory tested the enzymatic activity of METTL3-METTL14 in different concentrations of NaCl and found highest activity at low NaCl (0 – 5mM NaCl) concentration. Please see Figure 1C from Li et al., J of Biomol Screen. 2016 Mar; 21(3):290-7. PMID:26701100 .50 values at a higher salt concentration (50.0 mM NaCl). These results further strengthen our conclusion about ability of small structured RNAs to block m6dA activity in vitro, which is consistent at higher salt concentration. We have added the new results (panel B) in the lower panel of the revised Figure 2e.Regardless, we performed the inhibition of m6dA activity of METTL3-METTL14 by both rTCE23 and rNEAT2 RNA in a buffer that contains 50.0 mM NaCl. As shown in panel B of Figure 2e, both RNA oligos inhibited the m6dA activity, with only modest increment in ICThe title is misleading and it should state 'in vitro'.‘in vitro’ to the title as suggested.We agree with the reviewer and added In the abstract, the authors state: "We also show that the methyltransferase activity of METTL3-METTL14 on ssDNAs is largely regulated by structured RNA elements prevalent in long noncoding (lnc) RNAs but also found in other cellular RNAs." However, the experiments are done in vitro using short oligos. Also, WTAN is known to interact with this complex and affect its methylation activity. In the absence of an experiment to test the role of WTAN in the methyltransferase activity of METTL3-METTL14, it may be difficult to reach this conclusion. The authors have discussed the significance of the work in the process of transcription, DNA damage repair, and R loop metabolism, and also depicted in the model in Figure 3d. However, none of the substrates used in this study represent that of the model.METTL3-METTL14 enzymes have been studied extensively as RNA methyltransferases (m6A). Most of these major studies have delineated the physiological roles of this complex in human biology and diseases by genetic depletion of METTL3 and/or METTL14. METTL3-METTL14 can form up to ~1.0 mega Dalton size complex through its direct interaction with WTAP (as pointed out by the reviewer), and other accessory proteins (through WTAP) e.g., RBM15/15B, VIRMA, CBLL1 (HAKAI), ZC3H13 :17697-704; PMID: 8021282), :608-624; PMID: 31520073). While WTAP is an important accessory factor of METTL3-METTL14, it also harbors an uncharacterized N-terminal domain followed by a largely disordered C-terminus. We could not test the effect of WTAP mainly due to technical difficulties associated with the purification of the tag-free full length human WTAP protein in large enough quantities and in stable form.Woodcock et al.,  who first reported the DNA methyltransferase (m6dA) activity of METTL3-METTL14 also did not use WTAP in their methyltransferase assay. Given the essential role played by METTL3-METTL14 in human embryonic development :1002-6; PMID: 25569111) it is important to study the factors that regulate its enzymatic activity. Thus, our major finding that structured RNA elements can block m6dA activity is very important and may provide strong rationale for new in vivo or genetic studies.We agree with the reviewer that the substrates used in this work are not true representation of Rloops, and/or nucleic acids generated during DNA damage and repair. We have revised the figure 3d to rectify this concern. Moreover, we believe the addition of ‘in vitro’ to the revised title, as suggested by the reviewer, should alleviate the confusion, and convey our conclusions in a simplified form.2. The enzymatic reactions are done in buffer with 5 mM NaCl, while binding studies were done in 50 mM KCl. Why the difference in salt concentrations? What role the different salt conditions play in these experiments?A previous study has thoroughly examined the effects of various salts and concentrations and concluded that METTL3-METTL14 has comparable activities in both KCl and NaCl. Please see figure 1B and C from Li et al., J of Biomol Screen. 2016 Mar; 21(3):290-7. PMID: 26701100We used NaCl in methyltransferase assay because we wanted to examine the inhibition of m6dA activity of METTL3-METTL14 by RNA oligos under identical assay condition that was previously used for assaying m6dA activity on a single stranded DNA (d6T) . Moreover, the same group has recently reported the METTL3-METTL14’s activity on double stranded RNA containing DNA lesions :11629-11642; PMID: 34086966). In this study, they also used 5.0 mM NaCl in their methyltransferase reaction.As mentioned in our response to previous comment, we did examine the m6dA activity of METTL3-METTL14 and its dose dependent inhibition by RNA oligos in a buffer containing higher salt (50.0 mM) and did not find major effect of higher salt concentration.We routinely use KCl in the buffers for the binding studies performed by fluorescence polarization method. It is known that both sodium and potassium ions stabilize DNA with similar potency :5336-53; PMID: 18422348). Thus, there was no specific reason for using KCl other than a routine practice in the lab.3. Why rNEAT2 binds with higher affinity but has lower methylation than r6T, which binds with lesser affinity? What is the mechanism behind the inverse relationship of binding and methylation activity?off) of METTL3-METTL14. It is likely that METTL3-METTL14 when complexed with rNEAT2 has slower off rate than r6T. In line with this observation, the d6T* DNA yielded highest methylation but weaker binding than both r6T and rNEAT2. In the revised manuscript we included additional data in Figure 2. We tested a mutated version of rNEAT2 termed as rNEAT2* where we replaced the DRACH motif (AAACA) in its loop region to an ideal m6A target sequence (GGACU) for METTL3-METTL13. The rNEAT2* showed higher RNA methylation than rNEAT2 but could not achieve the methylation levels of single stranded d6T* DNA . Consistently, rNEAT2* showed 6-fold weaker binding (Kd = 13nM) to METTL3METTL14 than rNEAT2 (Kd = 2nM) . These results are in line with our model that both shape and sequence of nucleic acids dictate the efficiency of SAM-dependent methylation of RNA and DNA by METTL3-METTL14. Consistently, the addition of rNEAT2* to the reaction mixture of METTL3-METTL14-d6T* attenuated the overall methylation thus, corroborate our conclusions in this study that structured RNAs restrict the m6dA methylation in ssDNA by METTL3-METTL14 in vitro. We have added this description on page 9-10.The rNEAT2 RNA oligonucleotide is predicted to form a stem-loop structure as shown in figure 1g whereas the r6T RNA can only form a partial duplex with 8 nucleotide long 3’- overhang, but no stem-loop. Binding of METTL3-METTL14 to structured RNA oligos that harbor imperfect  or no m6A target sequence  is stronger than the unstructured nucleic acids while the r6T shows higher RNA methylation than rNEAT2. This inverse relationship of binding and methylation can be explained by differences in the off rate  to clarify this notion as follows:“This suggests that the RGG motifs participate in the binding of both RNA or DNA, while other parts of the enzyme  may also contribute to the overall binding of the substrate .”5. In the present form, the manuscript is difficult to read for a general reader. The manuscript text assumes the reader has a more detailed understanding of the subject. For example, the RGG motif and its role in substrate binding is not introducedWe agree with the reviewer and have rectified this by revising the main text. We have added a description of RGG motifs on page 5 (lines 102-109) as follows:37. In human METTL14, a total of six tri-RGG motifs are two RG motifs are present at its c-terminus tail (aa 408-457). As expected, this region also harbors a few aromatic amino acids, which can further stabilize the nucleic acids via hydrophobic or stacking interactions. Consistently, this region contributes to the substrate binding and m6A activity of METTL3-METTL14 enzyme complex38. The sequence encompassing the RGG motifs in METTL14 is well conserved in higher vertebrates17.”“The RGG motifs represent the clustered sequence of arginines and glycines. These motifs are commonly present in diverse set of RNA binding proteins that play important roles in physiological processes such as RNA synthesis and processingReviewer #3:[…]To enhance the manuscript and further the conclusions in this manuscript, authors need to further investigate the role of structured RNAs initially using molecular modelling to further inform functional roles. The authors in Figure 1 show structure of EcoP15I with a DNA substrate to show a model of the METTL3/METTL14 with an RNA substrate. It is not clear how the authors have gone about modelling this, what is the sequence similarity/ conservation of residues in the binding site, is the RGG domain involved in binding in this model, does the structural model support specificity to RNA or DNA, which residues might be involved in the active site? Authors need to undertake a structural analysis using the crystal structure available to perform molecular dynamics with DNA and RNA substrates to get a mechanistic understanding for this or do an experimental structure with a nucleic acid substrate to provide this understanding.As explained in our previous responses to the comments #4 (from Reviewer 2) and comment #1 (reviewer 1), we have included detailed description of our structural modeling and analyses. The substrate binding regions of EcoP15I and METTL-METTL14 are very different. For example, in EcoP15I a well folded target recognition domain  is present in addition to the canonical MTase motifs  in the class β MTases. While METTL3 and METTL14 both have conserved canonical MTase motifs they both lack an extensive TRD as in EcoP15I. In METTL3-METTL14 the substrate recognition can be accomplished by the combination of three relatively short loops encompassing the sequence intervening MTase motifs IV and V (loop 1), VIII and VIII’ (loop 2), and motifs IX and X (loop 3), two Zinc-finger domains of METTL3, and the RGG motifs of METTL14. In the absence of a single experimental structure of all these domains, modeling of a DNA and RNA substrate in a full length METTL3-METTL14 heterodimer would be highly speculative. We will be performing molecular dynamics simulations and validate our results by extensive mutagenesis in the future. We did attempt to co-crystallize METTL3-METTL14 with several DNA and RNA sequences mentioned in this study, but unfortunately could not grow co-crystals yet.The authors have tested the effect of structured RNAs on DNA methylation specifically, why not RNA substrates lacking structure. Is this a competition between substrates with different affinities and what could be the relevance in cellular processes specifically? Competition experiments haven't been performed here.We did include the unstructured single stranded RNA such as rL3-5, rc-myc-p5 and observed consistent results i.e., the attenuated methylation of DNA substrate (d6T*). However, the level of inhibition of DNA methylation was more pronounced when structured RNAs lacking an ideal m6A target sequence e.g., rNEAT2 and rTCE23 were added to the reaction mixture. As shown in Figure 2e-g, we also performed competition experiments to confirm that the m6dA DNA methylation decreased in the presence of structured RNA. Moreover, data shown in Figure 2e suggests that the structured RNA (with high affinity to METTL3-METTl14) can attenuate m6dA DNA methylation in a dose dependent manner. These results indicate a competition between the RNA and the DNA substrate. The emerging evidence suggests that the recruitment of METTL3METTL14 and/or the presence of m6A methylated RNA at the site of DNA damage and repair :573-576). A more recent study confirmed the activity of METTL3-METTL14 on DNAs that contain various types of DNA damages  11629-42, PMID: 34086966). METTL3-METTL14 is also active on chromatin associated RNAs . In the context of these studies, our results provide the first biochemical evidence for the interplay between METTL3-METTL14, DNA and RNA, and how the RNA and DNA methylation activities can be regulated in an environment enriched with all three molecules. Our work also presents strong rationale to decipher the physiological consequences of potentially new regulatory layer in the epitranscriptomic gene regulation.