Caenorhabditis elegans ortholog of mammalian TDP-43, which is strongly implicated in the etiology of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We discovered that deletion of the tdp-1 gene results in enhanced nuclear RNA interference (RNAi).TDP-1 is the  Caenorhabditis elegans ortholog of mammalian TDP-43, which is strongly implicated in the etiology of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We discovered that deletion of the tdp-1 gene results in enhanced nuclear RNA interference (RNAi). As nuclear RNAi in C. elegans involves chromatin changes moderated by HPL-2, a homolog of heterochromatin protein 1 (HP1), we investigated the interaction of TDP-1 and HPL-2. We found that TDP-1 and HPL-2 interact directly and that loss of TDP-1 dramatically alters the chromatin association of HPL-2. We showed previously that deletion of the tdp-1 gene results in transcriptional alterations and the accumulation of double-stranded RNA (dsRNA). These molecular changes are replicated in an hpl-2 deletion strain, consistent with HPL-2 acting in consort with TDP-1 to modulate these aspects of RNA metabolism. Our observations identify novel mechanisms by which HP1 homologs can be recruited to chromatin and by which nuclear depletion of human TDP-43 may lead to changes in RNA metabolism that are relevant to disease.TDP-1 is the  Pathological inclusions of the TDP-43 RNA binding protein are found in ∼97% of all amyotrophic lateral sclerosis (ALS) cases and ∼45% of all frontotemporal dementia (FTD) cases . The cyt3–10–Caenorhabditis elegans ortholog of mammalian TDP-43: it has significant sequence similarity to TDP-43 in the RNA recognition motif (RRM) domains, binds the canonical TDP-43 binding sequence [(UG)n] with high affinity , which is initiated by exposure to exogenous dsRNA, and endogenous RNAi (endo-RNAi), initiated by processing of endogenous transcripts  homolog HPL-2 (reviewed in reference The accumulation of dsRNA in the TDP-1 mutant raised the possibility that this protein plays a role in the RNA interference (RNAi) pathway. In nscripts . A compepathways . Indeed,pathways . Exo- anDrosophila HP1 homolog associates with genes in an RNA-dependent manner and copurifies with several pre-mRNA processing factors, including hnRNP proteins  and that TDP-1 facilitates HPL-2 association with active genes to maintain mRNA abundance. This novel function of TDP-1 may explain the HPL-2 specificity for a subset of genes and may have implications for the molecular functions of TDP-43 that are relevant to human disease.Here we show that loss of TDP-1 sensitizes tdp-1 null worms [tdp-1(ok803)] and wild-type worms on bacteria producing dsRNAs against a variety of somatic genes. Quantification of the percentage of progeny affected by each type of RNAi indicated that tdp-1 mutants had a mild but reproducible increased sensitivity to exo-RNAi (snb-1::TDP-1) were highly resistant to neuron-specific unc-73 RNAi: less than 1% of treated animals displayed an Unc phenotype, compared to ∼10% of wild-type animals  also decreased RNAi efficiency  mutant animals expressing muscle-specific green fluorescent protein (GFP) from an integrated transgene were subjected to GFP-specific RNAi by feeding (GFP feeding RNAi) or treated with an empty vector. Comparison of the GFP signals following 24 h of treatment indicated that RNAi against GFP was more effective in tdp-1 mutants than in wild-type animals, mirroring the results seen for endogenous genes , showed similar results  bacteria and then assaying GFP expression of these progeny at the L4 stage, as done previously (tdp-1(ok803) progeny displayed a dramatic reduction in GFP levels, with some animals displaying no observable GFP fluorescence . Because nuclear but not cytoplasmic RNAi results in inhibition of elongating RNA Pol II  animals to exo-RNAi was due to enhanced nuclear RNAi, as introduction of the nrde-3(gg66) mutation into the tdp-1(ok803) strain completely reversed the enhanced RNAi sensitivity . However, mutation of nrde-3 in the tdp-1(ok803) background reversed the sensitivity to unc-73 RNAi, to the level of the nrde-3 mutant alone, supporting the conclusion that enhanced sensitivity to unc-73 RNAi in tdp-1 mutants is dependent on nuclear RNAi.Pronounced sensitivity to heritable gene silencing in A Pol II , nucleare operon . We foun >2-fold , demonstsitivity . We did C. elegans genes involved in production of endo-siRNAs can result in enhanced exo-RNAi sensitivity. Therefore, we considered that tdp-1 mutants might be sensitive to nuclear exo-RNAi due to tdp-1 playing a role in the production of endo-siRNAs. To address this possibility, we deep sequenced small RNAs from tdp-1(ok803) and wild-type animals to look for decreases in endo-siRNA abundance in the mutants. We created ligation-independent small RNA sequencing libraries to capture molecules with either a 5′ monophosphate  or a 5′ triphosphate (secondary siRNAs).Mutation of tdp-1 mutant . Consistent with our previous finding that TDP-1 limits the abundance of dsRNA, which is the precursor to siRNAs, the large majority (>88%) of genes with a differential abundance of antisense siRNAs contained increased levels in tdp-1(ok803) animals compared to those in wild-type animals  . To identify siRNA target genes, the sequences were filtered for reads mapping antisense to annotated genes (excluding piRNAs and microRNAs). We identified ∼6,000 genes targeted by antisense siRNAs, in good agreement with the results of previous studies . Direct  animals . Figure tdp-1 associated with the nuclear RNAi/NRDE complex to limit nuclear RNAi. However, we were unable to detect an RNA-independent association between TDP-1 and NRDE-3 in the lysate of a strain expressing N-terminally FLAG-tagged NRDE-3 by coimmunoprecipitation with anti-TDP-1 or anti-FLAG antibodies , resulting in increased nuclear RNAi efficiency, analogous to the competition between endo- and exo-RNAi (see the introduction). To ask if tdp-1 functions in parallel to the NRDE complex, we asked if tdp-1(ok803); nrde-3(gg66) double mutants showed synthetic phenotypes. While we observed no obvious defects in tdp-1(ok803); nrde-3(gg66) animals at a normal growth temperature (20°C), shifting the animals to 25°C resulted in a maternal-effect decrease in brood size (tdp-1(ok803); nrde-3(gg66) animals showed complete maternal-effect sterility (tdp-1(ok803); nrde-3(gg66) double mutants grown from embryos at 25°C had clearly uncoordinated movement (Unc phenotype) and showed an approximately 2-fold decrease in thrashing rate compared to that for the wild type and/or each of the single mutants  and loss-of-function mutations in other chromatin factors, including set-2, met-2, lin-35, and hpl-2 (data not shown).If ood size  and delaterility . Additiohpl-2 deletion results in maternal-effect, temperature-sensitive sterility not observed in nrde-3(gg66) mutants (tdp-1(ok803); nrde-3(gg66) double mutants also displayed temperature-sensitive sterility, we hypothesized that tdp-1 may function in parallel to the NRDE complex to control HPL-2 function/recruitment. To determine if TDP-1 could modulate HPL-2 availability or chromatin association by a direct interaction, we asked if TDP-1 antibodies could immunoprecipitate HPL-2. As shown in eri-1 mutant background. As loss of eri-1 blocks endo-siRNA production and subsequent NRDE-3 nuclear localization  mutants and wild-type animals. Using the model-based analysis of ChIP-seq (MACS) peak-calling algorithm, we identified ∼14,000 distinct peaks of HPL-2 association in either or both wild-type and tdp-1(ok803) animals (Data Set S2), which showed high reproducibility  overlapped peaks previously identified by McMurchy et al.  HPL-2 associations between tdp-1(ok803) mutants and wild-type animals, with roughly equal numbers of regions with decreased and increased HPL-2 associations in the mutant (Data Set S2).To ask if the association of TDP-1 with HPL-2 is needed for HPL-2 recruitment to chromatin, we performed a chromatin immunoprecipitation (ChIP) assay with an antibody against HPL-2, followed by deep sequencing, for cibility . As prevcibility , 31, thetdp-1(ok803) mutants (Data Set S3), the majority of which (71%) showed a decrease in HPL-2 association in the mutant compared to that in the wild type  indicates that in the majority of regions where TDP-1 affects HPL-2 localization, TDP-1's role is to maintain HPL-2's chromatin association.In order to focus on HPL-2-bound regions likely to be affected directly by TDP-1, we filtered differentially bound HPL-2 ChIP peaks for regions also directly associated with TDP-1 . Remarkaild type . This hitdp-1-dependent HPL-2 association were also siRNA target genes (taken from this study). However, genes with TDP-1-dependent HPL-2 peaks showed no enrichment for targeting by endo-siRNAs , nor did we find an enrichment for genes with altered siRNA abundance in tdp-1 mutants among genes with tdp-1-dependent HPL-2 ChIP peaks . These results suggest that TDP-1-mediated HPL-2 localization is likely not facilitated by siRNAs. Interestingly, examination of HPL-2 ChIP signals for all siRNA target genes, regardless of colocalization with TDP-1, revealed a strong enrichment for siRNA target genes with increased HPL-2 ChIP signals in tdp-1 mutants   animals. Taken together, these observations support the ideas that TDP-1 maintains HPL-2 localization in an siRNA-independent manner and that deletion of tdp-1 leads to increased availability of HPL-2 to siRNA target genes and the nuclear RNAi complex.To ask if TDP-1-mediated localization of HPL-2 was facilitated by endo-siRNAs , we asked if genes with n [(TG)n/(AC)n in the DNA] (n repeat [(TG)n on the opposite strand] (tdp-1(ok803) mutants failed to identify (AC)n repeats as bound by HPL-2 (n/(TG)n repeats. Interestingly, we noted that a second binding motif, (AG)n, identified for HPL-2 in wild-type animals (tdp-1 mutants. While (AG)n repeats have not previously been characterized as a binding site for TDP-43 orthologs, it is possible that TDP-1 associates with another RNA binding protein specific to this site in order to recruit/maintain HPL-2 at chromatin (see Discussion). Regardless, (AC)n and (AG)n motifs were identified in over 50% of all regions bound by HPL-2 in wild-type animals, indicating that TDP-1 mediates specificity for the majority of HPL-2 binding locations.TDP-1 localization to chromatin is dependent on RNA , and TDPthe DNA] . To inveP = 2.68 × 10−48; chi-square test]). Specifically, we noted that the locations of these TDP-1-dependent HPL-2 ChIP peaks corresponded to intronic regions (71%) and promoters (20%). Indeed, TDP-1/TDP-43 orthologs have been shown to bind predominantly to introns in multiple organisms (tdp-1(ok803) animals compared to that in wild-type animals are shown in To determine what types of HPL-2-bound regions are mediated by TDP-1, we compared the locations of differentially bound HPL-2 peaks associated with TDP-1 to the locations of annotated genes. Interestingly, we found a significant enrichment of HPL-2/TDP-1 peaks in gene bodies compared to differentially bound HPL-2 peaks not associated with TDP-1  and wild-type animals for genes with TDP-1-dependent HPL-2 ChIP peaks. Of the 1,005 genes with TDP-1-dependent HPL-2 peaks that were expressed in our mRNA data sets, 161 showed a significantly changed RNA abundance in tdp-1 mutant animals compared to that in wild-type animals. Importantly, 137 of these genes (85%) showed decreased transcript abundance, representing a highly significant enrichment for decreased expression. This includes all transcripts altered for expression in tdp-1 mutants  and transcripts with altered expression from genes identified as cotranscriptionally bound by TDP-1 . This result is consistent with the hypothesis that when TDP-1 binds nascent RNA in concert with HPL-2, a function of this complex is to maintain transcript abundance.HP1 controls the abundance of transcripts from active genes , 28–30. 28–tdp-1 deletion strain. We therefore used transcriptome sequencing (RNA-seq) to characterize the transcriptome of a strain containing the hpl-2 null deletion allele tm1489 under the same conditions as those used previously to characterize the tdp-1(ok803) transcriptome (tdp-1(ok803) data were significantly overrepresented in the hpl-2(tm1489) data (among genes expressed in both data sets), with 72% of significant genes from the tdp-1 list present on the hpl-2 list (hypergeometric P = 1.7 × 10−25) (tdp-1 mutants and those underexpressed in hpl-2 mutants (75% codecreased) , supporting the idea that tdp-1-dependent HPL-2 association functions to maintain gene expression. To validate the hpl-2(tm1489) RNA-seq data, quantitative reverse transcription-PCR (RT-PCR) was performed on independent RNA preparations from hpl-2 and tdp-1 mutants, targeting genes with reduced abundance based on the RNA-seq analysis. Coordinate reduction was observed in both strains as well as an hpl-2(tm1489); tdp-1(ok803) double mutant for 9/10 genes tested  transcripts (1.54-fold). Deletion of hpl-2 similarly, and more dramatically, reduced gsa-1 transcript accumulation (6.8-fold).If HPL-2 acts in concert with TDP-1 to modulate RNA metabolism, a strong prediction is that loss of HPL-2 should replicate transcriptome changes we previously observed in the criptome . Loss of× 10−25) . Importas tested . Illustrhpl-2(tm1489) mutants. For tdp-1(ok803) mutants, 89% of transcripts in the dsRNA pool were more abundant than those in the wild-type dsRNA pool. We found for hpl-2(tm1489) mutants that 84% of dsRNA transcripts followed this pattern, indicating a similarly strong enrichment for increased dsRNA formation. Over half the transcripts  with increased dsRNA enrichment in tdp-1(ok803) mutants also showed increased dsRNA enrichment in hpl-2(tm1489) mutants, which is a highly significant overlap (hypergeometric P = 1.5 × 10−144). This pattern is also reflected for the gsa-1 locus: deletion of either tdp-1 or hpl-2 led to a significant increase in double-stranded transcripts corresponding to the gsa-1 gene (Loss of TDP-1 results in a large accumulation of double-stranded RNA (dsRNA), which was previously quantified by a RNA immunoprecipitation (RIP-seq) experiment using the J2 monoclonal antibody to specifically immunoprecipitate dsRNA . We emplC. elegans and are highly enriched in the dsRNA pool of tdp-1(ok803) mutants . Similahpl-2(tm1489) mutants. Using the rMATS algorithm, we detected splicing changes in 1,563 genes in hpl-2(tm1489) mutants (Data Set S6), a large fraction of which  also had altered splicing in tdp-1(ok803) mutants. Unexpectedly, the majority of splicing changes in the hpl-2 deletion mutants were not concordant with those observed in tdp-1(ok803) mutants. In fact, 74% of shared altered splicing events in tdp-1(ok803) and hpl-2(tm1489) mutants were discordant. Similarly, tdp-1(ok803) genes with altered splicing displayed no consistent pattern of increased or decreased HPL-2 binding. We concluded that altered splicing in tdp-1(ok803) mutants does not result from changes in HPL-2 chromatin association.Loss of TDP-1 results in changes in alternative splicing , as obseC. elegans. Indeed, we found that loss of TDP-1 sensitizes C. elegans to exogenous RNAi. This effect is due to enhanced efficiency of nuclear RNAi , as it is blocked by a loss-of-function mutation in nrde-3, a critical component of the nuclear RNAi machinery. The specific enhancement of nuclear exo-RNAi caused by loss of tdp-1 makes it unlikely that exo-RNAi efficiency is increased in the tdp-1 mutant simply because this mutation increases the accumulation of dsRNA, as this would presumably have an impact on both cytoplasmic and nuclear RNAi. Increased efficiency of exo-RNAi is also unlikely to be due to a defect in endo-siRNA biogenesis, as deep sequencing of small RNAs in tdp-1(ok803) mutants did not show decreased abundances of siRNAs directed against most genes or NRDE-3 target genes , we investigated whether loss of TDP-1 altered RNA interference (RNAi) in et genes  but, inset genes . If the pression .eri-1-dependent endo-siRNAs and nuclear NRDE-3 (eri-1 and rrf-3) that reduce cytoplasmic endo-RNAi can lead to increased availability of shared factors to the cytoplasmic exo-RNAi pathway, we hypothesize that tdp-1 deletion enhances nuclear RNAi by increasing the amount of available HPL-2. Supporting this idea, we show by ChIP assay that TDP-1 maintains localization of HPL-2 in hundreds of active genes but also indirectly limits the HPL-2 ChIP signal for siRNA target genes ; tdp-1(ok803) double mutants show synergistic phenotypes  pathway, as a class B gene (tdp-1(ok803) mutants in combination with either class A or class B synMuv genes, suggesting that HPL-2 function in vulva formation is independent of TDP-1.Why does loss of TDP-1 lead specifically to enhanced nuclear exo-RNAi? While we cannot rule out the possibility that TDP-1 is a novel negative regulator of nuclear exo-RNAi, we favor the idea that loss of TDP-1 increases the availability of chromatin-modifying proteins that contribute to nuclear exo-RNAi. In support of this, we show that TDP-1 coimmunoprecipitates with HPL-2 in the absence of r NRDE-3 , supportet genes . These renotypes  to E, in mutants . Interess B gene , we did in vivo, independent of RNA n binding motif was not enriched in HPL-2 ChIP assays of tdp-1 mutants, indicating that HPL-2's specificity for (TG)n is dependent on TDP-1. Interestingly, TDP-1 also facilitated HPL-2 association with (AG)n repeats. While TDP-1 is not known to bind (AG)n repeats, the consensus binding motif of another splicing factor, SFSR1, does contain (AG)n repeats n (tdp-1(ok803) mutant, with the majority of regions losing HPL-2 localization.HP1 homologs localize to active genes and coimmunoprecipitate with elongating RNA Pol II , but howt of RNA , and that of RNA , our datt of RNA , 48, as t of RNA . We spect of RNA . However repeats , and thi repeats , 29. Thean (UG)n . Regardltdp-1(ok803) and hpl-2(tm1489) deletion strains revealed a highly significant overlap in transcript abundance changes, although loss of HPL-2 appears to have a more dramatic effect on the global transcriptome. Similarly, many transcripts found to have increased dsRNA structure in tdp-1(ok803) mutants also had increased dsRNA structure in hpl-2(tm1489) mutants, including transcripts from repetitive elements. We interpret these observations to indicate that (i) the proximal cause of a significant fraction of the tdp-1(ok803) transcriptome changes we previously identified may in fact be due to altered HPL-2 chromatin association and (ii) HPL-2 also has additional, TDP-1-independent roles in RNA metabolism. It would be of significant interest to know the degree to which HP1 isoforms play a role in the transcriptome changes identified in TDP-43 knockdown  can act as an RNA chaperone to control the accumulation of double-stranded RNA . PerhapsDrosophila increases levels of the gypsy retrotransposon element, resulting in a reduced life span and in neurodegeneration. As the Drosophila HP1 homolog binds retrotransposon-derived RNA , and image brightness and contrast were digitally adjusted in Photoshop.Liquid thrashing assays were performed as described previously , using sFor each assay, 5 individual L4 worms of each indicated genotype were singly picked to a small plate spotted with OP50 bacteria. Each day, worms were moved to fresh plates and the progeny counted until no more progeny were laid. The total brood for each animal was determined by adding the numbers of progeny laid on all days. The assay was done twice in triplicate.Worms were broken by bead beating  in homogenization buffer  at a 1:1 ratio of 0.1-mm glass beads to packed worm volume for 3 cycles of 30 s each at 5,000 rpm. Extracts were used immediately for immunoprecipitations, with equal concentrations of extract (as determined by protein quantification) added to IP mixtures. ChIP extracts were prepared as described previously .C. elegans-specific Cstf64 rabbit polyclonal antibody  was diluted 1:7,500 in blocking solution. Horseradish peroxidase (HRP)-conjugated secondary antibodies  were developed with ECL Plus reagent (Amersham).For immunoblotting, 10 to 20 μg of protein per sample or equal volumes of immunoprecipitated protein samples were run at 180 V in NuPAGE 4 to 12% Bis-Tris gels (Invitrogen), using morpholineethanesulfonic acid (MES)-SDS running buffer (Invitrogen). The gels were transferred to 0.45-μm supported nitrocellulose membranes (GE Osmonics) by use of 20% methanol, 39 mM glycine, and 48 mM Tris base. Transfer conditions were 35 V for 70 min. Prestained Rainbow size markers  were used to size bands. Blots were blocked in Tris-buffered saline (TBS)–Tween  plus 5% milk. Blots were probed with a primary mouse monoclonal antibody against green fluorescent protein , rabbit polyclonal anti-TDP-1 , and anti-HPL-2 . For reprobing of blots to assay loading levels, a Twenty microliters of protein A magnetic beads  or anti-FLAG magnetic beads (Invitrogen) was washed, blocked, and bound by antibody (Dynabeads only) at a ratio of 10 μl/IP for anti-TDP-1 (made in-house) or 3 μl/IP for anti-HPL-2 , and 100 μg of protein was added to each IP mixture. RNase-treated extract was incubated with 1 to 5 μl of RNase T1 (single-stranded RNA specific) and RNase V1 (double-stranded RNA specific) for 30 min (room temperature) prior to addition to beads. Tubes were rocked at 4°C for 2 h. The supernatant was removed, and beads were washed three times in IP buffer , moved tGSE61581.ChIP was performed as described previously . A crosspmp-3, which shows no change in expression in mRNA-seq experiments between wild-type, tdp-1(ok803), and hpl-2(tm1489) strains. The fold changes in signal between the wild-type and mutant strains were calculated using the ΔΔTC method.Quantitative RT-PCR was performed on two independent biological replicates of each indicated strain. Total RNA was isolated using TRIzol, and oligo(dT)-primed cDNA was synthesized by use of SuperScript IV (Invitrogen) and amplified using the primers shown in tdp-1(ok803) analyses performed, we used previously reported RNA-seq data  for 30 min at 37°C in company-supplied buffer, phenol-chloroform extracted, ethanol precipitated, and brought up to 30 μl in RNase-free H2O. RNA was then incubated at 37°C for 30 min with polynucleotide kinase (NEB) in DNA ligase buffer (containing ATP), with the addition of 1 μl of RNaseout (Invitrogen). RNA was phenol-chloroform extracted, precipitated with isopropanol, washed, and dissolved in 12 μl RNase-free H2O. Library construction was done using an Illumina TrueSeq small RNA sample prep kit.For all seq data . Informahpl-2(tm1489) analyses, RNAs for poly(A) and total RNA sequencing libraries were extracted from whole animals by TRIzol extraction. Genomic DNA was removed using Turbo DNase (Invitrogen). Poly(A)-selected, single-ended, 125-bp strand-specific libraries were prepared by the UCCC Genomics Core  by use of a TruSeq stranded mRNA library prep kit (Illumina). For J2-IP analyses, RNAs were recovered from young adult worms  by immunoprecipitation with the J2 antibody, and RNAs as well as input material (as a loading control) were converted into strand-specific total RNA libraries by use of V2 Scriptseq (Epicenter) kits following the manufacturer's instructions, except that reverse transcription was done with SuperScript III (Invitrogen), using incrementally increasing temperatures from 42 to 59°C to allow for transcription though structured RNAs. rRNA was not removed from J2-IP RNA samples. Immunoprecipitated DNA from ChIP samples was converted into sequencing libraries by use of a ChIP-seq DNA sample prep kit. Cluster generation and sequencing were performed on an Illumina HiSeq 2500 platform. The reads were demultiplexed and converted to FASTQ format by use of CASAVA software from Illumina.For www.wormbase.org). Quantification of antisense siRNAs mapping to genes was done by separating SAM files into positive and negative reads and counting the numbers of siRNAs mapping to defined gene intervals. Only siRNAs mapping antisense to the annotated gene were counted. Expression differences between the wild-type and tdp-1 mutant strains were calculated using DESeq software, with cutoffs of an FDR of <0.05% and a >2-fold change.Small RNA reads were trimmed to remove adaptor sequences and filtered to allow only 1 base with a quality score below 20 and a minimum sequence length of 15 bp. Reads passing the filter were mapped to the WS220 genome by use of Bowtie1 , allowinq < 10) were trimmed from the 3′ ends of reads. Adaptor sequences and reads shorter than 40 nucleotides were removed. Reads were aligned to the C. elegans WS220 genome by use of TopHat2 (v2.0.14), with the following parameters: -b2-very-sensitive -i 30 -I 5000 -read-edit-dist 3 -N 3 -read-realign-edit-dist 0 -p 10 -segment-length 25 -segment-mismatches 2 -no-coverage-search -min-coverage-intron 30 -max-coverage-intron 5000 -min-segment-intron 30 -max-segment-intron 5000. For poly(A)-selected RNA-seq reads, uniquely mapped reads were used as input to obtain gene counts by using the WS220 annotation with Rsubread (v 1.18.0) (tdp-1(ok803) mutant were taken from the data available under accession number GSE61581. Details related to all sequencing experiments done in this work can be found in Low-quality bases ( 1.18.0) . Differe 1.18.0) . Signifi 1.18.0) . RNA-seqhttp://repeatmasker.org. The suggested parameters for running RepEnrich on single-end reads were used. The commands are available at https://github.com/nskvir/RepEnrich.Trimmed and filtered reads from total RNA and J2-IP samples were used as input into the RepEnrich program to determine repetitive element expression levels . The WS2P value of <0.10 were used to determine repeat significance. J2-IP RNA-seq data for the tdp-1(ok803) mutant were taken from the data available under accession number GSE61581.To determine if repetitive elements were significantly enriched in the J2-HPL-2 samples, we used DESeq2. Repeat counts were normalized by obtaining the difference between total mapped reads and the number of reads aligning to rRNA. DESeq2's likelihood ratio test was used, and a minimum mean expression value of 20 and an adjusted q < 0.05) for the wild-type and tdp-1(ok803) HPL-2 ChIP-seq samples, using the corresponding [N2 and tdp-1(ok803)] genomic sequencing samples as background controls. Parameters for MACS peak calling used for both the wild-type and tdp-1(ok803) strains were as follows: callpeak -t Strain_HPL2.bam -c Strain_genomic_control.bam -B -g ce -keep-dup 3 -n Strain_HPL2 -m 2 50. To call differentially represented peaks between wild-type and tdp-1(ok803) samples, we used the Bioconductor program Diffbind (version 1.10.2). To briefly elaborate, we first input the significant MACS peak calls for all samples, along with the corresponding HPL-2 ChIP-seq and genomic alignment files, into Diffbind. Diffbind identified a consensus set of peaks found in our MACS peak calls, with the requirement that at least 2 samples contain the peak. Read counts for each consensus peak were generated. Genomic read counts for the corresponding consensus peaks were subtracted from those for their corresponding samples . Differential analysis of the peak counts between the wild-type and tdp-1(ok803) strains was performed using DESeq. An FDR cutoff of <10% was used to determine the significance of differences.Chromatin immunoprecipitation sequencing (ChIP-seq) and genomic reads were trimmed and filtered in the same manner as that described above for RNA-seq data. ChIP-seq and genomic reads were uniquely mapped to the WS220 genome by use of Bowtie1, allowing 2 mismatches in a 45-bp seed. MACS, version 1.4.2, was used to identify significant peaks (http://repeatmasker.org), and one repeat overlap per peak was kept if at least one nucleotide overlapped.To determine the overlap of HPL-2 peaks with repetitive element sequences, consensus peaks were derived from our biological replicate wild-type HPL-2 ChIP-seq reads by use of Diffbind. The consensus wild-type HPL-2 peaks were intersected with the CE10 repeatmasker file .Permutation tests were done to compare called HPL-2 ChIP-seq peaks for wild-type animals to ChIP-seq peaks previously identified by McMurchy et al. . HPL-2 btdp-1(ok803) replicate HPL-2 ChIP-seq samples by using MACS. The consensus peak sets were then analyzed using HOMER, a software suite for ChIP-seq analysis and motif discovery. The UCSC ce10 genome was used for the HOMER motif search, with the option “-size 200.”We first derived consensus peak sets for wild-type and C. elegans. The STAR parameters used were as follows: -alignIntronMin 10 -alignIntronMax 5000 -outSJfilterIntronMaxVsReadN 10 20 40 -outSJfilterCountTotalMin 5 3 3 3 -outSJfilterCountUniqueMin 3 2 2 2. rMATS (v3.2.5) software was used to identify alternative splicing (AS) events, which corresponded to five types of AS patterns  containing an empty L4440 vector] grown to the same optical density. In each case, 250 μl of RNAi food was seeded onto a 10-mm or 3-mm worm growth plate containing solid worm growth agar freshly spread with 1 mM isopropyl-β-d-thiogalactopyranoside  and 0.11 mM ampicillin . IPTG and AMP were allowed to disperse through the solid agar for 1 h, and then the plates were immediately spotted with RNAi food. Bacteria were grown overnight at room temperature. Worms of the desired strain were placed onto each seeded plate at 20°C as L4 worms and allowed to lay eggs for 16 h. Parents were removed, and RNAi phenotypes were observed in hatched progeny. For heritable RNAi experiments, synchronized wild-type and tdp-1(ok803) animals expressing a muscle-specific GFP transgene were treated with GFP feeding RNAi for 1 generation. Embryos from these animals were collected and hatched on normal (OP50) bacteria. The resultant progeny were scored for GFP signals 2 days later, as L4 worms. The criteria applied to determine whether animals were affected by a given RNAi treatment were as follows: unc-22 RNAi- and unc-54 RNAi-treated animals were considered affected if the animals were unable to move away after prodding with a pick, act-5 RNAi-treated animals were considered affected upon L2 arrest, and unc-73 RNAi-treated animals were considered affected if they failed to back up and displayed a “kinked tail” upon prodding of the head region with a pick.Single colonies from the Ahringer library  were inoGSE100829.The raw data generated in this study can be found in the Gene Expression Omnibus (GEO) database under accession number


Thanks to a wide range of biological functions of RNA, and advancements in nanotechnology, RNA nanotechnology has developed in multiple ways for RNA-based therapeutics. In particular, among RNA engineering techniques, enzymatic self-assembly of RNA structures has gained great attention for its high packing density of RNA, with a low cost and one-pot synthetic process. However, manipulation of the overall size of particles, especially a reduction in size, has not been studied in depth. Here, we reported the enzymatic self-assembly of short hairpin RNA particles for the downregulation of target genes, and a rational approach to the manipulation of the resultant particle size. This is the first report of the size-controllable enzymatic self-assembly of short hairpin RNA nanoparticles. While keeping all the benefits of an enzymatic approach, the overall size of the RNA particles was controlled on a scale of 2 μm to 100 nm, falling within the therapeutically applicable size range. RNA nanotechnology has developed enormously by taking advantage of the intrinsic properties of RNA, and advancements in nanotechnology ,2,3. TheTo fully exploit RNA, RNA nanotechnology has been developed in many ways. For the synthesis of functional RNA structures, a simple hybridization approach was used for the generation of a polyhedral structure, while a crossover technique and RNA architectonics were used for the generation of more complex RNA structures ,9,10,11.Here, we report the size-controllable assembly of short hairpin RNA (shRNA) particles via an enzymatic approach. While the synthesis of size-controllable RNA nanoparticles was possible using complementary rolling circle transcription with two types of circular DNA templates ,20, thisTM spin desalting columns were purchased from Thermo Fisher Scientific . Cyanine 5-UTP was purchased from Enzo Life Sciences . Mica and Lacey Formvar/carbon-coated copper grids (01883-F) were obtained from Ted Pella . Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin (P/S), fetal bovine serum (FBS), and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Gibco . The StemfectTM RNA Transfection Kit was purchased from Stemgent . CelLytic M and MgCl2 solution were purchased from Sigma-Aldrich . A Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories . Tris-HCl buffer was purchased from Invitrogen .All oligonucleotides were purchased from Integrated DNA Technologies . T4 ligase was obtained from Promega . T7 RNA polymerase and ribonucleotide triphosphates (rNTPs) were purchased from New England BioLabs . ZebaTM Thermal Cycler, Bio-Rad, Hercules, CA, USA). T4 ligase (0.06 units/µL) was then introduced to the mixture with ligase buffer . The solution was incubated overnight at room temperature to ligate the nick in the circularized DNA.A 92-base-long phosphorylated linear DNA for anti-GFP shRNA was mixed with a 22-base-long primer DNA for T7 RNA polymerase at a final concentration of 3.0 μM in nuclease-free water , and 5 units/μL T7 RNA polymerase. For the rolling circle transcription (RCT) reaction, the reaction solution was incubated for 20 h at 37 °C. The final reaction solution was briefly sonicated, before the shRNA particles were purified with a ZebaTM Desalting Column, following the manufacturer’s protocol. For the synthesis of cy5-labeled shRNA-NPs, cy5-UTP  was added to the RCT reaction mixture at the beginning of the incubation process. To remove unincorporated cy5-UTP, the shRNA-NPs were purified with a ZebaTM Desalting Column after the RCT reaction. The circular DNA at final concentrations of 0.03 μM, 0.1 μM, or 0.3 μM were mixed with 8 mM ribonucleotide solution mix, reaction buffer . Nanoparticle tracking analysis was carried out with Nanoparticle Tracking Analysis . Transmission electron microscopy (TEM)  was employed to characterize the shRNA-NPs, operating at an accelerated voltage of 200 kV, before TEM-based energy dispersion X-ray (EDX) was used to analyze the chemical compositions of the shRNA-NPs. For the preparation of samples, the shRNA-NPs were deposited onto Lacey Formvar/carbon-coated copper grids, and then air-dried at room temperature.A field emission scanning electron microscope (FE-SEM)  and an atomic force microscope (AFM)  were used to obtain high-resolution digital images of the shRNA particles. The shRNA particles for SEM imaging were deposited onto a silicon wafer, and coated with Pt after being dried. For AFM imaging, 10 μL of the reaction mixture was diluted in nuclease-free water containing 5 mM Tris-HCl and 5 mM MgCl2. The cells were passaged routinely to maintain exponential growth. One day prior to transfection (~90% confluence), the cells were trypsinized, diluted with fresh medium, and transferred to 24-well plates . The cy5-labeled shRNA-NPs were covered with the delivery carrier, a StemfectTM RNA Transfection Kit, following the manufacturer’s instructions. Specifically, the cy5-labeled shRNA-NPs were mixed with the StemfectTM RNA Transfection reagent (RNA:reagent = 1:3 w/v) in PBS solution, and incubated for 10 min at room temperature. After diluting the shRNA-NPs/reagent solution with media, the samples were treated to cells for 4 h at a concentration of 2.5 μg/mL at 37 °C. After further incubation for 12 h in fresh serum-containing media, the cells were detached from the plates by treatment with trypsin-EDTA solution, and washed three times with PBS. The cells were analyzed by NucleoCounter . The data were analyzed using the FlowJo software.HeLa cells were grown in DMEM, supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 1% Antibiotic-Antimycotic, at 37 °C in a humidified atmosphere, supplemented with 5% COTM RNA Transfection Kit, prior to transfection according to the manufacturer’s instructions. Then, the cells were treated with various concentrations of the covered shRNA-NPs, ranging from 0.1 to 2.5 μg/mL. After 24 h of treatment, cells were washed with DPBS, and lysed with CelLytic M. The green fluorescence from each well containing the lysed cells was detected by a microplate reader , and then normalized with the green fluorescence from the well containing untreated HeLa-GFP cell lysates, to obtain relative GFP expression. Cell viabilities were assessed with CCK-8 according to the manufacturer’s instructions. HeLa-GFP cells were transferred to 96-well plates (7000 cells per well). The shRNA-NPs were covered with the transfection reagent from the Stemfectn = 4). Error bars indicated mean standard deviations of each experiment. Statistical analysis was performed with a Student’s t-test. Statistical significance was assigned for p < 0.05 (95% confidence level).Data in this study were represented as mean values of independent measurements  synthesized with 0.03 μM of circular DNA to further characterize their nature. The self-assembled shRNA-NPs had spherical structures that were 100 nm in diameter, revealed by scanning electron microscopy SEM, B, and naz-axial resolution, so as to determine the full three-dimensional structure . By introducing cy5-UTPs into the RCT reaction, T7 RNA polymerase incorporated these molecules into synthesized RNA strands. Thus, the resulting self-assembled shRNA-NPs emitted red fluorescence, resulting in an increased red fluorescence signal when analyzed by image cytometry A. AccordTo test the gene-silencing activities of shRNA-NPs, anti-GFP shRNA-NPs were treated to HeLa cells stably expressing GFP (HeLa-GFP). The expression levels of GFP went down by 50% when treated with 2.5 μg RNA per ml C, while In summary, we developed size-controllable synthesis of short hairpin RNA nanoparticles via an enzymatic approach. By controlling the concentrations of template circular DNAs while maintaining the same concentration of RNA polymerase in the RCT reaction, the sizes of the shRNA particles could be reduced from 2 μm to 100 nm. The resulting shRNA-NPs were fully characterized, and controlling their sizes was possible through controlling the level of entanglement of RNA strands by changing concentrations of template DNA in the RCT reaction. In addition, the shRNA-NPs had a favorable size for therapeutic applications. Through such a development in RNA engineering, we envision that we can step forward into the real-world applications of RNA therapeutics.


Maintaining a healthy proteome involves all layers of gene expression regulation. By quantifying temporal changes of the transcriptome, translatome, proteome, and RNA-protein interactome in cervical cancer cells, we systematically characterize the molecular landscape in response to proteostatic challenges. We identify shared and specific responses to misfolded proteins and to oxidative stress, two conditions that are tightly linked. We reveal new aspects of the unfolded protein response, including many genes that escape global translation shutdown. A subset of these genes supports rerouting of energy production in the mitochondria. We also find that many genes change at multiple levels, in either the same or opposing directions, and at different time points. We highlight a variety of putative regulatory pathways, including the stress-dependent alternative splicing of aminoacyl-tRNA synthetases, and protein-RNA binding within the 3’ untranslated region of molecular chaperones. These results illustrate the potential of this information-rich resource. Proteostasis, the integrated assembly of pathways that regulate transcription and translation, the proteins’ correct folding, localization, and - eventually - degradation, is a major challenge for the cell . Much ofPERK is autophosphorylated leading to the subsequent phosphorylation of the initiation factor eIF2α limiting its use for translation initiation, thereby decreasing the ER’s protein folding load , triggered by challenged proteostasis, affects all levels of gene expression regulation creating an intricate and highly dynamic network of responses . Initialing load .ATF4, where increased eIF2α phosphorylation allows ribosomes to bypass inhibitory upstream open reading frames (uORFs) in the 5’ untranslated region (UTR) during stress  produced during protein folding in the ER Figure 1. In addiTo provide new insights into these open questions we conducted one of the most comprehensive assessments of the regulatory landscape in response to ER stress available to date - revealing previously unknown and underappreciated aspects of the adaptive UPR. We collected replicate samples at four time points  from human cervical cancer cells treated with tunicamycin. Tunicamycin is a naturally occurring antibiotic that inhibits protein N-linked glycosylation and therefore prevents proper folding particularly in the ER, thus inducing the accumulation of misfolded proteins . Using RWe present a resource comprising four complementary data types collected for cervical cancer cells treated with tunicamycin and hydrogen peroxide: RNA and protein concentrations measured with RNA-seq and quantitative proteomics, respectively, and the binding profiles of ribosomes and non-ribosomal proteins along mRNAs measured with ribosome footprinting and protein occupancy profiling . The datTo account for the dynamic nature of the stress response, we monitor four time points 0, 1, 4, 8 hr). As illustrated in , 8 hr. Aand time points (change points). We expand the use of PECA to all four datasets and convert concentration and binding data into information on regulatory events. For the RNA data, assuming DNA copy number does not change, PECA informs on significant changes in transcription and RNA degradation (TRXP/RNA-DEG) to extra/RNA-DEG). For rib/RNA-DEG). All resHSPA5 (BiP/GRP78) and HSP90B1, the tryptophanyl-tRNA-synthetase Trp-tRS (WARS), and the DNA repair enzyme RAD51. HSPA5 is a key chaperone of the ER stress response (XBP1 results in a frame-shifted transcript (sXBP1) that produces the XBP1 transcription factor; this is one of the earliest steps in the ER stress response . The nonresponse . While wresponse . The sple 5’ UTR . GADD34 e 5’ UTR . According frame .ATF4, GADD34, and HSPA5 discussed above, as well as ATF3, ATF5, DDIT3 (CHOP), IBTK, and IFRD1. Across all three replicates, we identify significant translation upregulation based on the increase in ribosome footprints compared to RNA levels . Finally, we validate our ability to extract significant regulatory events through examination of genes with known uORF-medidated translation increase during ER stress . The genHSPA5, GADD34, and HSP90B1. This cluster also encompasses aminoacyl-tRNA synthetases (p<3×10^−9) and serine biosynthesis enzymes (p<3×10^−6) that are regulated in a manner similar to the UPR genes and are discussed later. Using an upstream regulator analysis  we also confirm that cluster 11 is enriched for genes that are themselves targets of established UPR regulators (e.g. ATF4 and ATF6)  .We observe a significant increase in protein expression during ER stress for a set of genes significantly enriched in the integrin signalling pathway . IntegriRAD51 mentioned above  cycle to one-carbon metabolism to feed oxidative phosphorylation. Translationally upregulated genes are enriched in subunits of Complexes I-IV from the oxidative phosphorylation pathway . While tAUGGCGGC) highly enriched among the translation initiation sites of Complex I-IV genes , but not amongst genes for ATP synthase subunits or TCA cycle enzymes .Examination of the 5’ UTR of the genes for Complex I-IV reveals a sequence element that might explain their differential translation increase during ER stress. Translation of mitochondrial genes is often regulated by the TISU element, the Translation Initiator of Short 5’ UTR . The TISSHMT2, MTHFD2, and ALDH1L2). While the TCA cycle generally supplies NADH that feeds into the oxidative phosphorylation pathway to generate ATP, production of mitochondrial NADH can also be achieved through one-carbon metabolism, particularly under conditions of stress . While eT1, PSPH), we findT1, PSPH). We alsoTIMM17A represents an exception as it significantly decreases at the protein level, consistent with previous findings . The impThese elements were identified based on similarity of sequence and predicted secondary structure across vertebrate genomes , howeverbona fide regulatory elements, for example the iron response element. Two predicted RNA structures with the highest abundance of ribosome footprints are located within the 5’ UTR of the ferritin heavy and light chain transcripts, FTH1 and FTL , as well as a hairpin without any predicted motif or known RNA-binding partner . As SXL e 3’ UTR . Footprie 3’ UTR , while Se 3’ UTR .TSC22D3 transcript . This transcript encodes the glucocorticoid-induced leucine zipper protein (GILZ), which has been shown to act as a pro-survival factor during ER stress . Remarkably, these structures are all within genes that have an established role in the UPR, including three molecular chaperones that act as critical sensors of misfolded proteins: and CALR . We obseand CALR .We present a high-quality, information-rich resource that provides new insights at multiple levels at which cancer cells respond to either ER or oxidative stress. In measuring not only RNA and protein abundances, but also ribosome and other protein footprints along mRNAs, we capture the adaptive landscape of various stress response pathways during the first eight hours of stress. We extract hundreds of genes with significant changes in transcription, translation, and degradation at individual time points and isolate regulatory signatures that have been hitherto unknown or underappreciated.Translation regulation is a key component of the mammalian UPR, as translational shutdown is one of the earliest reactions to ER stress. However the extent to which genes can evade this global inhibition and the ways in which they do is an active area of investigation. We identify many genes that significantly increase in translation during ER stress , expandiUsing these data, we provide new insights into how the different regulatory levels affect gene expression in response to protein misfolding. We show that transcription regulation often occurs early in the stress response, while post-transcriptional regulation takes place both early and late during stress  - expandSeveral key regulators of the UPR are upregulated concordantly in their transcription and translation. The timing of their regulation supports the concept of an adaptive Unfolded Protein Response, where after surviving the acute phase of stress the cell adjusts via continual modulation of translation to recover protein synthesis and cope with the burdened ER . TranslaIn comparison, we also find a statistically significant and unexpectedly large number of genes with discordance between their transcriptional and post-transcriptional regulation . These gPERK-mediated pathways , but also in genes from serine biosynthesis , which is upstream of one-carbon metabolism and localized to the cytosol. As serine biosynthesis diverts 3-phosphoglycerate from its use in glycolysis and the TCA cycle, the production of NADH via one-carbon metabolism may be used as an alternative to fuel Complex I and therefore drive oxidative phosphorylation  during ER stress . GSH is SHMT2 which synthesizes glycine that is required for the biosynthesis of GSH  that has multiple functions in the ER and stress response  with 10% fetal bovine serum  and 1X penicillin streptomycin solution (Corning cellgro) at 37°C and 5% CO2. At ~60% confluency, we treated the cells with 60 μM HWe authenticated the cell line identity by STR profiling from Genetica DNA Laboratories. No mycoplasma contamination has been detected, as confirmed by mycoplasma contamination test using PlasmoTest kit (Invivogen) and ATCC universal mycoplasma detection kit (ATCC) separately.All steps were performed according the the TruSeq Ribo Profile  kit protocol. Confluent plates of HeLa cells were first aspirated of their growth media and washed with fresh media supplemented with 0.1 mg/ml cycloheximide. The media was then removed and the cells were washed with 10 ml chilled phosphate buffer saline (PBS) containing 0.1 mg/ml cycloheximide. Following removal of the PBS, 800 μl of Lysis Buffer was added and the cells were extensively scraped off plate and transferred to a pre-chilled eppendorf tube, recovering ~1 ml of lysate per sample. To insure complete lysis, we passed the lysate through a 25 gauge needle and further incubated on ice for ~10 min. The lysate was then clarified by centrifugation for 10 min at 20,000, 4°C and ∼1 ml supernatant was recovered. For each treatment (tunicamycin and hydrogen peroxide), we aliquoted 100 μl of the cell lysate for total RNA extraction and 200 ul for ribosome footprinting.We used the TruSeq Ribo Profile  kit for both total RNA profiling and mapping of ribosome protected fragments (RPF). Briefly, for the total RNA samples, 100 μl aliquots of lysate were supplemented with 10 μl of 10% SDS and purified using a Zymo RNA Clean and Concentrator-25 Kits. For the RPF samples, we treated the cell lysate with 5 units of Truseq Ribo Profile Nuclease for each A260 of lysate and incubated for 45 mins at room temperature while agitating gently. We stopped reactions by adding SUPERase-In RNase inhibitor and placed the samples on ice. The nuclease-treated lysate (100 μl) was purified using Illustra Microspin S-400 columns as per instructions followed by addition of 10 μl of 10% SDS to the flow-through. The remaining 100 μl of the nuclease digested RNA was stored in −80C for future use. Samples were purified using Zymo RNA Clean and Concentrator-25 kits and separated by denaturing 15% Urea-polyacrylamide gel electrophoresis (PAGE). We excised the desired size - corresponding to the ~28 – 30 nt range - using a dark field trans illuminator and purified according to the manufacturer’s protocol.For library preparation, all samples were RiboZero treated and the total RNA samples were heat-fragmented at 94°C for 25 min according to the manufacturer’s protocol. Both the fragmented total RNA and RPF samples were then end-repaired and 3’ adapter ligated. Following adapter removal, the samples were reverse transcribed and the resulting cDNA was PAGE purified and circularized. We used one quarter of the circularized cDNA as a template for PCR amplification using Phusion (NEB) in a 50 μl volume using specific oligos (Truseq Riboprofile Forward PCR primer and Index PCR primer) and purified the samples with Agencount AMPure beads (Beckman Coulter). We purified the amplified libraries by using 8% Native PAGE and verified the final library size using the High Sensitivity DNA assay on the Agilent Bioanalyzer. We quantified samples using Qubit Fluorometric quantitation and sequenced them on a HiSeq 2500.After sample collection, cells were crosslinked with 365 nm UV light (0.2 J/cm2) on ice using a Stratalinker 2400 (Stratagene). We scraped crosslinked cells off the plates with a rubber policeman, collected by centrifugation, washed with ice-cold PBS once and flash-froze the samples in liquid nitrogen for long-term storage.+-RNA depletion. We washed beads 3 times in lysis/binding buffer and 3 times in NP40 washing buffer  and crosslinked poly(A)+-RNA-protein complexes were eluted in low-salt elution buffer (10 mM Tris-HCl at at 25°C) by incubation at 80°C for 2 min.Protein occupancy profiling was carried out as described previously . Briefly+-RNA depletion following the described procedure. Eluates from different rounds of poly(A)+-RNA depletion are combined, incubated with RNase I for 10 min at 37°C and precipitated with 4 volumes of ammonium sulfate. The resuspended precipitate was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. RNA-protein complexes were on-membrane incubated with Proteinase K for 30 min at 55°C to release protein-protected RNA fragments. RNA was recovered with phenol-chloroform extraction and subjected to small RNA library cloning procedure (32P]-ATP in a T4 Polynucleotide kinase (PNK) reaction followed by ligation of a pre-adenylated 3’ adapter and a 5’ adapter and reverse transcription to generate the cDNA library. In order to identify the RNA population with the desired fragment size, radiolabeled RNA size markers (24 and 50 nt) were used as ligation controls. The cDNA libraries were processed and subjected to sequencing on HiSeq 2500 following standard protocol rocedure . In shorprotocol .For proteomics analysis, we resuspended cell pellets for each sample in 50 μl ice-cold PBS containing 1:100 protease inhibitor cocktail. The cells were then sonicated with probe sonicator 2 × 30 s with amplitude 5. The samples were returned to ice for 30 s between sonication intervals. After sonication, we mixed the samples with 50 μl trifluororethanol, and the mixtures were kept at 60°C for 1 hr. Then the samples were reduced in 15 mM DTT at 55°C for 45 min, and alkylated in 55 mM iodoacetamide in the dark at room temperature for 30 min. Finally, we used 50 mM Tris (pH = 8) to adjust the sample volume to 1 ml, and 1 ug mass spectrometry grade trypsin (Sigma Aldrich) was added to digest the proteins into peptides at 37°C overnight.We measured peptide concentrations with the Pierce Quantitative Fluorometric Peptide Assay (ThermoFisher) kit a. Tandem mass tag (TMT) 10-plex reagents (Thermo Scientific) were dissolved in anhydrous acetonitrile (0.8 mg/40 μl) according to manufacturer’s instruction. We labeled 30 ug/100 μl peptide per sample labelled with 41 μl of the TMT 10-plex label reagent at final acetonitrile concentration of 30% (v/v). Following incubation at room temperature for 1 hr, we quenched the reactions with 8 μl of 5% hydroxylamine for 15 min. All samples were combined in a new microcentrifuge tubes at equal amounts and reduced to remove acetonitrile using an Eppendorf Concentrator Vacufuge Plus.TMT-labelled tryptic peptides were subjected to high-pH reversed-phase high performance liquid chromatography fractionation using an Agilent 1200 Infinity Series with a phenomenex Kinetex 5 u EVO C18 100A column . Mobile phase A was 20 mM ammonium formate, and B was 90% acetonitrile and 10% 20 mM ammonium formate. Both buffers were adjusted to pH 10. Peptides were resolved using a linear 120 min 0 – 40% acetonitrile gradient at a 100 ul/min flow rate. Eluting peptides were collected in 2 min fractions. We combined about 70 fractions covering the peptide-rich region to obtain 40 samples for analysis. To preserve orthogonality, we combined fractions across the gradient, that is each of the concatenated samples comprising fractions which were 40 fractions apart. Re-combined fractions were reduced using an Eppendorf Concentrator Vacufuge Plus, desalted with C18 stage-tip, and suspended in 95% mass spectrometry grade water, 5% acetonitrile, and 0.1% formic acid for subsequent low pH chromatography and tandem mass spectrometry analysis.For the first replicate, we used an EASY-nLC 1200 coupled on-line to a Fusion Lumos mass spectrometer (both Thermo Fisher Scientific). Buffer A (0.1% FA in water) and buffer B (0.1% FA in 80% ACN) were used as mobile phases for gradient separation. A 75 µm x 15 cm chromatography column  was packed in-house for peptides separation. Peptides were separated with a gradient of 5 – 40% buffer B over 110 min, 40 – 100% B over 10 min at a flow rate of 300 nL/min. Full MS scans were acquired in the Orbitrap mass analyzer over a range of 300 – 1500 m/z at a resolution of 120,000 at m/z 200. The top 15 most abundant precursors were selected in data-dependent mode with an isolation window of 0.7 Thomsons and fragmented by higher-energy collisional dissociation with normalized collision energy of 40. MS/MS scans were acquired in the Orbitrap mass analyzer at a resolution of 30,000. The automatic gain control target value was 1e6 for full scans and 5e4 for MS/MS scans respectively, and the maximum ion injection time is 60 ms for both.For the second replicate, we used an EASY-nLC 1000 coupled on-line to a Q Exactive spectrometer (both Thermo Fisher Scientific). Buffer A (0.1% FA in water) and buffer B  were used as mobile phases for gradient separation. An EASY Spray 50 cm x 75 µm ID PepMap C18 analytical HPLC column with 2 μm bead size was used for peptide separation. We used a 110 min linear gradient from 5% to 23% solvent B , followed by 20 min from 23% to 56% solvent B, and 10 min from 56% to 100% solvent B. Solvent B was held at 100% for another 10 min. Full MS scans were acquired with a resolution of 70,000, an AGC target of 1e6, with a maximum ion time of 120 ms, and scan range of 400 to 1500 m/z. Following each full MS scan, data-dependent high resolution HCD MS/MS spectra were acquired with a resolution of 35,000, AGC target of 1e5, maximum ion time of 250 ms, one microscan, 1.5 m/z isolation window, fixed first mass of 115 m/z, and NCE of 30.We used CM-H2DCFDA (Thermo Fisher Scientific) following protocols in . BrieflysXbp1 quantities by SYBR Green quantitative real-time PCR using Kapa Universal SYBR Green Supermix (Kapa biosystem) in a Roche Light Cycler 480 (Roche). All reactions were performed in triplicate. The expression of the spliced XBP1 was assessed by real time PCR (RT-PCR) in a Roche Light Cycler 480 (Roche) with KAPA universal SYBR green master mix. Relative gene expression was quantified using the ΔCT method with respective primers  and normalized to RPL19 . We used ∆∆CT method to determine the fold changes in the expression of sXbp1  and purified RNA with the RNAesay mini Kit (Qiagen). We then synthesized cDNA using the Super ScriptIII First Strand cDNA synthesis kit . We estimated of sXbp1 . BrieflyWe boiled all samples collected at different time points after indicated treatment in sample buffer (Bio-Rad) supplemented with B-mercaptoethanol as per manufacturer’s instruction. We used equal amount of protein (25 μg) from three independent grown cultures and treatments for blotting. The membrane was blocked using 5% BSA and incubated with respective antibodies , rabbit P-PERK , rabbit P- eIF2alpha , rabbit anti Oxidative Stress Markers (Abcam ab179843). β- Actin  served as a loading control. We captured signal intensities of the bands in the Western blot with Kwikquant Imager .We used an in-house R pipeline to process the sequencing data from total RNA, ribosome footprinting and protein occupancy profiling. The fastq files of individual samples were processed with FASTX-Toolkit v0.0.14 to remove adapters and trim reads based on a minimum quality score of 20, as well as discard reads with a trimmed length shorter than 20 nucleotides. Reads mapping to ribosomal DNA were removed using Bowtie2 . RemainiWe used MaxQuant Software version 1.5.5.1 with its integrated search engine Andromeda to analyse our raw files acquired from the mass spectrometer. Data was searched against the human sequence file  sequence file downloaded from the ENSEMBL database . All samTo derive the core dataset of 7,011 genes , we firshttps://www.qiagenbio- informatics.com/products/ingenuity-pathway-analysis), with an adjusted p-value cutoff of 0.0001.We visualized and analyzed the data matrix in PERSEUS version 1.5.5.1 , using hchange in the synthesis and degradation of the respective molecule since and until the time points before and after the time point in question. For the footprinting data, synthesis and degradation are replaced by association and dissociation of the respective molecules.To extract significant regulatory events, we adapted the statistical tool PECA that we developed and expanded recently . For PECThe paired concentration data for the four different levels was derived as follows: i) for transcription and RNA degradation  we paired the RNA abundance data with DNA abundance set to a constant; ii) for translation (TRL) we paired the ribosome footprinting with the RNA abundance data; iii) for translation and RNA degradation/localization/processing  we paired the protein footprinting with the RNA abundance data; and iv) for translation and protein degradation  we paired the protein and the RNA abundance data.PECA reports for each gene a putative change point score. Given the overall score distribution it also calculates a false discovery rate (FDR) for each data point. We reported a significant regulatory event for a gene if we observed a change point with a score corresponding to an FDR < 0.2 in both replicates, in the same direction (up or down), but regardless of the time point at which the event occurred. Stricter cutoffs resulted in very similar results . PECA reWe generated Sashimi plots  for the We obtained a list of conserved RNA secondary structures from reference . CoveragThe data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Juan Valcárcel as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Naama Barkai as the Senior Editor.Thank you for submitting your article "Systematic characterization of the adaptation to proteostatic challenges" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.2O2 (oxidative stress). The data represent a technical tour-de-force in the global characterization of the regulation of the response of human cells to proteostatic stress, and the authors provide good examples of the validity and usefulness of the datasets. The findings bring new insights in our understanding of the cellular response to stress and are expected to be of high value in a broad range of research fields that study physiological or pathological responses to stress conditions.The manuscript by Justin Rendleman and coauthors is an integrated quantitative study that systematically captures transcriptional and post-transcriptional changes induced in HeLa cells upon exposure to two cellular stresses, tunicamycin (which induces ER stress) and HMajor comments:1) The way in which the interplay between ER stress and oxidative stress is discussed in different points of the paper is problematic. At some points, such as when referring to Figures 1B and 2, oxidative stress is presented as having very little effect under the experimental conditions used by the authors . In contrast, somewhere else in the paper the opposite seems to be said, explicitly such as in the third paragraph of the Discussion ("All levels of regulation include genes that respond similarly in both stresses") or implicitly in many parts of the paper. This is confusing and should be clarified.2) The wide diversity of ways in which different results are displayed can be distracting. For instance, Figure 1A introduces the four data types and their representation in one way, but different representation methods are used in the results shown in Figure 1B on the one hand, Figure 1C-E on the other, and Figure 1F on yet another. The same can be said of the rest of the figures. Certainly, any paper that addresses a global analysis of cellular responses can be expected to have large amounts of data, but this highlights even more the importance of being clear in how this data is presented, in order to make the final take-home message of the paper as evident to the reader as possible.Drosophila protein without a homolog in human cells, although a number of poly-U binding proteins  share similar sequence motifs. Therefore the presence of a SXL motif should be explained in these terms in the text and in Figure 5B.3) Data in Figure 5 are based on predictions of RNA secondary structures, which are largely based on RNA stability estimates of relatively long fragments and phylogenetic conservation of base pairing potential; these predictions are necessarily imprecise and unlikely to reflect the actual structure of RNA within complex RNP particles at 5' and 3' UTRs. While detection of prominent structures like iron response elements is reassuring, how predictable other structural elements are in the absence of in vivo RNA structure data (e.g. using SHAPE) should be discussed properly and, if possible, stratified in terms of predicted stability of secondary elements/conservation. It will be also relevant to discuss the predicted local structure at the site of interaction with proteins displaying enriched sequence motifs: the proteins discussed in the text bind to single stranded RNA: is the local structure at these sites predicted to be open within the secondary structure of these elements? By the way, Sex-Lethal is a  Major comments:1) The way in which the interplay between ER stress and oxidative stress is discussed in different points of the paper is problematic. At some points, such as when referring to Figures 1B and 2, oxidative stress is presented as having very little effect under the experimental conditions used by the authors . In contrast, somewhere else in the paper the opposite seems to be said, explicitly such as in the third paragraph of the Discussion ("All levels of regulation include genes that respond similarly in both stresses") or implicitly in many parts of the paper. This is confusing and should be clarified.We restructured the manuscript to clarify our focus on the ER stress response and the connections to oxidative stress .We now discuss the results for the response to hydrogen peroxide in a separate section and explain that many changes are qualitatively similar but much less pronounced than in ER stress (subsection “The shared stress response involves few genes but highly similar patterns”).The revised manuscript also contains a schematic explaining the relationship between ER stress, oxidative stress, and cancer cells  and graphs with the measurements of the ROS levels as discussed below .2) The wide diversity of ways in which different results are displayed can be distracting. For instance, Figure 1A introduces the four data types and their representation in one way, but different representation methods are used in the results shown in Figure 1B on the one hand, Figure 1C-E on the other, and Figure 1F on yet another. The same can be said of the rest of the figures. Certainly, any paper that addresses a global analysis of cellular responses can be expected to have large amounts of data, but this highlights even more the importance of being clear in how this data is presented, in order to make the final take-home message of the paper as evident to the reader as possible.We reworked the figures to clarify the display of the data and results. Wherever possible, we now consistently show heatmaps and moved the original figures to the supporting materials .The new overview figure  explains the workflow with heatmaps and we moved the original Figure 1A – which explains PECA – to the supporting material . This change aims at clarifying that PECA was used to extract significant regulatory events (for each gene and time point), but algorithm of the method has been published elsewhere and should not be the focus of this manuscript.All validation experiments are now in a separate figure . While we moved the genomic traces of ATF4 and GADD34 to the supporting material , the other validation experiments show a variety of plots which we consider important orthogonal experimental approaches to confirm that we correctly induced ER stress and its relationship to the oxidative stress experiment.We adjusted the text according to these changes to the figures (Results subsections “Multi-layered data types identify new regulatory signatures during stress” and “The shared stress response involves few genes but highly similar patterns”). Other measures to increase clarity of the manuscript include editing of the Abstract and discussing the oxidative stress response in a separate section, addressing another comment below (subsection “The shared stress response involves few genes but highly similar patterns”).3) Data in Figure 5 are based on predictions of RNA secondary structures, which are largely based on RNA stability estimates of relatively long fragments and phylogenetic conservation of base pairing potential; these predictions are necessarily imprecise and unlikely to reflect the actual structure of RNA within complex RNP particles at 5' and 3' UTRs. While detection of prominent structures like iron response elements is reassuring, how predictable other structural elements are in the absence of in vivo RNA structure data (e.g. using SHAPE) should be discussed properly and, if possible, stratified in terms of predicted stability of secondary elements/conservation. It will be also relevant to discuss the predicted local structure at the site of interaction with proteins displaying enriched sequence motifs: the proteins discussed in the text bind to single stranded RNA: is the local structure at these sites predicted to be open within the secondary structure of these elements? By the way, Sex-Lethal is a Drosophila protein without a homolog in human cells, although a number of poly-U binding proteins  share similar sequence motifs. Therefore the presence of a SXL motif should be explained in these terms in the text and in Figure 5B.The revised manuscript discusses the validity of the RNA secondary structure prediction in several ways.First, we now explain in the text how these structures have been identified based on their similarity (conservation) in sequence and structure across vertebrate genomes .Second, we mapped all 5,504 predicted 3’ UTR RNA secondary structures to a publicly available dataset generated using Parallel Analysis of RNA Structure (PARS), which includes experimentally measured sensitivities to double stranded and single stranded ribonucleases to assess RNA structure. The predicted structures in our set do show overlap with the predictions from the PARS method suggesting an enrichment for real RNA secondary structures . The overlap is even more substantial when considering only the structures that are bound by proteins in our dataset . We discuss these validation tests in the main text (see the aforementioned subsection) and supporting material .HSP90B1, more closely by incorporating SHAPE reactivities into the minimum free energy folding algorithm. The SHAPE-based and predicted structure was consistent. We discuss these comparisons with SHAPE in the main text (subsection “High-resolution data generate hypotheses on new regulators of the stress response and disease-relevant pathways”) and include the adjusted predicted structure .Third, while structure predictions do not capture the complexity that occurs in vivo, we argue that agreement between different structure prediction tools, in particular those that include experimental data, increase the confidence in correct prediction of the overall structure. Therefore, we examined our primary example, Lastly, we edited the text to emphasize that the structures are predicted and individual structures would need further validation by in vivo studies to truly confirm the RNA structure.All these tests are now discussed in the text (subsection “High-resolution data generate hypotheses on new regulators of the stress response and disease-relevant pathways” and throughout) and supporting material .ELAVL1 is an RNA-binding protein that would share a similar motif (AU-rich). We edited the discussion on Pumilio binding to address how single/double strandedness may impact the ability of proteins to bind (subsection “High-resolution data generate hypotheses on new regulators of the stress response and disease-relevant pathways”).We also now clarify that there is no known SXL homolog in human . We also now discuss in the text that


Flavivirus replication occurs on the ER and is regulated by many mechanisms and factors. NS5, which consists of a C-terminal RdRp domain and an N-terminal methyltransferase domain, plays a pivotal role in genome replication and capping. The C-terminal RdRp domain acts as the polymerase for RNA synthesis and cooperates with diverse viral proteins to facilitate productive RNA proliferation within the replication complex. Here, we provide an overview of the current knowledge of the functions and characteristics of the RdRp, including the subcellular localization of NS5, as well as the network of interactions formed between the RdRp and genome UTRs, NS3, and the methyltransferase domain. We posit that a detailed understanding of RdRp functions may provide a target for antiviral drug discovery and therapeutics.Flaviviruses, most of which are emerging and re-emerging human pathogens and significant public health concerns worldwide, are positive-sense RNA viruses.  Flavivirus genus, and Flaviviridae family, are globally significant arthropod-borne viruses that cause disease in hundreds of millions of people annually across half of the world; flaviviruses include Dengue virus , Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV) [Flaviviruses, which belong to the flavivirus genome also contains a type I cap structure (m7GpppAmp) at its 5′ end, and characteristically lacks a poly(A) tail at its 3′ end. The virus enters the host cell by receptor-mediated endocytosis and is internalized, following which the endosome is acidified. The fusion of viral and vesicular membranes allows the release of genomic RNA that serves as the template for translation into the cytoplasm. The ORF is translated at the rough endoplasmic reticulum (ER) membrane and encodes a long single polyprotein that is co- and posttranslationally processed by viral and host cellular proteases, yielding the structural proteins C, prM, and E, and seven nonstructural (NS) proteins . The NS proteins cooperate with an array of host factors to form a membrane-bound replication complex (RC) where viral RNA (vRNA) synthesis takes place. NS5, the largest and most conserved protein among flaviviruses, harbors a C-terminal RNA-dependent RNA polymerase (RdRp) domain and an N-terminal RNA methyltransferase domain that are indispensable for viral RNA synthesis [Flaviviruses are enveloped viruses with a single-stranded RNA genome, composed of one large open reading frame (ORF), flanked at both its 5′ and 3′ ends by short noncoding sequences termed untranslated regions (UTRs); the Here, we emphasize the process of viral genome biogenesis within the replication complex and discuss molecular interactions between the RdRp and other viral proteins and genomic RNA. Knowledge of these processes may provide new targets for antiviral compound development and the design of vaccine candidates.flavivirus replication occurs in a RC within virus-induced vesicles in the perinuclear region of infected cells; this RC includes viral double-stranded RNA, nonstructural viral proteins, and host cell factors. Although the exact components of the replication complex are not yet well understood, all flaviviral NS proteins have been shown to participate in formation of the replication complex [flavivirus replication.Following the translation of viral RNA,  complex ,4,5,6,7. complex ,9,10; NS complex ; and NS5 complex . In addi complex ,14. AlthGenomic (+) stand RNA is first used as a template from which the RdRp synthesizes a complementary (−) strand RNA, resulting in a double-stranded RNA (dsRNA) replicative intermediate (RI) form. The -RNA then serves as a template for the production of a large excess of positive genomic RNA. The NS3 helicase specifically binds to the conserved 5′UTR sequences 5′-AGUUGUUAGUCU-3′, allowing NS3 to separate the RI into a single strand form in the 3′-5′ direction to release the newly generated viral genome and make the negative strand available as a template for the next round of viral genome synthesis . Severalflavivirus RdRp domain architecture resembles an encircle right hand with three channels , and can be divided into palm, fingers, and thumb subdomains surrounding the active site. A priming loop identified in the thumb subdomain is thought to play a major role in both ensuring correct de novo initiation and providing an initiation platform that stabilizes the de novo initiation complex [The architecture of the RdRp is well conserved across flaviviruses, as confirmed by the recently determined crystal structures of the RdRp from WNV, DENV, JEV, and ZIKV. Similar to the architecture of the RdRp domains from other RNA viruses, the  complex ,24,25,26Seven structural motifs (A to G) responsible for NTP binding and catalysis are shared by all viral RdRps with highly homologous sequences and/or viral RdRps that exhibit structural conservation A. MotifsThe RNA template entry tunnel and “N” pocket located at the junction of the thumb and palm subdomains are two conserved inhibitor-binding sites in the RdRp domains of both DENV and ZIKV, and a series of inhibitors have been identified ,35. NITDflavivirus replication occurs within the cytoplasm of infected cells; however, NS5 can translocate from the cytoplasm to the nucleus. In DENV-2, DENV-3, ZIKV, and YFV, a significant amount of NS5 accumulates within the nucleus, while NS5 in DENV-1, DENV-4, JEV, and WNV is predominantly localized within the cytoplasm of infected cells [KUN NS5 has been demonstrated to localize within the nucleus only in the presence of a specific nuclear export inhibitor [Interestingly, ed cells ,41,42,43nhibitor ,45. Howenhibitor .387KKK389 in DENV-2 NS5 abolished NS5 nuclear import and viral production, which suggested the integral role of nuclear NS5 during flavivirus infection [390KRPR393 in the monopartite NLS is necessary to direct NS5 to the nucleus, and its mutation to 390ARPA 393 changed NS5 localization from the nucleus to the cytoplasm [Site mutagenesis of NLS showed that the aNLS, rather than the bNLS, is essential for NS5 nuclear accumulation and viral replication, particularly as the mutation of nfection . In ZIKVytoplasm . In addiytoplasm . Althougytoplasm ,47,49 anytoplasm . In infeytoplasm . STAT1 iytoplasm .While inytoplasm ,53. Furtytoplasm . Analysiytoplasm .flavivirus NS5 nuclear import inhibitors with novel screening approaches, and many specific small-molecule inhibitors, some of which have apparent antiviral activities, have been analyzed. Ivermectin has been shown to inhibit importin α/β1, leading to a significant decrease in DENV production [N-(4-Hydroxyphenyl) retinamide (4-HRP), which can block the recognition of DENV NS5 by the host nuclear import proteins IMP-α/β1 and thus reduces viral RNA levels and titers, is equally effective in protecting against DENV-1-4- and ADE-mediated infection [Conventionally, NS5 translocation is thought to occur through the activities of intracellular nuclear transport proteins and members of the host importin (IMP) superfamily . Knowledoduction . N- and stem loop B (SLB), and SLA has been shown to function as a promoter for RNA synthesis during the replication process [The 5′- and 3′-terminal regions of the  process ,61. The  process . In addi process ,64,65.2 and NaCl concentrations in solution, and NS5 is able to bind SLAs from different DENV serotypes, which indicated that NS5 recognizes the overall shape of SLA, as well as specific nucleotides, to form an SLA-polymerase complex [flavivirus RNA, and a switch-like structure formed by genome cyclization has been shown to regulate dynamic RdRp binding for RNA synthesis [EMSA and footprinting assays documented the interaction between NS5 and the first hairpin element in the 5′ UTR, designated stem loop A (SLA). In this model, specific structures of the SLA, a top loop and a side stem loop, were found to be necessary for genome replication in infected cells and polymerase activity in vitro. Mutations of nucleotides at the top of SLA and in the side stem loop of the SLA element impaired promoter-dependent RNA synthesis, and revertant viruses restored viral replication when specific mutations were introduced at the top loop of SLA . Deletio complex . Recentlynthesis . Recentlynthesis . Studiesynthesis ,68.Generally, the 3′UTR is structurally subdivided into three autonomously folded regions (domains I–III) that show sequence and structural conservation across members of the genus to various extents; furthermore, the extreme 3′ region is a small hairpin 3′ stem loop (sHP-3′SL) ,70,71. DCritical residues in the thumb domain that participate in 3′SL-RdRp interactions were defined by RNA-protein interaction assays. R773, R770, Y838, and K841 likely create a TL RNA-accommodating platform, while Y838 seems to play a key role in side chain stacking during RNA recognition . A previAn in vitro RNA synthesis assay showed that recombinant full-length NS5 and its truncated RdRp domain alone are capable of initiating both de novo and primer-dependent (3′-end elongation) replication using various RNA templates in vitro ,75,76,77NS3 and NS5 are the two major enzymatic components required for viral RNA unwinding and subsequent RNA synthesis, respectively. How these two enzymes cooperate during viral replication is not known. NS3 contains an N-terminal serine protease domain and a C-terminal ATP/helicase domain . The NS3The interaction between the flaviviral NS3 and NS5 proteins has been demonstrated by immunoprecipitation assays with cultured cells and yeast two-hybrid studies, and the following interacting regions have been mapped: the C-terminal region of NS3 (residues 303–618) and the N-terminal region of RdRp domain (residues 320–368) . A previAlthough the NS3-RdRp interaction has been demonstrated in several flaviviruses, the exact function of cooperation between these two enzymatic proteins is unknown. NS5 or the RdRp may facilitate the localization of NS3 close to the entrance of the RNA template tunnel, so that these two catalytic components are in close spatial proximity to enable efficient RNA synthesis.flavivirus protein, consists of both an MTase involved in capping and the central replication enzyme, the RdRp, naturally fused through a 10-residue flexible linker. A longstanding question, however, has been whether these two domains cooperate to regulate viral replication and cap formation. Intramolecular interactions between the MTase and RdRp were first identified through analysis of the full-length NS5 structure by in silico docking of the WNV MTase domain onto the RdRp domain [NS5, the largest and most conserved p domain . The MTap domain ,83,84.flavivirus NS5, the MTase domain is stabilized by motif F in the RdRp domain, primarily by the conserved residue F466, which stacks against a pocket on the surface of the MTase domain formed by L115, L126, and P113. The interaction between F446 and P113 is the key feature of the MTase-RdRp interface among flavivirus NS5 [According to overall crystal structure analysis of irus NS5 . Among Yirus NS5 ,84. Mediirus NS5 .It was recently proposed that two molecules of NS5 inside the asymmetric unit form a dimer in which the MTase domains are oriented away from each other. In the NS5 monomer, the dsRNA exit site of the RdRp and the active site of the MTase face opposite directions in both the DENV and JEV NS5 structures. In contrast, in the NS5 dimer, the MTase and RdRp domains face the same direction . Six indThe impact of the MTase domain on RdRp activity was tested by comparing the RdRp activities of recombinant NS5 and RdRp proteins. Deletion of the ZIKV NS5 MTase domain decreased de novo-initiated RNA products to approximately half the level observed with full-length NS5, especially the products of RNA elongation . Full-leMTase-RdRp interactions potentially promote the adoption of different NS5 arrangements to facilitate positioning of the MTase near the site of the recently synthesized dsRNA exit and enable 5′-RNA cap transfer. However, adopting this conformation might be limited in the replication complex due to the multiple protein-protein interactions on the membrane. In the dimeric NS5 structure, the distance from the dsRNA exit of the RdRp of one monomer to the MTase active site of the other monomer is closer than the distance of the RdRp of one monomer to its own MTase; thus, the dsRNA product could more easily access the MTase active site of the neighboring RdRp.flavivirus replication because of not only its own polymerase activity, but also its interactions with other viral proteins and RNAs, which leads to efficient genomic RNA replication. However, understanding how these components cooperate and determining whether they interact in a distinct order or ratio requires further study.There are still gaps in our understanding of how the RdRp interacts with viral proteins and genomic RNA to efficiently coordinate their respective functions during RNA synthesis and capping. To date, there are no antiviral therapies available, and limited vaccines for flaviviruses. As the most conserved protein among flaviviruses, the RdRp plays a vital role in viral replication, which allows researchers to utilize this promising target for the development of antiviral inhibitors and therapeutics. In this review, we have attempted to comprehensively cover viral genome biogenesis mediated by the RdRp protein. The RdRp motifs, which are conserved among flaviviruses, participate in catalyzing de novo initiation, NTP binding and new RNA synthesis. In some sections, we emphasized the subcellular localization of NS5 and small-molecule inhibitors developed to target this region. Interestingly, the subcellular localization of NS5 from different flaviviruses varies. To some extent, the NLS within the RdRp domain leads to different subcellular NS5 distributions; however, this sequence is not well conserved, and further understanding of the mechanism of this difference and the distinct functions of nuclear NS5 is required. In addition, during the viral life cycle, RdRp regulates RNA replication via protein-RNA and protein-protein interactions to facilitate the efficient replication of genomic RNA. For example, the RdRp recognizes the initiation site of the genome via an RdRp-UTR interaction, the interaction between RdRp and NS3 promotes NTPase and helicase activity, and the interaction between the RdRp and the MTase is involved in new RNA synthesis. The RdRp is indispensable for