Cells respond and adapt to endogenous and exogenous stressors by activating stress response pathways that restore cellular homeostasis. The capacity of cells to respond to stress declines with age and has been associated with disease. However, the changes in cytoplasmic RNA metabolism during the cellular stress response are poorly characterized. In this work, we employ end-to-end direct RNA sequencing with nanopores, following the ligation of unique 5’ end adaptors, to assess the human transcriptome at single-molecule and -nucleotide resolution. To analyze this data, we developed NanopLen, a statistical tool to identify robust RNA length changes across conditions using linear mixed models. We find that upon cellular stress, RNAs are globally, subject to decay at their 5’ end. Stress-induced decay is coupled to translation and ribosome occupancy and can be inhibited by restoring translation initiation or preventing ribosome run-off. In contrast to models of RNA decay under normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but is independent of the poly(A) tail length. Surprisingly, we find that decaying RNAs persist in cells and are enriched in the stress granule transcriptome. Our results reveal the dynamics of post-transcriptional cell response that regulates the state of RNA upon cellular stress. The capacity of cells to activate these pathways deteriorates with age 3 and has been associated with many diseases, particularly age-related neurodegeneration 4. Oxidative stress (OS) caused by an imbalance between production and accumulation of reactive oxygen species (ROS) 5, is associated with aging and age-related disease, and has been a primary target for therapy and biomarker development 8. OS induces the Integrated Stress Response (ISR), a common adaptive pathway that eukaryotic cells activate to restore cellular homeostasis 25. ISR involves the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) that inhibits translation initiation and decreases the global protein synthesis 13. In turn, mRNAs exiting the translational pool are localized in stress granules (SGs), membraneless organelles that are formed when translation of numerous RNAs is arrested 17.Cells encounter and need to respond to a wide variety of exogenous and endogenous conditions that induce cellular stress. Stressed cells employ a cascade of molecular changes and activate specialized pathways that aim to restore homeostasis or induce apoptosis 21. Similarly, aberrant translation elongation that is induced by ribosome stalling on RNAs leads to RNA degradation via the No-Go decay (NGD) surveillance pathway 27. In yeast, ribosomal and other RNA binding proteins (RBPs) that contact mRNAs to facilitate translation under physiological conditions, instead show diminished mRNA association upon stress 11. Using short-read RNA-Seq, it has been shown that this reduction results in decreased mRNA abundance upon heat shock but not upon glucose withdrawal, mediated by the 5′–3′ exoribonuclease Xrn1 11. Similar studies have also shown that Xrn1 deletion leads to accumulation of oxidized RNAs bearing nucleotide adducts such as 8-oxoguanosine (8-oxoG), leading to translation inhibition and NGD 12. Thus, there are at least two distinct pathways that link stress-associated translation inhibition with reduced RNA stability in yeast. However, the dynamics of the involved pathways are still largely unexplored, particularly in human cell systems.Translation is intimately coupled to RNA stability and recent studies have shown that RNA can degrade co-translationally The global effects of stress on individual RNA molecules at single nucleotide resolution have been particularly elusive, with studies done almost exclusively using artificial reporters. Also, traditional short-read RNA sequencing approaches rely on artificial fragmentation of RNA thus convolving RNA abundance with decay. In this work, we globally profile the integrity of individual RNA molecules in human cells by full-length direct RNA sequencing with nanopores (dRNA-Seq). We develop a new computational framework for statistical interrogation of RNA decay and find that upon cellular stress, RNAs exhibit marked shortening at their 5′ end that is mediated by XRN1 independently of prior de-adenylation. Treating cells to restore translation initiation or prevent ribosome run-off during stress inhibits stress-induced RNA decay associating degradation to ribosome occupancy. Importantly, by decoupling RNA decay from abundance, we reveal that the decaying RNA molecules persist in cells and are enriched in the SG transcriptome. Our results highlight the dynamics of a cellular stress response mechanism regulating the state of RNA and establish a statistical tool for the analysis of long-read direct RNA sequencing data.28. However, how this balance between intact and partially degraded RNA adjusts, and is regulated in non-steady-state conditions, such as cellular stress, remains unclear. To explore this, we first triggered OS by subjecting HeLa cells to sodium arsenite (0.5 mM) for 60 minutes. We verified the anticipated formation of SGs and phosphorylation of eIF2α (discussed in subsequent sections) while cell viability was monitored at varied intervals up to a day after treatment with no major changes observed  compared to control  with no preference towards shorter or longer transcripts  that represents read length as a percent of annotated transcript length. We calculated normalized meta-length by splitting each annotated transcript in 20 equal bins, assigning the mapped read ends into individual bins, and defining the transcript meta-length as the difference of the 5’ and 3’ end bins . Our datnscripts  indicati2O2) for 2 hours followed by TERA-Seq. H2O2-induced OS also resulted in significant transcript shortening , similar to that observed with arsenite, indicating that OS results in RNA shortening irrespective of inducer , again independently of differential expression  to report statistical significance. We modeled the library as a random effect to adjust for variation across replicates (see methods). To test the model, we simulated sequencing data over varying RNA shortening proportions and expression counts. The simulated data showed that the model accurately predicted the true length difference for each tested shortening proportion and had a well-controlled false positive rate . As expe−256, 2O2 for 2 h and 300 μM NaAsO2 for 2 h followed by short-read RNA-Seq 29. Our analysis showed that, compared to control, all stress conditions resulted in significantly reduced read coverage at the 5’ end in agreement with our findings (Consistent with the requirement for the presence of poly(A) tail to sequence RNAs through the nanopore, careful inspection of significantly and non-significantly shortened transcripts showed that the 3’ ends of shortened transcripts were almost identical in OS and control whereas the corresponding 5’ ends were more heterogenous . In contfindings . ConclusXRN1 might be expected to have a general effect on RNA length at the 5’ end, we saw that the effects of reduced XRN1 were most prominently observed in the RNAs we identified as shortened during oxidative stress  that resulted in substantial reduction of protein level compared to non-targeted control (siCTRL) . We subse stress , strongl22. We reasoned that if 8-OxoG induced endonucleolytic cleavage were the major contributor of the transcript shortening we observed, then an increase in guanine prevalence should be expected at the vicinity of 5’ ends of sequenced RNAs upon stress. However, our data did not support this explanation, as no G-nucleotide enrichment difference between arsenite and control was observed , and endonucleolytic cleavage observed . This fiobserved , indicat23. We therefore wished to assess the possible role of the poly(A) tail in stress-induced RNA decay. We used nanopolish to quantify the poly(A) tail length of the RNA molecules in the TERA-Seq libraries 24. Contrary to the traditional model, our data showed a modest but significant increase of poly(A) tail length  in arsenite-treated compared to control cells  tail length to the corresponding aligned read length. Our data again showed that composite transcript length was reduced in arsenite-treated compared to control cells , untranslated regions (UTRs), and the total annotated length were predictive of the transcripts subject to stress-induced 5’ shortening. Our results showed no association between the expected length of these features and differential decay, except for the CDS and GC content, that were significantly reduced for significantly shortened transcripts , g. Sincortening , b indic31. Consistent with previous findings, ISRIB had no noticeable effect on the dose-dependent phosphorylation of eIF2α but inhibited the SG formation under stress, as expected  which has been shown to bypass the effects of eIF2α phosphorylation and to facilitate translation initiation under stress expected –c. Polyscted (25 .−156) essentially restoring transcript length to the level of the control  and compared against our dRNA-Seq data. As previously described , we found that SG-enriched genes were generally significantly longer, particularly in their coding and 3’UTR sequences, . This results in a swift detachment of scanning initiation factors from RNAs, eventually leading to ribosome run-off . Consequently, our results point towards a model where the occupancy time of ribosomes on RNAs, as determined by the CDS length, could thus play a pivotal role for RNA decay upon stress. Supporting this idea, ISRIB, an ISR inhibitor, re-initiates translation partially and curtails RNA decay. These findings indicate that ribosome run-off is a central factor in the observed RNA shortening under stress.17. In line with this idea, our results show that the SG transcriptome is enriched for significantly shortened RNAs. We and others have previously shown that ribonucleoprotein- and stress-granule formation is generally facilitated by stochastic RNA interactions thus, leaning towards an enrichment of longer RNAs 33. Assuming stress-induced decay relies on SG positioning, the shortened RNAs would be expected to reflect a similar length inclination. However, our data contradict this, implying that SGs aren’t decay centers during stress but likely temporary repositories for partially degraded RNA molecules.Interestingly, our end-to-end sequencing data show that the observed RNA shortening is not accompanied by a corresponding change in RNA abundance. This indicates that the RNA state captured in our experiments may correspond to a transient step towards complete RNA clearance. Alternatively, the observed shortened RNAs could be protected from further degradation, presumably by SGs as has previously been suggested 35.Past research has shown that cells counter stress by broadly halting translation via ISR activation . While the physiological role of stress-induced RNA decay is currently unknown an intriguing hypothesis could be that it evolved as a mechanism to further alleviate the burden on the translation machinery during stress. By breaking down the 5’ end of RNAs and eliminating translation initiation sites, cells may use an orthogonal strategy to comprehensively dial down translation. The mechanistic reason of concentrating these trimmed RNAs remains unclear as the loss of the 5’ end likely deems them unusable for translation after stress recovery. An intriguing hypothesis could be that these RNA fragments might act as sponges for post-transcriptional regulatory factors or simply become reserves for nucleotides post-stress. Future studies to test these hypotheses will be crucial, especially considering the emerging significance of SGs and RNA metabolism in neurodegenerative diseases and aging 2, 90% humidity in Dulbecco’s Modified Eagle Medium  supplemented with 10% heat-inactivated fetal bovine serum (GeminiBio #100–106), 2 mM L-Glutamine. The cells were tested for mycoplasma contamination using universal mycoplasma detection kit . Cells were treated with 500 μM of sodium arsenite  for 60 minutes and total RNA was isolated using TRIzol reagent  following the manufacturer’s recommendation. RNA concentration was quantified using a High Sensitivity (HS) RNA qubit assay  and Nanodrop ND-1000 (ThermoFisher). RNA integrity was assessed on a 2100 Bioanalyzer (Agilent Technologies) or Qubit™ RNA IQ assay (ThermoFisher).HeLa cells (ATCC CCL-2) were cultured at 37°C, 5% CO5 cells were resuspended in 100 μl of siRNA-R buffer mixture, electroporated  and immediately transferred to a 100 mm dish containing pre-warmed media. Cells were grown for 72 hours before treatment with indicated reagents and final harvesting for RNA and protein assays.Cells were transfected with control and gene-specific siRNAs at a final concentration of 20 nM using the Neon transfection system 100 μl kit  according to the manufacturer’s protocol. A commercially available siRNA specifically targeting the human XRN1 was used for XRN1 knockdown, and a non-targeting siRNA was used as control . Approxi28. 500–1000 ng of poly(A) RNA was used for library preparation using SQK-RNA002 sequencing kit (Oxford Nanopore Technologies). The final library was quantified using Qubit 1X dsDNA High Sensitivity (HS) assay kit (ThermoFisher # Q33231) and loaded on FLO-MIN106 or FLO-PRO002 flowcells.Library preparation was performed using the direct RNA sequencing kit  as previously described  with modifications. A minimum of 75 μg of total RNA was used as starting material to purify poly(A) RNAs using Oligo d(T)25 Magnetic Beads . 50 pmoles of linker (REL5) containing a 5′-Biotin-PC group and a 3’-OH  were lig3 cells per well) were seeded in a 96-well plate and incubated in humidified 5% CO2 incubator at 37°C for 24 hours. Following arsenite treatment, the cell viability was examined using MTS assay (100 μl of DMEM and 20 μl MTS incubated for indicated time points) for 10 minutes and incubated in a 5% CO2 incubator at 37 °C protected from light. Finally, the plate was subjected to shaking for 15 min followed by an optical density measurement (OD) at 590 nm using 1420 Multilabel Counter . The viable cells in arsenite treated samples were reported as the percentage of viable cells in control (untreated) samples.MTS assay was performed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) . HeLa cells  supplemented with 1x protease inhibitor cocktail  and 1 mM phenylmethylsulfonyl fluoride (PMSF) . The lysate was passed 10 times through a 25-gauge needle, heated at 95°C for 20 minutes and cleared by centrifugation at 16,500 × g for 10 minutes. Protein concentrations were measured using the Qubit protein assay . 25 μg of proteins was separated on NuPAGE 4–12% Bis-Tris Gel  and transferred to PVDF membrane . After blocking in TBS-T 5% milk for 2 hours, membranes were incubated with primary antibodies overnight at 4°C. Membranes were washed 3 times 5 min in TBS-T and incubated for 2 hours with secondary antibodies. Signals were developed using Chemiluminescence  and acquired on a ChemiDoc MP imaging system (Biorad). For studies of eIF2α phosphorylation (eIF2α-P), the lysis buffer was supplemented with phosphatase inhibitors  and 5% BSA was used instead of milk for blocking. Antibodies are listed in Cells were washed with cold PBS and pelleted by centrifugation 5 min at 4 °C 300 Cells were cultured at 70–80% confluency in Millicell EZ SLIDE 8-well glass  in the presence of 50, 100, 250, 500 μM of arsenite +/− 200 nM of ISRIB . After the indicated time of treatment, cells were washed 3 times with warm PBS, fixed for 10 min at room temperature (RT) in PBS / 3.7% paraformaldehyde and washed with PBS for 5 min with shaking. After permeabilization in PBS 0.5% Triton X-100 for 10 min at RT, cells were blocked in PBS 0.1% Tween-20 (PBS-T) supplemented with 1% BSA for 1 hour at 37°C with gentle shaking. Primary antibody diluted in 1% BSA were added to the cells and incubated at 4C overnight. Cells were washed in PBS-T three times 5 min with shaking before staining with secondary antibody for 1 hour at RT. Cells were washed four times for 5 min with PBS-T with shaking and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (1:1000 in PBS-T) for 5 min at RT. Slides were mounted with ProLong Glass Antifade Mountant  and images were acquired using a DeltaVision Microscope System (Applied Precision).36 with modifications. Briefly, HeLa cells (80–85% confluency) cultured in 100 mm dishes were treated with 500 μM sodium arsenite, 200 nM ISRIB or 25 μg/ml CHX as indicated. DMSO was used as a control. Immediately before harvesting, cells were treated with 100 μg/ml CHX for 10 minutes. After one wash with ice-cold PBS containing 100 μg/ml CHX, 500 μL of polysome extraction buffer  was added directly to the plate and lysates were harvested by scraping. The lysate was cleared by centrifugation at 14,000 × g for 10 min. The supernatant was loaded onto the 10–50% sucrose gradient followed by high-speed centrifugation . Using a density gradient fractionation system monitored by UV absorbance detector (A254), 12 fractions were collected.The polysome profiling was performed as described previously 37. Non-ribosomal reads were subsequently mapped against the human genome hg38 using minimap2 (version 2.17) 38 and parameters -a -x splice -k 12 -u b -p 1 --secondary=yes. They were also aligned against the human transcriptome using -a -x map-ont -k 12 -u f -p 1 --secondary=yes.Nanopore sequencing data were basecalled using Guppy (v3.4.5). The 5′ adaptors were identified and removed using cutadapt (v2.8). Reads were first aligned against ribosomal sequences obtained from SILVA The poly(A) tail lengths were extracted from sequenced reads using the nanopolish polya package . Only the poly(A) tail lengths which passed the software quality control scores and were tagged as “PASS” were used in our analysis.39. EnhancedVolcano (https://github.com/kevinblighe/EnhancedVolcano) was used for visualization. GO analysis was performed for significantly changed genes  using “clusterProfiler” 40.Transcript counts were quantified from the transcriptome alignments as the total number of reads aligning on each transcript. Differential expression analysis was performed using DESeq2 Ribosome profiling sequencing data were downloaded from GEO GSM2100595 . Adaptors were removed using cutadapt (version 2.8) and reads were aligned to the human genome using STAR (2.5.3a). Further processing to calculate counts per transcript was performed with in-house scripts using pysam (v0.15.4) and counts were converted to reads per kilobase of transcript per million (RPKM).For calculating the bin (meta) lengths, we divided the transcripts into 20 equal bins . Reads were assigned to each of these bins based on the corresponding location of their ends. The binned- (meta-) length is calculated as the difference of the binned 3’ end over the binned 5’ end.28. Briefly, all reads from all replicates were combined and the distribution of read 5’ ends within the 5’ UTR was quantified. The TSS was selected as the position with the highest read density withing the 5’ UTR with minimum 5 supporting reads.Transcription start site identification was performed as previously described NanopLen reads a file of read lengths with library identifiers and gene or transcript identifiers, and a metadata file describing the experimental design. The latter includes the library identifiers and the corresponding condition for each library. The software supports three models: t-test, Wilcoxon, and linear mixed model (LMM). In the t-test and LMM options, the user can also supply a customized model to use extra variables in the metadata. In this work we have used the LMM to adjust for putative batch effects across libraries.Given a vector Y of read lengths associated with a gene/transcript, the t-test is functionally equivalent to a linear regression model. The model also supports optional read-specific extra covariates but are not used in this work:Similarly, the LMM models Y using the condition as fixed effect and the library identifier as a random effect.To test NanopLen models, transcript length data with known shortening rates were simulated. Each simulation was parameterized with a “true” transcript length, a read count as a proxy for transcript expression, and a shortening proportion. The database of known human genic lengths from Ensembl (release 91) was used and the median was selected as “true” length. Since the simulated variance is in proportion of the true length, using any length will result in similar results so we do not simulate additional true lengths. Expression counts were simulated, ranging from 10 to 200 reads per transcripts to capture the dynamic expression range particularly towards small read depth. Varying shortening percentages were simulated from 50, 60, .., 100% of the true length  with the 100% corresponding to the null simulation of no-change.Given the parameters, the simulated lengths were generated based on the mixed model in NanopLen, with the number of sampled lengths being the selected expression counts. The library random effect was simulated as if imitating a fluctuation with standard deviation of 10% of the true length, and the global error having a standard deviation of 20% of the expected length mean. As with the real experiment, six libraries of three control and three condition were simulated, with 1000 genes per scenario. Using the LMM option with the logscale option selected, the resulting log2FCs and p-values were calculated and aggregated in https://github.com/maragkakislab/nanoplenNanopLen is available in ASCC3, FASTKD2, MTRR, HNRNPM and DSG2) for visualization. As control, 3 transcripts with non-significant, lower than 10 nt difference  were selected. Libraries were randomly downsampled to maximum 50 reads per window and the libraries were overlayed in Affinity Designer. All reads were used, irrespective of adaptor ligation status.Transcripts that were identified as significantly shortened at a false discovery rate of 0.05 by NanopLen, had at least 50 supporting reads in both conditions and had a difference of 200 nts in length were selected. From the list of 32 transcripts, 5 were randomly selected (Supplement 1Supplement 2Supplement 3Supplement 4Supplement 5


Drosophila cells. We observed that treatment with the anthracycline aclarubicin leads to elevated levels of RNA polymerase II and changes in chromatin accessibility. We found that promoter proximity and orientation affect chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally oriented tandem promoters. We also found that aclarubicin treatment changes the distribution of noncanonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. Our work suggests that the cancer-killing activity of aclarubicin is driven by the disruption of nucleosomes and RNA polymerase II.Anthracyclines are a class of widely prescribed anticancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, we used Cleavage Under Targets and Tagmentation (CUT&Tag) to profile RNA polymerase II during anthracycline treatment in  Anti-cancer drug aclarubicin promotes RNA polymerase II elongation, chromatin accessibility and G-quadruplex formation. Chromatin proteins shape the physical structure of DNA by regulating accessibility, torsion, and three-dimensional association . While paused RNA Pol II Ser5P showed minimal changes across all three drugs . Aclarubicin-treated samples showed an increase in total RNA Pol II levels relative to controls. Actinomycin D-treated samples, by contrast, showed a decrease in total RNA Pol II levels. These data for actinomycin D are similar to previous studies demonstrating that actinomycin D-treatment can lead to decreases in total chromatin-bound RNA Pol II  of RNA pol II’s C-terminal domain  result fWe next wondered whether the increase in RNA Pol II caused by aclarubicin might affect chromatin accessibility and G-quadruplex formation, both of which are associated with changes in RNA Pol II activity (Drosophila promoters and generated heatmaps to compare accessibility across different groups (n = 1306) promoters are highly accessible, cluster II (n = 5517) promoters are moderately accessible, and cluster III  promoters display background levels of accessibility. Doxorubicin-treated samples showed minimal changes across all clusters, while actinomycin D-treated samples showed subtle gains in accessibility at regions proximal to the TSS . ReLast, to assess changes in RNA Pol II at distinct gene clusters, we sorted RNA Pol II Ser5P and RNA Pol II Ser2P datasets using k-means clustering generated by CUTAC data. Similar to trends observed in CUTAC and G-quadruplex datasets, we observed that aclarubicin-treated samples showed gains in RNA Pol II Ser2P and RNA Pol II Ser5P at cluster II promoters . These gains in RNA Pol II signal were observable upstream and downstream of the promoter region . Actinomycin D-treated samples showed subtle gains in cluster I and cluster II, whereas doxorubicin-treated samples showed minimal changes across all clusters . Together, these data demonstrate that drug treatment differentially affects the distribution of RNA Pol II in distinct groups of genes.We sought to understand what features differentiate genes in cluster I from genes in cluster II. While examining gene promoters in each cluster, we noticed that cluster I promoters are frequently isolated in the genome, whereas cluster II promoters are frequently found close to gene neighbors. To assess whether proximity to neighboring genes correlates with differential responsiveness to anthracyclines, we sorted promoters by their distance to the nearest upstream promoter and generated heatmaps of chromatin features. Closely spaced promoters in drug-treated samples appear to show greater gains in chromatin accessibility, G-quadruplex formation, and RNA Pol II Ser2P/5P than more distant promoters  or divergent directions. We found that 58% (5372 out of 9288) of the promoters less than 2 kb apart are divergently oriented, with that percentage growing to 68% (4604 out of 6788) for promoters positioned closer than 1 kb. To determine whether accessibility differed in closely spaced divergent promoters versus closely spaced tandem promoters, we separated these two groups of promoters and plotted the accessibility changes during drug treatment  for eachAs previous studies have shown that highly expressed genes are sensitive to anthracycline-mediated chromatin disruption . We sortPrevious work from our laboratory proposed that negative supercoiling (underwound DNA) generated in the wake of transcribing RNA Pol II facilitates the intercalation of anthracyclines into the genome, leading to chromatin disruption . As negaThe histone locus cluster is a region of the genome with an especially high incidence of closely spaced divergent promoters . Given tD. melanogaster chromosome 3, can form noncanonical structures known as i-motifs from the repetitive runs of cytosines found within the repeat (Previous studies have shown that the Dodeca-satellite (CCCGTACTCGGT) tandem repeats, found exclusively in a pericentromeric region of e repeat . Notablye repeat , as wellDrosophila cells with the anthracycline aclarubicin results in gains in RNA Pol II and changes in chromatin accessibility and G-quadruplex formation at active gene promoters. Closely spaced divergent promoter pairs are particularly sensitive to drug treatment, likely due to high levels of negative supercoiling enriched at these promoter elements. Chromatin effects are not limited to active gene promoters and can also be found at certain repetitive sequences prone to forming noncanonical DNA structures. Our observations reveal molecular features underlying anthracycline-mediated chromatin disruption and demonstrate clear differences between doxorubicin and aclarubicin treatment, suggesting that these drugs may exert anticancer effects through different mechanisms.We have shown here that treatment of The predominant model for anthracycline cytotoxicity is that these drugs induce DNA damage by preventing topoisomerase II (topo II) from ligating together the double-strand breaks made during topoisomerase-mediated DNA cleavage  were grown to log phase in HYQ-SFX Insect medium (Invitrogen) supplemented with 18 mM l-glutamine and harvested as previously described (www.beckman.com). Doxorubicin , aclarubicin , and actinomycin D  were resuspended to 10 mM in dimethyl sulfoxide and frozen in aliquots. For cell treatments, compounds were added to a cell medium containing 1.5 × 106 cells per ml to a final concentration of 1 μM (aclarubicin and doxorubicin) or 5 μM (actinomycin D) and incubated at room temperature (RT). Cells were harvested after 30 min for imaging and CUT&Tag profiling, or 1 hour or 24 hours to assess growth rates and cell size, cell death, and nucleolus structure.Samples of 500,000 Kc167 cells were harvested, spun down, and resuspended in ice-cold phosphate-buffered saline (PBS) with 0.1% Triton X-100 (PBST) and 0.1% formaldehyde and incubated for 10 min on ice. Cells were spun down and resuspended in ice-cold PBST. Cells were then spun down onto a clean glass slide  using a Thermo Scientific Shandon Cytospin 4. Slides were washed twice with 50 ml of 1× PBS each time and placed in a humid chamber with 1 ml of blocking solution  for 30 min of pre-blocking. The blocking buffer was then drained, and primary antibodies were added for incubation overnight at 4°C. Slides were then washed twice with 50 ml of 1× PBS and incubated with secondary antibodies for 2 hours at RT. Slides were then washed twice with 50 ml of 1× PBS and mounted with ProLong Diamond Mounting Media . Cells were imaged on an EVOS auto 2.0 microscope (Thermo Fisher Scientific).Drosophila Kc167 cells were spun down and resuspended in 1 ml of ice-cold PBS containing 12,000 human K562 cells for spike-in normalization. Cells were spun down and resuspended in 1.5 ml of wash buffer  by gentle pipetting. Five microliters of concanavalin A–coated magnetic beads (Smart-Lifesciences) were activated and added to cells and incubated for 10 min on ice. The supernatant was then removed, and bead-bound cells were resuspended in 100 μl of dig-wash buffer  containing 2 mM EDTA and a 1:25 dilution of the BG4 primary antibody (0.1 mg/ml) (Sigma-Aldrich: MABE917). Primary antibody incubation was performed overnight at 4°C and then the liquid was removed. Cells were washed 3× in 200 μl dig-wash buffer. The wash buffer was then drained, and cells were resuspended in 100 μl of dig-wash buffer containing 2 mM EDTA and a 1:50 dilution of mouse anti-FLAG antibody  and incubated at RT for 1 hour. Cells were washed three times in 200 μl dig-wash buffer. The wash buffer was then drained, and cells were resuspended in 100 μl dig-wash buffer containing 2 mM EDTA and a 1:50 dilution of rabbit anti-mouse antibody  and incubated at RT for 1 hour with slow rotation. A 1:50 dilution of pA-Tn5 adapter complex was prepared in dig-300 buffer -3-aminopropanesulfonic acid (TAPS) with 16.5 mM EDTA and resuspended in 100 μl of buffer containing 10 mM TAPS, 16.6 mM EDTA, 0.1% SDS, and proteinase K (0.2 mg/ml) and incubated at 56°C for 1 hour. Two hundred microliters of 300 wash buffer  was then added to the tube, after which, the DNA was extracted via phenol-chloroform isoamyl alcohol (PCI) for library preparation. Twenty-one microliters of DNA was mixed with a universal i5 and a uniquely barcoded i7 primer and amplified with NEB Q5 high-fidelity 2× master mix . The libraries were purified with 1.1× volume of Sera-Mag carboxylate-modified magnetic beads and subjected to LabChip DNA analysis and Illumina sequencing. A similar methodology was used for profiling elongating RNA Pol II , initiating RNA Pol II , and total RNA Pol II  using 80,000 D. melanogaster whole cells. In some experiments, nuclei were extracted and profiled according to the CUT&Tag-direct protocol (CUT&Tag was performed as described (http://dx.doi.org/10.14806/ej.17.1.200) version 2.9 with the following parameters:Libraries were sequenced on an Illumina HiSeq instrument with paired-end 50 × 50 reads. Adapters were clipped by cutadapt . Normalized count tracks in bigwig format were also made by bedtools  by using the bedfile output of the plotHeapmap function as the “regions to plot” file for subsequent computeMatrix operations.https://epd.epfl.ch//index.php)  comparing relative total RNA Pol II levels between drug-treated and control conditions, the computeMatrix function of Galaxy was used to generate coverage plots of normalized read counts centered on the TSS. The values of these coverages were exported using the “save the matrix values underlying the heatmap” option. These matrices contained normalized counts for each promoter broken up into 10-bp bins. We then generated average normalized counts values for each drug treatment group for every 10-bp bin up to 100-bp upstream and 500-bp downstream of the TSS. Average values at each 10-bp bin were then divided  and the logTo generate plots shown in et al.  and lowly expressed genes (Q5). These lists were then used in the computeMatrix function with the “save the matrix values underlying the heatmap” option selected to plot RNA-seq values for each quintile (Q1 to Q5) and for each promoter class (tandem versus divergent). To generate the plots for fig. S3 (J and K), the RNA-seq quintile lists were used with the computeMatrix function with the “save the matrix values underlying the heatmap” option selected to generate matrices containing CUTAC data for aclarubicin and control samples. These matrices contained normalized counts for each promoter broken up into 10-bp bins. We then generated average normalized counts values for each drug treatment group, for each quintile, and for each promoter orientation (divergent versus tandem) for every 10-bp bin up to 100-bp upstream and 500-bp downstream of the TSS. Average values at each 10-bp bin were then divided  to generate a fold-difference ratio plot surrounding the TSS for each quintile sorted by RNA-seq values.To generate plots shown in fig. S3 (H and I), RNA-seq values from Cherbas


NUCB1 interacts with Galphai3 protein, cyclooxygenase, amyloid precursor protein, and lipids. It is involved in stress response and human diseases. In addition, this protein is a transcription factor that binds to the DNA E-box motif. Using surface plasmon resonance and molecular beacon approaches, we first showed the RNA binding and RNA melting activities of NUCB1. We suggest that NUCB1 could induce local changes in structured RNAs via binding to the GGNucleobindin 1 (NUCB1) is a ubiquitous multidomain protein that belongs to the EF-hand Ca Nucleobindin 1  is a highly conserved multi-domain DNA- and calcium-binding eukaryotic protein. NUCB1 was first found in the supernatant of a B lymphocyte cell line from systemic lupus erythematosus (SLE)-prone mice . Thus, oMoreover, NUCB1 can act as a chaperone-like amyloid-binding protein. It was shown that NUCB1 attenuates the aggregation of unfolded proteins and protects malate dehydrogenase, alcohol dehydrogenase, and catalase under thermal stress conditions . As a moNUCB1 is involved in the regulation of many processes via interactions with proteins: Gαi , carboxyThe NUCB1 gene is widely expressed in various human tissues , especia2+-binding EF-hand motifs, a C-terminal leucine zipper (Z), and a polyglutamine stretch. NUCB1 passes into various compartments of the cell and into the extracellular space due to the presence of a signal peptide [The protein consists of several independent functional domains: a signal peptide, a basic amino acid-rich DNA-binding domain (DBD), two Ca peptide .2+-binding induces structural changes in the EF-hand motifs [2+ ions affect NUCB1 folding [2+-bound state demonstrated low chaperone activity [EF-hand motifs of NUCB1 are very important for binding to proteins: Gαi1/3 , necdin d motifs , which c folding . It is wactivity .Due to the presence of the DBD and the leucine zipper, NUCB1 possesses features of a transcription factor. It binds to the canonical enhancer box (E-box) sequence of the Cripto-1 gene promoter, triggering cell epithelial–mesenchymal transition (EMT) . In addi2+-independent RNA-binding properties of NUCB1. NUCB1 was found to bind some dsRNAs and ssRNAs in vitro, including microRNAs associated with SLE and EMT. Based on our surface plasmon resonance (SPR) and chemical probing data, we suggest that NUCB1 specifically interacts with an ssRNA sequence containing guanines that may be followed by adenines or uridines. Using the molecular beacon technique, we revealed the ability of NUCB1 to melt RNA hairpin structures by means of the DBD and the leucine zipper. We suggest that NUCB1 could be involved in the regulation of translation of mRNAs associated with SLE and EMT via interaction with microRNAs.Here, we report the hitherto unknown CaThe binding of transcription factors (TFs) to RNAs is a broad phenomenon with potentially significant biological impact ,27. We hFirst, we investigated the interaction of NUCB1 with four ssRNA fragments containing 5′-GUGGUCAGUCGAGUGG-3′ followed by adenines (A-rich), uridines (U-rich), guanines (G-rich), or cytosines (C-rich). The 5′-end RNA adapter sequence was needed for the proper orientation of the RNAs on the sensor chip. NUCB1 interacted with the A-rich, U-rich, and G-rich with high affinity. NUCB1 binding to the C-rich was not detected .Gly, picornavirus mRNA IRES  type I element, a fragment of the SRP19 mRNA 3′-UTR (SRP19), and a transcript of an E-box DNA sequence (E-box)  . E-box R element .Interestingly, NUCB1 binding to RNAs did not depend on the presence of calcium ions , which iThen, we checked if the NUCB-RNA interaction is structure-specific and/or sequence-specific.We studied the NUCB1/E-box RNA and NUCB1/SRP19 mRNA complexes by RNA chemical probing a,b.Nucleotides, whose reactivity changes in the presence of NUCB1, are predominantly located in the single-stranded RNA regions that contain the motif GGAUAU d,e. NUCBAUAU . As in tGly (CCC in the anticodon loop) and the C-rich RNA. We modified the C-rich RNA sequence in order to introduce a possible NUCB1-binding motif by introducing AA after GG in the 5′-end of the C-stretch (C-rich+AA) (see D = 18 nM). Thus, one may assume that an ssRNA region containing the motif GGNUCB1 does not bind to the hairpin of the IRES mRNA, whose loop lacks such a motif . It also+AA) see . Such RNOur recent studies suggest that NUCB1 plays a role in SLE not only in mice , but alsInteractions of NUCB1 with some microRNAs associated with the development of the SLE and EMT containing the GGAA, GGUA, GGUU, or GGAU sequences were studied. Despite the differences in the primary structures , all theTo further dissect the NUCB1 binding site on RNA, we carried out chemical probing of the microRNA miR200a-3p NUCB1-RNA fragment in a complex with NUCB1 c,f. One 2+-binding TF with an N-terminal DNA-binding domain (DBD) and a C-terminal leucine-zipper (Z) domain (NUCB1 is a Ca) domain . It was The NUCB1 lacking the signal peptide (1–30 aa) was used in the following RNA-binding experiments. We obtained three truncated forms: NUCB1 ΔN (239–461 aa), with a deleted N-terminal region, including the DBD; NUCB1 ΔZ (31–320 aa), where the C-terminal region with the leucine zipper was deleted; and NUCB1 CaBD (228–326 aa), containing EF-hand motifs. The proteins were purified to homogeneity . The truWe studied the interaction of these truncated NUCB1 variants with the RNA fragments A-rich, U-rich, C-rich, G-rich, and miR-200a-3p. Corresponding dissociation constants are listed in Proteins that stabilize ssRNA regions can perform a chaperone function and unfold hairpin structures of RNA ,36,37. WIn our experiments, a molecular beacon was used which contained the sequence of miR-200a-3p with FAM (fluorophore) at the 5′-end and RTQ (quencher) at the 3′-end of the RNA hairpin (MB-miR). Upon addition of NUCB1 to the MB-miR solution in a 1:10 molar ratio, fluorescence significantly increased in one hour b. The shThus, we showed that the NUCB1 was capable of unfolding hairpin structures in RNA.Methanococcus vannielii (MvaSm), an archaeal RNA chaperone that interacts with single-stranded RNA regions containing oligoA or oligoU [For a negative control, BSA was added to MB-miR b. An Sm-r oligoU , was useInterestingly, the RNA-binding truncated NUCB1 variants did not exhibit RNA chaperone activity. The increase in fluorescence upon the addition of NUCB1 CaBD was indistinguishable from that of free MB-miR. The monomeric NUCB1 ΔZ, devoid of the Z domain, changed the molecular beacon fluorescence approximately as much as BSA. NUCB1 ΔN increased the fluorescence slightly more efficiently than NUCB1 ΔZ b.It is known that NUCB1 participates in epithelial–mesenchymal transition (EMT) via the binding to the E-box sequence of cripto promoter . PresumaThe increase in serum levels of NUCB1 in lupus-prone mice became known almost 30 years ago . RecentlWe dissected NUCB1 affinity for some EMT- and/or SLE-associated microRNAs that contain a GGAU motif . KineticAU motif . RNA cheAU motif c,f. ThisIt is known that two populations of extracellular microRNAs exist in biological fluids, such as plasma and serum. One of them can be associated with an RNA-induced silencing complex, and the other is found in exosomes, microvesicles, and apoptotic bodies . ExosomeThe ability of NUCB1 to bind to some microRNAs and stabilize their single-stranded regions may shed light on how the protein is involved in various cellular processes. For instance, the overexpression of NUCB1 in neuroblastoma N2a cells is associated with reduced levels of mRNA of the amyloid precursor protein (APP). At the same time, it was shown that the microRNAs miR-106a and miR-520c repress the translation of the APP mRNA by pairing to its 3′-UTR . These mAll of these factors confirm the importance of our studies of NUCB1-RNA interactions.NUCB1 is a multifunctional protein belonging to the family TF that binds to the E-box . There aGly, and picornavirus mRNA IRES element . Thus, single-stranded GGAs mentioned above, NUCB1 possesses high affinity for ssRNAs containing several guanines, or the GGIt was shown that two regions of NUCB1 interact with DNA: the DBD, which contains a cluster of positively charged amino acid residues, and the Z domain ,25. At tWe obtained three mutant NUCB1 variants that contained the calcium-binding domain . This doUsing the molecular beacon approach, we have shown that the NUCB1 possesses the RNA chaperone activity. This approach is widely used for studying nucleic acid chaperones. Adopting native functional conformations by cellular RNAs is a complex process that involves a number of proteins possessing an RNA chaperone activity. Such proteins destabilize intermediate secondary structures that are formed during RNA folding. By doing this, RNA chaperones promote the acquisition of thermodynamically stable conformations that are typically functionally active. RNA chaperone activity was found for a large number of proteins in various organisms. These proteins belong to evolutionary distinct protein families, and they are structurally diverse ,51.We synthesized a molecular beacon that contains a sequence of a potential NUCB1 target, miR-200a-3p, flanked by the 5′- and 3′-end paired extensions. The putative NUCB1-binding region is thus located within the loop structure a. Given The RNA chaperone MvaSm was used as a positive control because it is able to unfold hairpin RNA structures . HoweverAdding the calcium-binding domain of NUCB1 that shows high affinity for RNA does not affect the beacon structure; the fluorescence level corresponds to that of spontaneous beacon unwinding. The truncated NUCB1 variant lacking the Z domain also virtually does not change the fluorescence. The NUCB1 variant containing the calcium-binding domain and the C-terminal part, including the Z domain, forms a dimer. Adding this variant to the beacon slightly increased the fluorescence. The intact NUCB1 increases the fluorescence intensity dramatically within several minutes of incubation, and fluorescence further increases with time. The spatial structure of NUCB1 is unknown. However, according to the IsUnstruct 2.02 program that predicts disordered regions in proteins , a signiThus, we suggest with a high degree of confidence that NUCB1 possesses RNA chaperone activity in vitro. It is worth mentioning that both DNA-binding and RNA chaperone activity require the presence of all the domains of NUCB1. Further studies are needed to address the role of the RNA chaperone NUCB1 and find its targets in cells.The open reading frame of the human NUCB1  was PCR-amplified using primers the For NUCB1 and Rev NUCB1  and a cDE. coli BL21(DE3)/pRARE to express an N-terminal His6 tag protein. The transformants were grown in LB medium in the presence of kanamycin (50 µg/mL) and chloramphenicol (10 μg/mL) at 37 °C with agitation at 180 rpm. Protein expression was induced at the OD600nm = 0.6–0.8 o.u. by adding IPTG to a final concentration of 0.5 mM. The bacteria were harvested by centrifugation 3 h after induction.The pET28a vector carrying the NUCB1 gene (or the truncated form of the NUCB1 gene) was used for transformation in g for 30 min at 4 °C. Supernatant was loaded onto a Ni-NTA agarose  column equilibrated with a solution containing 500 mM NaCl, 50 mM Tris-HCl (pH 8.0), and 10 mM imidazole. NUCB1 variants were eluted using a step gradient of imidazole (40 mM and 150 mM) in a solution containing 500 mM NaCl and 50 mM Tris-HCl (pH 8.0). Fractions containing the protein were concentrated and dialyzed against a solution containing 150 mM NaCl and 50 mM Tris-HCl (pH 8.0). The final preparations were analyzed by size-exclusion chromatography on the Superdex 200 resin equilibrated with a solution containing 150 mM NaCl and 50 mM Tris-HCl (pH 8.0) , 10 mM imidazole, 5 mM β-mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were disrupted by sonication at 4 °C. Cell debris was removed by centrifugation at 15,000× (pH 8.0) .DNA fragments containing sequences of the T7 promoter and the E-box RNA, the SRP19 RNA fragment, and the miR200a-3p RNA sequences were prepared by PCR using pairs of overlapping primers: For E-box RNA and the Rev E-box RNA; For SRP19 and Rev SRP19; For miR-200a-3p and Rev miR-200a-3p, respectively . The PCRThe E-box, SRP19, and miR-200a-3p RNA fragments used for chemical probing experiments were obtained from linearized plasmid DNA by in vitro transcription with T7 RNA polymerase as described in .Kinetic analysis of protein interaction with specific RNA fragments was performed by surface plasmon resonance technique  using thGUGGUCAGUCGAGUGG-U18-3′U-rich: 5′-GUGGUCAGUCGAGUGG-A18-3′A-rich: 5′-GUGGUCAGUCGAGUGG-C6-3′C-rich: 5′-GUGGUCAGUCGAGUGG-AAC6-3′C-rich(+AA): 5′-GUGGUCAGUCGAGUGG-G6-3′G-rich: 5′-GUGGUCAGUCGAGUGGUAACACUGUCUGGUAACGAUGU-3′miR-200a-3p: 5′-GUGGUCAGUCGAGUGGAGUGGUUCUUAACAGUUCAACAGUU-3′miR-203a-5p: 5′-GUGGUCAGUCGAGUGGGGAUUCCUGGAAAUACUGUUCU-3′miR-145-3p: 5′-GUGGUCAGUCGAGUGGUUAAUGCUAAUCGUGAUAGGGGUU-3′miR-155-5p: 5′-mRNA: 5′-AGGUGCCGAAGUCGUGGGAGGAGGAAGUCGGGUAAUAG-3′E-box RNA: 5′-GCGCAGCGAGUUUCUCUCUUUUCACGUGGGGGAUAAUAAU-3′The RNA-binding properties of NUCB1, the intact protein, and its truncated forms were tested using 5′-biotinylated chemically synthesized RNA oligomers  listed below:Gly and IRES element mRNA (Sixteen nucleotides (underlined) at the 5′-end of oligo RNAs and miRNAs were added to increase the accessibility of short RNA fragments for interaction with proteins. As a negative control, biotinylated tRNAent mRNA  were obt2, followed by dialysis in a solution containing 50 mM Tris-HCl pH 8.0 and 200 mM NaCl to remove EDTA-Na2. Five different concentrations of the analyte samples (NUCB1 or truncated forms) were prepared by serial dilution in a solution containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 0.1 % Tween-20 (TNT) for each set of sensorgrams. The samples were injected at a flow rate of 30 µL/min. The injection step included a 300–480 s association phase followed by a 2000–6000 s dissociation phase in TNT buffer. To test the effect of Ca2+ on the ability of NUCB1 to bind RNA, all binding experiments were performed in TNT buffer containing 2 mM CaCl2. All binding experiments were performed at 25 °C. Each immobilization strategy and kinetic analysis was repeated at least three times.NUCB1 was treated with EDTA-NaKinetic analysis was performed by globally fitting curves describing the simple 1:1 bimolecular model to a set of three to five sensorgrams using BIAEvaluation v. 4.1 software.Structural analysis of RNAs alone  or in the presence of NUCB1 was performed using chemical probing of RNA as previously described ,62,63.For the chemical probing experiment, we used two types of samples: free RNAs and RNAs plus NUCB1.RNAs were heated to 65 °C for 10 min and cooled on ice before the addition of NUCB1. Then, RNAs (3 nmoles) were mixed with NUCB1 (6 nmoles) in a buffer containing 20 mM HEPES-NaOH (pH 7.5) and 100 mM NaCl. Samples were incubated at room temperature for 30 min. Control (free RNA) samples were incubated identically.Modification reactions (50 µL) with RNAs (750 pmoles) or RNAs with NUCB1 were performed in a buffer containing 20 mM HEPES-NaOH (pH 7.5) and 100 mM NaCl. 2. CMCT stop-buffer contained 3 M Na-Acetate. Kethoxal stop-buffer contained 150 mM Na-Acetate and 250 mM K3BO3 (pH 7.0).Modification was performed by the addition of freshly prepared DMS (1 µL of a 1/5 dilution in ethanol), kethoxal (1 µL of a 1/5 dilution in ethanol), or CMCT (50 µL of 100 mg/mL in the reaction buffer) followed by incubation at 37 °C for 10 min. Reactions were stopped by the addition of stop-buffers, followed by ethanol precipitation. DMS stop-buffer contained 1 M Tris-HCl, pH 7.5, 1 M β-mercaptoethanol, and 100 mM EDTA-Na3BO3 (pH 7.0), 300 mM Na-Acetate, 0.5% SDS, and 5 mM EDTA-Na2, followed by phenol–chloroform extraction and ethanol precipitation. RNA pellets were dissolved in 10 µL of 25 mM K3BO3 (pH 7.0).The RNAs were concentrated by ethanol precipitation, and the pellets were washed with cold 70% ethanol. Pellets were dissolved in a buffer containing 25 mM KControl (unmodified) samples were treated in the same way as modified samples, except that the modification step was skipped.Unmodified RNAs were used as templates for the reference sequencing reactions and to monitor artifact stops or pauses in reverse transcription.RNA fragments used in chemical probing experiments contain identical 3′-end, which correspond to the pUC18 sequence between restriction sites Sma I and EcoR I (5′-GGGUACCGAGCUCGAAUUC-3′). A Cy5 fluorescent reverse transcription primer complementary to the 3′-end of the studied RNA fragments was purchased from Evrogen .2, and 2 mM each dNTP. M-MuLV Reverse Transcriptase 5 units  were added, and reactions were incubated at 37 °C for 60 min. For sequencing samples, 1 µM ddATP, ddCTP, ddGTP, or ddTTP were added to the RT reactions of each sample. Reactions were stopped by adding formamide with 10 mM EDTA, 0.3% bromophenol blue, and 0.3% xylene cyanol. Aliquotes of 6–8 µL were analyzed in an 8% polyacrylamide gel in the presence of 8 M Urea. Gels were visualized using the ChemiDoc MP Imaging System .For reverse transcription (RT), RNAs or modified RNAs (4 µL of a 1 µM solution) were added to the Cy5-primer (4 µL of a 2 µM solution) in an annealing buffer (250 mM Tris-HCl (pH 8.3), 200 mM KCl), heated to 90 °C for 3 min, and slowly cooled to 37 °C. RT reactions were performed in 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgClA Cary Eclipse fluorescence spectrometer  was used in the assay. All measurements were performed in a 0.3×0.3 cm cuvette at a constant temperature of 22 °C; the excitation wavelength was 496 nm, and emission wavelength was 519 nm.To study the RNA chaperone properties of NUCB1 protein, chemically synthesized molecular beacon-microRNA-200a-3p (MB-miR) were used . A MB-miR is a hairpin RNA containing the microRNA-200a-3p RNA sequence in a loop with a FAM fluorophore at one end and an RTQ1 quencher  at the other end a. In theAnalysis of protein-induced changes in fluorescence intensity was performed for NUCB1, its truncated forms, MvaSmAP1 as a positive control, and BSA as a negative control.Proteins were added at 1:5 and 1:10 molar ratios to 200 nM MB-miR in 100 µL of 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Three independent melting curves were obtained for each MB-miR-protein combination.In this study, we have shown for first time that NUCB1 possesses RNA-binding and RNA-melting activities. Apparently, the protein can bind to the ssRNA GG


RNA translation is tightly controlled in eukaryotic cells to regulate gene expression and maintain proteome homeostasis. RNA binding proteins, translation factors, and cell signaling pathways all modulate the translation process. Defective translation is involved in multiple neurological diseases including amyotrophic lateral sclerosis (ALS). ALS is a progressive neurodegenerative disorder and poses a major public health challenge worldwide. Over the past few years, tremendous advances have been made in the understanding of the genetics and pathogenesis of ALS. Dysfunction of RNA metabolisms, including RNA translation, has been closely associated with ALS. Here, we first introduce the general mechanisms of translational regulation under physiological and stress conditions and review well-known examples of translation defects in neurodegenerative diseases. We then focus on ALS-linked genes and discuss the recent progress on how translation is affected by various mutant genes and the repeat expansion-mediated non-canonical translation in ALS. Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease mainly affecting motor neurons . In the RNA translation is tightly controlled in eukaryotic cells to regulate gene expression and maintain proteome homeostasis, which is important for cell function and survival . RNA binIn this review, we will first introduce the general mechanisms of translational regulation, and examples of translation defects in neurodegenerative diseases. We will then focus on ALS-associated genes and discuss the recent progress in understanding the dysregulated translation in ALS.iMet) to form the 43S pre-initiation complex (PIC). In canonical translation, the 43S PIC is then recruited to the 5’ end of mRNAs through the cap-binding complex eIF4F to form the 48 S initiation complex, which begins to scan the mRNA in the 5’ to 3’ direction until it reaches the start codon and establishes the codon-anticodon base pairing. The eIF2-bound GTP is then hydrolyzed, eIFs are released from the complex, and the 60 S large ribosomal subunit is recruited to assemble the 80 S ribosome at the start codon, which marks the end of the initiation phase [Translation is the last step in the flow of genetic information which involves the decoding of the triplet codons in the mature mRNAs and the synthesis of corresponding proteins by ribosomes. It is one of the most complex and fundamental processes in cells and can be broadly divided into three steps: initiation, elongation, and termination  Fig. 1)1). Inition phase , 17-site of the ribosome. Elongation begins with the delivery of cognate aminoacylated tRNA to the A (aminoacyl)-site of the ribosome by a ternary complex formed with eukaryotic elongation factor 1 A (eEF1A), GTP and aminoacyl-tRNA  and eRF3. The ternary complex formed by tRNA-shaped eRF1  and GTP-Regulating protein synthesis at the level of translation has obvious advantages over other layers of manipulation. As the final step of protein synthesis, translational regulation allows more immediate change on protein level from pre-existing mRNA. It enables cells to respond rapidly to stimuli. From the perspective of energy-consuming, translation regulation is much more efficient. As described above, every step of translation requires the usage of high-energy phosphate bonds. Indeed, it is estimated that around 30–50% of the cellular energy is consumed by translation –24. ReduTranslational control happens at the levels of both global and specific mRNAs. The initiation phase is the rate-limiting step where most regulation is exerted. Global regulation mainly occurs through influencing the activity of general initiation factors. The cap-binding protein eIF4E is important for cap-dependent translation initiation. The post-translational modifications of eIF4E could influence translation, such as the phosphorylation of Ser209, which is generally believed to promote initiation . The moseIF2 is a heterotrimer composed of three subunits: α, β, and γ. The α-subunit (eIF2α) is one of the most important targets for global translation regulation. The phosphorylation of eIF2α at the Ser51 residue increases its binding affinity to eIF2B , the guaeIF2α phosphorylation is the core component of the integrated stress response (ISR), which is an evolutionarily conserved signaling network that helps cells maintain homeostasis under stress circumstances. There are four different kinases that catalyze the phosphorylation of eIF2α to reduce global translation. PKR (double-strand RNA-dependent protein kinase) is initially found to be activated by virus infection , but hasISR plays important roles in aging and neurodegenerative diseases. For example, oxidative stress, ER stress, proteotoxic stress, and neuroinflammation are characterized in many neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), ALS, frontal temporal dementia (FTD), and Huntington’s disease (HD) –43. IndeMutations in genes encoding the five subunits of eIF2B cause the autosomal recessive inherited neurological disorder leukoencephalopathy with vanishing white matter (VWM) . Loss ofAlthough initiation phase is the primary focus of translation regulation, emerging evidence demonstrates that elongation also plays important roles. Modifications of elongation factors such as eEF1A1 and eEF2 influence translation elongation broadly , the essential enzymes that charge tRNAs with cognate amino acids, cause many diseases including neurological disorders. Charcot-Marie-Tooth disease (CMT), one of a group of disorders that cause damage to the peripheral nerves, is the first disease that linked to mutations in AARSs. Until now mutations in at least six AARSs  have been linked to CMT . Multipleduction , 77. Proeduction . Indeed,eduction . Geneticeduction . Restoriuropathy . Thus, turopathy , 79, 80.nmf205 develop cerebellar, hippocampal, cortical, and retinal neuron degeneration. This was identified to be caused by a null mutation in the gene encoding GTPBP2, a translational GTPase [n-Tr20, exacerbates the neuronal death when combined with Gtpbp2 mutation [Mice with homozygous N-ethyl-N-nitrosourea (ENU)-induced mutation l GTPase . Interesmutation . This tRmutation . OverallAsymmetric mRNA localization and localized translation provide the opportunity to fine-tune localized protein concentration which plays an important role in development , cell faACTB mRNA which is important for the proper transport of ACTB mRNA [Many neurological disease-related proteins have been shown to participate in the process of mRNA transport and localized translation in neurons. The survival motor neuron (SMN) protein whose loss is linked to the neuromuscular disease spinal muscular atrophy (SMA) is recently found to participate in mRNA transport . It has CTB mRNA . SMN depCTB mRNA  includinCTB mRNA . KIF5A, CTB mRNA , is a meCTB mRNA . Many neCTB mRNA .Expansions of short nucleotide sequence repeats account for more than 50 neurological or neuromuscular diseases . The patC9ORF72 gene is the most common genetic cause of both ALS and FTD [Hexanucleotide GGGGCC repeat expansion in the first intron of the  and FTD , 114. Th and FTD –14, 115. and FTD . An apprC9ORF72 repeat RNA translation mechanisms and regulatory pathways. The traditional method is using ensembled assays, such as luciferase, fluorescence proteins or short tags fused to the repeats to monitor the DPR levels translated from the repeats. This allows straightforward test and identification of novel modifiers or pathways that can modulate the DPR production. But one limitation is that the final protein product level can be influenced by many RNA/protein regulation steps besides translation, thus it is sometimes challenging to elucidate the exact molecular mechanisms. Alternatively, recently developed imaging approaches allow direct visualization of single RNA molecule dynamics in live cells, which can be used to assess translation directly. Finally, the measurement of endogenous DPRs in patient cells is critical to validate the findings from reporter systems.Many advances have been made on C9ORF72 expanded repeats are located in the intron. Both the spliced intron and not fully processed intron-containing pre-mRNA are normally excluded from cytoplasm under multiple surveillance mechanisms [Earlier examples of RAN translation occur on repeats in the UTR or ORF regions of mature mRNAs, which are naturally exported to cytoplasm for translation. However, the chanisms , 118. ThC9ORF72 intron 1 or all exons. Furthermore, by combining smFISH with exonuclease RNase R treatment, it was suggested that the cytoplasmic repeats containing introns mainly existed in a circular form, due to the defective debranching of spliced lariat intron induced by the GGGGCC repeats, which is believed to be more stable compared with linear RNAs [The single-molecule Fluorescence in Situ Hybridization (smFISH) provides an opportunity to directly visualize the spatial localization of single RNA transcripts in cells. The intron- and exon-containing molecules can be visualized simultaneously using orthogonal RNA tags (MS2 and PP7), which allows the examination of the molecular identity and spatiotemporal dynamics of the repeat RNA . It is dear RNAs . It is near RNAs –121. Altear RNAs , there iVarious approaches have been used to identify modifiers of the repeat RNA metabolisms. Many RBPs have been reported to modulate the repeat RNA nuclear export and translation. Through RNA-affinity pulldown assay, several RBPs with functions in RNA transport such as serine and arginine-rich splicing factors (SRSF), ALYREF, and transcription-export complex (TREX) subunits were identified as GGGGCC repeat RNA binding proteins , 123. ThFMR1 requires the 5’ 7-methylguanosine (m7G) cap on the mRNA [Although the canonical translation initiation requires 5’ cap, many viral RNAs and some cellular RNAs can start translation in a cap-independent manner, using the internal ribosome entry site (IRES), which usually contains complex RNA structures that directly recruit certain translation initiation factors to start translation . It has the mRNA . Howeverthe mRNA , the capthe mRNA . Studiesthe mRNA –134.The small ribosomal protein subunit 25 (RPS25), a non-essential protein component of the small (40 S) ribosomal subunit which has been shown to facilitate the recruitment of 40 S ribosomal subunit to IRES RNAs , 136, caC. elegans model of C9ORF72-associated ALS/FTD, functional loss of non-canonical initiation factor eIF2D but not eIF2A reduces DPR production and ameliorates lifespan and locomotion defects [Many translation factors have been shown to regulate GGGGCC associated RAN translation. In the  defects . Depleti defects  but has  defects  in HEK29 defects . Two oth defects .As RNA secondary structure is important for RAN translation, RNA helicases are also implicated in RAN translation. The GGGGCC repeat RNA has been shown to form both hairpin and G-quadruplex structures , 143. FrThe initiation of RAN translation can also be regulated by cell signaling pathways. As noted in the previous section, ISR is associated with many neurodegenerative diseases. The ISR activation leads to global translation repression, but a subset of mRNAs has increased translation instead, which usually use the non-canonical initiation mechanisms . Using sIn addition to ISR, changes in neuronal activity can also influence C9ORF72 GGGGCC RAN translation. Activation of glutamate receptors by various agonists increases RAN translation in both primary cortical neurons and in patient-derived iPSNs . ElevatiRibosomal frameshifting is an evolutionarily conserved biological process that has been found in many organisms such as viruses, bacteria, and eukaryotes. Programmed ribosome frameshifting is used to produce distinct proteins from the same RNA, which is a fundamental mechanism for viruses to maintain their infection efficiency . FrameshChimeric GA:GP protein has also been detected in C9ORF72-ALS/FTD patient postmortem tissues including the frontal cortex and cerebellar cortex by an immunoassay using GA antibody for capture and GP antibody for detection . This coAs the pure repeated DPRs and the chimeric DPRs probably have different properties and toxicities, it is important to understand the efficiency of translation frameshifting on the repeats and to what extent it contributes to the generation of various DPR species. Recently a new technology that uses multicolor probes to visualize the translation of different reading frames of a single RNA provides the opportunity to directly quantify the frameshifting activities in live cells. The study using the established HIV-1 frameshift sequence revealed that only a small subset (8%) of translating RNAs showed robust frameshifting event . ApplicaOnce initiated, the translation of the repeats follows the canonical elongation mechanisms. The GGGGCC repeat-containing RNAs form sophisticated secondary structure , 143 and100 mice and C9ORF72-ALS/FTD patient postmortem brain tissues [Multiple lines of evidence indicate that arginine-rich DPRs (poly-GR and poly-PR) can cause translation defects Fig. . Earlier tissues , suggest tissues , 175. In tissues . Overexp tissues . Overexp tissues . More re tissues . This stFUS account for around 5% of familial ALS [FUS/TLS is an RNA-binding protein that predominantly localizes in the nucleus and shuttles between the nucleus and cytoplasm. FUS protein is involved in many cellular processes including gene expression regulation, DNA repair, alternative splicing, RNA degradation, alternative polyadenylation and translation regulation . Mutatiolial ALS . Most oflial ALS . FALS-aslial ALS . It is wlial ALS .R) motifs [FUS contains four RNA-binding domains (RBDs): two arginine-glycine-glycine boxes (RGGs), an RNA-recognition motif (RRM), and a zinc finger domain (ZnF) . Stable ) motifs , have be) motifs –189. The) motifs , 191. Pr) motifs  Fig. 3)R) motifsFUS is also involved in localized translation Fig. . It has TAR DNA binding protein 43 kDa (TDP43) is encoded by the TARDBP gene located at chromosomal locus 1p36.22 and is an evolutionarily highly conserved protein belonging to the heterogeneous nuclear ribonucleoprotein (hnRNP) family . TDP43 iTDP43 contains 414 amino acids and can bind to both DNA and RNA. As an hnRNP protein, TDP43 contains all the classical domains of this family including RRM domains, N-terminal domain (NTD), and C-terminal domain (CTD) . TDP43 bTDP43 can bind to mRNA and modulate its translation directly. It has been demonstrated that the translation of genes essential for neurodevelopment and neuron plasticity, such as Rac1 , 209, MaGlobal translation can also be influenced by TDP43  that can bind to IRES sequences and regulate ribosomal entry for cap-independent translation . Human fAngiogenin (ANG) is a vertebrate-specific ribonuclease and was first identified and characterized due to its important role in angiogenesis . ANG hasANG plays an important role in translation regulation under physiological and stress conditions. ANG enhances ribosomal RNA synthesis through multiple mechanisms. First, ANG can directly bind to the promoter of rDNA and alter histone modifications, which increases rRNA transcription . Second,Under stress conditions such as oxidative stress, hypoxia, and starvation, ANG may translocate to cytoplasm and cleave tRNAs which will result in global translation inhibition Fig. . CleavagALS-related ANG variants have been shown to have cytotoxic effects on motor neurons and lack neuroprotective activity under stress . Many muT-cell intracellular antigen 1 (TIA1) is an RNA binding protein that plays diverse roles in many aspects of RNA metabolism and is ubiquitously expressed . TIA1 coTIA1 has been shown to directly modulate the translation of target mRNAs. TIA1 directly binds to the AU-rich element in the 3’UTR of target mRNAs and represses their translation –260.In aTranslation elongation can be slowed down under many circumstances, such as the presence of mRNA and rRNA damage, suboptimal codons, mRNA secondary structures, and environmental stresses. This could increase ribosome stalling and cause inefficient ribosome recycling, which is deleterious to cells and needs to be resolved quickly to maintain cell homeostasis. Ribosome-associated protein quality control (RQC) pathway is a dedicated surveillance mechanism that is used to monitor ribosome stalling. During RQC, stalled ribosomes can be detected and dissociated into subunits by specific factors for recycling , 263. NEThrough ENU-mediated random mutagenesis, mice with mutations in both LTN1  and NEMFMuch progress has been made over the past few years in the etiology of ALS including the discovery of a handful of RBPs whose mutations and pathology have been strongly linked with ALS. As mentioned above, under physiological conditions those proteins have been demonstrated to either play essential roles or carry regulatory functions in RNA translation and their ALS-linked mutations or pathology may disrupt their normal function in translational control. The fact that many ALS-associated RBPs can influence the function of other RBPs by regulating their expression and/or activity adds another layer of complexity in translation dysregulation. ISR is dysregulated in many neurological diseases including ALS. Many RBPs that are linked with ALS are components of stress granules. ALS-linked mutations in those RBPs can influence the dynamics of stress granule assembly and disassembly which contributes to ISR activation. Thus, the RBP dysfunction may also induce global translation dysregulation besides the subsets of RBP-specific targets. Depending on the individual functions of different RBPs, the translation perturbation could contribute to neurodegeneration via different mechanisms.There has been a lot of progress understanding the repeat associated translation of the C9ORF72 repeat expansion. Previous studies identified the cis-acting elements, trans-acting factors, as well as cell signaling pathways that can modulate the repeat translation initiation efficiency. The potential RNA species that is subjected to the nuclear export and translation in the splicing context was also revealed. These studies on C9ORF72 repeat RNA translation mechanisms also shed light on other repeat expansion diseases. There are also many questions remain to be answered. The translation of the antisense repeats could be less complicated, as there is no splicing involved. The antisense transcript likely contains the 5’ cap and is more efficiently exported to cytoplasm for translation. It is noted that there are AUG start codons in the GP and PR frames, 194 and 273 nt upstream of the antisense repeat expansion respectively . If the Although many translation initiation factors and RBPs have been reported to regulate the GGGGCC repeat translation, these factors were usually identified using non-neuronal cell types. It will be interesting to explore whether there are neuron-specific factors modulating the repeat translation and whether there are cell type differences of DPR production. Furthermore, as repeat RNA can be found in synapse , whetherFurthermore, it is intriguing to understand the pathophysiological role of translational defects caused by ALS-linked genes, including RBPs and poly-GR/PR. Translatome studies could help reveal the substrate specificity in vivo, such as whether certain mRNA and amino acid sequences are preferentially influenced, and any different impact on localized translation in synapse, etc. Recent studies also suggest heterogeneity in the translational machinery in different cell types and developmental stages . AdditioTaken together, increasing evidence suggests that the delicate RNA translation regulation plays important roles in neuronal homeostasis. Accumulating studies indicate that dysregulation of RNA translation could preferentially result in neurological and neurodegenerative diseases. Recent development of novel technology and experimental approaches allows advances in understanding the various molecular mechanism of translation regulation at different steps and at the genome-wide level. Despite the progress on the fundamental process in all cells, how the pathways and translatome are fine-tuned in neurons for the highly specialized morphology and function is relatively less studied. Moving forward, it is important to dissect translational changes in specific neuronal and glial cell types and at specific pathological conditions. For example, a cascade of cell type-specific and age-dependent translatome changes caused by mutant SOD1 in mouse spinal cord was identified using the TRAP coupled RNA-seq approach . Similar


PIWI-interacting RNAs (piRNAs) are responsible for preventing the movement of transposable elements in germ cells and protect the integrity of germline genomes. In this review, we examine the common elements of piRNA-guided silencing as well as the differences observed between species. We have categorized the mechanisms of piRNA biogenesis and function into modules. Individual PIWI proteins combine these modules in various ways to produce unique PIWI-piRNA pathways, which nevertheless possess the ability to perform conserved functions. This modular model incorporates conserved core mechanisms and accommodates variable co-factors. Adaptability is a hallmark of this RNA-based immune system. We believe that considering the differences in germ cell biology and resident transposons in different organisms is essential for placing the variations observed in piRNA biology into context, while still highlighting the conserved themes that underpin this process. The genomes of our germ cells live on in our children. Changes to the sequence of germline genomes impact the genetic make-up of future generations and the identity of a species. Genomes are vulnerable to threats by mobile genetic elements (transposons) that move or copy themselves into novel genomic locations –5. GermlPIWI proteins, the ZUC-processor nuclease (ZUC/PLD6), and the RNA helicases Vasa/DDX4 and Armi/MOV10L1 are conserved core-components of piRNA pathways and have long been known as essential germ cell factors . Co-factDrosophila melanogaster, a complex composed of the HP1 homolog rhino (rhi) and its co-factors deadlock (del) and cutoff (cuff) are required to initiate transcription and suppress splicing across dual-stranded piRNA clusters [PiRNA precursors are fast evolving and result in an everchanging repertoire of piRNA sequences , 32. In clusters –39. In aclusters , 40, 41.clusters . Until nclusters . In maleclusters , 43, 44.clusters . Male geclusters . PiRNA gDrosophila and later shown to be essential for primary piRNA biogenesis in flies and mice [Drosophila ZUC and mouse PLD6 were the first eukaryotic HKD-enzymes to be characterized as nucleases [The endonuclease Zucchini/PLD6 (ZUC) cleaves long single-stranded precursors into RNA fragments with 5’ monophosphorylated ends that can be loaded onto PIWI proteins to become primary pre-piRNAs. The ZUC/PLD6 nuclease is anchored into the outer mitochondrial membrane with its RNA-binding surface facing the cytoplasm. ZUC has been originally identified in a screen for female fertility in and mice , 46–51. and mice . Substraand mice . The bacand mice . In contand mice . Drosophucleases . Both enDrosophila piRNAs and upon loss of the trimming exonuclease PNLDC1 in mice, and implicates the ZUC-processor in 3’end formation of piRNAs [The preferences of primary piRNAs to harbor a uridine in the 5’ most position (1-U bias) is established in two steps during piRNA processing and PIWI-piRNA complex formation . The ZUCf piRNAs , 56. If Drosophila melanogaster (D.m.) is the prototype for ‘heterotypic ping-pong’ whereby Aubergine (Aub)-piRNA complexes generate slicing fragments that are loaded into Argonaute-3 (Ago3) and vice versa. Only Aub but not Ago3 can be loaded with primary piRNAs and initiates ping-pong. Ago3 can only be loaded with secondary (Aub-generated) piRNAs. Ago3-piRNA complexes generate secondary Aub-piRNAs and can induce further ‘phased’ piRNA production by the ZUC-processor complex [D.m. ovarian somatic cells (OSC) [Drosophila species do not operate ping-pong at all [PIWI proteins are piRNA-guided nucleases that can cleave the target RNA across nucleotide 10 and 11 of the piRNA-guide , 23. The complex , 62. In  complex . Ping-pols (OSC) , 64, andg at all .Drosophila and germ granules  in mammals [ZUC/PLD6 resides on the surface of mitochondria. Its hydrophobic N-terminus is anchored in the outer mitochondrial membrane and its substrate binding surface faces the cytoplasm. Substrate binding and cleavage require a homodimer, and while little is known about the regulation of primary piRNA biogenesis, controlling dimer formation could be a key event. Secondary piRNA processing (ping-pong) is also linked to the surface of mitochondria and electron dense structures termed ‘inter-mitochondrial cement’. These dense, membrane-less RNA–protein compartments are called nuage (French for cloud) in  mammals . The sur mammals . In vert mammals .Contribution of membranes and electron-dense subcellular compartments has hampered the purification of piRNA processing complexes and identification of co-factors. Most co-factors have been identified in genetic screens for female fertility and transposon restriction in flies, and revealed a few conserved and many species-specific genes , 69–71.Drosophila ovaries, Aubergine (Aub)-associated piRNAs are mostly antisense to transposon sequences and guide slicing of complementary transposon transcripts [Drosophila testes, Aub-piRNAs derived from Suppressor of Stellate (Su(Ste)) induce degradation of the complementary Stellate transcripts, and loss of Su(Ste) or Aub results in male sterility [PiRNA-guided slicing of RNA targets requires the nuclease activity of PIWI proteins and induces target RNA degradation. Slicer activity has been originally observed for AGO-clade Argonaute proteins during RNA interference (RNAi) and later been attributed to an RNase H fold in the PIWI-domain of Argonaute proteins , 73. SiRnscripts . In Drosterility –79. In mterility . The criterility .In the presence of the germ cell specific helicase Vasa/DDX4, piRNA-guided target cleavage can produce secondary piRNAs during ping-pong . It remaPiRNAs establish lasting epigenetic silencing. Select PIWI-piRNA complexes translocate to the nucleus and mediate the de novo establishment of heterochromatin to inhibit transcription. Nuclear PIWI-piRNA complexes recognize nascent RNAs co-transcriptionally by base-pairing complementarity and recruit epigenetic modifiers. In flies, Piwi-piRNA complexes recruit the histone methyltransferase Eggless (Egg)/SETDB1 to establish trimethylation at Lysine 9 of Histone H3 (H3K9me3) and initiate transcriptional repression . EfficieThe combination of biogenesis and effector modules differs for individual PIWI proteins and in different organisms Fig. . PIWI prDrosophila melanogaster encodes three PIWI proteins: Piwi, Aubergine (Aub) and Argonaute-3 (Ago3) [3 (Ago3) , 76  [Drosophila ovary express a single PIWI protein, Piwi, that is loaded with primary piRNAs and establishes co-transcriptional silencing of endogenous gypsy retroviruses. The restriction of gypsy elements in the ovarian somatic cells depends on the Flamenco piRNA cluster and is essential for germ cell health and fertility [Drosophila ovary and has not been observed in mammals.Different types of transposons invade germline genomes, adapt to distinct developmental stages and call for adaptations of the piRNA pathway for efficient defense –103. In s (ERVs) , 105. Ths (ERVs) –108. To s (ERVs) . The somertility , 40, 42.Stage-specific adaptations by resident transposons result in variable phenotypes of piRNA pathway mutants. Adaptations of active transposons to specific developmental stages impact the phenotype of core piRNA pathway mutants. For example, loss of the RNA helicase MOV10L1 during mouse spermatogenesis results in unleashed transposons during meiosis , 93. In Piwil1/Miwi gene results in male sterility and germ cells arrest at the round spermatid stage [Developmental timing separates two different classes of piRNAs in mammalian spermatogenesis Fig. . Pre-pacid stage . The mutid stage . PIWIL1/id stage , 114. Thde-novo from somatic cells. A signal from neighboring cells determines a small group of embryonic cells to become a primordial germ cell. Primordial germ cells proliferate and actively migrate to the developing gonads, where they commit to develop into an egg or sperm, based on the sex origin of the gonad [The mammalian germline is induced he gonad , 120.Drosophila germline is pre-formed by germ plasm that is composed of maternal determinates [In contrast, the rminates , 122. Norminates , 124. Ifrminates , 126. Inrminates . The difrminates , 129.Nos mRNA via a transposable element sequence in its 3' untranslated region (UTR) and thus impact patterning of the embryo [Mov10l1 or Piwil1 produce embryos that do not develop beyond the 2-cell stage. However, zygotes with homozygous deletions of Mov10l1 or Piwil1 develop into adults. Thus, the developmental arrest of embryos from mutant oocytes is suggested to be a maternal effect [Activation of the zygotic genome occurs as early as the 2-cell stage in mice and hamsters, whereas it takes several rounds of division for this event to occur in flies . While me embryo . PiRNA pl effect , 132. ExThe RNAi pathway is an ancient double-stranded (ds)RNA-sensing mechanism and the main anti-viral defense in fungi, plants and invertebrates –137. VirIn mosquitos, viral infection induces siRNA and piRNA production , and virDicer null mutant [While mammals generally lack an effective RNAi response, siRNAs are efficiently generated from long dsRNA by a specific Dicer isoform in mouse oocytes , 145. Thl mutant , 148. ThSiRNA-dependent transposon restriction in mouse oocytes represent an exception to the rule. The importance of the piRNA pathway in the mammalian female germline has been demonstrated by recent studies in golden hamsters –100. ResPiRNA pathways integrate highly conserved and fast evolving genes and adapt to the ever-changing landscape of resident transposons . While PThe modular framework for piRNA biogenesis and function can be extended to other small RNA silencing pathways. Three classes of small silencing RNAs are conserved in eukaryotes: microRNAs (miRNAs), small interfering RNAs (siRNAs), and piRNAs . MiRNAs The discovery of ping-pong amplification of a primary piRNA trigger defined ‘secondary piRNAs’ , 23, 30.In contrast to miRNAs, which exhibit a large degree of sequence conservation , piRNAs Finally, additional piRNA biogenesis and effector modules have been identified in other organisms. C. elegans piRNAs (21U RNAs) are processed from individual short single-stranded RNA precursors by a novel processing mechanism , 162 and