C9orf72 gene. The accumulation of repetitive RNA sequences can mediate toxicity potentially through the formation of intranuclear RNA foci that sequester key RNA-binding proteins (RBPs), and non-ATG mediated translation into toxic dipeptide protein repeats. However, the contribution of RBP sequestration to the mechanisms underlying RNA-mediated toxicity remain unknown. Here we show that the ALS-associated RNA-binding protein, Matrin-3 (MATR3), colocalizes with G4C2 RNA foci in patient tissues as well as iPSC-derived motor neurons harboring the C9orf72 mutation. Hyperexpansion of C9 repeats perturbed subcellular distribution and levels of endogenous MATR3 in C9-ALS patient-derived motor neurons. Interestingly, we observed that ectopic expression of human MATR3 strongly mitigates G4C2-mediated neurodegeneration in vivo. MATR3-mediated suppression of C9 toxicity was dependent on the RNA-binding domain of MATR3. Importantly, we found that expression of MATR3 reduced the levels of RAN-translation products in mammalian cells in an RNA-dependent manner. Finally, we have shown that knocking down endogenous MATR3 in C9-ALS patient-derived iPSC neurons decreased the presence of G4C2 RNA foci in the nucleus. Overall, these studies suggest that MATR3 genetically modifies the neuropathological and the pathobiology of C9orf72 ALS through modulating the RNA foci and RAN translation.The most common genetic cause of amyotrophic lateral sclerosis (ALS) is a GGGGCC (G4C2) hexanucleotide repeat expansions in first intron of the  Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that is characterized by degeneration of upper and lower motor neurons, leading to progressive atrophy and weakness in skeletal muscles, eventually resulting in death due to respiratory failure , 2. An eExtensive investigation into the mechanisms underlying C9-ALS has identified three potentially pathogenic mechanisms resulting from the G4C2 hexanucleotide repeat expansion (G4C2-HRE) –8—(1) LoMATR3 have been discovered in a subset of familial and sporadic ALS cases [Accumulation of repeat-bearing RNA in the neurons and glia is also observed in other neurological disorders caused by intronic repeat expansions, such as myotonic dystrophy and fragile X tremor ataxia syndrome, are also characterized by RNA accumulation or RNA foci , 20. TheLS cases –33, and LS cases , 34. HowDrosophila, mediated by its RNA-binding domain. In addition, genetic manipulation of MATR3 levels modulated the RAN-translation product levels and RNA foci in mammalian cells and C9-ALS patient-derived motor neurons, respectively. Collectively, our data presents evidence that MATR3 is a novel genetic modifier of neuropathologies and motor dysfunctions associated with G4C2 repeats through modulating the RNA foci and RAN translation products.Here, we demonstrate that MATR3 is an important component of C9orf72-associated disease neuropathology in C9-ALS patient postmortem brain tissues and iPSC motor neurons. We show colocalization between MATR3 and pathogenic G4C2 foci in C9-ALS patient-derived neurons and in post-mortem brain tissues. Additionally, we found that MATR3 sub-cellular localization and levels are altered in C9-ALS patient neurons. Furthermore, we show that ectopic expression of MATR3 strongly suppressed G4C2-HRE toxicity in Proteomic studies have suggested physical interaction between G4C2 RNA and MATR3 , 25, how10, indicating that there is physical interaction between G4C2 RNA and MATR3  , 30 repeats (30R), 36 repeats (36R) and 58 repeats (58R) of G4C2 [Our results so far indicated that G4C2-HRE in C9orf72 not only physically associates with MATR3, but also significantly impacts the levels and sub-cellular localization of MATR3. So, we sought to determine whether MATR3 and G4C2 HRE genetically interact in vivo in  of G4C2 –37. WhenTo determine if ectopic expression of MATR3 in G4C2-HRE flies can suppress motor deficit, we assessed locomotion ability in adult flies expressing G4C2-30R in neurons. Expression of G4C2-30R alone results in severe motor degeneration in these animals, with only ~ 10% flies able to climb at all , known to have DNA-binding and putative RNA-binding properties , 38, and60), with a fluorescent Dendra2 reporter in the GR-frame which is only detected if the transcript undergoes RAN translation. We co-transfected HEK293T cells with the G4C260-Dendra2 reporter plasmid along with either FLAG or FLAG-MATR3 plasmid. Using RNA-FISH combined with ICC, we confirmed that overexpression of the FLAG-empty vector and G4C260-Dendra2 reporter in HEK293T cells results in formation of G4C2 RNA foci in the nucleus, and importantly, that a subset of these cells (~ 59%) produced GR-Dendra2 RAN-translated product   to significantly knockdown endogenous MATR3 in C9-ALS iPSC MNs  that interact with the G4C2 repeat RNA, including hnRNP-family proteins , ALYREF, ADARB2, RanGAP1 and MATR3 , 24, 25.Our data from post-mortem human brain tissues also indicate that sub-cellular localization of MATR3 is perturbed in C9-ALS neurons. MATR3 is a predominantly nuclear protein under physiological conditions, whereas, in C9-ALS patients, MATR3 exhibits significant cytoplasmic mislocalization. Disruption in MATR3 subcellular localization has previously been reported in other forms of ALS cases, including in C9-ALS and sporadic-ALS patient brain tissues , 34. HowDrosophila models that produce DPR products through canonical translation of codon-optimized transcripts that don’t bear the G4C2 repeats, MATR3 expression had no effect on the polyGR- or polyPR-mediated toxicities. This suggests that overexpression of MATR3 has no impact on post-translational toxicities conferred by DPR products in C9orf72-ALS, and that it is likely acting through upstream mechanisms which might be dependent on its RNA-binding function. Interestingly, we also demonstrated that in patient-derived neurons, modulating MATR3 levels directly impacts G4C2 RNA foci formation, raising a possibility that RNA foci and RAN translation/DPR mechanisms are linked through MATR3.In addition to linking MATR3 with neuropathology in C9-ALS patient-derived neurons, we identified MATR3 as a strong suppressor of G4C2-HRE toxicity in vivo. Specifically, we found that deletion of the RNA-binding domain of MATR3 (MATR3-ΔRRM2) significantly reduced the ability of MATR3 to suppress G4C2 toxicity in vivo. Importantly, our data is consistent with a model of RNA-mediated suppression of G4C2 toxicity. Furthermore, utilizing a reporter for RAN translation, we showed that overexpression of MATR3 suppresses DPR production in human cells dependent on the RRM2 domain, suggesting that MATR3 interaction with G4C2 RNA is important for suppressing the C9-DPR toxicity. However, a caveat is that the cells overexpressing MATR3-ΔRRM2 also have endogenous wildtype MATR3 with, presumably, a fully functional RNA-binding domain. Thus, the true effects of RRM2 deletion upon DPR production would have to be further investigated in the context of MATR3 knockout or point mutations in the RNA-binding domain of endogenous MATR3 that debilitate its RNA-binding function. Nevertheless, in Drosophila models of C9-ALS, including ones used in this study, have identified that depletion of nuclear export adapters, such as ALYREF and SRFS1, is protective in these models [Deriving from the patient pathology showing physical- and colocalization-interaction between MATR3 and G4C2 RNA, and genetic interaction between MATR3 and G4C2, both in vivo and in C9-ALS patient-derived neurons, we suggest two possible ways in which this interaction could be involved in C9-ALS pathobiology: (1) Decreased levels of nuclear MATR3 and/or cytoplasmic localization of MATR3 that could perturb its physiological functions due to alteration in the subcellular distribution; and, (2) nuclear sequestration of G4C2 RNA foci and suppression of RAN translation. It is possible that loss of nuclear MATR3 and/or cytoplasmic localization of MATR3 in C9-ALS neurons could be promoting RAN translation of G4C2-HRE repeat RNA into toxic DPRs. We speculate that in cells expressing sufficient MATR3 or overexpressing MATR3, it interacts with G4C2 RNA to sequester the expanded repeat RNA within the nucleus, and inversely, decreases availability of G4C2-HRE RNA for RAN-translation. Therefore, MATR3 could be imparting a neuroprotective role to G4C2 RNA foci in C9-ALS. MATR3 may possibly aid in G4C2 RNA foci formation through mediating post-transcriptional modifications that increase the stability of G4C2 higher-order secondary structures and thus, serving to stabilize the foci. Our data also raises the possibility that MATR3 could be inhibiting nuclear export of G4C2 repeat RNA to the cytoplasm and this, in turn, reduces the production of RAN proteins. The benefit of inhibiting nuclear export of repeat expansion RNA has been previously explored in C9-ALS , 54, as e models , 59. IndThe patients and controls were sampled from a similar age range. At the time of death, the age of control individuals (n = 3) ranged between 54 and 76 years, C9-ALS patients (n = 6) ranged between 51 and 67 years and that of non-C9 ALS patients (n = 3) ranged between 46 and 58 years. Clinical and neuropathological information of C9-ALS, non-C9 ALS and age-matched non-neurological control post-mortem human brain tissues is listed in Table Formalin-fixed, paraffin-embedded human entorhinal cortex sections of ALS/FTD subjects were retrieved from the Neurodegenerative Brain Bank at the University of Pittsburgh, following protocols approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents (CORID).Six µm thick sections were deparaffinized in xylene and rehydrated through graded ethanol , followed by a 30-min incubation in methanol/hydrogen peroxide. Antigen retrieval was performed with citrate buffer, pH 6.0, for 20 min at 95 °C, followed by rinses with water and PBS/Tween. Sections were then blocked for 6 min at room temperature with Power Block . Sections were incubated in primary antibody  diluted in Antibody Diluent (ScyTek) for 45 min at room temperature, rinsed in PBS and then incubated with biotinylated anti-rabbit secondary antibody  for 30 min, Slides were then immersed in ABC solution for 30 min  and visualized with Nova Red . After counterstaining with Mayer’s hematoxylin , slides were dipped in 1% Lithium carbonate bluing solution , dehydrated in a series of ethanols, cleared in xylene, and mounted with Permount mounting medium .Quantification of the MATR3 IHC immunostaining was performed in a blinded fashion. Bright-field images from multiple fields of the entorhinal cortex were used for analysis. Briefly, cells with MATR3 signal (brown/DAB) in the cytoplasm were counted and plotted as a percentage of the total number of cells in the field.2, and one 5-min wash in 1× PBS were then performed prior to mounting slides with Prolong Antifade Mounting Media with DAPI (Invitrogen).Human control and ALS motor cortex sections  were then permeabilized with 0.4% Triton X-100 for 15 min at room temperature following two 1× PBS washes and subsequently equilibrated in 2× SSC for 10 min at room temperature. Sections were then incubated in 50% formamide solution (in 1× SSC) for 10 min at 85 °C while 5′ TYE 563-labeled LNA probes  were incubated at 95 °C for 5 min. Probe hybridization was then performed by first adding 200 µL probe solution  and 200 uL hybridization buffer  per section and incubation for 1 h at 66 °C. Sections were then washed first in 50% formamide (in 2× SSC) at 80 °C for 15 min prior to three 5-min washes in 2× SSC at room temperature, one 5-min wash in Tris–glycine solution  at room temperature, and one 5-min wash in TBS-50  at room temperature. Following UV crosslinking, sections were blocked overnight at 4 °C . The next day, sections were incubated with primary antibodies  in IF buffer (0.5% Protease-free BSA Fraction V (Roche), 0.5% Heat-shocked BSA Fraction V (Roche), 0.1% Triton X-100 in TBS-50) overnight at 4 °C. Sections were then exposed to four 5-min washes in IF buffer at room temperature prior to incubation with secondary antibodies  for 1 h at room temperature. Four 5-min washes in IF buffer, two 5-min washes in TBS-50, two 5-min washes in 1× PBS + 2 mM MgClFor quantification of G4C2 RNA Foci and MATR3 colocalization in human tissue, Z stack images of 12–20 nuclei per patient from three C9 ALS/FTD patient motor cortex embedded paraffin sections were acquired using a Nikon A1R confocal microscope. Images were then deconvolved using 10 Landweber iterations on the Nikon element program. The single middle plane of the reconstructed RNA foci was then selected and analyzed for Matrin3 protein signal overlap used to calculate the Pearson’s colocalization in Nikon elements. G4C2 RNA:MATR33 Pearson’s coefficient values from 70 RNA foci were binned in values of 10 and colocalization was classified using < − 0.79 inverse colocalization; − 0.8 to 0.19 no colocalization; 0.20 to 0.59 moderate colocalization  and C9-ALS iPSC lines used in this study are listed in Table 2 in mTESR1 media substituted with a 10 µM ROCK inhibitor . The next day, media was replaced with N2B27 medium  supplemented with SB431542 , LDN-193189 , Retinoic Acid  and Smoothened-Agonist  and fed daily with the same media for 6 days. On day 7, media was switched to N2B27 supplemented with 1 µM RA, 1 µM SAG, 5 µM DAPT  and 4 µM SU5402  and fed daily with the same media for 7 days. Cell were then dissociated using TrypLE Express (Gibco) supplemented with DNase I (Worthington), plated onto pre-coated Matrigel-coated surfaces (BD Biosciences) and cultured in Neurobasal medium supplemented with NEAA, Glutamax, N2, B27, Ascorbic acid (0.2 µg/mL) and BDNF, CNTF and GDNF . For imaging analysis, motor neurons (MNs) were seeded on pre-plated mouse glia cells.iPSC differentiation into motor neurons was performed as previously described . Briefly, iPSCs were dissociated with Accutase and plated at a density of 100,000 cells/cmTwo serial siRNA treatments were applied beginning 65–70 days after initiating MN differentiation. Each treatment was applied for 24 h. The second treatment began 4 days after application of the first siRNA treatment and FISH was performed on freshly fixed cells 4 days after the application of the second siRNA treatment.E. coli tRNA , 25 mL 80% formamide, and 0.4 mL 25 mM 50 digoxigenin (DIG)-labeled CCCCGGCCCCGGCCCC locked nucleic acid probe . Probe mixture for detection of poly (A) RNA, consisted of 1 mL salmon sperm , 0.5 mL E. coli tRNA , 25 mL 80% formamide, and 0.8 mL 25 mM 30 digoxigenin (DIG)-labeled polyT(25) locked nucleic acid probe . Tris-buffered saline comprised of 50 mM Tris and 15 mM NaCl solution, pH 7.4. Tris–glycine buffer comprised of 0.75% glycine and 200 mM Tris solution, pH 7.4. Blocking buffer consisted of 1% normal donkey serum (Jackson Immunoresearch) and 5% heat-shocked BSA in Tris-buffered saline. Immunofluorescence buffer consisted of 2% heat-shocked BSA in Tris-buffered saline. DIG-labeled probe was detected with a fluorescein-conjugated sheep anti-DIG antibody . All buffers were made with RNase-free water or PBS.FISH analysis was performed as previously described . BrieflyMotor neurons, matured for 50 days, and transiently transfected HEK293T cells at 72-h post transfection, were used for immunocytochemistry. Cells were washed and fixed with 4% paraformaldehyde and blocked for 1 h in Phosphate-buffered saline (PBS) containing 0.2% Triton X-100 and 10% normal donkey serum for iPSC-MNs and 5% normal goat serum for HEK293Ts respectively (Jackson ImmunoResearch). Samples were then incubated overnight at 4 °C with primary antibodies. The next day, cells were washed with PBS with 0.1% Triton X-100. Samples were then incubated with the appropriate secondary antibodies (diluted in blocking solution) conjugated to Alexa488, Alexa555 or Alexa647 fluorophores (1:500 to 1:1000 Molecular Probes) for 1 h at RT. Cell nuclei were labeled by DNA staining using DAPI or Hoechst (Life Technologies).Primary antibodies Chicken anti-MAP2 ; Goat anti-ChAT (1:500); Rabbit anti-MATR3 ; Mouse Anti-digoxin ; Rabbit anti-FLAG ; Rabbit anti-Histone H3 Images used for quantification were acquired at matched exposure times or laser settings and processed using identical settings. Quantifications were normalized within each respective experiment with n = 3 independent experiments unless otherwise specified in figure legends. Image acquisition was performed on a Leica DMI4000B laser scanning confocal microscope  or a Nikon W1 dual camera spinning disk confocal microscope (Northwestern University Center for Advanced Microscopy). Unless otherwise specified, image acquisition was performed through the z-dimension at 0.3–0.5 mm intervals and individual planes were projected into maximum intensity images.2 as calculated by the NIS-Elements Advanced Research v5 software . Fold change was determined by averaging the values from all control neurons and normalizing to this average.The nuclear region of interest was defined by DAPI and the mean signal intensity of MATR3 as the mean pixel intensity per mmFISH images were acquired in a blinded manner using a Plan Apo l 60× 1.45 NA oil immersion objective through the z dimension at 0.3 mm intervals and set to equal contrast. Images were cropped to single neuron dimensions and analyzed by individual plane for nuclear G4C2 foci in a blinded manner by the primary operator in one sitting. We defined “G4C2 foci” as signals that met the following criteria: (a) diameter > 0.8 µm (b) signal intensity > 1.5 fold higher than diffuse nuclear signal (c) punctate morphology and (d) nuclear localization.The assessment of the % of G4C2 RNA foci that colocalized with MATR3 was performed by identifying nuclear G4C2 foci as described above and tested for the presence or absence of total MATR3 signal (punctuate and diffuse). The assessment of the % of MATR3 puncta that colocalized with G4C2 RNA foci was performed by identifying nuclear MATR3 puncta  and tested for the presence or absence of a G4C2 RNA focus.Drosophila lines used in this study and their respective sources are outlined in Table Drosophila stocks were cultured on standard dextrose media on a 12-h light/dark cycle.The detailed list of Drosophila eyes was driven using the GMR-gal4 driver at 28 °C. One day old F1 adults (or 30-day old adults for aging experiments) were collected and external images of the eye were using Leica M205C microscope equipped with a Leica DFC450 camera. Percent external eye degeneration was quantified in an unblinded manner using a previously published scoring system [Expression of transgene in g system , 58, 60.For conditional pan-neuronal expression, G4C2-30R and UAS-MATR3 lines were crossed with inducible driver, ElavGS-Gal4. Day 1 adults from the F1 progeny were collected every 24 h and moved to standard media mixed with 20 mM RU486 to induce transgene expression in adults. The adults were cultured at 28 °C and death was recorded every other day. Motor function was assessed on day-30 of their lifespan. Locomotion was assessed using the RING assay, as previously described  with a fDrosophila lines, FLAG-MATR3 constructs were cloned into pUASTaTTB vector between NotI-XhoI restriction sites. All sequences were verified by Sanger sequencing. The pcDNA3.0-IRES2X-G4C260-Dendra2 construct was a generous gift from Dr. Aaron Haeusler at Thomas Jefferson University. Ambion Silencer™ Select siRNA for MATR3 (s18897) and scrambled negative control (4390843) were obtained from Thermofisher.FLAG-tagged MATR3, ΔRRM1, ΔRRM2, ΔZNF1 and ΔZNF2 in pCMV-Tag2B mammalian expression vector were a gift from Yossi Shiloh . For generating ® CRL-3216™) were cultured in Advanced DMEM supplemented with 10% FBS and 1% Glutamax and grown at 37 °C and 5% CO2. HEK293T cells were transiently transfected using Lipofectamine 3000 (Invitrogen L3000001) following manufacturer instructions.HEK293T cells (ATCC10 oligomer biotinylated at the 5′ end was synthesized by Integrated DNA Technologies Inc. (IDT) and RNA pull-down was performed as previously described [2) were pre-cleared with 50 µL of pre-washed Dynabeads Streptavidin Magnetic Beads (Thermofisher 11205D) at 4 °C overnight on a rotary shaker. 4 µg of biotinylated RNA was heated to 90 °C for 2 min in RNA structure buffer  and incubated at for 20 min at RT. Biotin-G4C210 RNA oligomer was added to the pre-cleared nuclear extract and incubated at 4 °C for 4 h. 50 µL of pre-washed Dynabeads Streptavidin Magnetic Beads was added to each reaction and incubated at 4 °C for 1 h. After incubation with the nuclear extracts, beads were washed 5 times in wash buffer . Co-precipitated proteins were eluted from bound biotinylated RNAs in wash buffer and boiled in 1× NuPage LDS-Sample buffer (Invitrogen NP0007) at 95 °C for 5 min. Co-precipitated proteins were detected by Western Blot. All buffers were freshly supplemented with 0.5 mM DTT, 0.1 U/mL RNase inhibitor, 0.1 µg/µL tRNA and 5 mM EDTA.G4C2escribed . NuclearDrosophila tissue was first flash frozen on dry ice and crushed using pestles. For preparation of lysate, crushed tissue or cells were incubated in RIPA buffer: 150 mM NaCl, 1% NP40, 0.1% SDS, 1% sodium deoxycholate, 50 mM NaF, 2 mM EDTA, 1 mM DTT, 2.5 mM Na orthovanadate, 1× protease inhibitor cocktail (Roche 11836170001), pH 7.4. The samples were sonicated in an ultrasonic bath and centrifuged down at 12,000×g for 10 min. The supernatant was boiled in 1× NuPage LDS-Sample buffer (Invitrogen NP0007) at 95 °C for 5 min.Primary antibodies: mouse anti-FLAG ; mouse anti-α tubulin Drosophila) and GAPDH (for human) using Integrated DNA Technologies PrimeTime qPCR Assay (www.idtdna.com). The comparative Ct method was used for analyzing the fold change differences [RNA was isolated using RNeasy Mini Kit (Qiagen) according to manufacturer instructions. cDNA synthesis was performed using the iScript Select cDNA Synthesis Kit  and was subsequently run using the Bio-Rad iQ™ Supermix 170-8862) on a 96-well plate  on Applied Biosystems StepOnePlus Real-Time PCR system. Primer pairs and probes were designed for each target of interest and housekeeping control α-tubulin  Representative confocal images of control iPSC-MN indicated by MAP2 (gray). White dotted box represents area of high magnification in the images on the right. FISH-IF staining of G4C2 RNA foci (green) and MATR3 (red) showed no signal for G4C2 foci in control iPSC neurons, and thus no colocalization between MATR3 puncta (yellow arrow) and G4C2. (B) FISH-IF of control post-mortem brains are negative for G4C2 foci. White-dotted circle demarcates the nucleus. Asterisks denote autofluorescence due to lipofuscinControl iPSC-derived motor neurons and post-mortem brain tissues are negative for G4C2 foci.  Representative confocal images of C9-ALS patient-derived iPSCs that were differentiated to neurons  showing colocalization between G4C2 RNA foci (green) with MATR3 (red) in diffuse form (yellow arrows). Dotted-white boxes show G4C2 foci represented in the high-magnification panels on the right (B) The percentage distribution of MATR3 puncta that co-localize with G4C2 foci (blue) and are independent of G4C2 foci (red) in two independent C9-ALS iPSC MNs (C9-ALS #1 and C9-ALS #2) and the combined averageG4C2 colocalization with punctate and diffuse MATR3 in C9-ALS iPSC MNs.  Representative confocal images of C9-ALS iPSC differentiated to neurons  stained for Histone H3 nuclear protein (green/gray) (B) Quantification of endogenous Histone H3 immunofluorescence levels in MAP2+ neurons revealed no significant differences in the levels of nuclear Histone H3 in C9-ALS iPSC-MNs compared to that in control iPSC-MNs (Unpaired t test) n = 3 (C) Quantification of HIST1H3D mRNA fold change in control and C9-ALS iPSC MNs showed no significant differences in the mRNA levels in C9-ALS iPSC-MNs compared to that in control (Unpaired t-test) n = 10-12. Error bars indicate S.E.MLevels of nuclear Histone H3 not altered in C9-ALS patient neurons.  Representative images of Drosophila eyes from flies expressing G4C2-30R along with UAS-EGFP (control for GAL4 dilution), UAS-MATR3, or (B) UAS-MATR3-ΔRRM2. Flies expressing G4C2-30R develop exacerbated eye degeneration upon aging, indicated by increase in de-pigmentation, ommatidial fusion and development of new necrotic patches. Ectopic expression of MATR3 continued to strongly suppress eye degeneration upon aging. Ectopic expression of MATR3-ΔRRM2 also suppressed eye degeneration in G4C2-30R flies at 30-days, however to a much lesser extent compared to full-length MATR3. (C) Quantification of external eye degeneration showed statistically significant suppression of G4C2-30R- mediated eye degeneration upon MATR3 expression, and to a milder extent, upon MATR3-ΔRRM2 expression  n ≥ 50 flies per genotype. Error bars indicate S.E.M. ****p value < 0.0001MATR3-mediated suppression G4C2-30R toxicity retained upon aging.  Representative images and (B) quantification of external eye degeneration of Drosophila eyes from flies expressing RNA-binding proteins TDP43, FUS and EWSR1 on its own (top) or co-expressed with G4C2-30R (bottom). Expression of either TDP43, FUS or EWSR1 on its own resulted in external eye degeneration , that was further exacerbated when co-expressed with G4C2-30R  suggesting compounded toxicities. n ≥ 50 flies per genotype. Error bars indicate S.E.M. ***p value < 0.001; ****p value < 0.0001Ectopic expression of TDP43, FUS and EWSR1 do not suppress G4C2-30R toxicity in Additional file 6: Figure S6.A) Representative western blot of full-length MATR3 and deletion variants: ΔRRM1, ΔRRM2, ΔZF1 and ΔZF2. α-tubulin was used as loading control. Deletion variants are lower in size compared to full-length MATR3. (B) Representative images Drosophila eyes from flies expressing MATR3 deletion variants that showed normal (non-degenerative) eye phenotypeDeletion in functional domains of MATR3 do not cause external eye degeneration.  Representative western blot of endogenous MATR3 in HEK293T cells that have been treated with either scrambled control siRNA or MATR3 siRNA. (B) Quantification of 3 independent blots showed statistically significant reduction of MATR3 protein levels in cells treated with MATR3 siRNA compared to scrambled control. (Mann–Whitney U-test). (C) Representative confocal images of C9-ALS patient derived iPSC-MNs  transfected with either scrambled siRNA or MATR3 siRNA and stained for MATR3 (red)Validation of siRNA mediated knockdown of


We recently reported “CapZyme-seq,” a 5′-RNA-seq method that enables the differential detection and quantitation of relative yields of NCIN-capped RNA and uncapped 5′-triphosphate RNA. Here we provide the protocol for constructing cDNA libraries for CapZyme-seq.Nucleoside-containing metabolites such as the oxidized and reduced forms of nicotinamide adenine dinucleotide , 3′-desphospho-coenzyme A (dpCoA), and flavin adenine dinucleotide (FAD) can be incorporated as RNA 5′ end caps by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase. We recently reported “CapZyme-seq,” a 5′-RNA-seq method that enables the differential detection and quantitation of relative yields of NCIN-capped RNA and uncapped 5′-triphosphate RNA. Here we provide the protocol for constructing cDNA libraries for CapZyme-seq.Nucleoside-containing metabolites such as the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD CRITICAL: Use nuclease-free H2O at all steps whenever H2O is needed.CRITICAL: Perform all steps in a nuclease-free environment.in vitro (part A) or in vivo (part B) as input.Libraries can be prepared using RNA generated in vitro, the experimenter controls the identity of the NCIN added to transcription assays, and thus controls the identity of the NCIN-capped RNA products used as input. Transcription assays are performed in the presence and absence of an NCIN; results obtained for assays performed in the absence of an NCIN are used as a negative control, or background correction .For CapZyme-seq analysis of RNA products generated rrection . We recoTIMING: ∼3 h1.in vitro transcription assays as indicated below.Mix reagents for 2.Incubate the mixture at 37°C for 10 min to form RNAP-promoter open complexes.3.+) as indicated below and incubate at 37°C for 10 min.Prepare NTP mixtures with or without an NCIN  to the solution containing RNAP-promoter open complexes (prepared in 2). Incubate at 37°C for 15 min and stop the reactions by adding 0.5 M EDTA (pH 8) to a final concentration of 50 mM.5.Add 10 μL of 3 M NaOAc, mix by vortexing for 30 s, add 330 μL of 100% EtOH, and precipitate nucleic acids at −80°C for 16 h.6.g at 4°C, wash pellets 3 times with cold 80% EtOH, resuspend in 30 μL of nuclease-free water, and add 30 μL of 2x RNA loading dye.Recover the nucleic acids by centrifugation for 30 min at 21,000 x TIMING: ∼4 h1.Load sample (from step 6 above) on a 10% TBE-urea slab gel along with a ssRNA size ladder. Run gel in 1x TBE buffer until bromophenol blue dye front reaches ∼3/4 of the length of the gel. Samples for distinct transcription reactions should be run using separate gels.2.Incubate gels with SYBR Gold nucleic acid gel stain and visualize bands with UV transillumination. Excise RNA products of the expected size from the gel using a scalpel. Gels for distinct transcription reactions should be stained in separate containers.3.x g for 2 min or until all gel fragments have passed through the holes in the upper 0.5 mL tube into the lower 1.7 mL tube.Assemble two tubes to crush the gel pieces. Pierce one to three holes in the bottom of a 0.5 mL low binding microfuge tube with a 23-gauge needle. Place this tube into a 1.7 mL low binding microfuge tube. Place gel slices into the upper tube and centrifuge the tube-in-tube assembly at 14000 4.x g for 1 min, and transfer the eluate into a 1.7 mL tube.Add 400 μL of gel elution buffer  to the crushed gel and incubate the slurry at 70°C for 10 min. Mix by vortexing for 30 s, transfer the slurry to a Spin-X tube filter, centrifuge at 14,000 5.Remove the crushed gel from the Spin-X tube, mix with 400 μL of gel elution buffer, and repeat steps described in step 4 above.6.Combine the eluates from steps 4 and 5, add 80 μL of 3M NaOAc and 8.8 μL of glycogen (10 mg/mL), mix by vortexing for 30 s, add 650 μL of isopropanol, mix by vortexing for 30 s, and precipitate RNA at −80°C for 16 h.PAUSE POINT: RNA can be stored at −80°C for ∼6 months.7.g to pellet RNA. Wash pellets 3 times with 1 mL of 80% ice-cold EtOH.Centrifuge tubes at 4°C for 30 min at 21,000 x 8.Allow the pellet to air-dry for ∼5 min. Resuspend RNA in 10 μL of nuclease-free water. Pipette the suspension and incubate on ice for at least 20 min to fully resuspend the RNA.Note: The amount of RNA generated in four 100 μL reactions is sufficient for use in two separate enzymatic treatments (Rpp and NudC or Rai1) and two distinct control reactions .3.g at 25°C. Remove supernatant and freeze cell pellets rapidly on dry ice. Store cell pellets at −80°C.Harvest 2 mL of cell suspension into 2-mL tubes and collect cells by centrifugation for 1 min at 21,000 x PAUSE POINT: Cell pellets can be stored at −80°C for ∼6 months.Note: Use one cell pellet for RNA isolation, store the remaining pellets at −80°C as a back up.TIMING: ∼3 h1.g at 4°C to remove insoluble material. Transfer supernatant to a fresh tube.Resuspend cell pellet in 600 μL of TRI Reagent solution. Incubate at 70°C for 10 min and centrifuge for 10 min at 21,000 x 2.Add 100% EtOH to a final concentration of 60.5% and divide the mixture equally between two Direct-zol spin columns (Zymo Research). Perform DNase I treatment on-column according to the manufacturer’s recommendations.3.Elute RNA from the column using 30 μL of nuclease-free water heated to 70°C. Repeat the elution two more times. Pool the eluates.4.Add 10 μL of TURBO DNase 10x buffer and 1 μL of TURBO DNase to the recovered RNA. Incubate the mixture at 37°C for 30 min to remove residual DNA.5.a.2O to TURBO DNase-treated RNA to a total volume of 200 μL, mix with 200 μL of acid phenol/chloroform pH 4.5. Mix by vortexing for 10 s.Add nuclease-free Hb.g at 25°C and recover the upper aqueous phase containing RNA.Centrifuge for 30 s at 21,000 x c.g at 25°C and remove the majority of the lower organic phase. Centrifuge for 1 min at 21,000 x g at 25°C and remove the residual organic phase.Add 200 μL of chloroform to remove residual phenol, mix by vortexing for 10 s. Centrifuge for 30 s at 21,000 x d.PAUSE POINT: RNA can be stored at −80°C for ∼6 months.Add 20 μL of 3M NaOAc and 2.2 μL of glycogen (10 mg/mL), mix by vortexing for 30 s, add 670 μL of 100% EtOH, mix by vortexing for 30 s, and precipitate RNA at −80°C for 16 h.e.g to pellet RNA. Wash pellets 3 times with 1 mL of 80% ice-cold EtOH.Centrifuge at 4°C for 30 min at 21,000 x f.Note: The typical yield of RNA isolated from 2 mL of an E. coli cell suspension is ∼60 μg.Air-dry the pellet for ∼5 min. Resuspend RNA by addition of 50 μL of nuclease-free water. Pipette the suspension and incubate on ice for at least 20 min to permit complete resuspension of the RNA. Measure the concentration using a NanoDrop.Purify RNA using acid phenol:chloroform pH 4.5.TIMING: ∼3 h1.Use MICROBExpress Kit to deplete rRNAs from 9 μg of recovered RNA according to the manufacturer’s recommendations.2.Measure the volume of rRNA-depleted RNA separated from beads. Add 10% volume of 3M NaOAc and 1% volume of glycogen (10 mg/mL), mix by vortexing for 30 s, add 3 volumes of 100% EtOH, and precipitate RNAs at −80°C for 16 h.PAUSE POINT: RNA can be stored at −80°C for ∼6 months.3.B, RNA isolation, step 5e-f). Resuspend RNA in 30 μL of nuclease-free water. Measure the concentration with a NanoDrop.Recover RNA as in  to remove 5′ end phosphates prior to performing the NudC or Rai1 treatments described below.1.Assemble the reaction as indicated below.2.Incubate the reaction at 37°C for 1 h.3.2O to 200 μL, mix with 200 μL of acid phenol/chloroform (pH 4.5), and purify RNA using the procedure described in .Add nuclease-free H4.Resuspend the RNA pellet in 10 μL of nuclease-free water. Pipette the suspension and incubate on ice for at least 20 min to ensure the complete resuspension of RNA. Samples can be stored at −80°C prior to use in subsequent enzymatic reactions.Note: The protocol for construction of cDNA libraries in CapZyme-seq enables single-nucleotide-resolution analysis of RNA 5′ ends. In particular, single-stranded oligonucleotide adapters are ligated to RNA 5′ ends and the 5′-adapter-ligated products are used as templates for generation of cDNAs that are analyzed by high-throughput sequencing. For each sequencing read, the precise identification of the RNA 5′ end sequence is accomplished by using the sequence of the 3′ end of the 5′ adapter as a point of reference.TIMING: ∼1-2 hCapZyme-seq relies on ligation of single-stranded oligonucleotide adapters to RNA 5′ ends prior to cDNA synthesis. This ligation step requires that the RNA have a 5′-monophosphate. Thus, analysis of RNAs without a 5′-monophosphate requires enzymatic processing of the RNAs to yield RNAs having a 5′-monophosphate. In CapZyme-seq the processing enzymes NudC and Rai1, which are specific for NCIN-capped RNA, along with RNA 5′ polyphosphatase (Rpp), a processing enzyme specific for uncapped, 5′-triphosphate RNA, are employed to enable differential detection and quantitation of NCIN-capped RNA and uncapped, 5′-triphosphate RNA.+, NADH, dpCoA, and FAD  followed by the remainder of the RNA . Rai1 pr and FAD .a.Set hea.Assemble the reactions as indicated below.b.Incubate at 37°C for 30 min.For selective processing of uncapped, 5′-triphosphate RNA to 5′-monophosphate RNA we use the RNA processing enzyme RNA 5′ polyphosphatase (Rpp), which cleaves the phosphodiester bond between the triphosphate β and α phosphates, yielding products comprising 5′-p- followed by the remainder of the RNA . Reactio+) followed by the remainder of the RNA .Elute RNAs from gel using the procedure described in .Add nuclease-free Hf.Resuspend RNA in 10 μL of nuclease-free water. Pipette the suspension and incubate on ice for 20 min to permit solubilization of RNA.TIMING: ∼18-20 hNudC involves quantitative comparisons between RNA samples treated with Rpp or NudC, along with two “mock” treated samples. CapZyme-seqRai1 involves quantitative comparisons between RNA samples treated with Rpp or Rai1, along with two “mock” treated samples. Thus, to enable quantitative comparisons between the four samples in CapZyme-seqNudC or CapZyme-seqRai1, a unique, barcoded 5′-adaptor oligonucleotide is used in ligation reactions performed with each of the four individual samples .Place gel slices into 4 to 8 tube-in-tube assemblies and recover nucleic acids using the procedure described in (7.in vitro and 30 μL for RNA generated in vivo). Pipette the suspension and incubate on ice for at least 20 min to enable full resuspension of the 5′-adaptor-ligated products.Resuspend adaptor-ligated products in nuclease-free water. .8.Resuspend cDNAs in 20 μL of nuclease-free water. Pipette the suspension and incubate on ice for at least 20 min to enable the complete resuspension of cDNA. Measure the concentration of nucleic acids in each solution using Qubit ssDNA Assay kit (ThermoFisher).PAUSE POINT: cDNA can be stored at −80°C for at least one week.Note: Ensure that any primers used for RT contain sequence complementary to the Illumina 3′ PCR primer.Mix 15 μL of 5′-adaptor-ligated products with 0.3 μL of 100 μM of the reverse transcription (RT) primer .TIMING: ∼7-8 h1.9 molecules/μL.Dilute the cDNA with nuclease-free water to a concentration of ∼102.Prepare emulsion PCR (ePCR) reaction mix as indicated below.3.Perform ePCR reactions using the recommended cycling conditions: 10 s at 90°C, 20 cycles of 5 s at 90°C, 5 s at 60°C, and 15 s at 72°C, followed by 5 min at 72°C.4.PAUSE POINT: cDNA can be stored at −80°C for at least one week.Isolate cDNA from ePCR reactions using Micellula DNA Emulsion and Purification Kit. Elute cDNA from the column filter with 150 μL of the elution buffer provided with the kit. Add 15 μL of 3M NaOAc, 1.5 μL of glycogen (10 mg/mL) and 500 μL of 100% EtOH. Mix solution by vortexing and precipitate cDNA at −80°C for 16 h.5.Centrifuge tubes at 4°C for 30 min to pellet cDNAs. Wash pellets 3 times with 1 mL of 80% ice-cold EtOH and resuspend cDNAs in 20 μL of nuclease-free water.6.Mix 1 μL of the resuspended cDNA with 6x Orange DNA load dye and run a non-denaturing 10% TBE gel to check the library quality. Load O’Gene Ruler Ultra Low Range DNA ladder as size standard.7.A, Isolation of RNA products).If residual primers are present, mix the rest of cDNA with 6x Orange DNA load dye and isolate amplified cDNAs by gel electrophoresis using a non-denaturing 10% TBE gel. Isolate products by gel excision using the procedure described in , and an equal volume of isopropanol. Precipitate cDNAs at −80°C for 16 h.10.Centrifuge samples at 4°C for 30 min to pellet cDNAs. Wash pellets 3 times with 1 mL of 80% ice-cold EtOH and resuspend cDNAs in 20 μL of nuclease-free water.11.Measure the concentration of the amplified libraries using Qubit dsDNA HS Assay kit. Submit libraries for sequencing.Note: Use of ePCR minimizes the generation of amplicons derived from “template switching.”Note: Optimization of the amplification of cDNAs by ePCR is critical for successful library sequencing. In our experience, use of a Qubit ssDNA Assay kit to prepare ssDNA dilution of ∼109 molecules/μl provides an imprecise measure of ssDNA concentration, likely because of chemicals in the solution that interfere with quantitation.Note: Barcoded libraries generated by CapZyme-seq are suitable for sequencing on Illumina NextSeq and Illumina HiSeq platforms in high-output mode. Sequencing primers provided by Illumina typically contain a mixture of several oligos. To avoid potential complications that may arise due to the presence of additional primers in the Illumina reagents we use a custom sequencing primer (s1115).+-capped and uncapped RNA and to define preferred transcription start-site positions and consensus promoter sequences for NAD+ capping by bacterial RNAP in vitro and in vivo  underscores the need to determine, for each NCIN, the promoter-sequence determinants for NCIN capping. The CapZyme-seq method, either using the same decapping enzymes described here or using alternative decapping enzymes with alternative decapping specificities, should enable analysis of determination of NCIN-capping efficiencies and promoter sequence determinants thereof for each nucleoside-containing metabolite that can serve as an NCIN.The prevalence of nucleoside-containing metabolites that can function as NCINs . A goalMultiple bands are visible on the gel below the 5′ adaptor during size selection of 5′-adaptor-ligated products.Have adaptor resynthesized or perform gel purification of adaptor prior to ligation .Insufficient amount of 5′-adaptor-ligated products.Increase amount of starting material; collect all of the upper aqueous phase during the acid phenol/chloroform purification step.Insufficient yield of ePCR amplicons.If the library after ePCR amplification is < 2.5 ng/μl as detected by Qubit dsDNA Assay kit, (i) perform two ePCR reactions per library; (ii) increase the volume of diluted ssDNA used in ePCR reaction by two-fold; (iii) increase the number of amplification cycles up to 30.A large portion of the library is comprised of a ∼150 bp product.After 5′ adapter-ligated products or ssDNA have been subjected to gel electrophoresis, place the gel in SYBR Gold nucleic acid gel stain, incubate for 5 min with low agitation, and carefully excise products of the desired size. Alternatively, prior to incubation with SYBR Gold nucleic acid gel stain, remove the lower part of the gel containing an excess of 5′ adapters or an excess of RT primer(s), stain the remaining part of the gel, and carefully excise products of the desired size. For the final cDNA library, also perform gel electrophoresis, gel staining, and careful excision of products of the desired size.


Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides.Bacteria of the genus  Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.Carbapenem-resistant IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance. Acinetobacter baumannii have become increasingly problematic, particularly among immunocompromised patients and patients in intensive care units, due to its ability to acquire and develop resistance to multiple antimicrobials and thereby severely limit treatment options  complex due to difficulties in identifying these bacteria by traditional biochemical methods  were A. baumannii followed by A. nosocomialis (10.4%), with multidrug resistance much more prevalent in A. baumannii  due to the association of blaNDM-1 with other resistance genes  or Ambler class B enzymes, such as the New Delhi metallo-β-lactamase (NDM) group, represents another class of acquired carbapenemases that have been found in om India  and Chinom India . MBLs, iom India . The bla-encoded . NDM-1 c-encoded , 23, 24.ce genes , 22.Acinetobacter clinical isolates, A. baumannii AC1633 and A. nosocomialis AC1530, obtained from the main tertiary hospital in the state of Terengganu in Peninsular Malaysia. We show that in these two isolates, the blaNDM-1 gene is colocated with blaOXA-58 on a large ca. 170-kb plasmid along with various other antimicrobial-resistance genes, and that the carriage of this plasmid in these two isolates likely led to their MDR status. We also show sequence evidence that this plasmid was likely derived from two plasmids that separately encoded blaNDM-1 and blaOXA-58 in a Malaysian A. pittii isolate and that they combined via an IS1006-mediated recombination event.Here, we report the whole-genome sequences of two NDM-1-producing, carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530 are part of our collection of Acinetobacter spp. clinical isolates that were obtained since 2011 from Hospital Sultanah Nur Zahirah (HSNZ), the main public tertiary hospital in the state of Terengganu, Malaysia , cephalosporins , β-lactam/β-lactamase inhibitor combinations (piperacillin-tazobactam and ampicillin-sulbactam), trimethoprim-sulfamethoxazole, ciprofloxacin, and tetracycline. AC1633 also showed resistance to gentamicin but was susceptible to the other aminoglycosides tested , as well as to levofloxacin, doxycycline, and the polymyxins (polymyxin B and colistin). A. nosocomialis AC1530 was resistant to the carbapenems , cephalosporins , trimethoprim-sulfamethoxazole, and gentamicin, but was susceptible to all other antibiotics tested. Thus, both AC1633 and AC1530 are categorized as MDR following the criteria proposed by the joint commission of the U.S. Centers for Disease Control and Prevention (CDC) and the European Centre for Disease Prevention and Control (ECDC) (l (ECDC) .A. baumannii A1633 and A. nosocomialis AC1530 indicated the presence of the blaNDM-1 and blaOXA-58 carbapenemase genes. Production of the NDM-1 metallo-β-lactamase (MBL) in both isolates was validated by testing with the Etest MBL kit (bioMérieux). Further analyses of the assembled genome data of AC1530 and AC1633 revealed the possibility that the blaNDM-1 and blaOXA-58 genes could be harbored in either one or two large plasmids in both isolates; however, this was difficult to ascertain as there were more than 20 assembled contigs from each isolate’s genome data that could potentially belong to these plasmids. Thus, the genomic DNA of these two isolates was subjected to PacBio sequencing and hybrid assembly was then performed on the PacBio and Illumina reads. The resulting assembled genome features of these two isolates are listed in Analysis of the Illumina-sequenced genomes of A. baumannii AC1633 is nearly 4.4 Mb in size and is comprised of a single chromosome of 4.36 Mb (accession no. CP059300) and four plasmids designated pAC1633-1 , pAC1633-2 , pAC1633-3 , and pAC1633-4 . A. baumannii AC1633 is typed as ST2089 under the Oxford multilocus sequence type (MLST) scheme and ST126 under the Pasteur MLST scheme. AC1633 does not belong to any of the major A. baumannii global clonal lineages, and phylogenetic analysis of the whole-genome sequence in comparison with the sequences of selected A. baumannii isolates (A. baumannii CIP70.10 (ATCC 15151), which was isolated in France in 1970 and is an important reference strain due to its susceptibility to most antimicrobials  lineage /ST2 (Pasteur)  biosynthetic genes  . A recenic genes , AC1633 10.1128/mSphere.01076-20.1TABLE S1Acinetobacter baumannii strains that were used in comparative genomics and phylogenetic tree construction in Acinetobacter nosocomialis strains that were used in comparative genomics and phylogenetic tree construction in Table S1, DOCX file, 0.1 MB.(A) Characteristics of the Copyright © 2021 Alattraqchi et al.2021Alattraqchi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A. nosocomialis AC1530 has a single chromosome of 3.98 Mb (CP045560.1) and a plasmid of 173.9 kb designated pAC1530 (CP045561.1). AC1530 was assigned by the curators of the PubMLST database  and Oxford ST2195 . Phylogenetic analyses showed that the closest relative of AC1530 is A. nosocomialis T228 (accession no. JRUA01000001.1), a clinical isolate obtained from Bangkok, Thailand in 2010  and two alleles in the Pasteur scheme  with T228.database  to the P in 2010 . HoweverA. baumannii AC1633 and A. nosocomialis AC1530 came from the large ca. 170-kb plasmid, pAC1633-1 and pAC1530, respectively  gene blaADC-25 (accession no. EF016355.1) (blaOXA-51/blaOXA-51-like or blaADC genes through insertion of ISAba1 or related IS elements that harbor outward-directing promoters (blaOXA-116 and blaADC-25 genes in A. baumannii AC1633. Tetracycline resistance in AC1633 is likely mediated by the tetA(39) gene that was carried on the smaller 12.6-kb plasmid, pAC1633-2 (adeABC operon that encodes the multidrug resistance-nodulation-cell division (RND) family efflux system along with its two-component regulatory system, adeRS, which is located upstream and transcribed divergently from adeABC. This efflux system is usually chromosome encoded in Acinetobacter and the multidrug resistance phenotype has been shown to correlate with overexpression of adeABC (A. baumannii AC1633 chromosome harbors another copy of adeRS and adeAB (nucleotides [nt] 1011642 to 1016860), but adeC was absent. There were no obvious signs of mobile elements or genomic islands in the vicinity of the chromosomal adeRS-adeAB genes that could explain the deletion of adeC. The chromosome of AC1633 also harbors genes encoding the other Acinetobacter RND family efflux pumps adeFGH and adeIJK, along with their respective regulatory genes adeL and adeN  family efflux pump  . In someromoters , 35, 36;AC1633-2 , and thaf adeABC , 38. Intand adeN , 39, thrlux pump .A. nosocomialis AC1530, very few antimicrobial resistance genes are found in its chromosome, and the only resistance gene encoding an antibiotic-inactivating enzyme is a blaADC-encoding cephalosporinase that shared 94% amino acid sequence identity with ADC-68 (accession no. AGL39360.1)  . Howeverily abeS . The samily abeS  and, theA. baumannii AC1633 and pAC1530 from A. nosocomialis AC1530 were nearly identical except at five regions as follows (Aba11 into an open reading frame (ORF) encoding an 85-aa (amino acid) hypothetical protein  which contains a ribosomal protein L7/L12 C-terminal domain (pfam00542) in pAC1530 at nt 40824 (nt 40825 to 41927 of pAC1633-1); this ORF is upstream of the traMNO genes and lies within a cluster of genes that are proposed to be part of the conjugative transfer region for the plasmid; (ii) an IS4 family transposase at nt 58863 to 60133 in pAC1633-1 with no matches to existing IS elements in the ISFinder database ; (iii) a 255-bp insertion within a hypothetical ORF at nt 89589 of pAC1530 in pAC1633-1 (nt 91978 to 92233); no characteristic signature sequences of mobile elements could be detected within this short fragment; (iv) insertion of ISAba11 into an ORF encoding a putative toxin of the Zeta toxin family in pAC1530; this insertion, at nt 101270 of pAC1633-1, led to a 5-bp direct repeat (TATAG) in pAC1530 (nt 98631 to 99731); and (v) addition of an relBE toxin-antitoxin (TA) system along with a downstream ORF encoding a protein of the SMI1/KNR4 family in pAC1530 at nt 164446 of pAC1633-1; this 1,192-bp fragment is flanked by pdif (XerD/XerC) recombination sites, more of which will be discussed in a later section. pAC1633-1 and pAC1530 belong to a group of diverse Acinetobacter plasmids that do not have an identifiable replication initiator or replicase (Rep) protein (Plasmids pAC1653-1 from  follows : (i) insn pfam005 in pAC15 protein .blaNDM-1 and blaOXA-58 carbapenemase genes (aac(3)-IId group is a subclass of the AAC(3) enzymes that catalyze acetylation of the NH2 group at the 3-position of the aminoglycoside 2-deoxystreptamine nucleus and usually confers resistance to gentamicin, sisomicin, and fortimicin , aph(6)-Id and aph(3”)-Ib, and both genes confer resistance to streptomycin (Acinetobacter strains). Both genes were adjacent to each other and are flanked by IS elements, with ISAba1 upstream of aph(3”)-Ib and IS1006 downstream of aph(6)-Id -Id .sul2 gene that encodes dihydropteroate synthase, which confers sulfonamide resistance, is sandwiched between ISAba1 upstream and ISCfr1 downstream in both pAC1633-1 and pAC1530  genomic island designated GIsul2 -IId, aph(6)-Id and aph(3′’)-Ib, along with the sul2 gene, were found to be in a 42,125-bp fragment in pAC1530 that was flanked by ISAba1 with characteristic 9-bp direct repeats of the target sequence in a typical composite transposon-like structure. This 42-kb fragment also included the blaOXA-58, msrE, mphE, and adeRS-adeABC resistance genes that were nested in a 29,670-bp fragment flanked by IS1006 , as has been previously reported for this transposon  separated by a 6-bp spacer, which are recombination targets for the XerC and XerD proteins (–A. baumannii pABVA01a plasmid (dif modules (named for plasmid dif) which comprise a pair of inverted pdif sites surrounding a gene or several genes, have been described , which are in turn flanked by a pair of inverted pdif sites, an arrangement that has been previously reported for the A. johnsonii-encoded plasmid pXBB1-9, but without the presence of ISAba11 (Aba11 led to a characteristic 5-bp direct repeat (ATTTA) of the target sequence. In some Acinetobacter, a full ISAba3 or ISAba3-like element is found upstream of blaOXA-58, while in other instances this upstream ISAba3 is disrupted by other IS elements  were found within this region and they were each flanked by a pair of inverted pdif sites. As mentioned earlier, one of the differences between pAC1633-1 and pAC1530 is the addition of a relBE TA system and an ORF encoding a protein of the SMI1/KNR4 family in pAC1530, upstream of higBA-2  sequences showed that in pAC1633-1, the pdif (XerD/C) sequences upstream of higBA-2 are a hybrid of the pdif (XerD/C) sequences that flanked the relBE-SMI1/KNR4 module in pAC1633-1, i.e., the sequences of the XerD site are identical to the sequences found upstream of relBE, while the 6-bp spacer and the XerC site sequences are identical to the sequences upstream of higBA-2 in pAC1633-1   . Currentucidated . Deletiointed at , 56, andAcinetobacter pittii AP882 designated pNDM-1_AP882 (accession no. CP014478) and pOXA-58_AP882 (accession no. CP014479). Notably, A. pittii AP882 was isolated from Peninsular Malaysia in 2014 but from a different state (Perak)  that encodes two transposases characteristic of the IS3 family flanked by 21/22-bp imperfect inverted repeats and a 5-bp direct repeat (ACCTG) of the target sequence  that is characteristic of this IS element -Ib, aph(6)-Id, and aac(3)-IId. Tn6948 has an overall GC content of 50.6% compared to 38 to 39% for the surrounding genes, thus supporting its possible non-Acinetobacter origin.BLASTN analysis of pAC1633-1 and pAC1530 showed they are most similar in sequence to two plasmids found in  (Perak)  comparedsequence . Further element . The tar10.1128/mSphere.01076-20.2FIG S1Acinetobacter pittii AP882 (accession no. CP014478). The single IS1006 copy is indicated in red. The 1,940-bp region adjacent to IS1006 that differed from pAC1530/pAC1633-1 and contained an IS-like element of the IS3 family is indicated in a pale yellow box. The two ISAba1 elements that flank the composite transposon Tn6948 are highlighted in blue boxes. Antimicrobial resistance genes, including blaNDM-1, are shown as gold and orange arrows; other IS elements are shown as gray boxes with their encoded transposases in blue arrows. Green arrows are genes/ORFs that are likely involved in conjugative gene transfer, with darker green arrows showing genes that have homology with known conjugative transfer genes. Sky blue arrows are genes involved in DNA recombination and repair. Maroon arrows are ORFs/CDS encoding hypothetical proteins. (B) Circular map of plasmid pOXA-58_AP882 from Acinetobacter pittii AP882 (accession no. CP014479) showing distribution of pdif sites. The two copies of IS1006 are depicted in red, IS-encoded transposases are shown by blue arrows. The 7,191-bp fragment of pOXA-58_AP882 that is absent from pAC1530 and pAC1633-1 is labeled and highlighted in yellow. This region contains the mobA/L gene, the repBGR12 gene encoding a Rep3 family plasmid replicase of the Acinetobacter GR12 family, an ORF encoding a helix-turn-helix putative DNA-binding protein (depicted as a dark green arrow downstream of repBGR12), and a putative relE-xre toxin-antitoxin system shown as black arrows. Other TA systems are also indicated as black arrows and labeled accordingly. pdif sites are depicted as horizontal bars and are labeled as per the orientation of their XerD- and XerC-binding sites . The blaOXA-58 gene is shown as a yellow arrow and labeled. Download FIG S1, TIF file, 2.8 MB.(A) Circular map of plasmid pNDM-1_AP882 from Copyright © 2021 Alattraqchi et al.2021Alattraqchi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the blaOXA-58, msrE, mphE, and adeABC-adeRS, is flanked by two copies of IS1006  but from the same hospital is suggestive of plasmid transmissibility. Sequence analysis also indicated the presence of several conjugative transfer-related genes, most of which share between 50 to 70% amino acid sequence identities with the corresponding translated proteins of the conjugative plasmid pA297-3 from A. baumannii isolate A297  plasmids that were isolated from two different ate A297   tetracycline resistance gene which was adjacent to and divergently transcribed from a tetR(39) regulatory gene (tetAR(39) genes that made up a pdif module in plasmids pS30-1, pRCH52-1, and pAB1-H8 (dif sites that flank this tetAR(39) region in pAC1633-2 differed from those in pS30-1, pRCH52-1, and pAB1-H8 at the 6-bp spacer and the XerC-recognition site   .mobA/L gene that encodes a relaxase of the MOBQ family, indicating the possibility of the plasmid being mobilized should a suitable conjugative plasmid be present in the host cell. Since AC1633 also harbored the potentially conjugative pAC1633-1 plasmid, the ability of pAC1633-1 to mobilize pAC1633-2 was tested in conjugation experiments by selecting for transconjugants that exhibit tetracycline resistance in addition to azide resistance. Despite repeated experiments, no such transconjugants were detected, indicating that pAC1633-1 was unable to mobilize pAC1633-2. This, however, does not rule out the possibility that pAC1633-2 could be mobilized by a different type of conjugative plasmid to pAC1633-1.AC1633-2 also harbors a A. baumannii AC1633, the 9,950-bp pAC1633-3 and the 5,210-bp pAC1633-4. Neither plasmid carries any antimicrobial or metal resistance genes, nor any genes that could confer a specific phenotype to their host, and are thus characterized as cryptic plasmids. Both plasmids encode replicases of the Rep3 superfamily that are common in Acinetobacter plasmids, with pAC1633-3 belonging to the GR28 group where pAC1633-4 belongs to the GR7 group . escribed , 71. Antter spp.  and by dter spp. . ProductA. baumannii MLST database (https://pubmlst.org/abaumannii/)  trees using FastTree   following the manufacturer’s recommended protocol and the extracted DNA quality was evaluated using a Qubit 2.0 fluorometer . Genome sequencing was performed on the Illumina NextSeq  and PacBio RSII  platforms by a commercial service provider  and hybrid assembly was carried out using SPAdes (version 3.11.1) . Gene prmannii/) . Pan genFastTree  with 100mbl.de/) . The A. https://cge.cbs.dtu.dk/services/ResFinder/) (https://card.mcmaster.ca/) (https://isfinder.biotoul.fr/) (https://bioinfo-mml.sjtu.edu.cn/TADB2/index.php) (https://db-mml.sjtu.edu.cn/SecReT4/) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) to validate the genes/open reading frames (ORFs) that were predicted by the annotation and other programs. Pfam searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the nonredundant (nr) nucleotide collection and initially optimized for highly similar sequences (MegaBLAST). Regions of high sequence identities with multiple hits (such as the blaNDM-1-encoded Tn125) were then subsequently omitted from subsequent searches, which were optimized for more dissimilar sequences (discontiguous MegaBLAST) and somewhat similar sequences (BLASTN). The presence of pdif sites in pAC1530, pAC1633-1, and related plasmids was determined by BLASTN screening using known XerC/XerD and XerD/XerC sites in published reports (–Antibiotic resistance genes were identified using ResFinder (Finder/)  and the ter.ca/) , whereasoul.fr/)  was useddex.php) , and putecReT4/) . All plasearches  and the searches  were als reports –54 and mhttp://mjsull.github.io/Easyfig/)   was used to visualize and manipulate the sequences studied. Figures were drawn to scale using EasyFig 2.2.3 (asyfig/)  and CGViserver/) .A. nosocomialis AC1530 and A. baumannii AC1633, to the appropriate susceptible isolates, A. baumannii ATCC 19606 and A. baumannii AC1529. A. baumannii ATCC 19606 is the type strain of A. baumannii that has been widely used in various studies, is resistant to sulfonamides due to the presence of the sul2 gene in its chromosome, but remains susceptible to a wide range of other antibiotics  agar plates overnight. Cells were resuspended and diluted in 0.9% NaCl and selected on LB agar plates supplemented with 300 μg/ml sodium azide and 10 μg/ml imipenem. Conjugation assays were also repeated with different ratios of donor to recipient cells . To investigate if the tetA(39)-harboring plasmid pAC1633-2 could be mobilized by pAC1633-1 in A. baumannii AC1633, conjugation experiments involving AC1633 as donor were also plated on LB agar plates supplemented with 300 μg/ml sodium azide and 4 μg/ml tetracycline.A. baumannii AC1633 chromosome was deposited in GenBank under accession no. CP059300, whereas its four plasmids were deposited under the following accession numbers: pAC1633-1 (CP059301), pAC1633-2 (CP059303), pAC1633-3 (CP059304), and pAC1633-4 (CP059302). The A. nosocomialis AC1530 chromosomal sequence was deposited under accession no. CP045560.1, whereas its plasmid pAC1530 was deposited under accession no. CP045561.1.The complete sequence of the


Transitivity in plants is a mechanism that produces secondary small interfering RNAs (siRNAs) from a transcript targeted by primary small RNAs (sRNAs). It expands the silencing signal to additional sequences of the transcript. The process requires RNA-dependent RNA polymerases (RDRs), which convert single-stranded RNA targets into a double-stranded (ds) RNA, the precursor of siRNAs and is critical for effective and amplified responses to virus infection. It is also important for the production of endogenous secondary siRNAs, such as phased siRNAs (phasiRNAs), which regulate several genes involved in development and adaptation. Transitivity on endogenous transcripts is very specific, utilizing special primary sRNAs, such as miRNAs with unique features, and particular ARGONAUTEs. In contrast, transitivity on transgene and virus (exogenous) transcripts is more generic. This dichotomy of responses implies the existence of a mechanism that differentiates self from non-self targets. In this work, we examine the possible mechanistic process behind the dichotomy and the intriguing counter-intuitive directionality of transitive sequence-spread in plants. The proper regulation of gene expression is essential for the development and the adaptation of plants to their environment. Gene silencing mediated by small RNAs (sRNAs) plays a major role in this process, leading to the downregulation of gene activity at the transcriptional  or post-transcriptional level  . In addivia the vasculature can be dramatically increased by means of transitivity , serve as templates for RDR6-dependent synthesis of dsRNAs, which are subsequently processed by DCLs  gene by co-suppression was positively correlated with the strength of the 5′ regulatory sequence used, with silencing efficiency increasing when stronger promoters were employed  35S regulatory sequence. Indeed, several reports showing transitivity in plants involved the expression of a transgene using the 35S promoter . In petuemployed . In addie effect . Analysie effect , or the e effect  gives rie effect . Neverthe effect . Despitee effect . Furthersequence . Nonethesequence .OCTOPINE SYNTHASE (OCS) and the NOPALINE SYNTHASE (NOS) regulatory sequences. This specific configuration is strongly related to prokaryotic genes, and could thus, be recognized by plants as a foreign nucleic acid that needs to be shut down. The idea that such features in a transgenic sequence are perceived by the plant cell and differently handled when compared to an endogenous gene is supported by studies using a GFP reporter construct designed to mimic an endogene  tail to the nascent transcript and triggering the end of the polymerization process by the RNA polymerase II . A growiEukaryotic cells need to keep an appropriated balance of their mRNAs and quickly eliminate aberrant transcripts that might interfere with the proper cell function. An important mechanism to achieve this is through the RNA decay pathway, which degrades mRNAs missing their poly(A) tail or the 5′ cap. In plants, uncapped transcripts are efficiently degraded by XRN4, while mRNAs missing the poly(A) became targeted by the RNA exosome, which is a multimeric complex that includes SKI2, SKI3 and SKI8, among others . The RNAHEN2 (HUA enhancer 2) is another gene that when mutated leads to increased S-PTGS  tail  tail . LikewistasiRNAs . TherefoNonetheless, other factors need to be taken in consideration and probably contribute for the preferred spreading of transitivity toward the 3′ region of the target transcript. With RDR6 transcription initiating at the 3′ extremity of a sequence, siRNA production from parts of the transcript located closer to the 5′ end of the RNA would rely on the complementary strand synthesis continuing until it reaches such regions. Different studies have estimated the range of RDR6 processivity and indicate that the enzyme could support RNA synthesis spanning at least 750 nt . Howeverski3 (one of the components of the exosome) and xrn4 mutants revealed that the former has a weaker effect on the onset of S-PTGS than the latter . In principle, this should not contribute for the over-representation of siRNAs being produced from the 3′ region, since both cleavage fragments are produced at the same ratio, and the 5′ one could also support RDR6 activity in ways similar to the one seen for the 3′. But this seems not to be the case. As discussed before, each of these fragments is degraded by different processes. The 5′ fragment, which no longer carries a poly(A) tail, is degraded by the exosome, while the uncapped 3′ fragment is targeted by XRN4, with both processes competing with RDR6 for the target transcript . Therefoe latter . The autDespite the importance of transitivity to plant defense and development, several aspects of the mechanism are still poorly understood. In this review, we focused on two characteristics that are unique to this process: the considerable bias toward the production of secondary siRNAs from transgenic transcripts, and the preferential spreading of the silencing signal to the 3′ end of the gene.Several factors have been identified and used to explain the susceptibility of transgenes to transitivity, especially when triggered by siRNAs. Among them, the proper formation of the mRNA 3′ end seems to be of major importance, directly influencing the recruitment of RDR6 and compensating for the negative effect that might be associated with other elements, such as the high expression levels of some transgenes and the origin of their regulatory sequences. The competition between the RNA decay pathway and the silencing pathway also seems to play a major role, and that defects introduced into the RNA degradation machinery strongly induce PTGS. However, the biological relevance of this interplay still needs to be tested. Given the universal role of the exosome and XRN4 in degrading aberrant mRNAs, it seems unlikely that changing the efficiency of this process would be a reliable way for the cell to control gene expression, since it would probably affect several unrelated genes. Nonetheless, the identification of cell types or physiological conditions where the activity of the RNA decay pathway is altered would be a good indication that cells do indeed modulate the degradation of mRNAs to control gene expression.Despite all the advances in this field, some aspects of siRNA-triggered transitivity are still elusive. For instance, while many features affecting the onset of transitivity occur in the nucleus, such as splicing and transcription termination, RDR6 and other factors involved with secondary siRNA generation are mainly present at the cytoplasm, most specifically at specialized foci known as siRNA bodies . This diThere is no doubt that transitivity plays a central role in many different processes in plants. It adds more plasticity to the gene regulation mediated by sRNAs and at the same time boosts the intensity of the silencing signal. However, this process needs to be strictly regulated to avoid sRNA silencing going out of control and spreading to loci that should not be downregulated. The onset of transitivity has most likely evolved to be a rare event, occurring only in certain conditions. This is probably reflected in the dominance of DCL4 over DCL2 and in the relative low abundance of 22 nt sRNAs, which are known to be triggers of secondary siRNA biogenesis. The fact that transgenes are more sensitive to transitivity might be a consequence of the participation of this process in the defense against exogenous sequences, and therefore, part of the mechanism allowing plants to recognize “self” from “non-self.”FFF and PMW conceived, performed literature review, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA binding proteins (RBPs) not only control which transcripts are translated, but also determine the speed, location, and concentration of mRNA translation, through controlling multiple layers of gene regulation. Base-dominant interaction and backbone-dominant interaction categorize the two main modes of the way RBPs interact with RNA.Protein–RNA interaction: experimental techniques and computational methods  and in vivo  assays, and structural biology approach. However, both technologies have clear limitations: the assay experiments can reveal statistical patterns  of the binding RNAs to an RBP, but cannot elucidate where and how the RNA interacts with the RBP, whereas the structural biology approach can only capture a snapshot of a specified RNA binding to the RBP, without revealing any statistical property. The computational approach, on the other hand, is still at the early development stage. All the existing machine learning-based methods try to make binary predictions. That is, the state-of-the-art resolution is on predicting if a residue of an RBP is a binding residue or not. Such methods often have high false positive rates, even for known RBPs. For a previously unknown RBP, the predictions from the existing machine learning-based methods and docking-based methods are even less reliable, which hamper their applications in guiding the downstream experimental design.There are mainly two approaches to understand protein methods . The forhttp://sfb.kaust.edu.sa) at the King Abdullah University of Science and Technology (KAUST), in collaboration with groups in China and USA, is the first-of-its-kind to predict protein–RNA interactions at the RNA-constituent resolution. They formulated the problem as a seven-class classification problem, where the label space includes non-site, ribose, phosphate, and four different bases.The new tool, NucleicNet –RNA complex structures in the Protein Data Bank (PDB), and carefully removed the redundant structures and redundant chains, which resulted in a stringent dataset of 175 RNA-binding protein chains. The surface grid points are extracted and the labels are assigned to the grid points by considering the nearest RNA constituents or assigned 0 if the grid point is outside the bound RNA. The FEATURE framework is applied on a contour-manner to extract spatial, physicochemical properties of the local protein surface environment. A 16-level residual network is trained to learn the mapping between the input features and the seven classes. To optimize the problem in a more efficient way, they applied a number of techniques, such as down-sampling of the negative dataset, hierarchical classification, batch normalization, and weight decay.For any deep learning approach, data are the most critical component. Lam et al. composed a dataset that contains all the solved proteini.e., to predict if a surface residue is an RNA-binding residue or not. Although NucleicNet was trained on the 7-class classification task, when rounding the prediction results to binding/nonbinding, it can still outperform all the sequence-based predictors by a large margin. They then evaluated 7-class classification performance, to which there is no precedent method to compare, through both micro- and macro-performance measures, where ‘micro’ is sample-averaged and ‘macro’ is class-averaged.The authors tested NucleicNet on various tasks, starting from the traditional binary classification, Aquifex aeolicus ribonuclease III (Aa-RNase III), which binds to double-stranded RNA , which binds to RNA through base contacts, human Argonaute 2 (hAgo2), which binds to RNA through backbone, and nded RNA . For FBFnded RNA A, Nucleith hAgo2 B and Aa-th hAgo2 C, Nucleiin vitro and in vivo assay data. On the in vitro RNACompete datasets, NucleicNet scores on different RNA-binding sequences showed a remarkable level of agreement with the RNACompete position weight matrix scores. NucleicNet was also able to differentiate top scoring sequences from the bottom scoring ones. On the in vivo Ago2 immunoprecipitation and siRNA knockdown datasets from different cell lines of both humans and mice, NucleicNet correctly predicted asymmetry in guide strand loading for majority of the cases. These results become even more significant when considering the fact that NucleicNet was never trained on any assay data, and yet it reached a remarkable level of consistency with such high-throughput experiments.They further validated NucleicNet on In the case of known RBPs, NucleicNet can be applied to score any given RNA-binding sequence, design the most preferred binding sequence, and draw sequence logos. In the case of proteins with unknown RNA-binding functions, NucleicNet can be applied to check if the protein has a proper RNA-binding site, and if so, what the preferred RNA-binding sequences are. They further provided a webserver for the community to use NucleicNet.–RNA interactions. The deep learning model clearly does much more than ‘memorizing’ the training data. NucleicNet can be potentially applied to understand the binding mechanism and design RNAs for some important RBPs, such as Ago2, m6A-responsive RBPs, and RBPs for single guide RNA (sgRNA) in the CRISPR-Cas9 system.The aforementioned experiments demonstrated the ability of NucleicNet to capture both the statistical and physicochemical properties underlying protein–RNA interaction. Thus the input apo structure may undergo a large-scale conformational change to accommodate the binding of the RNA, which would cause the extracted physicochemical features to be imprecise. Second, the idea of NucleicNet can be naturally transferred to model other interactions, such as protein–DNA interaction, protein–drug interaction, and protein–ligand binding. Finally, some ablation studies might help simplify the network.Despite the success of NucleicNet, there are two future directions. First, NucleicNet does not consider the conformational change caused by proteinThe author has declared no competing interests.