Cells spatially organize their molecular components to carry out fundamental biological processes and guide proper development. The spatial organization of RNA within the cell can both promote and result from gene expression regulatory control. Recent studies have demonstrated diverse associations between RNA spatial patterning and translation regulatory control. One form of patterning, compartmentalization in biomolecular condensates, has been of particular interest. Generally, transcripts associated with cytoplasmic biomolecular condensates—such as germ granules, stress granules, and P-bodies—are linked with low translational status. However, recent studies have identified new biomolecular condensates with diverse roles associated with active translation. This review outlines RNA compartmentalization in various condensates that occur in association with repressed or active translational states, highlights recent findings in well-studied condensates, and explores novel condensate behaviors. The spatial organization of cells has fascinated scientists since the advent of the microscope. Observations as early as the 1890s documented dyes concentrating within cytoplasmic aggregates of insect germ cells, structures now known as germ granules . Even asAs dozens of large, micrometer-scale ribonucleoprotein (RNP) aggregates similar to those described in the germline—were characterized, focus returned to these mysterious germline granules. Seminal studies determined that germ granules and similar RNP structures are often phase-separated organelles, coined biomolecular condensates . BiomoleBiomolecular condensates typically contain RNA and protein. As RNA detection methods improved, so have observations of mRNA accumulated within condensates. Great efforts have been made to identify the components of condensates through unbiased and candidate-based screens as well as by isolating condensates . HoweverIn recent years, many biological fields have found biomolecular condensates in their systems resulting in diverse posited functional roles for these structures. Biomolecular condensates of the nucleus—the nucleolus, Cajal bodies, and nuclear speckles—are associated with coordination of ribosome assembly, RNA processing, enhancing gene expression, or still unresolved functions, respectively . Those oThough experimental data can indicate the function of a biomolecular condensate, it is rare to find experimental evidence that definitively demonstrates condensation is essential for a proposed functional role. One reason for the challenge in definitively assigning functional consequences to condensation is the difficulty in separating the effects of their physical disruption from the perturbation of their components. Often, the elements that promote condensate formation are inseparable from elements proposed to perform functions within them. For instance, depletion of the stress granule proteins TIA-1 and TIAR-1 reduce stress granule formation under many conditions, which correlates with decreased cell viability after stress . HoweverC. elegans often lead to sterility; however, circumstances exist where fertility does not depend on germ granule formation , were first observed as fluorescent foci of the mouse 5′–3′ exonuclease, XRN-1 . As furtDrosophila demonstrated that irradiation of the germ granule-containing posterior pole plasm abolishes germline development but can be rescued by injection of healthy pole plasm  accumulates in P granules early in embryogenesis  interactions with its 3′UTR  lineage as it concentrates within P granules  during stages prior to zygotic genome activation when transcription is paused . Second,mulation , the conmulation , and RNAmulation . These rmulation .Though the specific names of germ granules, their individual components, and their posited functions are diverse across the animal kingdom, they share several features.The role of germ granules as hubs of RNA regulatory activity and organization is universal . Many ofC. elegans, the Argonaute PRG-1 is implicated in piRNA regulation and germ granule structure, whereas the Drosophila homolog, Aubergine, has an additional role in recruiting mRNA to germ granules through a piRNA-dependent templating mechanism  domain. This distinct environment promotes CD47 protein interaction with the effector protein, SET, in a splice-variant-specific manner. The extended mesh-like structure of TIS granules may provide an expanded surface area for these interactions to occur . UltimatPCNT mRNA to the pericentriolar material (PCM).Post-transcriptional regulation in condensates can transcend the protein-effector interactions observed in TIS granules and support the organization of entire organelles. One example is the cotranslational localization of PCNT mRNA to centrosomes in a translation- and dynein-dependent manner during centrosome maturation  to direct PCM and centrosome formation during the short period of early mitosis.At the onset of mitosis, the PCNT protein promotes condensation of PCM surrounding the centrosome . This coturation . PCNT mRin vivo. Additionally, the N-terminal low complexity region of the protein displays rapid recovery after photobleaching, has a distinct phase boundary, and granules can coalesce and deform , are essential for assembling these dynein complexes . DynAPs These granules demonstrate standard condensate behaviors. DynAPs components recover rapidly from photobleaching, readily undergo fission and fusion, and even share some, but not all, components with stress granules . The conIn vivo Axin has been shown to have condensate behavior as evidenced by its fusion and recovery after photobleaching, while further support comes from its salt sensitivity and concentration-dependent phase-separation (One particularly striking form of condensate-mediated post-transcriptional regulation is the ß-catenin “destruction complex.” The destruction complex is a condensate formed through the multivalent interaction of Axin and APC proteins . In vivoparation .The striking aspect of the destruction complex is that ß-catenin mRNA perpetually translates throughout the cell cycle within the destruction complex, but the ß-catenin protein is rapidly degraded in these condensates. Within the destruction complex, ß-catenin phosphorylation by Axin-recruited kinases promotes subsequent proteasomal degradation. However, upon the activation of Wnt signaling, destruction complex proteins are recruited to the cell membrane . The seqOverall, cytoplasmic biomolecular condensates represent dynamic environments. Germ granules, stress granules, and P bodies are enriched for translationally inactive transcripts, but newly discovered roles for condensates in post-transcriptional regulation adds complexity to these models with many examples of condensates that form in association with active translation or for the purpose of regulating translational outcomes. Even condensates long thought to house translationally repressed RNAs, such as stress granules, can host the translation of a subset of their constituents. As a result, the functional purpose of organizing RNA and protein into biomolecular condensates is an active field of research with many remaining questions.Though the exact functions of many biomolecular condensates remain elusive, the fact remains: mRNA localization, transcript function, and translational control are intimately linked to these phase-separated organelles. It is advantageous to translate proteins when and where they are required and to maintain translational repression when translation would be hazardous to cell viability. It is increasingly apparent that mRNA accumulation within biomolecular condensates is integral to this regulation.Drosophila, localization is clearly important in development as loss of germ granules causes severe morphogenic phenotypes and lethality (Many unanswered questions remain. A key challenge will be to determine where RNA condensation itself is essential. In some classic examples, such as the germ granule RNAs in ethality . Howeverethality . Thus, iin vivo imaging will reveal which mRNAs are true long-term residents of condensates, which are merely migrating through them, and how this correlates with their translational regulation.Another challenge is understanding the relationships between the cytoplasmic RNP condensates such as stress granules, P-bodies, and germ granules given that many of their protein and RNA components overlap . Do theyRPS28B, which recruits its own protein to scaffold P-bodies, and the Ashbya gossypia mRNAs CLN3, BNI1, and SPA2 that assist in determining the specificity of Whi3 droplets (BglG mRNA in E. coli may localize to cell membranes through a PolyU tract, which can interact electrostatically with membranes in vitro (in vivo or self-segregate in vitro (Additionally, it will be interesting to continue exploring the structural role of RNA itself in mediating phase separation. Several studies have demonstrated that mRNA can scaffold condensates, such as droplets . Incrediin vitro . Furtherin vitro . UnderstDrosophila and Xenopus, defects in germ granules prevent germline development, while in C. elegans, loss of P granules causes germline transcriptomic changes and can result in immediate, temperature-sensitive, or multi-generational progressive sterility (Though a complete understanding of condensates remains unclear, their defects can result in pronounced phenotypes and human disease. Defects in mouse germ granule components impair spermatogenesis . In Drosterility . Stress terility . These tterility . Neuroloterility . Furtherterility .in vivo. An improved understanding will also lead to advances in the treatments of human diseases. In medicine, understanding the mechanisms underlying misregulation of condensates implicated in neurological disorders can impact human health by supporting the search for treatments. Perhaps more importantly, it may reveal the underlying genetics and environmental conditions that contribute to the progression of these diseases allowing for more preventative measures to be taken.By understanding the components and mechanisms cells use to organize RNA and regulate local translation we can begin to better design experiments. In the lab, developing nuanced genetic tools to control the temporal availability of proteins in living cells could provide new tunable or inducible expression systems. Further, identifying cis-acting elements sufficient for sequestration of transcripts away from their usual destination will allow for dissecting the functions of RNA localization As this field matures, insights will continue to emerge. The theme that multiple modes of mRNA regulation can occur concurrently within condensates is likely to continue. The interrelatedness between mRNA localization, translation regulation, decay, and small RNA-mediated regulation will continue to come into focus. Discoveries of highly specialized biomolecular condensates are likely to accelerate as we determine how the biophysical properties of these structures impact the biochemistry of mRNA regulatory control. Finally, the linkages between coordinated translational control at each distinct level of translation initiation, elongation, termination, and recycling are all likely to be important. The field is rich for potential discoveries as mRNA condensation and translation regulatory control emerge from a niche field, studied in a few systems, to a generalizable feature of cell biology.


Our results show that SAF-A mutants cause dramatic changes to cytological chromatin condensation through dominant negative effects on C0T-1 RNA’s association with euchromatin, and likely other nuclear scaffold factors. In contrast, depletion of SAF-A by RNA interference (RNAi) had no discernible impact on C0T-1 RNA, nor did it cause similarly marked chromatin changes as did three different SAF-A mutations. Overall results support the concept that repeat-rich, chromatin-associated RNAs interact with multiple RNA binding proteins (RBPs) in a complex dynamic meshwork that is integral to larger-scale chromatin architecture and collectively influences cytological-scale DNA condensation.Here we provide a brief review of relevant background before presenting results of our investigation into the interplay between scaffold attachment factor A (SAF-A), chromatin-associated RNAs, and DNA condensation. SAF-A, also termed heterogenous nuclear protein U (hnRNP U), is a ubiquitous nuclear scaffold protein that was implicated in XIST RNA localization to the inactive X-chromosome (Xi) but also reported to maintain open DNA packaging in euchromatin. Here we use several means to perturb SAF-A and examine potential impacts on the broad association of RNAs on euchromatin, and on chromatin compaction. SAF-A has an N-terminal DNA binding domain and C-terminal RNA binding domain, and a prominent model has been that the protein provides a single-molecule bridge between XIST RNA and chromatin. Here analysis of the impact of SAF-A on broad RNA-chromatin interactions indicate greater biological complexity. We focus on SAF-A’s role with repeat-rich CThe online version contains supplementary material available at 10.1007/s00335-021-09935-8. Interphase chromosomes are associated with a diverse abundance of RNAs, ranging from highly produced pre-mRNAs to thousands of low-level lncRNAs, and evidence increasingly suggests this collection of chromatin-associated hnRNA broadly influences large-scale nuclear chromosome architecture. XIST RNA is the preeminent example of an RNA whose influence on local chromatin packaging involves recruiting histone modifying enzymes , which is essential for X-chromosome inactivation in mammalian female cells. This ~ 17 kb non-coding RNA is transcribed exclusively from the inactive X-chromosome where it accumulates and spreads to physically “paint” the chromosome territory in interphase nuclei . This enrichment of SAF-A on the Xi first linked SAF-A to XIST RNA  are abundant across euchromatin, and absent on the inactive X-chromosome and peripheral heterochromatin . Hence, during the cell-cycle, changes in the chromatin association of both SAF-A and RNA roughly coincide, detaching during mitosis, and reappearing on chromatin in early G1.SAF-A is released from chromosomes in early prophase of mitosis, as is XIST RNA, C0T-1 DNA probe, strictly localizes to its parent chromosome territory in interphase, and resists extraction by classical nuclear matrix isolation procedures , which lacks all but the C-terminal 280 amino acids and still contains the RGG which binds RNA , detected using a Cres Fig.   . These results indicated that the localization of C0T-1 hnRNA to chromatin was not directly dependent upon tethering by SAF-A, as had been proposed and debated for XIST RNA (summarized above).To investigate whether SAF-A is required to maintain C0T-1 RNA localization than SAF-A depletion provided the first indication that the mutant acts more indirectly via a dominant negative effect on some other factor. As further considered below regarding analysis of other mutants, there is some heterogeneity of SAF-A mutant effects within a cell population, particularly in transformed cell lines which have inherent variability, but this can also be impacted by expression levels, or, as explained below, whether a cell divided in the presence of the mutant. Control comparisons with GFP-tagged wild-type SAF-A indicated that cells with very high expression of even wild-type (GFP-tagged) SAF-A caused aberrant nuclear/chromatin structure in a subset (~ 20%) of cells; therefore, throughout these experiments analysis of results excluded cells with extremely bright GFP (protein), as well as very weakly expressing cells. This became most important for quantification of results on other mutants below for which more heterogeneity was observed. While there was some variability in the patterns observed with the C280 mutant expression, this highly truncated SAF-A consistently had obvious effects on nuclear chromatin structure and RNA distribution, further suggesting likely dominant negative effects on nuclear structure.The fact that the C280 mutant has a much greater effect on XIST . Hence, it was of interest to determine if perturbing SAF-A would impact other nuclear structural proteins. Here we examined C280 SAF-A mutant effects on NuMA, Lamin B1, SAFB1, FUS, and hnRNP C. Figure We therefore surveyed whether expression of the C280 SAF-A mutant leads to perturbation of other important nuclear structural proteins. Creamer et al. recently revealed that in normal cells, IF for SAF-A as well as other well-known nuclear matrix proteins, NuMA and Matrin-3, co-distribute with C0T-1 RNAs.These results show that the mutant C280 SAF-A impacts certain other major structural proteins, bolstering the evidence that the effects of the C280 mutant protein are likely indirect and involve broader perturbation of other factors that interact with C0T-1 RNA localization using defined mutations of SAF-A’s DNA and RNA binding domains. While the C280 mutation deleted multiple domains of SAF-A , with greater effect in cells with higher expression , appearing to sequester C0T-1 RNA away from chromatin in DNA depleted regions.To better understand the interplay of SAF-A on localization of RNA to chromatin, we examined the effects on CF-A Fig. A. Remarkion Fig. B, C. We 0T-1 RNA’s localization to chromatin, however, further scrutiny suggests an alternative explanation. As illustrated in Fig. 0T-1 RNAs co-localize with the G29A-SAF-A protein and very likely still bind the mutant SAF-A’s RGG domain. However, that protein no longer binds DNA. Hence, it may be SAF-A’s ability to bind RNA, in the absence of its capacity to bind DNA, that impacts C0T-1 RNA’s chromatin association. Unbound SAF-A that contains a functioning RGG domain causes displacement of C0T-1 RNA, in effect “stripping” it from chromatin. This finding is key to understanding why in our experiments the robust depletion of SAF-A (via RNAi), to often cytologically undetectable levels, did not discernibly mis-localize C0T-1 RNA, whereas the presence of this SAF-A mutant does. That this effect is mediated by effects on RNA is further suggested by results below.These results may initially appear to suggest that SAF-A’s ability to bind DNA is required for C0T-1 RNA localization of a mutant SAF-A lacking the RGG domain but retaining the DNA binding domain (ΔRGG-GFP)   Fig. A. Our SAF-A Fig. G, H, sup0T-1 RNA to localize to chromatin, the question becomes: why does the ΔRGG SAF-A impact C0T-1 RNA-chromatin association, even in cells that still contain endogenous SAF-A? To address this, we examined localization of endogenous SAF-A with an antibody that does not recognize the ∆RGG mutant. Strikingly, this revealed that in 96% of ∆RGG-GFP-positive cells with abnormal C0T-1 RNA distribution, the majority of endogenous SAF-A no longer associates with chromatin but co-localizes with released C0T-1 RNA in “DAPI holes” lacking substantial DNA . Importantly, in examining mitotic cells we discovered that the GFP-tag causes abnormal binding of SAF-A throughout mitosis (of both full-length and mutant SAF-A). As shown in Fig. 0T-1 hnRNA is released   Fig. D, E. As NA) Fig. D–F. As mNA) Fig. , the tot0T-1 RNA has been displaced  is with an RNP “scaffold” that retains structural integrity independent of chromatin , and the RNA is apparently displaced along with mutant SAF-A (lacking DNA binding) or endogenous SAF-A displaced by the mutant SAF-A. These findings strengthen the evidence that RNA itself is a key determinant of physical chromosome structure, and also highlight the importance of considering a mutant protein’s effects on RNA in interpreting its role in chromatin.This broader concept informs how we interpret results here to now “make sense” of what were initially perplexing and seemingly contradictory results; our endeavor to understand this began with our earlier results that confounded expectations that human SAF-A was required to tether XIST RNA. Kolpa et al.  earlier 0T-1 RNA is pre-mRNA undergoing synthesis, it will be of interest to determine whether SAF-A disrupts the RNA polymerase II (Pol II) transcriptional machinery. SAF-A has been reported to interact with RNA Pol II although some evidence indicates it is not required to maintain ongoing transcription  and the ΔRGG and G29A mutants made by PCR-based mutagenesis. Dominant negative C280-GFP was cloned into pEGFP-NLS (Clontech) after PCR amplification. Flag tags were added by restriction digest to cut GFP from pEGFP-N1and ligating with annealed oligo adaptor. For stable lines, Flag-tagged constructs were cloned into pcDNA/FRT/TO (Invitrogen). Mutagenesis to SAF-A siRNA target sites was done by Genscript: GGC → GGA (Glycine), TCG → TCA (Serine), GTT → GTA , and GTT → GTA . See Supplemental Experimental Procedures for primer sequences.ΔRGG: Fwd: 5′-TCCGTTAAACTGGTTCTTGCCACTCTTA-3′.Rev: 5′-TCCGGCGGTGGAGGAAGTGGTGGAATC-3′.G29A: Fwd: 5′-ACGCCTTTCTGACAAGGCCCTCAAGGCCGAGCTC-3′.Rev: 5′-GAGCTCGGCCTTGAGGGCCTTGTCAGAAAGGCGT-3′.C280: Fwd: 5′-AGATCGA ATTCTGTTTAAGAAGCAAATG GCAGAT-3′.Rev: 5′-AGATCGGATCCGAATAATATCCTTGGTGATA ATGC-3′.Flag oligo adaptors:sense: 5′-AGCTTGATTACAAGGATGACGACGATAAGATCTGAGCGGCCGCGGTAC-3′.antisense: 5′-CGCGGCCGCTCAGATCTTATCGTCGTCATCCTTGTAATCA-3′.RNase: we permeabilized unfixed cells on coverslips with 0.1% Triton-X in CSK buffer (4 °C for 3 min) then treated with 5 µL/mL DNase-free RNase (Roche #11119915001) or RNasin Plus RNase inhibitor (Promega), in CSK . Our standard cell fixation protocols have been previously described  were transfected with SMARTpool siRNAs and DharmaFECT 1 transfection reagent (GE Dharmacon) according to manufacturer’s instructions and fixed after 72 h. siGLO siRNA (GE Dharmacon) was used as a control which had no impact on RNA localization. For plasmid transfections, 2–4 μg of DNA was mixed with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions and added to 80–90% confluent cells in a 6-well plate. Cells were fixed and assayed 24, 48, or 72 h after transfection.As previously described  was nick translated with either biotin-11-dUTP or digoxigenin-16-dUTP (Roche). Hybridizations were done overnight at 37 °C in 2 × SSC, 1 U/μl of RNasin Plus RNase inhibitor (Promega), and 50% formamide with 2.5 µg/ml DNA probe. Cells were washed with 50% formamide/2 × SSC at 37 °C for 20 min, 2 × SSC at 37 °C for 20 min, 1 × SSC at RT for 20 min, and 4 × SSC at RT for 1 min. Detection was with antidigoxigenin or fluorescein-conjugated avidin in 1% BSA/4 × SSC for 1 h at 37 °C. Three 10 min washes were done in 4 × SSC, 4 × SSC with 0.1% Triton, and 4 × SSC at RT in the dark. Coverslips were counterstained with DAPI and mounted with Vectashield (Vector Laboratories).RNA hybridization was performed under non-denaturing conditions as previously described  were acquired using immersion oil with a refractive index of 1.518. Images were reconstructed using Wiener filter settings of 0.001 and optical transfer functions (OTFs) measured specifically for each channel with SoftWoRx 6.1.3  to obtain super-resolution images with a two-fold increase in resolution both axially and laterally. Images from different color channels were registered using parameters generated from a gold grid registration slide  and SoftWoRx 6.5.2 . Huygens was used to measure Chr 4 territory voxel volumes, Fiji to quantify SAF-A signal intensity (before and after SAF-A RNAi) in individual nuclei, and when necessary, Photoshop (Adobe) or Fiji to enhance images for brightness and contrast.Experiments were performed a minimum of three times , and a minimum of 50 and typically 150 cells were scored in each experiment. To account for potential effects of protein over-expression, transiently transfected cells were scored according to whether they expressed dim, moderate, bright, and very bright SAF-A-GFP levels, and the grossly overexpressing cells were eliminated. Digital imaging and analysis was performed using an Axiovert 200 microscope  with a 100 × NA 1.4 Plan-Apochromat objective and multi-bandpass dichroic and emission filter sets  set up in a wheel to prevent optical shift. We used AxioVision software  and an Orca-ER camera (Hamamatsu Photonics). A narrow bandpass fluorescein filter was inserted to eliminate cross talk between channels. Super-resolution 3D-SIM images were acquired on a DeltaVision OMX V4  equipped with a 60x/1.42 NA PlanApo oil immersion lens (Olympus), 405, 488, 568, and 642 nm solid state lasers and sCMOS cameras (pco.edge). Image stacks of 7–9 µm with 0.125 µm thick We have noted that SAF-A binding to chromatin appears to be affected by the type of tag fused to the protein. Most of this was discovered during work done for XIST RNA localization studies  but the residual SAF-A is clearly associated with chromatin before release (SIM image). B Antibody labeling for endogenous SAF-A sometimes appears diminished over heterochromatin, including the Barr body (arrow). C With antigen retrieval methods to expose embedded epitopes, SAF-A labeling increases over heterochromatin, including enrichment over the Barr body . D Both C0T-1 RNA and SAF-A are released from chromatin at mitosis (metaphase cell). E–F C0T-1 RNA remains bound to chromatin before (E) and after (F) SAF-A RNAi, and DAPI DNA morphology remains unaffected (TIF 24155 kb)Supplementary file1 Supplemental figure 1: Endogenous SAF-A is enriched on the Xi, released during mitosis, binds RNAs, but is not necessary for RNA localization. For all images: color channels are separated in black and white. Scale bars 5 μm. Cell types: Normal human fibroblasts (Tig-1). A  Additional examples of C280-GFP effects on NuMA. Neighboring cells lacking C280-GFP are normal controls. B Additional examples of the lack of effect of C280-GFP on LaminB1 nuclear distribution. Neighboring cells lacking C280-GFP are normal controls. C More examples of C280-GFP effects on FUS. D Additional example of the pattern seen for how C280-GFP affects hnRNP C. E Additional examples of C280-GFP effects on SAFB1 (TIF 30314 kb)Supplementary file2 Supplemental figure 2: C280 SAF-A displaces specific nuclear proteins and hnRNAs to disrupt DNA structure. For all images: color channels are separated in black and white. Scale bars 5 μm. Cell types: Normal human fibroblasts (Tig-1). 0T-1 RNA and alter DNA morphology For all images: color channels are separated in black and white. Scale bars 5 μm. Cell types: Normal human fibroblasts (Tig-1) & mouse/human hybrid cells with human chromosome 4 (Hybrid). A Two neighboring fibroblast nuclei, with (top) and without (bottom) expression of the G29A SAF-A mutant. The top cell shows changes to mutant protein distribution, C0T-1 RNA distribution and DNA morphology. B A linescan histogram of all color channels in the path indicated in image (A). C More examples of G29A-GFP effects on C0T-1 RNA. Neighboring cells lacking G29A-GFP are normal controls. D More examples of DRGG-GFP effects on C0T-1 RNA in hybrid cells. Neighboring cell lacking DRGG-GFP is a normal control. E More examples of C280-GFP effects on C0T-1 RNA (TIF 29306 kb)Supplementary file3 Supplemental figure 3: SAF-A mutants displace CBelow is the link to the electronic supplementary material.


Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA–RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA–RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA–mRNA interactions are recovered and we find that an mRNA encoding a long 3′ untranslated region (UTR) (termed vigR 3′UTR) functions as a regulatory ‘hub’ within the RNA–RNA interaction network. We demonstrate that the vigR 3′UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA–mRNA base-pairing. Deletion of the vigR 3′UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3′UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.Treatment of methicillin-resistant  Regulatory small RNA (sRNA) interact with mRNAs to regulate their stability, transcription, and translation via diverse mechanisms. Here, Mediati et al. apply RNase III-CLASH to multidrug-resistant Staphylococcus aureus to characterise the network of RNA–RNA interactions associated with RNase III and identify a regulatory mRNA 3′UTR, named vigR-3′UTR, involved in the regulation of genes relevant for vancomycin sensitivity. Staphylococcus aureus is a highly adaptable opportunistic pathogen capable of causing a wide range of infections. S. aureus is a leading cause of osteomyelitis, infective endocarditis, and bacteraemia. Methicillin-resistant S. aureus (MRSA) has become common in both community and healthcare settings with global prevalence ranging from 13 to 74% of S. aureus infections2. Mortality rates are high among patients that develop MRSA bacteraemia2 and treatment options are often limited to last-line antibiotics1. The treatment of choice for patients with MRSA bacteraemia is the glycopeptide vancomycin3. Vancomycin resistant S. aureus  is relatively rare and vancomycin treatment failure is most commonly associated with S. aureus strains that have vancomycin-intermediate resistance, termed VISA (MIC 4–8 μg/mL)3. The genetic determinants that lead to VISA are heterogeneous and incompletely defined, although it is clear that these strains do not acquire additional genetic elements. Genome sequencing has identified single nucleotide polymorphisms (SNPs) that arise during repeated vancomycin exposure and commonly lead to cell wall thickening, reduced autolysis, and reduced acetate catabolism3. Cell wall thickening is thought to reduce diffusion of vancomycin to the division septum however the underlying mechanism of cell wall thickening remains unclear.4. These two isolates belong to ST239, a sequence-type representing a global multi-drug resistant, hospital-associated S. aureus clone. The VISA isolate JKD6008 has acquired at least nine SNPs when compared to the parental vancomycin-sensitive (VSSA) isolate JKD6009, including mutations in the transcriptional regulators graRS and walKR4. Consistent with many VISA isolates, JKD6008 exhibited reduced autolysis and cell wall thickening.In this study, we have used the MRSA isolate JKD6009 which was isolated from a patient in New Zealand with bacteremia. Over 42 days of treatment with vancomycin, a second isolate from the patient presented a VISA phenotype (designated strain JKD6008)5. More recently, Dejoies et al. identified a coordinated expression profile of specific sRNAs under acute biocide stress6. The transcriptome of S. aureus contains hundreds of sRNAs, most of which have unknown biological functions. The sRNA SprX represses the transcriptional regulator SpoVG and alters intermediate glycopeptide tolerance7. More recently, the non-coding antisense RNA, SprF1, was shown to bind ribosomes and reduce protein synthesis to favour ciprofloxacin and vancomycin persister cell development8.Regulatory small RNA (sRNA) are approximately 50-500 nucleotide gene regulators that form base-paired interactions with mRNAs and inhibit or activate mRNA translation, transcription, and stability through a range of mechanisms. Using a collection of VSSA and VISA isolates, Howden et al. demonstrated that sRNA expression is correlated with exposure to last-line antibiotics, suggesting that sRNAs may constitute an acute antibiotic responseS. aureus, the double-stranded RNA-specific endonuclease RNase III plays a major role in rRNA maturation and mRNA processing. RNase III typically binds 22-nt RNA duplexes9 with a preference for at least one GC or CG base pair at cleavage sites10. RNA duplexes can be formed in cis or trans and many sRNA-mRNA pairs in S. aureus are processed by RNase III. Interstingly, RNase III also recognises a co-axially stacked interaction between RNAIII and coa mRNA that pairs through a short kissing loop interaction12. This interaction mimics an extended RNA duplex and is processed by RNase III indicating that shorter regions of complementarity may also be RNase III substrates.In 13 to MRSA and captured RNA–RNA interactions associated with RNase III. RNase III-CLASH identifies RNase III binding sites enriched within the untranslated regions (UTRs) of mRNAs and recovers many RNAs that act as regulatory ‘hubs’. We reveal the in vivo targets for many uncharacterised sRNAs providing insight into their function. We also utilise differential RNA-seq (dRNA-seq) and Term-seq to accurately map the 5′ and 3′ RNA boundary ends and identified many sRNAs not previously identified in S. aureus. We use this RNA interaction network to identify regulatory RNAs that may contribute to intermediate vancomycin resistance and find that a previously unknown regulatory mRNA, here termed vigR, is required for vancomycin-intermediate tolerance. The vigR mRNA encodes an unusually long 3′UTR that acts in trans to positively regulate isaA and folD mRNAs through direct base-pairing interactions. IsaA is a lytic transglycosylase involved in cell wall peptidoglycan turnover and expansion, and we demonstrate that it also plays a role in intermediate glycopeptide tolerance. Deletion and CRISPRi knockdown of isaA significantly reduces cell wall thickness of the VISA isolate JKD6008, while the vigR 3′UTR deletion has a modest reduction in cell wall thickness. Our study has uncovered a previously unknown but important mechanism of intermediate vancomycin resistance through a regulatory mRNA 3′UTR that promotes cell wall thickening in a clinical VISA isolate.In this work, we have adapted our proximity-dependant RNA ligation technique (RNase-CLASH) for profiling sRNA-mRNA interactionsS. aureus JKD6009 we utilised dRNA-seq14 and Term-seq15 to map RNA 5′ and 3′ boundaries, respectively. Total RNA was extracted from cells grown in liquid Mueller–Hinton (MH) to an OD600 ~0.6 and sequenced using the dRNA-seq and Term-seq protocols (Supplementary Methods). Analysis of the dRNA-seq data using the ANNOgesic workflow16 identified 1399 transcription start sites (TSS) and 17 RNA processing sites  as bait to capture sRNA–RNA interactions using an UV-crosslinking and RNA proximity-dependant ligation technique termed CLASH13. While S. aureus does not encode an orthologue of rne, many sRNA–mRNA pairs are substrates for the double-stranded RNA-specific endoribonuclease RNase III25 that interacts with the sRNA–mRNA duplex. To facilitate high-stringency purification of UV-crosslinked RNA-RNase III complexes we created a translational fusion of the chromosomal copy of RNase III (rnc) with the dual affinity tag, His6-TEV-FLAG (HTF) called JKD6009 rnc-HTF. RNase III is not essential in S. aureus26 and JKD6009 rnc-HTF produced wild-type levels of mature 16S and 23S ribosomal RNA (rRNA)  media to OD578nm 3.0. BHI was chosen as a rich medium to better facilitate capture of RNA–RNA duplexes associated with RNase III in exponential growing S. aureus. Our initial experiments (replicates 3–6) were prepared using a W5 small diameter UV-crosslinking unit   while replicates 1–2 were prepared using a Vari-X-linker  that allowed faster UV-crosslinking with lower doses 27.RNA-RNase III complexes were covalently crosslinked using UV-C in wild-type (untagged) and 29. Coding sequences (3–29%), 3′UTRs (1.5–3.8%), sRNAs (0.9–2.6%), and 5′UTRs (0.4–2.3%) were the next most abundantly recovered RNA classes  Fig. . The majses Fig. . Plottinses Fig.  and plotses Fig. . RNase IUTR Fig. . These r13. We used the software package hyb31 to extract hybrid reads that represent RNA–RNA interactions and filtered for interactions with an FDR < 0.05 (detailed in Supplementary Methods). We collated our hybrid reads with additional RNase III-CLASH data generated in a parallel study by McKellar et al.32 utilising TSB and RPMI-1640 media  is able to repress SpoVG expression consistent with earlier work7. Interestingly, the sRNA SprD is required for S. aureus pathogenicity33 and we show it is also able to repress SpoVG expression through an imperfect 34-nt base-pair interaction at the 5′ UTR of the mRNA that likely occludes the RBS  ons Fig. . We indeRBS Fig. . The sRN 34 Fig. . Point m 34 Fig. . Many noion Fig.  and RNAIRNA Fig.  indicatiS. aureus sRNA interactome.These data indicate that RNase III-CLASH detects in vivo RNA–RNA interactions including functional sRNA-mRNA interactions providing a condition-specific snapshot of the 4. Regulatory RNA interactions with mRNAs that are linked to the development of vancomycin tolerance3, or up-regulated during vancomycin treatment5 were selected for further analysis. CRISPR interference35 (CRISPRi) was used to knockdown expression of six putative regulatory RNAs and the reduced expression was confirmed by Northern blot  was reduced 1000-fold  in the presence of vancomycin, similar to VSSA strain JKD6009  is required for vancomycin intermediate resistance in JKD6008, and that knocking-down expression of vigR 3′UTR reverts the strain to a vancomycin-sensitive phenotype.We next asked if sRNA interactions identified within our collated RNase III-CLASH network contributed to increased susceptibility to vancomycin in the VISA strain JKD6008009 Fig. . These dvigR 3′UTR and our dRNA-seq data indicated a primary TSS upstream of the uncharacterised YtxH-domain protein, E0E12_RS09390 (vigR)  indicating that sRNA275 is the 3′UTR of the E0E12_RS09390 mRNA . Northern blot analysis indicated that vigR expression is up-regulated in the VISA strain JKD6008 compared to JKD6009, consistent with a role in intermediate vancomycin tolerance  Fig. . We did RNA Fig. . Based once Fig. . NorthervigR 3′UTR may confer vancomycin tolerance through cis regulation of the VigR protein. To determine the relative contribution of each region to intermediate-vancomycin tolerance, clean deletions of both the 3′UTR (vigR∆3′UTR) and CDS (vigR∆CDS), and a chromosomally repaired vigR∆3′UTR  were constructed in JKD6008  in both the VISA . The addition of sub-inhibitory vancomycin (2 μg/mL) for 10 min did not reveal additional transcriptional changes , involved in folate metabolism, produces tetrahydrofolate which forms as a key metabolite for amino acid (histidine) and nucleotide (purine) biosynthesis. The vigR and folD mRNAs are predicted to form an extensive 145-nt RNA–RNA duplex including 63-nts of perfect Watson–Crick base-pairing  and knockdown strain  , confirming that the vigR 3′UTR promotes isaA mRNA expression independently of transcriptional regulation  that included the GC-rich RNase III-CLASH hybrid read  or isaA frag-C (C5–C8) tiled across the duplex site were added into the EMSA reaction (500× excess concentration). This analysis revealed that competitor oligonucleotides at the C1 and C5 site antisense to vigR and isaA, respectively, were able to effectively compete away radiolabelled vigR 3′UTR-isaA frag-C duplex formation  is crucial in vitro for vigR 3′UTR-isaA frag-C duplex formation. To complement the EMSA, we titrated isaA frag-C with vigR 3′UTR in the presence of RNase T1 and ShortCut RNase III followed by 32P-labelled-primer extension  Fig. , and we 69) Fig. . To confion Fig. . EMSA wasaA Fig. . These aead Fig. . To furtion Fig. . Additio063 Fig. .Fig. 6ThisaA, a deletion of rnc was constructed in JKD6009. Using qRT-PCR, we confirm the increased abundance of the isaA transcript in the ∆rnc strain , JKD6008 (VISA), vigR∆3′UTR, ∆isaA, and JKD6008 pSD1 and pSD1-isaA knockdown  when compared to the isogenic VISA parent strain, suggesting that vigR 3′UTR influences cell wall architecture in S. aureus  reverted the clinical VISA isolate JKD6008 to vancomycin-sensitive. Northern analysis, dRNA-seq, and Term-seq mapping of RNA 5′ and 3′ ends demonstrated that sRNA275 is the 3′UTR of the upstream coding sequence E0E12_RS09390 (SAA6008_01724) encoding a hypothetical YtxH-domain protein (here termed VigR). The vigR 3′UTR is unusually long for a prokaryotic 3′UTR at 657-nt 42, and is substantially longer than the 378-nt CDS encoded within the vigR mRNA. We demonstrate that the 3′UTR, but not the CDS is required for intermediate vancomycin tolerance indicating that the mRNA, and not the protein mediates these regulatory effects. Bacterial 3′UTRs can have both cis-45, and trans-acting regulatory activity. Examples of trans-acting regulatory sRNAs encoded within 3′UTRs have recently been described47. These 3′UTR sRNAs can be transcribed as independent transcripts or released from the mRNA by RNase cleavage, and function independently of the parent mRNA48. Here we find that the vigR 3′UTR is neither independently transcribed, nor processed from the vigR mRNA indicating that it is a regulatory mRNA. To our knowledge, vigR is the fourth example of a bacterial mRNA that has trans-acting regulatory functions. In S. aureus, the CDS region of gdpS was shown to directly interact and stabilise the 5′UTR of sarS, however, the mechanism was not determined49. In the gram-positive pathogen, Listeria monocytogenes, interactions between the 3′UTR of hly and the 5′UTR of prsA block exonucleolytic digestion of prsA mRNA by RNase J150. In Streptococcus mutans, the 5′UTR of irvA interacts with the CDS of gbpC where it occludes an RNase J2 endonucleolytic cleavage site51. In both cases, the mRNA-mRNA interaction stabilise the target mRNA by protecting the transcript from ribonuclease processing or degradation. In a slight variation on this Gram-positive theme, we show that the vigR 3′UTR interaction with the CDS of folD and isaA mRNAs also stabilise these transcripts likely by blocking RNase III endonucleolytic processing within these mRNAs.Methicillin-resistant vigR 3′UTR led to a modest reduction in cell wall thickness and notable changes in the transcriptome. RNA-seq analysis of the vigR∆3′UTR and knockdown strain indicated differential expression of 117 transcripts with many that clustered within the ontological term ‘cell wall, membrane, and envelope biogenesis’. The addition of sub-inhibitory concentrations of vancomycin did not significantly alter the mutant transcriptome and vigR was not up-regulated by vancomycin, indicating that it promotes an intrinsically resistant physiology rather than an antibiotic-dependant response. This is supported by upregulated of vigR expression levels in the VISA strain JKD6008 when compared to the VSSA strain JKD6009. The transcriptional response to deletion or knockdown of the vigR 3′UTR was quite broad and we cannot rule out that interactions beyond those recovered by RNase III-CLASH and confirmed in vitro, may be due to secondary effects on transcription factors or general stress responses. We note that the lytic transglycosylases isaA and sceD, and autolysin lytM are positively regulated by the two-component sensor/kinase WalKR40 and additional, indirect regulation of WalKR may be occurring. Indeed for isaA, indirect activation of WalKR and direct post-transcriptional activation of isaA mRNA though vigR would represent a feed-forward loop that is an increasingly common motif in regulatory RNA circuits52.Deletion of the S. aureus RNase III-CLASH dataset contains a surprisingly large proportion of interactions between coding sequences. Some of these may represent interactions with sRNAs that are embedded within CDSs and were missed by our transcriptome annotation54. However, given the abundance of CDS-CDS interactions, these results suggest that regulatory mRNA interactions, like vigR-isaA and vigR-folD, may be a much more widely utilised mechanism for coordinating gene expression in S. aureus. This may be due to the presence of 5′-3′ exoribonuclease activity (RNase J1 and J2) found in Gram-positive Firmicutes, which readily degrades free 3′UTR intermediates and may represent an evolutionary barrier to the evolution of 3′UTR-derived sRNA–mRNA interactions in S. aureus (discussed in ref. 55). Regulatory mRNA 3′UTRs like vigR that are protected from processing at the 5′ end is likely a more widespread regulatory mechanism in S. aureus than previously appreciated.Our methicillin-resistant 57. ASOs targeting regulatory RNAs like vigR may represent an effective therapeutic approach to re-sensitise VISA isolates to last-line vancomycin treatment.Peptide-conjugated antisense oligonucleotides (ASOs) have gained renewed interest in recent years and allow specific targetting of RNAs for repressionS. aureus RN4220 and the JKD6009/JKD6008 (VSSA/VISA) pair strains were routinely cultured at 37 °C on solid or in liquid brain heart infusion  or Mueller–Hinton  media. Antibiotics were routinely used in this study to select for plasmids in S. aureus at 10 μg/mL chloramphenicol and/or 10 μg/mL erythromycin, unless otherwise specified. E. coli DH5α and IM08B strains were routinely cultured at 37 °C on solid or in liquid Luria-Bertani (LB) media. Antibiotics were routinely used to select for plasmids in E. coli at 100 μg/mL ampicillin and 15 μg/mL chloramphenicol, unless otherwise specified. All bacterial strains were stored at −80 °C as stationary phase cultures with 16% (v/v) glycerol.The bacterial strains, plasmids, and oligonucleotides used in this study are listed in Supplementary Data S. aureus deletions for vigRCDS, vigR3′UTR, and isaA and chromosomal repair for vigR3′UTR were constructed using the pIMAY-Z vector and allelic exchange58. At least 500-nt flanking regions using primer pairs detailed in Supplementary Data 59. Mutants were passaged and selected on solid BHI and confirmed using allele-specific PCR  transcriptional knockdown constructs in S. aureus JKD6009 was diluted 1/100 and grown in liquid MH at 37 °C with 200 rpm shaking to an OD600nm 0.6. vancomycin (2 μg/mL) was added to antibiotic-treated cultures and allowed to grow for a further 10 min. Media-based cultures were additionally grown for a further 10 min at 37 °C to reflect the treated samples. Growth was halted by the addition of RNAProtect bacterial reagent (Qiagen) and incubated on ice for 10 min. Cells were harvested by centrifugation (3200 × g for 10 min) and underwent GTC-phenol:chloroform RNA extraction procedures60. cDNA libraries were prepared by Vertis Biotechnologie  as described previously61 and sequenced on an Illumina NextSeq500 platform (75-cycle single-end reads) .An overnight stationary culture of S. aureus JKD6009 was prepared for total RNA extraction as described above, with the exception of using cold transcriptional stop solution (1:9 phenol:ethanol) to halt culture growth. Total RNA was DNase-treated using 10 U of RQ1 RNase-free DNase (Promega) at 37 °C for 30 min and ethanol precipitated. cDNA libraries were prepared by Vertis Biotechnologie  as described previously15 and sequenced on an Illumina NextSeq500 platform (75-cycle single-end reads) . Detailed protocols for the analysis of the Term-seq data are presented in Supplementary Methods.16 was used to integrate transcriptomics data generated from RNA-seq, dRNA-seq61, and Term-seq15 to provide a detailed annotation of the S. aureus JKD6009 genome. In brief, the transcription start sites (TSS) were identified using the TSSPredator62 module within the ANNOgesic pipeline. Rho-independent transcription termination sites were identified from the Term-seq data using custom R scripts and the ‘peakPick’ R library63. The ANNOgesic pipeline was used to analyse the transcriptomics data and the analyses resulted in a GFF file containing detailed annotations of S. aureus JKD6009 transcriptome. This GFF file was used in subsequent CLASH data analyses. The scripts from TermPick pipeline for the analysis of Term-seq data are available in the following GitHub repository (https://github.com/IgnatiusPang/TermPick). Detailed protocols for the annotation of the JKD6009 genome using ANNOgesic are presented in Supplementary Methods.The ANNOgesic pipelinernc) in S. aureus JKD6009 was tagged with the dual affinity tag, His6-TEV-FLAG (HTF). RNase III-CLASH was performed as described previously13 using the JKD6009 WT and rnc.HTF strains with a total of six replicates. For a detailed description of RNase III-CLASH performed on initial replicates 3–6 (protocol A) and then replicates 1–2 (protocol B) and modifications to Waters et al. refer to Supplementary Methods. Briefly, JKD6009 and rnc.HTF strains were used to inoculate pre-warmed liquid BHI and grown at 37 °C to an OD578nm 3.0 with 200 rpm shaking. Cultures were crosslinked with UV irradiation and immediately harvested. Cells were lysed and debris clarified by centrifugation. Supernatants were added to equilibrated M2 anti-FLAG resin (Sigma) and incubated at 4 °C with gentle rotation for 2 h. Resin-bound RNase III.RNA complexes were washed and underwent GST.TEV protease digestion at 18 °C with gentle rotation for 2 h. Eluates were collected by filtration through a Bio-spin chromatography column (Bio-Rad) and limited RNase digestion was performed using 0.15 U of RNace-IT (Agilent) to trim back overhang RNA. Eluates were added to equilibrated Ni-NTA resin and incubated at 4 °C with gentle rotation for at least 16 h. Resin-bound RNase III.RNA complexes were washed and 5′ RNA phosphate groups removed using 8 U of thermosensitive alkaline phosphatase . RNA was radiolabelled using a combination of 20 U T4 polynucleotide kinase  and 30 μCi 32P-ATP. Linker aptamers were ligated consecutively on the 5′ (unique barcoded index) and 3′ ends of RNA using 40 U of T4 ssRNA ligase I (NEB). 32P-labelled RNase III.RNA complexes were eluted and resolved on a NuPAGE 4–12% gradient Bis-Tris PAGE gel (Invitrogen). Radiolabelled RNase III.RNA complexes were visualised by autoradiography and gel excised. Gel pieces were fragmented, recovered, and digested using 100 μg of Proteinase K at 55 °C for 2 h. RNA was phenol:chloroform extracted and cDNA libraries were constructed by performing reverse transcription and PCR amplification. Amplicons were separated on a 1.5% metaphor agarose gel and excised using the MiniElute gel extraction kit (Qiagen). Libraries were pooled, quantified on an Agilent Bioanalyzer, and sequenced using Illumina NextSeq500 platforms. Sequence data has been deposited at NCBI GEO under accession number GSE158830.The chromosomal copy of RNase III 13. The pipeline for CLASH data analysis, called Hyb-CRAC-R, has been implemented in Snakemake workflow management language and is available in the following GitHub repository (https://github.com/IgnatiusPang/Hyb-CRAC-R). Detailed protocols for the analysis of RNase III-CLASH data are presented in Supplementary Methods.Analysis of the CLASH data was similar to those described in Waters et al. (2017) and Tree et al. (2018)S. aureus JKD6009, JKD6008, and JKD6008 containing pSD1 constructs were used to inoculate 5 mL of liquid MH, supplemented with 10 μg/mL chloramphenicol where appropriate and grown 16 h at 37 °C with 200 rpm shaking. Cultures were aliquoted into a 96-well microtitre plate, serially diluted (up to 10−7) into liquid MH, and spotted onto solid MH plates supplemented with 100 ng/mL anhydrotetracycline (aTC), with and without 3 μg/mL vancomycin. Spot plates were air dried at room temperature and incubated at 37 °C for 24 h. Plates were imaged on a Bio-Rad Chemi-doc using the default trans-white light setting.spoVG and mgrA were amplified from JKD6008 gDNA  to include the TSS, 5′UTR and exclude amino acids 2–29 as this incorporated the predicted signal peptide region. This was cloned into the BglII/EcoRV sites of pCN33. The PtufA promoter from JKD6008 was cloned at the PaeI/PstI sites of pICS3 to enable constitutive expression. RNAs encoding sprX2, sprD, rsaA and vigR3′UTR were cloned into the resulting pICS3::PtufA vector using the PstI/EcoRI sites (Supplementary Data S. aureus RN4220 was transformed with either the pCN33::PtufA-mRNA::gfp or pICS3::PtufA-sRNA constructs and stored at −80 °C as stationary phase cultures with 16% (v/v) glycerol. The transformed strains were then made electrocompetent and transformed a second time with the alternative vector, resulting in co-transformed cultures. These were then stored at −80 °C as stationary phase cultures with 16% (v/v) glycerol.Target mRNAs S. aureus clones. Individual colonies were used to inoculate 1 mL of 0.45 μm filtered liquid BHI and grown 16 h at 37 °C with 200 rpm shaking. Cultures were then diluted to an OD578nm 2.0 into 0.45 μm filtered PBS (pH 7.4) and at least 100,000 events were sampled on a LSRFortessa SORP (BD Biosciences) using a 530/30 nm bandpass filter. The PMT was adjusted so that background fluorescence was detectable and within range. The median fluorescence intensity (MFI) for the entire population for each test and control culture was determined using FlowJo software (version 8). The events were not gated and all events were recorded in the MFI. The MFI from S. aureus RN4220 (without GFP expressing plasmid) was used to determine background fluorescence and was subtracted from the MFI of test cultures. A two-sided Student’s t-test assuming unequal variance was used to determine significance.Co-transformed RN4220 GFP translation fusion constructs were streaked out onto solid BHI supplemented with 5 μg/mL erythromycin and 5 μg/mL chloramphenicol. RN4220 strain was chosen as it provided better fluorescence intensity within S. aureus JKD6008 and derivative cultures were diluted 1:100 into 10 mL fresh liquid BHI and grown at 37 °C with 200 rpm shaking to OD578nm 3.0. Cells were harvested by spinning at 3,800 × g for 10 min at 4 °C. A total of 5 U of RNasin (Promega) and 10 U of RQ1 RNase-free DNase (Promega) was added and RNA purified using the GTC-phenol:chloroform extraction proceedure60. At least 1 μg of RNA was reverse-transcribed using SuperScript III (Thermo), according to the manufacturer’s instructions. qRT-PCR was performed on a RotorGene Q (Eppendorf) using SensiFAST SYBR Hi-ROX mastermix (Bioline), according to the manufacturer’s instructions. A total cDNA concentration of 50 ng in combination with 400 nM oligonucleotides were used per reaction  transcript as a reference control67.600nm ~0.02. Vancomycin , teicoplanin (2 μg/mL), fosfomycin (2 μg/mL), ampicillin (5 μg/mL) and tigecycline (2 μg/mL) were added to assess sensitivity. Strains containing the pSD1 vector were also supplemented with 100 ng/mL aTC and 5 μg/mL chloramphenicol. The plate was incubated and analysed with a Bioscreen C spectrophotometer (Growth Curves USA) at 37 °C for 20 h with continuous low shaking, measuring OD600nm at 20 min intervals. Growth curves were obtained by plotting biological triplicates. The DMFit  growth curve modelling software68 was used to obtain values for the lag phase, growth rate (μ) and maximum OD.JKD6008, JKD6009, and strain derivatives were plated onto solid MH supplemented with the appropriate antibiotics and incubated at 37 °C for 16 h. Single colonies were used to inoculate 5 mL of pre-warmed liquid MH and cultures incubated at 37 °C with 200 rpm shaking for a further 16 h. A volume of 300 μL of fresh liquid MH was added into each well of a sterile 100-well honey-comb microtiter Bioscreen plate (Thermo) in triplicates to give a starting OD32P-ATP-labelled probe  and run for ~1 h at 100 V in 1× BPTE buffer. Intact rRNA was confirmed on a Bio-Rad Chemi-doc and washed consecutively in 200 mL of 75 mM NaOH, 200 mL of neutralising solution , and 200 mL of SSC buffer  for 20 min each. RNA was capillary transferred onto a Hybond-N + nylon membrane  and UV-crosslinked in a Stratagene Auto-Crosslinker with 1200 mJ UV-C. The membrane was equilibrated in Ambion ULTRAhyb hybridisation buffer (Thermo) for 1 h at 42 °C and then incubated with 10 pmol of 20 μCi γS. aureus JKD6009, JKD6008, and JKD6008 derivative constructs were streaked onto Columbia horse blood agar and grown at 37 °C for 16 h. Colonies were scraped from the agar and resuspended in 1 mL of PBS. Cultures were then centrifuged (50 × g for 5 min) and pellets resuspended in paraformaldehyde fixation solution and prepared for TEM as described previously by Howden et al.69. Cells were viewed and imaged on a FEI Tecnai G2 20 microscope at 15,000-22,000× magnification, with specific images taken at >100,000× magnification to focus on the cell wall. To determine cell wall thickness, 100 measurements of individual horizontally-planar cells were recorded using ImageJ and the mean and SEM reported. A two-sided Student’s t-test assuming unequal variance was used to determine significance.S. aureus JKD6008, JKD6008 ΔvigR, JKD6008 pSD1, and pSD1-vigR3′UTR were grown in liquid MH with and without vancomycin (2 μg/mL) to an OD600nm 0.8. Strains containing the pSD1 vector were grown in the presence of 100 ng/mL of aTC and 5 μg/mL chloramphenicol. Cultures were harvested by centrifugation  and RNA purified using the GTC-phenol:chloroform protocol. This was repeated for three independently performed experiments, generating a total of 24 RNA samples. At least 3 μg of purified RNA underwent rRNA depletion, library preparation and then were sequenced on either an Illumina HiSeq150 platform generating 300-cycle paired-end reads  or Illumina NextSeq500 generating 150-cycle single-end reads at the Ramaciotti Centre for Genomics . The received demultiplexed sequence reads underwent quality control analysis and filtering using FastQC software. Poor quality nucleotides were trimmed using the trimmomatic software tool (v0.38) to reveal high-quality reads of at least 50 bp. Reads were aligned to the JKD6008 genome (Genbank NC_017341.1) using Bowtie2 (v2.3.4.2), allowing for 1 mismatch. The frequency of reads in each generated SAM file that mapped to the “gene” or “sRNA” feature of the JKD6008 genome GFF was calculated using HTseq-count70. The tabular datasets were used as input into DESeq271 to compare differential expression between biological replicate conditions. Any feature below a read count of 5 was filtered out using DESeq2 before differential expression analyses. The complete list of all differentially expressed genes and sRNAs are available at NCBI GEO under accession number GSE158830. Clusters of orthologous (COG) classes detailed for differentially expressed transcripts found in both vigR∆3′UTR and CRISPRi knockdown strains (adjusted p < 0.05) were determined using the protein function annotation by COG on WebMGA72.isaA and folD mRNA, and vigR 3′UTR were in vitro transcribed (IVT) using 40 U of T7 RNA polymerase (Roche). IVT products were RQ1 DNase treated (Promega) for 15 min at 37 °C, phenol-chloroform extracted and ethanol precipitated, and then separated on a 6% polyacrylamide TBE-6M urea gel. Products were excised, crushed, and incubated in 500 μL RNA gel elution buffer  for 16 h at 4 °C. RNA was extracted from the eluate using phenol-chloroform extraction and ethanol precipitation. Approximately 50 pmol of RNA was dephosphorylated using Quick calf intestinal alkaline phosphatase , then extracted using phenol-chlorofom and ethanol precipitation. The 5′ ends of the RNA were radiolabelled with γ32P-ATP using T4 polynucleotide kinase (NEB). Radiolabelled products were separated from free nucleotides using a MicroSpin G-50 column (Cytiva), and purified on a denaturing PAGE gel as above. To analyse vigR binding to folD, increasing excess amounts of vigR 3′UTR were annealed to 50 fmol of either radiolabelled full-length folD mRNA or a sub-fragment of folD in 1× duplex buffer  in a 10 μL reaction. These were incubated at 95 °C for 5 min, then at 37 °C for 2 h. Samples were run on a 4% polyacrylamide 0.5X TBE gel containing 5% glycerol for ~4 h at a maximum of 16 V/cm or 1.33 mA/cm. Gels were then dried and visualised using a Fuji BAS-MP 2040 phosphorscreen and Typhoon FLA9500. To analyse vigR binding to isaA, increasing excess amounts of full-length isaA mRNA or sub-fragments of isaA were added to 25 fM of radiolabelled vigR 3′UTR in 1× duplex buffer. Where appropriate 1.25 μM of antisense competitor oligonucleotides  for 1 h at 37 °C. Radiolabelled products were separated from free nucleotides using a MicroSpin G-50 column (Cytiva), and purified on a 10% denaturing PAGE gel as above. Gel purified, in vitro transcribed vigR 3′UTR and isaA Frag-C (5 pmol) were renatured by heating to 95 °C for 5 min and vigR 3′UTR was then incubated with (250× and 500× excess) and without isaA Frag-C in 1× duplex buffer as above, in the presence of 1 μg of yeast RNA (Invitrogen). Following incubation at 37 °C for 2 h, 0.2 U of RNase T1 (Invitrogen) and 0.4 U of ShortCut RNase III (NEB) were added and incubated at 25 °C for 10 min and 37 °C for 3 min, respectively. Reactions were stopped by placing on ice and performing phenol-chloroform extraction followed by ethanol precipitation. The pellet was hydrated in nuclease-free water and underwent reverse-transcription using SuperScript SSIV (Thermo), according to the manufacturer’s instructions. Approximately 2.5 μM of the radiolabelled DNA oligonucleotide was used per reaction. Sequencing ddNTP ladders were obtained by reverse-transcription of 2 pmol of denatured vigR 3′UTR RNA with either 0.3 mM ddCTP, 0.9 mM ddTTP or 0.1 mM ddGTP termination mix and 2.5 μM of radiolabelled oligonucleotide. Following reverse-transcription, 1 μL of 4 M NaOH was added and incubated at 70 °C for 10 min to hydrolyse and degrade template RNA. Loading dye was added and samples denatured at 90 °C for 5 min and separated on a 4% polyacrylamide TBE-6M urea gel in 1× TBE buffer for ~2 h at a maximum of 40 W. Gels were dried and analysed using a Fuji BAS-MP 2040 phosphorscreen and Typhoon FLA9500.Approximately 100 μM of DNA oligonucleotide “All Northern blots and EMSA analyses were performed a minimum of twice and the images presented here are representative of results from replicate experiments.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supporting InformationSupplementary data 1Supplementary data 2Supplementary data 3Supplementary data 4Reporting Summary


Alternative splicing (AS) is a common and pivotal process for eukaryotic gene expression regulation, which enables a precursor RNA to produce multiple transcript variants with diverse cellular functions. Aberrant AS represents a hallmark of cancer, engaged in all stages of tumorigenesis from initiation to metastasis. Accumulating pieces of evidence have revealed the involvement of non-coding RNAs (ncRNAs) in regulating AS in human cancers. In this review, we overview the underlying mechanisms of non-coding RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) modulated AS at diverse levels in human cancers, and summarize their regulatory functions in tumorigenesis. Over 90% of human intron-containing genes undergo alternative splicing (AS) to form multiple mRNA isoforms . AS is gA series of evidence has demonstrated that aberrant AS is engaged in multiple biological processes, including cancer initiation, development and metastasis . For insThe majority of the human genome is actively transcribed into a diverse group of non-coding RNAs (ncRNAs) that are not translated into proteins ,21. NcRNMicroRNAs (miRNAs) are small non-coding RNA molecules that are approximately 22 nucleotides in length with high conservation across species ,29. MiRNMiRNAs modulate AS by targeting SFs or RNA-binding proteins (RBPs) in cancer ,31 Fig.. For insMiRNAs can also function by specifically targeting alternatively spliced transcripts . For exaLong non-coding RNAs (lncRNAs) are an abundant class of endogenous RNA molecules with more than 200 nucleotides ,35. LncRLncRNAs interact with pre-mRNAs via complementary sequences ,39. The in cis [cis-regulatory role in modulating AS is the lncRNA EGOT (Eosinophil granule ontogeny transcript) [Antisense lncRNAs always bind to the parental pre-mRNAs to regulate AS in cis ,40. For in cis   . EGOT isnscript) . Mechaninscript) . EGOT-trnscript) .in trans via RNA-RNA interaction [Some lncRNAs exert the function of modulating AS eraction ,43,44. Feraction   and the histone demethylase KDM2a to the FGFR2 genomic region [LncRNAs have been shown to participate in AS mediated by chromatin structure and histone modifications . For ins nucleus . asFGFR2c region   could modulate AS to engage in cancer progression and metastasis [Circular RNAs (circRNAs) are naturally endogenous covalently closed RNA molecules back-spliced from pre-mRNA or other RNA circularization mechanisms ,67. In ctastasis  Fig. 1GcircURI1 in vitro and in vivo [e7IN) [e7IN possesses a more remarkable ability to prevent the promoting effect of circURI1 silencing on GC cell invasion than exon 7 exclusion of VEGFA (VEGFAe7EX) [The circRNA circURI1 back-spliced from exons 3–4 of URI1 is identified from circRNA profiling of 5 paired gastric cancer (GC) and adjacent non-cancerous (paraGC) specimens . CircURI in vivo . The RBP in vivo . The Alu in vivo . Mechani in vivo . VEGFA io [e7IN) . CircURIGFAe7EX) . This stOne of the common mechanisms for circRNAs is to serve as miRNA sponges or competing endogenous RNAs (ceRNAs) . Once thAlthough ncRNA-mediated AS in physiological and pathological conditions has drawn more attention, the corresponding functional and mechanistic characterization is still in its infancy ,27. Up ttrans delivery of SFs such as U2 small nuclear RNA auxiliary factor 2 (U2AF65) to the targeted pre-mRNAs [Given that AS events act in cell type-, tissue- and developmental stage-dependent manners, and specific AS contributes to tumorigenesis, manipulating AS appears to be a promising therapeutic strategy for cancer treatment ,80–83. Sre-mRNAs . AlthougIn conclusion, a better understanding of the underlying molecular mechanisms of ncRNA-mediated AS expands the energetic roles of ncRNAs and AS in human diseases.


Transmembrane transport of small organic and inorganic molecules is one of the cornerstones of cellular metabolism. Among transmembrane transporters, solute carrier (SLC) proteins form the largest, albeit very diverse, superfamily with over 400 members. It was recognized early on that xenobiotics can directly interact with SLCs and that this interaction can fundamentally determine their efficacy, including bioavailability and intertissue distribution. Apart from the well-established prodrug strategy, the chemical ligation of transporter substrates to nanoparticles of various chemical compositions has recently been used as a means to enhance their targeting and absorption. In this review, we summarize efforts in drug design exploiting interactions with specific SLC transporters to optimize their therapeutic effects. Furthermore, we describe current and future challenges as well as new directions for the advanced development of therapeutics that target SLC transporters. For example, nasal delivery of therapeutics through the nasal cavity is attractive because olfactory neurons exposed in the nasal cavity provide direct access to the CNS, thus bypassing the BBB while also avoiding initial metabolism in the liver . To dateal route , these mal route .L-carnityl ester conjugate of prednisolone for pulmonary administration against bronchial asthma [Similarly, the pulmonary route of administration through inhalation could be an interesting alternative because of the thin epithelial barrier and large surface area of the lungs , and lowl asthma . This nol asthma . HoweverAlong the same lines, many other transporters besides those mentioned in this review could also be potential targets for tissue- and barrier-specific targeting of medications. Specifically, in the intestines, several known uptake systems are available, including the cholesterol transporter SLC65A2/NPC1L1 (Niemann–Pick C1-like 1) , the fatRecently, many SLC transporters have been shown to have very specific expression patterns, in contrast to non-transporter protein families . NeverthIn terms of the exploitation of individual transporters, the lack of specific binders to transporters with similar substrate specificity can be a bottleneck . HoweverTransporters can also modulate the efficacy of nanoparticle uptake without directly taking part in the process. In this context, it is important to note that systematic database analyses suggest that many more transporter-like proteins may be encoded in mammalian genomes than previously thought . For exaIn addition to their advantages, many of the current transporter-targeted delivery formulations still have to overcome certain challenges. One such obvious barrier in targeting nutrient transporters is the occurrence of off-target effects due to the abundant expression of these transporters in healthy cells in a variety of tissues . On the While the efficacy of absorption and a phenotypic readout are often used to evaluate the success of transporter-targeted delivery, the underlying mechanism of uptake is often not known in detail, especially with respect to the recycling of the targeted transporters ,29. In a


Lupus autoantibodies directed at neuroregulatory BC200 RNA cause seizure susceptibility in mice, but sequestration with antigen prevents seizures, indicating utility in therapeutic interventions. Lupus autoimmunity frequently presents with neuropsychiatric manifestations, but underlying etiology remains poorly understood. Human brain cytoplasmic 200 RNA (BC200 RNA) is a translational regulator in neuronal synapto-dendritic domains. Here, we show that a BC200 guanosine-adenosine dendritic transport motif is recognized by autoantibodies from a subset of neuropsychiatric lupus patients. These autoantibodies impact BC200 functionality by quasi irreversibly displacing two RNA transport factors from the guanosine-adenosine transport motif. Such anti-BC autoantibodies, which can gain access to brains of neuropsychiatric lupus patients, give rise to clinical manifestations including seizures. To establish causality, naive mice with a permeabilized blood–brain barrier were injected with anti-BC autoantibodies from lupus patients with seizures. Animals so injected developed seizure susceptibility with high mortality. Seizure activity was entirely precluded when animals were injected with lupus anti-BC autoantibodies together with BC200 decoy autoantigen. Seizures are a common clinical manifestation in neuropsychiatric lupus, and our work identifies anti-BC autoantibody activity as a mechanistic cause. The results demonstrate potential utility of BC200 decoys for autoantibody-specific therapeutic interventions in neuropsychiatric lupus. In neurons, regulatory brain cytoplasmic (BC) RNAs implement translational control of gene expression in synapto-dendritic domains  in one oNoncanonical C-loop motif structures in BC RNA 3′ stem-loop domains, the RNAs’ business centers, implement translational control by interacting with eukaryotic initiation factors (eIFs) 4A and 4B , 2014. BA noncanonical guanosine-adenosine (GA) motif structure, so called because it features G and A residues in non–Watson–Crick base pairs, in the rodent BC1 RNA 5′ stem-loop domain is required for dendritic targeting as part of a dendritic targeting element (DTE). This DTE, the RNA’s transport center, specifies synapto-dendritic delivery through interactions with transport factor heterogeneous nuclear ribonucleoprotein (hnRNP A2) , 2011. IThe GA motif in the rodent BC1 RNA DTE is targeted by autoimmune antibodies from patients with systemic lupus erythematosus (SLE) . Such anCurrent treatments of lupus typically focus on various nonspecific anti-inflammatory or immunosuppressive approaches. Such therapies are not usually directed at neutralization or depletion specifically of disease-causing autoantibodies, and serious adverse effects are therefore not uncommon . For thiIn the case of SLE anti-BC abs, the autoantigen is human BC200 RNA. In this study, we present data to demonstrate utility of interventions directed at the specific neutralization of SLE autoantibodies against human BC200 RNA and its DTE-containing 5′ domain. This work had to take into consideration the fact that rodent BC1 RNA and primate BC200 RNA are not phylogenetic orthologs but have developed through unrelated evolutionary lineages . Thus, eHere, we functionally dissect the motif structure of the BC200 RNA DTE, its interactions with RNA transport factors hnRNP A2 and Purα, and long-term displacement of such factors from the DTE by SLE anti-BC autoantibodies. We identify strong anti-BC reactivity in CSF of NPSLE patients, results to indicate that these autoantibodies can gain access to the brain. We demonstrate that severe seizure susceptibility is elicited by human NPSLE anti-BC abs in animal models and that this seizure phenotype is completely precluded by application of the BC200 autoantigen. Our work provides proof-of-principle evidence that target-specific therapeutic interventions using BC200 decoys are feasible in the treatment of NPSLE patients with anti-BC reactivity.We posit that BC RNA DTEs can become autoantigenic in neuropsychiatric SLE (NPSLE). To develop treatment options, it will be essential to arrive at an in-depth understanding of the human BC200 RNA DTE motif structure and its interactions with RNA transport factors and, as an autoantigenic epitope, with SLE anti-BC abs. We report here that Purα is one such RNA transport factor that is required for BC RNA dendritic delivery. We structurally and functionally dissect the human BC200 RNA DTE and identify an apical GA motif that specifies dendritic targeting. The motif interacts with transport factors hnRNP A2 and Purα, both of which are irreversibly displaced from the BC200 RNA DTE by SLE anti-BC abs. We replicate the seizure phenotype of NPSLE patients in animals by i.v. injection of SLE anti-BC abs into mice with a permeabilized blood–brain barrier (BBB). Significantly, the resulting seizure phenotype is completely precluded by co-application of BC200 RNA or derivatives. We suggest that BC200 decoys, based on the autoantibody-interacting BC200 DTE epitope structure, will be valuable in the autoantibody-specific treatment of NPSLE.To ascertain the role of Purα in dendritic BC RNA targeting, we performed knockdown experiments with neurons in primary culture using siRNA . After P35S-labeled BC1 RNA appeared normal as the labeling signal extended along the entire dendritic arborization (P < 0.0001) and Dunnett’s post hoc test , P > 0.5 for sample points 50 μm, P < 0.001 for sample points 100 μm, and P < 0.0001 for sample points 150 μm and beyond. Analogous results were obtained when Purα antisense oligonucleotides were used instead of Purα siRNA (not illustrated). The results indicate that Purα knockdown in cultured neurons causes significantly reduced dendritic delivery of BC1 RNA, thus providing evidence that Purα is a BC RNA dendritic transport factor that is required for dendritic delivery.We used siRNA directed against the translational start site region of Purα. In sympathetic neurons exposed to nontargeting control (NC) siRNA, dendritic delivery of microinjected rization . In contrization , in comprization  was perfIn regulatory BC RNAs, dendritic targeting competence in DTEs is encoded by architectural motifs that feature noncanonical  purine•purine base pairings . We hypoIn our work toward elucidation of transport-relevant Purα–BC1 RNA interactions, we performed electrophoretic mobility shift assay (EMSA) supershift assays , 2011 wiNext, the EMSA supershift experiment was repeated with a BC1 RNA mutant in which the DTE noncanonical GA motif had been abolished by an AAG-to-UCU conversion, resulting in a switch from non-WC base pairings to standard WC base pairings. This GA-mutant BC1 RNA was only negligibly supershifted in the presence of anti-Purα antibodies . This reRNA transport factor hnRNP A2, binding to the BC1 RNA GA motif, is required for dendritic delivery of the RNA , 2011. TIn the experiment shown in Earlier work on regulatory BC RNAs has mostly used rodent BC1 RNA as an experimental model. However, recent data concerning relevance in human disease, in particular in SLE, have motivated a redirection of focus toward human BC200 RNA, necessitating the molecular dissection of its structure–function relationships as a precondition for the development of target-specific therapeutic approaches.In subtypes of neuropsychiatric SLE (NPSLE), patients generate anti-BC RNA autoantibodies (anti-BC abs) that engage human BC200 RNA. After crossing the BBB, anti-BC abs are taken up by neurons where they interfere with BC RNA dendritic delivery . HoweverContrasting with rodent BC1 RNA, human BC200 RNA features a 5′ stem-loop domain that contains two putative GA motifs with candidate DTE functionality  followed by Dunnett’s multiple comparison analysis . The results indicate that BC200 RNA apical GA motif GA1, but not basal GA2, is required for dendritic targeting.When GA1-mutant BC200 RNA was microinjected instead of WT BC200 RNA, a strikingly different result was obtained: the labeling signal remained in this case somatically restricted with little or no signal in dendrites except for soma-proximate segments . It is cThe data of The human BC200 RNA apical GA1 motif, but not the basal GA2 motif, shares a structural feature with the rodent BC1 RNA GA motif: a noncanonical A•A/G•A element . The resIn the experiments shown in EMSA methodology is uniquely suited for the analysis and quantification of interactions between RNAs and proteins . It has 32P-radiolabeled RNAs were incubated with SLE sera autoantibodies immobilized on Dynabeads Protein G for magnetic separation  to examine whether the factor was able to displace the SLE anti-BC IgG from BC200 RNA. Vice versa, in a second set of experiments, radiolabeled BC200 RNA was first incubated with a transport factor (hnRNP A2 or Purα) to allow binding after which an SLE anti-BC IgG was added to the reaction mix to examine its long-term ability to displace the transport factor from BC200 RNA. Transport factors and SLE anti-BC IgGs were used at 1:1 molar ratios. The course of displacement was monitored for a total period of 106 h at 37°C.Working with IgG SLE S6, we observed that this SLE anti-BC ab bound BC200 RNA rapidly and avidly and that it was not displaced from the RNA by transport factor hnRNP A2 over the course of 106 h >4 d, . When coWe conclude that SLE anti-BC IgG is able to displace transport factors hnRNP A2 and Purα from human BC200 RNA and to disallow re-engagement of those factors with the RNA over at least several days.How do SLE anti-BC abs, causing the described molecular–cellular alterations, give rise to phenotypic manifestations? Two approaches were used to address this question. In the first approach, correlative by nature, we worked with SLE patient material. In the second approach, to be detailed in the following section, we worked with animal models to establish causality.A 24-yr-old male with a diagnosis of SLE  was admitted to University Hospital of Brooklyn (UHB) of SUNY Downstate Health Sciences University (DHSU) in March 2020 with fever, new severe tonic-clonic seizures, new proteinuria, and pancytopenia. Workup to assess the etiology of his seizures included normal brain MRI and MRA and negative assessment for infection including bacterial cultures of serum and CSF, negative CSF cryptococcal antigen, and a negative COVID-19 test. Treatment was initiated with anti-seizure medication, intravenous pulse corticosteroids, and intravenous cyclophosphamide. The patient responded well to treatment, clinical manifestations of active lupus resolved, and steroids were slowly tapered.The first blood sample was obtained from the patient in March 2020, before treatment was initiated. The second blood sample was obtained during a routine follow-up visit to UHB in May 2021, at that time when the patient had been seizure-free for more than a year.EMSA analysis revealed high serum levels of anti-BC200 reactivity at the time of first admission but barely detectable levels at the time of the follow-up visit . While tFor SLE anti-BC autoantibodies to cause neuropsychiatric manifestations, they would be expected to have gained at least temporary access to the brain. To test this prediction, we obtained paired samples of serum and CSF from NPSLE patients at the Division of Rheumatology, Department of Medicine, Queen Elizabeth II Health Sciences Center and Dalhousie University. Results from a total of 24 SLE patients, including those presented in Patients of category 4 (lines 20–24) are those where no anti-BC200 reactivity was recorded in either serum or CSF. Two of these patients were free of SLE-attributable neuropsychiatric symptoms, whereas the other four had presented with seizures or psychosis. It is concluded that these NP symptoms were caused by reactivities directed at autoimmune antigens other than BC200 RNA.In summary, the data of We will now address the question whether SLE anti-BC abs are causing neuropsychiatric manifestations in lupus. To this end, we worked with an animal model in which naive WT mice were i.v. injected with SLE anti-BC sera or IgGs. Injected animals were inspected for signs of macroscopic or microscopic pathology, in particular brain pathology. None were noted see also .We recall that model animals in which endogenous BC1 RNA is lacking cell-wide or locally at the synapse are highly seizure-susceptible upon sensory stimulation . We therP < 0.0001 . As controls, we used sera from a healthy subject (HS4) and from patients with non-SLE autoimmune disorders . In a further control, WT mice were injected with serum from an SLE patient without neuropsychiatric manifestations (patient SLE 10); serum from this patient was lacking anti-BC reactivity . When su< 0.0001 .Seizures were preceded by a brief period of wild running. Qualitatively, wild running and seizures were similar if not identical to the seizure phenotype of BC1 KO animals , 2010. HWe next injected WT animals with purified IgG preparations from NPSLE patients S1, S6, or S13 . These eVideo 1https://www.videolan.org/vlc/). Download videoAnti-BC RNA NPSLE autoantibodies cause seizure susceptibility. WT animals were i.v. injected with lupus IgG S6  or with P < 0.0001 . The respective numbers for non-injected BC1 KO animals are 84% (seizure rate) and 23%  . The resP < 0.0001).In summary, our data show that SLE anti-BC IgGs from NPSLE patients with seizures are highly potent epileptogenic agents when introduced into WT animals with a permeabilized BBB. However, a key question remains to be addressed. Can the seizure phenotype that is elicited by SLE anti-BC abs be prevented by sequestration of autoantibodies with antigen or derivatives? We used BC200 RNA and the BC200 5′ domain as decoys, testing their efficiencies in engaging and neutralizing SLE anti-BC abs in vivo. Anti-BC autoantibodies, in the form of SLE patient sera or purified IgG, were i.v. coinjected with BC200 decoys into naive WT mice, in conjunction with LPS i.p. injection for BBB permeabilization. Mice so injected never seized or showed any indication of seizures . MortaliVideo 2https://www.videolan.org/vlc/). Download videoAnti-BC RNA NPSLE autoantibodies cause seizure susceptibility. WT animals were i.v. injected with lupus IgG S6  or with The above rescue data show that the seizure phenotype elicited by NPSLE autoantibodies can be averted by application of BC200 decoy autoantigen. As phenotype rescue was quantitatively complete, it is concluded that the observed seizure manifestations are solely caused by SLE anti-BC abs. The use of total IgG in BC200 rescue experiments in these experiments was critical: it warrants the conclusion, given the 100% rescue of the seizure phenotype by BC200 decoys, that any non–anti-BC IgG present in total IgG did not contribute to the phenotype, and therefore only anti-BC IgG did. The data indicate potential utility of BC200 decoys in autoantibody-specific therapeutic interventions with neuropsychiatric lupus patients.In neurons, local protein synthesis in synapto-dendritic domains is a key mechanism in the spatiotemporal regulation of synaptic function and plasticity . Select A requisite precondition for local translational control mechanisms is the targeted delivery of RNAs, including mRNAs and regulatory BC RNAs, to synapto-dendritic sites of function . In rodeWe report here that dendritic delivery of human BC200 RNA is dependent on apical GA motif GA1 but not on basal GA2. The tandem noncanonical GA1 base-pairing type A•A/G•A is also found in the targeting-determinant 5′ GA motif of rodent BC1 RNA. We have previously suggested  that theRecent evidence suggests that dysregulation of BC RNA dendritic targeting is associated with disease , 2019. IInteractions of NPSLE autoantibodies with human BC200 RNA were assessed using two independent experimental assays: EMSA and IP. Both approaches reveal that recognition of BC200 RNA by NPSLE abs depends on the 5′ GA1 architectural motif that is required for dendritic targeting  and 6. NTransport factors hnRNP A2 and Purα are effectively displaced from human BC200 RNA by SLE anti-BC IgGs . EarlierEstablishing SLE anti-BC ab binding determinants in BC200 RNA will be important for the development of therapeutic BC200 decoys. We plan to design minimal BC200 decoy structures that will act as high-affinity epitopes for the specific depletion of SLE anti-BC abs from the systemic circulation. BC RNAs are afforded high intrinsic stability by their 5′ and 3′ stem-loop domains with noncanonical motif content . In addiWith the exception of some of the autoimmune samples listed in The rescue data suggest therapeutic utility. Current treatments of lupus typically involve application of anti-inflammatory or immunosuppressive agents which are not specific for disease-driving autoantibodies and are therefore prone to causing side effects . In contA further caveat is exemplified by NPSLE patients 700, 1327, and 2591 of Our work suggests that human BC200 RNA can become a target of NPSLE autoantibodies. Consequently, we posit that BC200 decoys will lend themselves to autoantibody-specific treatment of NPSLE seizure disorder, thus avoiding the type of adverse effects that are caused by nonspecific chronic immunosuppressive agents . In addiBC200 RNA has been implicated in a number of human malignancies, with particularly high expression levels in mammary carcinoma cells . Care wiIn summary, our work provides support for the thesis that SLE anti-BC abs can cause neuropsychiatric phenotypic manifestations including seizures, and it indicates that such manifestations can specifically be mitigated by application of epitope-presenting BC200 decoys.IL-A:WC to generate BC1 RNA in which the apical GA motif has been eliminated by conversion to standard WC base pairings ; (6) BC200ΔGA2 to generate BC200 RNA in which the basal GA motif GA2 has been eliminated by conversion to standard WC base pairings (GAGA-to-UAUC exchange); (7) BC200ΔGA1/ΔGA2 to generate BC200 RNA in which both GA motifs, GA1 and GA2, have been eliminated by conversion to standard WC base pairings; and (8) pSP6-U6 to generate U6 RNA (We generated transcripts from the following BC RNA plasmid constructs: (1) pBCX607 to generate full-length rat BC1 RNA , 2011; ; (3) pBCpairings ; (4) pBCking U22 ; (5) pBCe U6 RNA .35S- or 32P-labeled transcripts were generated using T3, T7, or SP6 RNA polymerase as described , from patients with rheumatoid arthritis (RA), and from patients with multiple sclerosis (MS) . Some coPaired serum and CSF samples from NPSLE patients were collected at the Division of Rheumatology, Department of Medicine, Queen Elizabeth II Health Sciences Center and Dalhousie University. In most cases, paired serum and CSF samples were obtained from a patient on the same day. Exceptions are noted as follows, listing Nova Scotia patient numbers and numbers of interval days separating serum from CSF collection : 700, 3; 1202, 1; 2267, 2; 2317, 4; 2532, - 3; 2591, - 4; 2779, - 9; 2827, - 1.32P-labeled at 3–5 × 106 cpm/μl by in vitro transcription. RNAs were incubated with transport factors or IgGs, or with serum or CSF, as described below  for 30 min at 37°C. To minimize unspecific RNA–protein interactions, heparin was added at 5 mg/ml and incubated for 10 min at room temperature. When desired, unbound RNA was digested using RNase T1 at 30°C for 10 min. RNA–protein complexes were resolved by native PAGE on 8% gels (ratio acrylamide/bisacrylamide 19:1) in 90 mM Tris-borate, pH 8.3, 15 mM MgCl2, at 35 mA for 2 h at room temperature.Before incubation, RNAs were heated at 70°C for 10 min and allowed to cool for 10 min at room temperature. Purified transport factors or IgGs (10 nM) were incubated with RNA in a total of 20 μl binding buffer  was preincubated with RNA in 20 μl binding buffer at 37°C for 1 h. IgG or transport factor was then added at a 1:1 molar ratio with the respective preincubated transport factor or IgG, and incubation was continued for a total of 106 h. Aliquots of the reaction mixture were taken at sample time points as indicated in the figures.6 cpm/μl) was mixed with serum (1 μl RNA plus 2 μl undiluted serum) or CSF (1 μl RNA plus 3 μl undiluted CSF) and incubated in binding buffer  for 1 h at 37°C. After treatment with heparin and RNase T1, RNA–protein complexes (bound RNA) and non-complexed RNA  were resolved by PAGE as described above.For EMSA analysis with human sera or CSF, radiolabeled BC200 RNA  were used for serum IgG mobilization  and incubated for 2 h at 4°C with 2.5 μl of serum. Beads were again washed five times and subsequently incubated with 32P-labeled RNAs at 4°C overnight. Beads were again washed five times and treated with proteinase K (1 mg/ml) in proteinase K buffer , following which RNAs were phenol-chloroform–extracted and ethanol-precipitated. Purified RNAs were resolved on 8% polyacrylamide gels.IP assays were performed with serum SLE S6 (from an NPSLE patient with seizures) and serum SLE 24 (from an SLE patient without neuropsychiatric involvement). Three lization . Beads wIn a second type of IP assay, beads were washed five times after incubation with serum and subsequently incubated with 3 μg of total RNA prepared from MCF-7 cells. MCF-7 cells are breast cancer cells expressing high levels of BC200 RNA . After fBC200 forward: 5′-CCTGGGCAATATAGCGAGAC-3′BC200 reverse: 5′-GGGTTGTTGCTTTGAGGGA-3′The reverse-transcription step was performed as follows, using random hexamer primers and SuperScript III (Thermo Fisher Scientific): 5 min at 25°C, 1 h at 50°C, and 15 min at 70°C. PCR amplification reactions were carried out in a final volume of 25 μl using 10 U of Platinum Taq DNA Polymerase (Invitrogen). PCR amplification conditions were as follows: 1 cycle of 1 min at 94°C, 45 s at 57°C, 1 min at 72°C, followed by 37 cycles of 30 s at 94°C, 45 s at 57°C, 1 min at 72°C, and a final cycle of 1 min at 94°C, 45 s at 57°C, and 15 min at 72°C. Primers for the amplification of BC200 RNA were as follows (expected PCR product size 98 nucleotides):U6 forward: 5′-GCTTCGGCAGCACATATACTAAAAT-3′U6 reverse: 5′-CGCTTCACGAATTTGCGTGTCAT-3′Primers for the amplification of U6 RNA were as follows (expected PCR product size 89 nucleotides):PCR products were resolved on 1% agarose gels and visualized by ethidium bromide staining. Parts of the samples were used for protein analysis to verify IgG binding to the beads. In this case, after the last wash, beads were suspended in sample buffer  and heated at 95°C for 5 min. Protein samples were resolved by SDS–PAGE and subjected to Coomassie staining for analysis.We maintained sympathetic neurons in low-density primary cultures as described , 2014. EPurα siRNA treatment was performed as previously described . Purα si35S-radiolabeled (∼5 × 106 cpm/μl) by in vitro transcription. 10–20 fl was injected into a cell’s perikaryal region per pulse. To monitor the injection process and to ascertain that experimental manipulations did not cause alterations in neuronal cell morphology , we coinjected Lucifer yellow  in all RNA injections of cultured neurons . Cells were exposed at 4°C for 7 d before photographic development  as described , 2011.A Nikon Microphot-FXA microscope was used for dark-field and phase-contrast microscopy , 2019. PWork with vertebrate animals was approved by the SUNY DHSU Institutional Animal Care and Use Committee (IACUC) and is reported in accordance with ARRIVE guidelines. Animals were housed in the Division of Comparative Medicine (DCM) of SUNY DHSU.Animals of both sexes were used. I.v. injections of SLE anti-BC sera or IgGs, or control sera or IgGs, were performed with WT mice and BC1 KO mice. In our previous work , i.v. inBC1 model mice and WT mice injected with SLE anti-BC abs are sensitive to auditory stimulation and may undergo seizures upon exposure to loud sounds. These animals are therefore housed in a designated “quiet room” (room 9-035B) where such exposure is minimized. The room is separated from other animal colonies (including a macaque colony) and is physically isolated by dual-door entry. Access to this room is restricted to authorized personnel trained in the husbandry of these animals, and animal occupancy is limited to a single-PI IACUC protocol. The DCM quiet-room requirement  is a formal component of the approved IACUC protocol. Food (standard pellets) and water were available ad libitum. All animals were housed in compliant cages.Sera and IgGs, obtained from NPSLE patients SLE S1, S6, and S13, were i.v. injected into mice as described  using thFull-length BC200 RNA and the BC200 5′ domain were used as decoys. BC200 decoys were i.v. coinjected with SLE sera or purified IgG. The molar ratio of IgG:decoy was 1:1.Seizure susceptibility that results from dysregulated translational control in neurons has in the past typically been analyzed using auditory stimulation . For thiInjected animals—females and males were used—were examined singly in a plastic cage equipped with a 120-dB personal alarm device (TBO-Tech) mounted under the cage cover. Auditory stimulation was applied for 15 min. End-point experimenters of in vivo seizure susceptibility tests were not cognizant of animals’ injection protocols . All in vivo seizure susceptibility tests were videotaped, and all videos were independently viewed by staff without knowledge of the animals’ experimental histories. The following parameters were scored: time to onset of wild running, time to onset of seizures, percentage of animals undergoing tonic-clonic seizures, time to death, and percent mortality during seizures. Whenever scored parameters did not significantly differ between groups of animals , they were analyzed jointly.Sympathetic neurons in primary culture were used for RNA dendritic transport experiments. The overall experimental design followed previously published protocols , 2014. TSilver grain densities were established along the dendritic extent at 50-μm interval sample points, up to 250 μm. A 0-μm sample point was defined at the base of a dendrite and was assigned a relative signal intensity of 100%. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test for multiple comparisons  , 2014. DNaive WT mice were i.v. injected with SLE anti-BC sera or IgGs from NPSLE patients with seizures or with control sera or IgGs. We asked if SLE anti-BC abs so injected would give rise to seizure susceptibility. Statistical significance of seizure data was examined using Fisher’s exact test, analyzing seizure versus no-seizure and death versus survival outcomes of animals injected with NPSLE sera or IgGs, with NPSLE sera or IgGs in the presence of BC200 decoys, with non-NP SLE sera, or with non-SLE sera or IgGs. Confidence intervals were calculated using the Agresti-Coull method .P < 0.05, **P < 0.01, ***P < 0.001.Quantitative and statistical analyses were performed using Prism (GraphPad), SPSS Statistics , and the R Project for Statistical Computing (The R Foundation). Significance levels are identified as follows: *