Characterization of long G4-rich enhancer-associated genomic regions engaging in a novel loop:loop 'G4 Kissing' interaction.

Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.

Characterization of novel small RNAs (sRNAs) contributing to the desiccation response of Salmonella enterica serovar Typhimurium.

Noncoding RNA (ncRNA) modulation of gene expression has now been ubiquitously observed across all domains of life. An increasingly apparent role of ncRNAs is to coordinate changes in gene expressions in response to environmental stress. Salmonella enterica, a common food-born pathogen, is known for its striking ability to survive, adapt, and thrive in various unfavourable environments which makes it a particularly difficult pathogen to eliminate as well as an interesting model in which to study ncRNA contributions to cellular stress response. Mounting evidence now suggests that small RNAs (sRNAs) represent key regulators of Salmonella stress adaptation. Approximately 50-500 nucleotides in length, sRNAs regulate gene expression through complementary base pairing with molecular targets and have recently been suggested to outnumber protein-coding genes in bacteria. In this work, we employ small RNA transcriptome sequencing to characterize changes in the sRNA profiles of Salmonella in response to desiccation. In all, we identify 102 previously annotated sRNAs significantly differentially expressed during desiccation; and excitingly, 71 novel sRNAs likewise differentially expressed. Small transcript northern blotting and qRT-PCRs confirm the identities and expressions of several of our novel sRNAs, and computational analyses indicate the majority are highly conserved and structurally related to characterized sRNAs. Predicted sRNA targets include several proteins necessary for desiccation survival and this, in part, suggests a role for desiccation-regulated sRNAs in this stress response. Furthermore, we find individual knock-outs of two of the novel sRNAs identified herein, either sRNA1320429 or sRNA3981754, significantly impairs the ability of Salmonella to survive desiccation, confirming their involvements (and suggesting the potential involvements of other sRNAs we identify in this work) in the Salmonella response to desiccation.

Characterization of Disinfection By-Products from Chromatographically Isolated NOM through High-Resolution Mass Spectrometry.

As levels of natural organic matter (NOM) in surface water rise, the minimization of potentially harmful disinfection by-products (DBPs) becomes increasingly critical. Here, we introduce the advantage that chromatographic prefractionation brings to investigating compositional changes to NOM caused by chlorination. Fractionation reduces complexity, making it easier to observe changes and attribute them to specific components. Under the conditions tested (0.1-0.4 g of Cl to g of C without further additives), the differences between highly and less oxidized NOM were striking. Highly oxidized NOM formed more diverse Cl-containing DPB, had a higher propensity to react with multiple Cl, and tended to transform so drastically as to no longer be amenable to electrospray-ionization mass spectral detection. Less-oxidized material tended to incorporate one Cl and retain its humiclike composition. N-containing, lipidlike, and condensed aromatic structure (CAS)-like NOM were selectively enriched in mass spectra, suggesting that such components do not react as extensively with NaOCl as their counterparts. Carbohydrate-like NOM, conversely, was selectively removed from spectra by chlorination.

Characterization of a novel sRNA contributing to biofilm formation in Salmonella enterica serovar Typhimurium.

Small RNAs (sRNAs) are short noncoding RNAs of ~50-200 nucleotides believed to primarily function in regulating crucial activities in bacteria during periods of cellular stress. This study examined the relevance of specific sRNAs on biofilm formation in nutrient starved Salmonella enterica serovar Typhimurium. Eight unique sRNAs were selected for deletion primarily based on their genomic location and/or putative targets. Quantitative and qualitative analyses confirm one of these, sRNA1186573, is required for efficient biofilm formation in S. enterica further highlighting the significance of sRNAs during Salmonella stress response.

Global Switch from DICER-dependent MicroRNA to DICER-independent SnoRNA-derived RNA Biogenesis in Malignancy.

SnoRNAs are frequently processed into snoRNA-derived RNAs (sdRNAs) that function much like traditional microRNAs (miRNAs). That said, our analyses suggest a global switch from DICER-dependent (predominately miRNA) to DICER-independent (predominately sdRNA) biogenesis/gene regulation in colon cancer. Whereas the expressions of 259 of 288 appreciably expressed miRNAs are significantly decreased (avg. 6.4% of WT) in human colon cancer DICER-KOs, 95 of 103 sdRNAs are conversely, significantly increased (avg. 679.3%) in DICER-KOs as compared to WT. As many diseases are characterized by DICER deficiency, this putative global switch to DICER-independent sdRNA regulations may contribute to an array of human diseases.

Immunomodulatory LncRNA on antisense strand of ICAM-1 augments SARS-CoV-2 infection-associated airway mucoinflammatory phenotype.

Noncoding RNAs are important regulators of mucoinflammatory response, but little is known about the contribution of airway long noncoding RNAs (lncRNAs) in COVID-19. RNA-seq analysis showed a more than 4-fold increased expression of IL-6, ICAM-1, CXCL-8, and SCGB1A1 inflammatory factors; MUC5AC and MUC5B mucins; and SPDEF, FOXA3, and FOXJ1 transcription factors in COVID-19 patient nasal samples compared with uninfected controls. A lncRNA on antisense strand to ICAM-1 or LASI was induced 2-fold in COVID-19 patients, and its expression was directly correlated with viral loads. A SARS-CoV-2-infected 3D-airway model largely recapitulated these clinical findings. RNA microscopy and molecular modeling indicated a possible interaction between viral RNA and LASI lncRNA. Notably, blocking LASI lncRNA reduced the SARS-CoV-2 replication and suppressed MUC5AC mucin levels and associated inflammation, and select LASI-dependent miRNAs (e.g., let-7b-5p and miR-200a-5p) were implicated. Thus, LASI lncRNA represents an essential facilitator of SARS-CoV-2 infection and associated airway mucoinflammatory response.

MicroRNA-like snoRNA-Derived RNAs (sdRNAs) Promote Castration-Resistant Prostate Cancer.

We have identified 38 specifically excised, differentially expressed snoRNA fragments (sdRNAs) in TCGA prostate cancer (PCa) patient samples as compared to normal prostate controls. SnoRNA-derived fragments sdRNA-D19b and -A24 emerged among the most differentially expressed and were selected for further experimentation. We found that the overexpression of either sdRNA significantly increased PC3 (a well-established model of castration-resistant prostate cancer (CRPC)) cell proliferation, and that sdRNA-D19b overexpression also markedly increased the rate of PC3 cell migration. In addition, both sdRNAs provided drug-specific resistances with sdRNA-D19b levels correlating with paclitaxel resistance and sdRNA-24A conferring dasatinib resistance. In silico and in vitro analyses revealed that two established PCa tumor suppressor genes, CD44 and CDK12, represent targets for sdRNA-D19b and sdRNA-A24, respectively. This outlines a biologically coherent mechanism by which sdRNAs downregulate tumor suppressors in AR-PCa to enhance proliferative and metastatic capabilities and to encourage chemotherapeutic resistance. Aggressive proliferation, rampant metastasis, and recalcitrance to chemotherapy are core characteristics of CRPC that synergize to produce a pathology that ranks second in cancer-related deaths for men. This study defines sdRNA-D19b and -A24 as contributors to AR-PCa, potentially providing novel biomarkers and therapeutic targets of use in PCa clinical intervention.

Characterization of 475 Novel, Putative Small RNAs (sRNAs) in Carbon-Starved Salmonella enterica Serovar Typhimurium.

An increasingly apparent role of noncoding RNA (ncRNAs) is to coordinate gene expression during environmental stress. A mounting body of evidence implicates small RNAs (sRNAs) as key drivers of Salmonella stress survival. Generally thought to be 50-500 nucleotides in length and to occur in intergenic regions, sRNAs typically regulate protein expression through base pairing with mRNA targets. In this work, through employing a refined definition of sRNAs allowing for shorter sequences and sRNA loci to overlap with annotated protein-coding gene loci, we have identified 475 previously unannotated sRNAs that are significantly differentially expressed during carbon starvation (C-starvation). Northern blotting and quantitative RT-PCRs confirm the expressions and identities of several of these novel sRNAs, and our computational analyses find the majority to be highly conserved and structurally related to known sRNAs. Importantly, we show that deletion of one of the sRNAs dynamically expressed during C-starvation, sRNA4130247, significantly impairs the Salmonella C-starvation response (CSR), confirming its involvement in the Salmonella CSR. In conclusion, the work presented here provides the first-ever characterization of intragenic sRNAs in Salmonella, experimentally confirms that sRNAs dynamically expressed during the CSR are directly involved in stress survival, and more than doubles the Salmonella enterica sRNAs described to date.

A long noncoding RNA antisense to ICAM-1 is involved in allergic asthma associated hyperreactive response of airway epithelial cells.

Epithelial cells of the conducting airways are a pivotal first line of defense against airborne pathogens and allergens that orchestrate inflammatory responses and mucociliary clearance. Nonetheless, the molecular mechanisms responsible for epithelial hyperreactivity associated with allergic asthma are not completely understood. Transcriptomic analysis of human airway epithelial cells (HAECs), differentiated in-vitro at air-liquid interface (ALI), showed 725 differentially expressed immediate-early transcripts, including putative long noncoding RNAs (lncRNAs). A novel lncRNA on the antisense strand of ICAM-1 or LASI was identified, which was induced in LPS-primed HAECs along with mucin MUC5AC and its transcriptional regulator SPDEF. LPS-primed expression of LASI, MUC5AC, and SPDEF transcripts were higher in ex-vivo cultured asthmatic HAECs that were further augmented by LPS treatment. Airway sections from asthmatics with increased mucus load showed higher LASI expression in MUC5AC+ goblet cells following multi-fluorescent in-situ hybridization and immunostaining. LPS- or IL-13-induced LASI transcripts were mostly enriched in the nuclear/perinuclear region and were associated with increased ICAM-1, IL-6, and CXCL-8 expression. Blocking LASI expression reduced the LPS or IL-13-induced epithelial inflammatory factors and MUC5AC expression, suggesting that the novel lncRNA LASI could play a key role in LPS-primed trained airway epithelial responses that are dysregulated in allergic asthma.

ADAR Mediated RNA Editing Modulates MicroRNA Targeting in Human Breast Cancer.

RNA editing by RNA specific adenosine deaminase acting on RNA (ADAR) is increasingly being found to alter microRNA (miRNA) regulation. Editing of miRNA transcripts can affect their processing, as well as which messenger RNAs (mRNAs) they target. Further, editing of target mRNAs can also affect their complementarity to miRNAs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes being largely unclear. In addition, numerous reports have now identified an array of miRNAs that directly contribute to various malignancies although the majority of their targets remain largely undefined. Here we propose that modulating the targets of miRNAs via mRNA editing is a frequent occurrence in cancer and an underappreciated participant in pathology. In order to more accurately characterize the relationship between these two regulatory processes, this study examined RNA editing events within mRNA sequences of two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits could modulate miRNA associations. Computational analyses of RNA-Seq data from these two cell lines identified over 50,000 recurrent editing sites within human mRNAs, and many of these were located in 3' untranslated regions (UTRs). When these locations were screened against the list of currently-annotated miRNAs we discovered that editing caused a subset (~9%) to have significant alterations to mRNA complementarity. One miRNA in particular, miR-140-3p, is known to be misexpressed in many breast cancers, and we found that mRNA editing allowed this miRNA to directly target the apoptosis inducing gene DFFA in MCF-7, but not in MDA-MB-231 cells. As these two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and physiological responses, we hypothesized that the differential RNA editing of DFFA in these two cell lines could contribute to their phenotypic differences. Indeed, we confirmed through western blotting that inhibiting miR-140-3p increases expression of the DFFA protein product in MCF-7, but not MDA-MB-231, and further that inhibition of miR-140-3p also increases cellular growth in MCF-7, but not MDA-MB-231. Broadly, these results suggest that the creation of miRNA targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity.

Human snoRNA-93 is processed into a microRNA-like RNA that promotes breast cancer cell invasion.

Genetic searches for tumor suppressors have recently linked small nucleolar RNA misregulations with tumorigenesis. In addition to their classically defined functions, several small nucleolar RNAs are now known to be processed into short microRNA-like fragments called small nucleolar RNA-derived RNAs. To determine if any small nucleolar RNA-derived RNAs contribute to breast malignancy, we recently performed a RNA-seq-based comparison of the small nucleolar RNA-derived RNAs of two breast cancer cell lines (MCF-7 and MDA-MB-231) and identified small nucleolar RNA-derived RNAs derived from 13 small nucleolar RNAs overexpressed in MDA-MB-231s. Importantly, we find that inhibiting the most differentially expressed of these small nucleolar RNA-derived RNAs (sdRNA-93) in MDA-MB-231 cells results primarily in a loss of invasiveness, whereas increased sdRNA-93 expression in either cell line conversely results in strikingly enhanced invasion. Excitingly, we recently determined sdRNA-93 expressions in small RNA-seq data corresponding to 116 patient tumors and normal breast controls, and while we find little sdRNA-93 expression in any of the controls and only sporadic expression in most subtypes, we find robust expression of sdRNA-93 in 92.8% of Luminal B Her2+tumors. Of note, our analyses also indicate that at least one of sdRNA-93's endogenous roles is to regulate the expression of Pipox, a sarcosine metabolism-related protein whose expression significantly correlates with distinct molecular subtypes of breast cancer. We find sdRNA-93 can regulate the Pipox 3'UTR via standard reporter assays and that manipulating endogenous sdRNA-93 levels inversely correlates with altered Pipox expression. In summary, our results strongly indicate that sdRNA-93 expression actively contributes to the malignant phenotype of breast cancer through participating in microRNA-like regulation.