While RNA secondary structure prediction from sequence data has made remarkable progress, there is a need for improved strategies for annotating the features of RNA secondary structures. Here, we present bpRNA, a novel annotation tool capable of parsing RNA structures, including complex pseudoknot-containing RNAs, to yield an objective, precise, compact, unambiguous, easily-interpretable description of all loops, stems, and pseudoknots, along with the positions, sequence, and flanking base pairs of each such structural feature. We also introduce several new informative representations of RNA structure types to improve structure visualization and interpretation. We have further used bpRNA to generate a web-accessible meta-database, ‘bpRNA-1m’, of over 100 000 single-molecule, known secondary structures; this is both more fully and accurately annotated and over 20-times larger than existing databases. We use a subset of the database with highly similar (≥90% identical) sequences filtered out to report on statistical trends in sequence, flanking base pairs, and length. Both the bpRNA method and the bpRNA-1m database will be valuable resources both for specific analysis of individual RNA molecules and large-scale analyses such as are useful for updating RNA energy parameters for computational thermodynamic predictions, improving machine learning models for structure prediction, and for benchmarking structure-prediction algorithms.

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway.

Long non-coding RNAs (lncRNAs) have emerged as important in cancer development and progression. The impact of diet on lncRNA expression is largely unknown. Sulforaphane (SFN), obtained from vegetables like broccoli, can prevent and suppress cancer formation. Here we tested the hypothesis that SFN attenuates the expression of cancer-associated lncRNAs. We analyzed whole genome RNA-sequencing data of normal human prostate epithelial cells and prostate cancer cells treated with 15 μM SFN or DMSO. SFN significantly altered expression of ~100 lncRNAs in each cell type, and normalized the expression of some lncRNAs that were differentially expressed in cancer cells. SFN-mediated alterations in lncRNA expression correlated with genes that regulate cell cycle, signal transduction, and metabolism. LINC01116 was functionally investigated because it was overexpressed in several cancers, and was transcriptionally repressed after SFN treatment. Knockdown of LINC01116 with siRNA decreased proliferation of prostate cancer cells, and significantly upregulated several genes including GAPDH (regulates glycolysis), MAP1LC3B2 (autophagy) and H2AFY (chromatin structure). A 4-fold decrease in the ability of the cancer cells to form colonies was found when the LINC01116 gene was disrupted through a CRISPR/CAS9 method, further supporting an oncogenic function for LINC01116 in PC-3 cells.. We identified a novel isoform of LINC01116 and bioinformatically investigated the possibility that LINC01116 could interact with target genes via ssRNA:dsDNA triplexes. Our data reveal that chemicals from the diet can influence the expression of functionally important lncRNAs, and suggest a novel mechanism by which SFN may prevent and suppress prostate cancer.

While RNA secondary structure prediction from sequence data has made remarkable progress, there is a need for improved strategies for annotating the features of RNA secondary structures. Here we present bpRNA, a novel annotation tool capable of parsing RNA structures, including complex pseudoknotcontaining RNAs, to yield an objective, precise, compact, unambiguous, easily-interpretable description of all loops, stems, and pseudoknots, along with the positions, sequence, and flanking base pairs of each such structural feature. We also introduce several new informative representations of RNA structure types to improve structure visualization and interpretation. We have further used bpRNA to generate a webaccessible meta-database, "bpRNA-1m", of over 100,000 single-molecule, known secondary structures; this is both more fully and accurately annotated and over 20-times larger than existing databases. We use a subset of the database with highly similar (≥90% identical) sequences filtered out to report on statistical trends in sequence, flanking base pairs, and length. Both the bpRNA method and the bpRNA-1m database will be valuable resources both for specific analysis of individual RNA molecules and large-scale analyses such as are useful for updating RNA energy parameters for computational thermodynamic predictions, improving machine learning models for structure prediction, and for benchmarking structure-prediction algorithms.

Homopolymeric adenosine RNA plays numerous roles in both cells and non-cellular genetic material, and for lack of evidence to the contrary, it is generally accepted to form a random coil under physiological conditions. However, chemical mapping data generated by the Eterna Massive Open Laboratory indicates that a poly (A) sequence of length seven or more, at pH 8.0 and MgCl concentrations of 10 mM, develops unexpected protection to selective 2’-hydroxyl acylation read out by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing. This protection first appears in poly(A) sequences of length 7 and grows to its maximum strength at length ~10. In a long poly(A) sequence, substitution of a single A by any other nucleotide disrupts the protection, but only for the 6 or so nucleotides on the 5’ side of the substitution. The authors are grateful for pre-publication comments; please use https://docs.google.com/document/d/14972Q36IDTYMglwMXTOrqd4P9orQ6-P3bPbCuITdv6A.

Homopolymeric adenosine RNA plays numerous roles in both cells and non-cellular genetic material, and for lack of evidence to the contrary, it is generally accepted to form a random coil under physiological conditions. However, chemical mapping data generated by the Eterna Massive Open Laboratory indicates that a poly (A) sequence of length seven or more, at pH 8.0 and MgCl concentrations of 10 mM, develops unexpected protection to selective 2'-hydroxyl acylation read out by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing. This protection first appears in poly(A) sequences of length 7 and grows to its maximum strength at length ~10. In a long poly(A) sequence, substitution of a single A by any other nucleotide disrupts the protection, but only for the 6 or so nucleotides on the 5' side of the substitution. The authors are grateful for pre-publication comments; please use https://docs.google.com/document/d/14972Q36IDTYMg lwMXTOrqd4P9orQ6-P3bPbCuITdv6A.Homopolymeric poly(A) plays many regulatory roles in eukaryotes, prokaryotes 1 , organelles 2 3 , retroviruses 4 and retrotransposons. 5 Perhaps the most well-studied roles are those involving the lengthening and shortening of poly(A) tails added to various types of RNA sequences. In the case of mRNA, poly(A) tails are involved in transcription, nuclear export, translation initiation, protein synthesis regulation and, ultimately, decay of the mRNA.Given all these roles, together with the observation that there is no information in a poly(A) sequence other than its length, it is reasonable to suspect that there is something about its structure that makes it so ubiquitous in vivo. Indeed, starting in the 1950's, many experiments were done to determine the structure. Evidence was found of a single stranded structure forming under neutral pH, 6 7 8 and specific molecular level single stranded models for it were proposed. 9 10 More recently, new experimental techniques such as atomic force microscopy, 11 nanoscale pores 12 and vibrational circular dichroism 13 have provided additional supporting evi-