Massive parallel-sequencing-based hydroxyl radical probing of RNA accessibility

Hydroxyl Radical Footprinting (HRF) is a tried-and-tested method for analysis of the tertiary structure of RNA and for identification of protein footprints on RNA. The hydroxyl radical reaction breaks accessible parts of the RNA backbone, thereby allowing ribose accessibility to be determined by detection of reverse transcriptase termination sites. Current methods for HRF rely on reverse transcription of a single primer and detection by fluorescent fragments by capillary electrophoresis. Here, we describe an accurate and efficient massive parallel-sequencing-based method for probing RNA accessibility with hydroxyl radicals, called HRF-Seq. Using random priming and a novel barcoding scheme, we show that HRF-Seq dramatically increases the throughput of HRF experiments and facilitates the parallel analysis of multiple RNAs or experimental conditions. Moreover, we demonstrate that HRF-Seq data for the Escherichia coli 16S rRNA correlates well with the ribose accessible surface area as determined by X-ray crystallography and have a resolution that readily allows the difference in accessibility caused by exposure of one side of RNA helices to be observed

Use of tiling array data and RNA secondary structure predictions to identify noncoding RNA genes

<p>Abstract</p> <p>Background</p> <p>Within the last decade a large number of noncoding RNA genes have been identified, but this may only be the tip of the iceberg. Using comparative genomics a large number of sequences that have signals concordant with conserved RNA secondary structures have been discovered in the human genome. Moreover, genome wide transcription profiling with tiling arrays indicate that the majority of the genome is transcribed.</p> <p>Results</p> <p>We have combined tiling array data with genome wide structural RNA predictions to search for novel noncoding and structural RNA genes that are expressed in the human neuroblastoma cell line SK-N-AS. Using this strategy, we identify thousands of human candidate RNA genes. To further verify the expression of these genes, we focused on candidate genes that had a stable hairpin structures or a high level of covariance. Using northern blotting, we verify the expression of 2 out of 3 of the hairpin structures and 3 out of 9 high covariance structures in SK-N-AS cells.</p> <p>Conclusion</p> <p>Our results demonstrate that many human noncoding, structured and conserved RNA genes remain to be discovered and that tissue specific tiling array data can be used in combination with computational predictions of sequences encoding structural RNAs to improve the search for such genes.</p

Pharmaco-miR:linking microRNAs and drug effects

MicroRNAs (miRNAs) are short regulatory RNAs that down-regulate gene expression. They are essential for cell homeostasis and active in many disease states. A major discovery is the ability of miRNAs to determine the efficacy of drugs, which has given rise to the field of ‘miRNA pharmacogenomics’ through ‘Pharmaco-miRs’. miRNAs play a significant role in pharmacogenomics by down-regulating genes that are important for drug function. These interactions can be described as triplet sets consisting of a miRNA, a target gene and a drug associated with the gene. We have developed a web server which links miRNA expression and drug function by combining data on miRNA targeting and protein–drug interactions. miRNA targeting information derive from both experimental data and computational predictions, and protein–drug interactions are annotated by the Pharmacogenomics Knowledge base (PharmGKB). Pharmaco-miR’s input consists of miRNAs, genes and/or drug names and the output consists of miRNA pharmacogenomic sets or a list of unique associated miRNAs, genes and drugs. We have furthermore built a database, named Pharmaco-miR Verified Sets (VerSe), which contains miRNA pharmacogenomic data manually curated from the literature, can be searched and downloaded via Pharmaco-miR and informs on trends and generalities published in the field. Overall, we present examples of how Pharmaco-miR provides possible explanations for previously published observations, including how the cisplatin and 5-fluorouracil resistance induced by miR-148a may be caused by miR-148a targeting of the gene KIT. The information is available at www.Pharmaco-miR.org

Excess primer degradation by Exo I improves the preparation of 3' cDNA ligation-based sequencing libraries

RNA sequencing library construction using single-stranded ligation of a DNA adapter to 3′ ends of cDNAs often produces primer–adapter byproducts, which compete with cDNA–adapter ligation products during library amplification and, therefore, reduces the number of informative sequencing reads. We find that Escherichia coli Exo I digestion efficiently and selectively removes surplus reverse transcription primer and thereby reduces the primer–adapter product contamination in 3′ cDNA ligation-based sequencing libraries, including small RNA libraries, which are typically similar in size to the primer–adapter products. We further demonstrate that Exo I treatment does not lead to trimming of the cDNA 3′ end when duplexed with the RNA template. Exo I digestion is easy to perform and implement in other protocols and could facilitate a more widespread use of 3′ cDNA ligation for sequencing-based applications

Identification of miRNA targets with stable isotope labeling by amino acids in cell culture

miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection. This repressed set of genes significantly overlaps with miRNA-1 regulated genes that have been identified with DNA array technology and are predicted by computational methods. Moreover, we find that the 3′-untranslated region for the repressed set are enriched in miRNA-1 complementary sites. Our findings demonstrate that SILAC can be used for miRNA target identification and that one highly expressed miRNA can regulate the levels of many different proteins

SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA–RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing