Analysis Of Small Non-coding RNAs Research Articles

A Non-Canonical Pathway Induced by Externally Applied Virus-Specific dsRNA in Potato Plants.

The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18-30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection.

Integrative analysis of small non-coding RNAs predicts a piRNA/miRNA-CCND1/BRAF/HRH1/ATXN3 regulatory circuit that drives oncogenesis in glioblastoma.

The high-grade astrocytoma, glioblastoma multiforme (GBM), is the most common primary tumour of the brain, known for being aggressive and developing drug resistance. The non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), have critical functions in tumorigenesis and cancer drug resistance. Hence, we profiled miRNAs, piRNAs, and genes in U-87 MG GBM cells by next-generation sequencing and performed target prediction, pathway enrichment, protein-protein interaction, co-expression studies, and qRT-PCR validations to predict their possible roles in the malignancy. The study identified 335 miRNAs, 665 piRNAs, and 4286 genes differentially expressed (DE) in GBM. Among them 128 DE genes (DEGs) were targeted by both miRNAs and piRNAs, while 1817 and 192 were targeted solely by miRNAs or piRNAs, respectively. Interestingly, all the DEG targets enriched in cancer processes were overexpressed in GBM. Among these, BRAF was solely targeted by two piRNAs and this was found to be co-expressed with 19 sole targets of 5 miRNAs, including CCND1, and both were found to regulate cell proliferation in cancer. We conjectured that upregulated HRH1 and ATXN3 were targeted by both piRNAs and miRNAs, and along with BRAF and CCND1 might induce cell proliferation in GBM through G-protein-coupled receptor or Akt signalling pathways due to downregulation of the respective targeting small RNAs. These targets were also linked to the progression and overall survival of GBM patients, suggesting that they could be used as biomarkers. Overall, this study has identified a few novel ncRNA targets, which might aid in a better understanding of GBM pathogenesis.

Analysis of Small Non-coding RNAs as Signaling Intermediates of Environmentally Integrated Responses to Abiotic Stress.

Research to date on abiotic stress responses in plants has been largely focused on the plant itself, but current knowledge indicates that microorganisms can interact with and help plants during periods of abiotic stress. In our research, we aim to investigate the interkingdom communication between the plant root and the rhizo-microbiota. Our investigation showed that miRNA plays a pivotal role in this interkingdom communication. Here, we describe a protocol for the analysis of miRNA secreted by the plant root, which includes all of the steps from the isolation of the miRNA to the bioinformatics analysis. Because of their short nucleotide length, Next Generation Sequencing (NGS) library preparation from miRNAs can be challenging due to the presence of dimer adapter contaminants. Therefore, we highlight some strategies we adopt to inhibit the generation of dimer adapters during library preparation. Current screens of miRNA targets mostly focus on the identification of targets present in the same organism expressing the miRNA. Our bioinformatics analysis challenges the barrier of evolutionary divergent organisms to identify candidate sequences of the microbiota targeted by the miRNA of plant roots. This protocol should be of interest to researchers investigating interkingdom RNA-based communication between plants and their associated microorganisms, particularly in the context of holobiont responses to abiotic stresses.

TRNA-derived fragment tRFLys-CTT-010 promotes triple-negative breast cancer progression by regulating glucose metabolism via G6PC.

tRNA-derived fragments (tRFs) are a novel class of small non-coding RNAs whose biological roles are not well defined. Here, using multiple approaches, we investigated its role in human triple-negative breast cancer (TNBC). Our genome-wide transcriptome analysis of small non-coding RNAs revealed that tRFLys-CTT-010 was significantly increased in human TNBC. It promoted TNBC proliferation and migration. It also closely associated with starch and sucrose metabolism pathways (Kyoto Encyclopedia of Genes and Genomes analysis) and positively regulated the expression of glucose-6-phosphatase catalytic subunit (G6PC), one of the related genes in the pathway. G6PC, a complex of glucose-6-phosphatase in gluconeogenesis and glycogenolysis, is upregulated in human TNBC samples. Further studies demonstrated that overexpression of G6PC in tRFLys-CTT-010 inhibitor-transfected TNBC cell lines can reverse malignant biological behavior and knockdown of G6PC in TNBC cell lines inhibited tumor progression and reversed the oncogenic function of tRFLys-CTT-010. In addition, tRFLys-CTT-010 interacted with G6PC to regulate cellular lactate production and glycogen consumption, resulting in cell survival and proliferation. Thus, fine-tuning glucose metabolism and the tRFLys-CTT-010/G6PC axis may provide a therapeutic target for TNBC treatment.

309 - Analysis of small non-coding RNAs in urinary exosomes accurately classifies prostate cancer into low grade (GG1) and higher grade (GG2-5)

309 - Analysis of small non-coding RNAs in urinary exosomes accurately classifies prostate cancer into low grade (GG1) and higher grade (GG2-5)

Analysis of small non-coding RNAs in urinary exosomes to classify prostate cancer into low-grade (GG1) and higher-grade (GG2-5).

277 Background: To develop a new predictive test for prostate cancer, based on the interrogation of small non-coding RNAs (sncRNA) isolated from urinary exosomes. We report the development and performance of the miR Sentinel PCa Test, that distinguishes patients with prostate cancer from those with no evidence of prostate cancer (NEPC) and the miR Sentinel CS Test, that distinguishes low grade from higher grade disease. Methods: Affymetrix miR 4.0 arrays were used to identify informative sncRNAs isolated from urinary exosomes. sncRNA from 233 subjects undergoing a prostate biopsy [89 men with benign biopsies, 88 with grade group 1 (GG1) cancer and 56 patients with grade group 2-5 (GG2-5)] were interrogated on these arrays. A custom OpenArray platform was designed to interrogate the 280 most informative sncRNAs, identified using a data-driven selection algorithm. The platform was designed to categorize patients as either no cancer or cancer using the miR Sentinel PCa Test, and subclassify the patients with cancer into GG1 or GG2-5 cancer using the miR Sentinel CS Test. The performance of the miR Sentinel PCa and CS Tests was validated in an independent cohort. Results: In 233 men, theSentinel PCa Test correctly classified 89/89 subjects with no cancer and 144/144 with cancer. The Sentinel CS Test correctly identified 55/56 patients with GG2-5 and 87/88 patients with GG1. Sensitivity was 98%, Specificity 98%, NPV 98% and PPV 93%. For validation, a prospective observational study of 329 subjects (NEPC = 139; GG1= 88; GG2-5 = 102) with elevated PSA correctly classified 134/139 as no cancer [Sensitivity 98% (195/199); Specificity 96% (134/139), PPV 98% and NPV 97%]. The Sentinel CS Test classified 87/88 as GG1 and 102/102 as GG2-5 [Sensitivity 100% (102/102), Specificity 99% (87/88), PPV 99%, and NPV=100%]. Conclusions: Initial evaluation of the miR Sentinel PCa and CS Tests demonstrated the high precision of these tests to detect prostate cancer and distinguish high grade (GG2-5) disease. Further validation is ongoing.[Table: see text]

In silico prediction and expression profile analysis of small non-coding RNAs in Herbaspirillum seropedicae SmR1

BackgroundHerbaspirillum seropedicae is a diazotrophic bacterium from the β-proteobacteria class that colonizes endophytically important gramineous species, promotes their growth through phytohormone-dependent stimulation and can express nif genes and fix nitrogen inside plant tissues. Due to these properties this bacterium has great potential as a commercial inoculant for agriculture. The H. seropedicae SmR1 genome is completely sequenced and annotated but despite the availability of diverse structural and functional analysis of this genome, studies involving small non-coding RNAs (sRNAs) has not yet been done. We have conducted computational prediction and RNA-seq analysis to select and confirm the expression of sRNA genes in the H. seropedicae SmR1 genome, in the presence of two nitrogen independent sources and in presence of naringenin, a flavonoid secreted by some plants.ResultsThis approach resulted in a set of 117 sRNAs distributed in riboswitch, cis-encoded and trans-encoded categories and among them 20 have Rfam homologs. The housekeeping sRNAs tmRNA, ssrS and 4.5S were found and we observed that a large number of sRNAs are more expressed in the nitrate condition rather than the control condition and in the presence of naringenin. Some sRNAs expression were confirmed in vitro and this work contributes to better understand the post transcriptional regulation in this bacterium.ConclusionsH. seropedicae SmR1 express sRNAs in the presence of two nitrogen sources and/or in the presence of naringenin. The functions of most of these sRNAs remains unknown but their existence in this bacterium confirms the evidence that sRNAs are involved in many different cellular activities to adapt to nutritional and environmental changes.

Abstract 2466: A comprehensive genome-wide analysis of small noncoding RNAs in T-cell prolymphocytic leukemia

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare type of neoplasm with an adverse prognosis due to poor responsiveness to conventional chemotherapies. Accurate and rapid diagnosis of T-PLL is problematic due to the heterogeneous nature of the disease. Therefore, further molecular insights of T-PLL are needed. Most of the T-PLL cases (80%) harbor chromosome 14 inversions and translocations or activating mutations in the JAK-STAT pathway. Small non-coding RNAs (sncRNA) are defined as single stranded RNAs that are smaller than 200nt in length and include e.g. microRNAs (miR), small nucleolar RNA (snoRNA) and PIWI-interacting RNAs (piRNA). To increase the molecular understanding of T-PLL, we performed sncRNA expression profiling with the Illumina platform on a well-characterized panel of 21 T-PLL samples. Notably, detailed molecular, cytogenetic, morphologic and clinical data are available of all T-PLL patients included in this study. Of the selected samples, we performed gene expression profiling by microarrays, cytogenetics, VDJ-recombination and multi-color flow cytometric analysis. In short, we sorted CD4+ and CD8+ naïve (CD45RA+, CD27+), effector (CD45RA+, CD27-) and memory (CD45RO+, CD27+) T-cells, and TCRγδ+ T-cells from Ficoll separated PBMCs of buffy coat blood. Total RNA of leukemic cells isolated from the blood was extracted and cDNA libraries for sequencing sncRNAs were generated. For the annotation of the sequence reads we used the DASHR database of sncRNAs and miRbase database of mature miRs. SnoRNA (16%) and ribosomal (r) RNAs (25%) were the most abundant, whereas miRs were only 12% of the total sncRNA fraction. Next, we performed a correlation study using the expression profiles of the different types of sncRNAs and found a clear separation of T-PLL samples from the normal T-cell fractions when miRNA, piRNA and snoRNA expression profiles were used in the analysis. In addition, based on these data we could already identify potential subgroups of T-PLL, indicating that T-PLL is not a homogeneous disease. Finally, we noted aberrant expression of different types of sncRNAs compared to normal T-cell fractions, which may be indicative for essential roles of sncRNA species in the pathogenesis of T-PLL. Currently, we are in the process of characterizing sncRNA species that are aberrantly regulated specifically in T-PLL. Finally, we are selecting candidate sncRNAs for further functional characterization and for testing their diagnostic and/or prognostic value. Citation Format: Leticia G. Leon, Steven C. Koetzier, Fabiënne van Opstal, Joyce Schilperoord-Vermeulen, Anton W. Langerak, Stefan J. Erkeland. A comprehensive genome-wide analysis of small noncoding RNAs in T-cell prolymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2466.

Novel platform for monitoring bladder cancer recurrence using expression analysis of small non-coding RNAs.

12070Background: Bladder cancer patients are routinely monitored after treatment by cystoscopy due to a high rate of recurrence. In the absence of an accurate non-invasive screening test to monitor...

Small noncoding RNAs in FSHD2 muscle cells reveal both DUX4- and SMCHD1-specific signatures.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by insufficient epigenetic repression of D4Z4 macrosatellite repeat where DUX4, an FSHD causing gene is embedded. There are two forms of FSHD, FSHD1 with contraction of D4Z4 repeat and FSHD2 with chromatin compaction defects mostly due to SMCHD1 mutation. Previous reports showed DUX4-induced gene expression changes as well as changes in microRNA expression in FSHD muscle cells. However, a genome wide analysis of small noncoding RNAs that might be regulated by DUX4 or by mutations in SMCHD1 has not been reported yet. Here, we identified several types of small noncoding RNAs including known microRNAs that are differentially expressed in FSHD2 muscle cells compared to control. Although fewer small RNAs were differentially expressed during muscle differentiation in FSHD2 cells compared to controls, most of the known myogenic microRNAs, such as miR1, miR133a and miR206 were induced in both FSHD2 and control muscle cells during differentiation. Our small RNA sequencing data analysis also revealed both DUX4- and SMCHD1-specific changes in FSHD2 muscle cells. Six FSHD2 microRNAs were affected by DUX4 overexpression in control myoblasts, whereas increased expression of tRNAs and 5S rRNAs in FSHD2 muscle cells was largely recapitulated in SMCHD1-depleted control myoblasts. Altogether, our studies suggest that the small noncoding RNA transcriptome changes in FSHD2 might be different from those in FSHD1 and that these differences may provide new diagnostic and therapeutic tools specific to FSHD2.

Analysis of small non-coding RNAs of poorly annotated species in Galaxy with the help of ortholog information of well annotated species

Analysis of small non-coding RNAs of poorly annotated species in Galaxy with the help of ortholog information of well annotated species

Microarray analysis of small non-coding RNAs.

Microarray technology has evolved to efficiently profile the expression of RNAs. However, analysis of small non-coding RNAs (ncRNAs) is challenging due to their short length and highly divergent sequences with large variation in GC content leading to very different hybridization properties. To overcome these challenges, LNA-modified oligonucleotides have been used to enhance and normalize the melting temperature (Tm) of capture probes, which allows sensitive profiling of small ncRNAs regardless of their sequence. Here, we describe the isolation and labeling of small non-coding RNAs, as well as their hybridization to microarrays with LNA-modified oligonucleotide probes using a semi-automated hybridization device.

Abstract 522: Revealing the complexity of cancer associated small non-coding RNAs by next generation sequencing (NGS) and low-density array

Abstract Background. The recent development of high-throughput sequencing technologies (Next Generation Sequencing - NGS) provided instruments to reveal the complexity of nucleic acids, highlighting the existence of numerous species of non-coding RNAs (ncRNAs) that play a significant regulatory role in complex organisms, impacting on both physiology and disease. In particular, microRNAs (miRNAs) have been proposed as “next-generation” markers for their ability to unambiguously distinguish cellular states, including SCs, progenitors and differentiated cells, as well as tumour types, even among closely related cancers or in different biological fluids. However, a comprehensive approach for the identification and characterization of small non-coding RNAs from pathological samples (blood, FFPE, primary cultures) has not been established nor compared with current available methodologies (i.e. QPCR). Aim: Our aim is to develop a simultaneous and comparative protocol for the analysis of small non-coding RNAs from pathological samples, which encompasses both high-throughput QPCR and sequencing analysis in order to reveal the complexity of cancer associated small ncRNAs. Results: An optimized protocol to analyse small ncRNAs from different pathological samples has been developed and compared with high-throughput QPCR. Both platforms resulted as highly efficient and quantitative, although NGS manages to score many molecules and RNA species that could not be analysed by current QPCR platforms, thus revealing a major complexity of cancer associated non-coding RNAs. Conclusion: We propose that the combined use of NGS and QPCR platforms would allow a wider and more detailed analysis of ncRNAs, expanding our ability to fish out robust and efficient molecular markers for diagnostic (early detection), prognostic or therapeutic use. Note: This abstract was not presented at the meeting. Citation Format: Francesca Montani, Matteo Marzi, Rose Mary Carletti, Pier Paolo Di Fiore, Francesco Nicassio. Revealing the complexity of cancer associated small non-coding RNAs by next generation sequencing (NGS) and low-density array. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 522. doi:10.1158/1538-7445.AM2014-522

IMir: an integrated pipeline for high-throughput analysis of small non-coding RNA data obtained by smallRNA-Seq.

BackgroundQualitative and quantitative analysis of small non-coding RNAs by next generation sequencing (smallRNA-Seq) represents a novel technology increasingly used to investigate with high sensitivity and specificity RNA population comprising microRNAs and other regulatory small transcripts. Analysis of smallRNA-Seq data to gather biologically relevant information, i.e. detection and differential expression analysis of known and novel non-coding RNAs, target prediction, etc., requires implementation of multiple statistical and bioinformatics tools from different sources, each focusing on a specific step of the analysis pipeline. As a consequence, the analytical workflow is slowed down by the need for continuous interventions by the operator, a critical factor when large numbers of datasets need to be analyzed at once.ResultsWe designed a novel modular pipeline (iMir) for comprehensive analysis of smallRNA-Seq data, comprising specific tools for adapter trimming, quality filtering, differential expression analysis, biological target prediction and other useful options by integrating multiple open source modules and resources in an automated workflow. As statistics is crucial in deep-sequencing data analysis, we devised and integrated in iMir tools based on different statistical approaches to allow the operator to analyze data rigorously. The pipeline created here proved to be efficient and time-saving than currently available methods and, in addition, flexible enough to allow the user to select the preferred combination of analytical steps. We present here the results obtained by applying this pipeline to analyze simultaneously 6 smallRNA-Seq datasets from either exponentially growing or growth-arrested human breast cancer MCF-7 cells, that led to the rapid and accurate identification, quantitation and differential expression analysis of ~450 miRNAs, including several novel miRNAs and isomiRs, as well as identification of the putative mRNA targets of differentially expressed miRNAs. In addition, iMir allowed also the identification of ~70 piRNAs (piwi-interacting RNAs), some of which differentially expressed in proliferating vs growth arrested cells.ConclusionThe integrated data analysis pipeline described here is based on a reliable, flexible and fully automated workflow, useful to rapidly and efficiently analyze high-throughput smallRNA-Seq data, such as those produced by the most recent high-performance next generation sequencers. iMir is available at http://www.labmedmolge.unisa.it/inglese/research/imir.

Prediction and Characterization of Small Non-Coding RNAs Related to Secondary Metabolites in Saccharopolyspora erythraea

Saccharopolyspora erythraea produces a large number of secondary metabolites with biological activities, including erythromycin. Elucidation of the mechanisms through which the production of these secondary metabolites is regulated may help to identify new strategies for improved biosynthesis of erythromycin. In this paper, we describe the systematic prediction and analysis of small non-coding RNAs (sRNAs) in S. erythraea, with the aim to elucidate sRNA-mediated regulation of secondary metabolite biosynthesis. In silico and deep-sequencing technologies were applied to predict sRNAs in S. erythraea. Six hundred and forty-seven potential sRNA loci were identified, of which 382 cis-encoded antisense RNA are complementary to protein-coding regions and 265 predicted transcripts are located in intergenic regions. Six candidate sRNAs (sernc292, sernc293, sernc350, sernc351, sernc361, and sernc389) belong to four gene clusters (tpc3, pke, pks6, and nrps5) that are involved in secondary metabolite biosynthesis. Deep-sequencing data showed that the expression of all sRNAs in the strain HL3168 E3 (E3) was higher than that in NRRL23338 (M), except for sernc292 and sernc361 expression. The relative expression of six sRNAs in strain M and E3 were validated by qRT-PCR at three different time points (24, 48, and 72 h). The results showed that, at each time point, the transcription levels of sernc293, sernc350, sernc351, and sernc389 were higher in E3 than in M, with the largest difference observed at 72 h, whereas no signals for sernc292 and sernc361 were detected. sernc293, sernc350, sernc351, and sernc389 probably regulate iron transport, terpene metabolism, geosmin synthesis, and polyketide biosynthesis, respectively. The major significance of this study is the successful prediction and identification of sRNAs in genomic regions close to the secondary metabolism-related genes in S. erythraea. A better understanding of the sRNA-target interaction would help to elucidate the complete range of functions of sRNAs in S. erythraea, including sRNA-mediated regulation of erythromycin biosynthesis.

Comparative transcriptome analysis of small noncoding RNAs in different stages of Trypanosoma brucei

Trypanosoma brucei, a pathogen of human and domestic animals, is an early evolved parasitic protozoan with a complex life cycle. Most genes of this parasite are post-transcriptionally regulated. However, the mechanisms and the molecules involved remain largely unknown. We have deep-sequenced the small RNAs of two life stages of this parasite--the bloodstream form and the procyclic form. Our results show that the small RNAs of T. brucei could derive from multiple sources, including NATs (natural antisense transcripts), tRNAs, and rRNAs. Most of these small RNAs in the two stages were found to share uniform characteristics. However, our results demonstrate that their variety and expression show significant differences between different stages, indicating possible functional differentiation. Dicer-knockdown evidence further proved that some of the small interfering RNAs (siRNAs) could regulate the expression of genes. Based on the genome-wide analysis of the small RNAs in the two stages of T. brucei, our results not only provide evidence to study their differentiation but also shed light on questions regarding the origins and evolution of small RNA-based mechanisms in early eukaryotes.

Unbiased approach to profile the variety of small non-coding RNA of human blood plasma with massively parallel sequencing technology

Objective: Understanding structures of circulating RNA expands fundamental knowledge of cell communications and signaling pathways as well as allows developing new molecular diagnostic approaches. The aim of this study was to deploy a new approach to sequencing cDNA library construction which expands the capabilities of high-throughput sequencing analysis of small non-coding RNAs. With the approach, we performed massively parallel sequencing of human blood plasma RNA to document profile of common and peculiar RNA species normally circulating in blood of healthy individuals.Methods: Total RNA was extracted from blood plasma samples of eight apparently healthy individuals. To obtain comprehensive cDNA libraries RNA was dephosphorylated and then 5′-phosphorylated. 5′-Phosphorylated total plasma RNA was ligated with adapters, reverse transcribed and eight personalized cDNA libraries were constructed. Libraries were sequenced with SOLiD™ technology.Results/conclusion: Fragments of rRNA, mitochondrial transcripts, microRNAs, fragments of scRNAs, snRNA and snoRNA, fragments of several mRNAs as well as the set of newly discovered transcripts were found to be permanent representatives of human blood plasma RNAs. Advanced mapping allowed to identify circulating herpes virus and enterobacterial transcripts. Documented profile of circulating RNA of healthy individuals provides basis for development of new approaches in research and diagnosis of human pathology.

Deep sequencing analysis of small non-coding RNAs reveals the diversity of microRNAs and piRNAs in the human epididymis

The epididymis plays a crucial role in regulating the development of sperm motility and fertilizing capacity. Small non-coding RNAs (sncRNAs), especially microRNAs (miRNAs), can participate in the regulation of various physiological pathways. However, their abundance and whether they are involved in the regulation of gene expression in the human epididymis are unknown. By adopting the Solexa deep sequencing approach, we systematically investigated the sncRNAs in the adult human epididymis. A total of 4903 unique sequences representing 527 known miRNA were discovered. Eighteen novel miRNA genes encoding 23 mature miRNAs were also identified and the expression of some of them was confirmed by qRT-PCR. The presence of Piwi-interacting RNAs (piRNAs) in the library also adds to the diversity of the sncRNA population in the human epididymis. This research will contribute to a preliminary database for their functional study in male reproductive system.

Selective 2′-Hydroxyl Acylation Analyzed by Protection from Exoribonuclease

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a powerful approach for characterizing RNA structure and dynamics at single-nucleotide resolution. However, SHAPE technology is limited, sometimes severely, because primer extension detection obscures structural information for approximately 15 nts at the 5' end and 40-60 nts at the 3' end of the RNA. Moreover, detection by primer extension is more complex than the actual structure-selective chemical interrogation step. Here we quantify covalent adducts in RNA directly by adduct-inhibited exoribonuclease degradation. RNA 2'-O-adducts block processivity of a 3'-->5' exoribonuclease, RNase R, to produce fragments that terminate three nucleotides 3' of the modification site. We analyzed the structure of the native thiamine pyrophosphate (TPP) riboswitch aptamer domain and identified large changes in local nucleotide dynamics and global RNA structure upon ligand binding. In addition to numerous changes that can be attributed to ligand recognition, we identify a single nucleotide bulge register shift, distant from the binding site, that stabilizes the ligand-bound structure. Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) should prove broadly useful for facile structural analysis of small noncoding RNAs and for RNAs that have functionally critical structures at their 5' and 3' ends.