RNA Framework for Assaying the Structure of RNAs by High-Throughput Sequencing

RNA structure is a key player in regulating a plethora of biological processes. A large part of the functions carried out by RNA is mediated by its structure. To this end, in the last decade big effort has been put in the development of new RNA probing methods based on Next-Generation Sequencing (NGS), aimed at the rapid transcriptome-scale interrogation of RNA structures. In this chapter we describe RNA Framework, the to date most comprehensive toolkit for the analysis of NGS-based RNA structure probing experiments. By using two published datasets, we here illustrate how to use the different components of the RNA Framework and how to choose the analysis parameters according to the experimental setup.</p

Computational approaches for RNA structure ensemble deconvolution from structure probing data

RNA structure probing experiments have emerged over the last decade as a straightforward way to determine the structure of RNA molecules in a number of different contexts. Although powerful, the ability of RNA to dynamically interconvert between, and to simultaneously populate, alternative structural configurations, poses a nontrivial challenge to the interpretation of data derived from these experiments. Recent efforts aimed at developing computational methods for the reconstruction of coexisting alternative RNA conformations from structure probing data are paving the way to the study of RNA structure ensembles, even in the context of living cells. In this review, we critically discuss these methods, their limitations and possible future improvements

High-throughput single nucleotide variant discovery in E14 mouse embryonic stem cells provides a new reference genome assembly

Mouse E14 embryonic stem cells (ESCs) are a well-characterized and widespread used ESC line, often employed for genome-wide studies involving next generation sequencing analysis. More than 2×10(9) sequences made on Illumina platform derived from the genome of E14 ESCs were used to build a database of about 2.7×10(6) single nucleotide variants (SNVs). The identified variants are enriched in intergenic regions, but several thousands reside in gene exons and regulatory regions, such as promoters, enhancers, splicing sites and untranslated regions of RNA, thus indicating high probability of an important functional impact on the molecular biology of these cells. We created a new E14 genome assembly reference that increases the number of mapped reads of about 5%. We performed a Reduced Representation Bisulfite Sequencing on E14 ESCs and we obtained an increase of about 120,000 called CpGs and avoided about 20,000 wrong CpG calls with respect to the mm9 genome reference

SHAPE-guided RNA structure homology search and motif discovery

The rapidly growing popularity of RNA structure probing methods is leading to increasingly large amounts of available RNA structure information. This demands the development of efficient tools for the identification of RNAs sharing regions of structural similarity by direct comparison of their reactivity profiles, hence enabling the discovery of conserved structural features. We here introduce SHAPEwarp, a largely sequence-agnostic SHAPE-guided algorithm for the identification of structurally-similar regions in RNA molecules. Analysis of Dengue, Zika and coronavirus genomes recapitulates known regulatory RNA structures and identifies novel highly-conserved structural elements. This work represents a preliminary step towards the model-free search and identification of shared and conserved RNA structural features within transcriptomes.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo

In vivo probing of nascent RNA structures reveals principles of cotranscriptional folding

Defining the in vivo folding pathway of cellular RNAs is essential to understand how they reach their final native conformation. We here introduce a novel method, named Structural Probing of Elongating Transcripts (SPET-seq), that permits single-base resolution analysis of transcription intermediates' secondary structures on a transcriptome-wide scale, enabling base-resolution analysis of the RNA folding events. Our results suggest that cotranscriptional RNA folding in vivo is a mixture of cooperative folding events, in which local RNA secondary structure elements are formed as they get transcribed, and non-cooperative events, in which 5'-halves of long-range helices get sequestered into transient non-native interactions until their 3' counterparts have been transcribed. Together our work provides the first transcriptome-scale overview of RNA cotranscriptional folding in a living organism

Role of CD133 Molecule in Wnt Response and Renal Repair

Renal repair after injury is dependent on clonal expansion of proliferation‐competent cells. In the human kidney, the expression of CD133 characterizes a population of resident scattered cells with resistance to damage and ability to proliferate. However, the biological function of the CD133 molecule is unknown. By RNA sequencing, we found that cells undergoing cisplatin damage lost the CD133 signature and acquired metanephric mesenchymal and regenerative genes such as SNAIL1, KLF4, SOX9, and WNT3. CD133 was reacquired in the recovery phase. In CD133‐Kd cells, lack of CD133 limited cell proliferation after injury and was specifically correlated with deregulation of Wnt signaling and E‐cadherin pathway. By immunoprecipitation, CD133 appeared to form a complex with E‐cadherin and β‐catenin. In parallel, CD133‐Kd cells showed lower β‐catenin levels in basal condition and after Wnt pathway activation and reduced TCF/LEF promoter activation in respect to CD133+ cells. Finally, the lack of CD133 impaired generation of nephrospheres while favoring senescence. These data indicate that CD133 may act as a permissive factor for β‐catenin signaling, preventing its degradation in the cytoplasm. Therefore, CD133 itself appears to play a functional role in renal tubular repair through maintenance of proliferative response and control of senescence