Project description:To characterize RNA-capsid binding sites genome-wide within mature RNA virus particles, we have developed a Next-Generation Sequencing (NGS) platform: viral Photo-Activatable Ribonucleoside CrossLinking (vPAR-CL). In vPAR-CL, 4-thiouridine is incorporated into the encapsidated genomes of virus particles and subsequently UV-crosslinked to adjacent capsid proteins. We demonstrate that vPAR-CL can readily and reliably identify capsid binding sites in genomic viral RNA by detecting crosslink-specific uridine to cytidine transitions in NGS data. Using Flock House virus (FHV) as a model system, we identified highly consistent and significant vPAR-CL signals across virus RNA genome, indicating a clear tropism of the encapsidated RNA genome. Certain interaction sites coincide with previously identified functional RNA motifs. We additionally performed dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to generate a high-resolution profile of single-stranded genomic RNA inside viral particles. Combining vPAR-CL and DMS-MaPseq reveals that the predominant RNA-capsid interaction sites favored double-stranded RNA regions. We disrupted secondary structures associated with vPAR-CL sites using synonymous mutations, resulting in varied effects to virus replication, propagation and packaging. Certain mutations showed substantial deficiency in virus replication, suggesting these RNA-capsid sites are multifunctional. These provide further evidence to support that FHV packaging and replication are highly coordinated and inter-dependent events.
Project description:Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.
Project description:The goal of this set of experiments is to determine the structure of HIV-1 NL4-3 and NHG RNA structure using a novel algorithm that allows for the identification of multiple RNA folding conformations. The input data used for this algorithm is DMS-MaPseq data. We focused on two specific areas of the HIV-1 genome, the Rev Response Element (RRE) and A3 splice acceptor site, as well as probing the whole HIV-1 genome. RNA is DMS-modified in vitro and in vivo as indicated. Overall design: DMS-Modification and targeted sequencing of the HIV-1 A3 splice acceptor site in CD4+ T-cells or transfected HEK293t cells. 1 sample in CD4 T cells, 3 in HEK293t. DMS-modification and targeted sequencing of the HIV-1 A3 splice acceptor site in transfected HEK293t cells using two designed mutant viruses. 1 sample per mutant. HEK293t cells were transfected with a plasmid containing HIV-1 NHG. The cells were DMS-modified and RNA was extracted and sequenced. RNA strucural models were generated using a novel algorithm for regions of interest across the HIV-1 genome. Two HIV-1 RRE mutants were transfected in a HIV-1NHG plasmid into HEK293. Structure of both mutants were analyzed and mixed computationally in order to validate the DREEM pipeline. HIV-1 RNA structure of RRE was analyzed after DMS modification in vitro, in vivo and in the virion. For in vitro samples, HIV-1 RRE was in vitro transcribed. For in vivo and in virion, isolated CD4+ T-cells were infected with HIV-1 NL4-3.
Project description:Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase (TGIRT). DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features for each molecule, and allows genome-wide studies as well as focused investigations of low abundance RNAs. We apply DMS-MaPseq to Drosophila melanogaster ovaries—the first experimental analysis of RNA structure in an animal tissue—and demonstrate its utility in the discovery of a functional RNA structure involved in the non-canonical GUG translation initiation of the human FXR2 mRNA. Additionally, we use DMS-MaPseq to compare the in vivo structure of messages in their pre-mRNA and mature forms. These applications illustrate DMS-MaPseq’s capacity to dramatically expand our ability to monitor RNA structure in vivo. Overall design: Development and application of novel RNA structure probing method in mammalian cells and Drosophila ovaries. Cells were treated with DMS in vivo and RNA was isolated. TGIRT was used to reverse transcribe either randomly fragmented mRNA or gene specific products. TGIRT inserts mismatches at the DMS modified RNA, which are read our mutations when sequencing dsDNA. Samples were prepared using standard library generation approaches (attaching a 3'linker, followed by RT, circlularization and PCR for attaching sequencing adapters) or Nextera XT kit/ LM-PCR for gene specific samples.
Project description:Recent high-throughput structure-sensitive genome-wide sequencing-based assays have enabled large-scale studies of RNA structure, and robust transcriptome-wide computational prediction of individual RNA structures across RNA classes from these assays has potential to further improve the prediction accuracy. Here, we describe HiPR, a novel method for RNA structure prediction at single-nucleotide resolution that combines high-throughput structure probing data (DMS-seq, DMS-MaPseq) with a novel probabilistic folding algorithm. On validation data spanning a variety of RNA classes, HiPR often increases accuracy for predicting RNA structures, giving researchers new tools to study RNA structure.
Project description:RNA is emerging as a key regulator of a plethora of biological processes. While its study has remained elusive for decades, the recent advent of high-throughput sequencing technologies provided the unique opportunity to develop novel techniques for the study of RNA structure and post-transcriptional modifications. Nonetheless, most of the required downstream bioinformatics analyses steps are not easily reproducible, thus making the application of these techniques a prerogative of few laboratories. Here we introduce RNA Framework, an all-in-one toolkit for the analysis of most NGS-based RNA structure probing and post-transcriptional modification mapping experiments. To prove the extreme versatility of RNA Framework, we applied it to both an in-house generated DMS-MaPseq dataset, and to a series of literature available experiments. Notably, when starting from publicly available datasets, our software easily allows replicating authors' findings. Collectively, RNA Framework provides the most complete and versatile toolkit to date for a rapid and streamlined analysis of the RNA epistructurome. RNA Framework is available for download at: http://www.rnaframework.com.
Project description:Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) prepared from leaves of adult plants in Arabidopsis. Chromatin remodeling factor 2 (CHR2) positively regulates transcription of MIR loci whereas repressing microRNA (miRNA) accumulation in vivo. CHR2 can directly bind to and unwind primary miRNAs (pri-miRNAs) and inhibit their processing; and this inhibition entails its remodeling activity in vitro and in vivo. Overall design: Use DMS-MaPseq to probe in vivo RNA structure.
Project description:The structure of 5' untranslated regions (5' UTRs) of bacterial mRNAs often determines the fate of the transcripts. Using a dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) approach, we developed a protocol to generate sequence libraries to determine the base-pairing status of adenines and cytosines in the 5' UTRs of bacterial mRNAs. Our method increases the sequencing depth of the 5' UTRs and allows detection of changes in their structures by sequencing libraries of moderate sizes. For complete details on the use and execution of this protocol, please refer to Ignatov et al. (2020).