Project description:We develop an enhanced MaP protocol based on MarathonRT and bioinformatic optimizations which enables robust DMS probing of all four RNA nucleotides within living cells. We demonstrate this on RNA from E. coli and HEK293 cell lines.
Project description:Structure probing experiments were performed on in vitro transcripts and E. coli and human cell cultures under natively extracted (cell-free) and in-cell conditions to benchmark the performance of the newly introduced PAIR-MaP correlated chemical probing strategy for detecting RNA duplexes. Multiple-hit dimethyl sulfate (DMS) probing was done using new buffer conditions that facilitate DMS modification of all four nucleotides.
Project description:Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) from Methanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing in M. acetivorans. Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure within M. acetivorans.
Project description:Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes. In a genetic screen in E. coli, we discovered a novel rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNA) to rescue upstream ribosomes. SmrB is recruited by ribosome collisions. Cryo-EM structures of collided disomes from E. coli and B. subtilis reveal interactions between the 30S subunits and a possible SmrB binding site. These findings show that ribosome collisions trigger ribosome rescue in bacteria and reveal the mechanism by which this occurs.
Project description:mRNA molecules are generally thought to be messengers of genetic information in the cell. Stretches of RNA that are complementary in sequence have a propensity to pair, forming elements of secondary structure within RNA molecules. Although these structures will exist in every mRNA molecule, the role they play in gene regulation is not well understood. Currently two techniques are available to profile the cell RNA structure, in-vivo, in an unbiased manner. We applied one of those techniques, DMS-seq, for probing the human mRNA structure in primary foreskin fibroblasts (HFFs) along human cytomegalovirus (HCMV) infection. As a proof of concept, using DMS-seq, we managed to predict the already solved human 28S rRNA structure with high accuracy. Using our data, we are able to show for the first time in-vivo, that human coding sequences (CDSs) are less structured relative to UTRs. Additionally, we provide systematic in-vivo evidences for unwinding of the mRNA by the ribosomes during translation. Intriguingly, we also found structural changes in human CDSs around the start and stop codon, and also in 3’UTRs. The combination of accurate measurements of translation regulation and mapping changes in mRNA structure along a dynamic process can be used as a platform for deciphering cis-regulatory elements that control gene expression in various cell types, organisms and biological processes.
Project description:Ribosomes are among the largest folded RNAs, whose function depends on their structure. Nonetheless, in vitro studies indicate a propensity of rRNAs to misfold. We use a combination of DMS-MaPseq, structural analyses, biochemical experiments, and yeast genetics to dissect the final RNA folding steps of the small ribosomal subunit head.
Project description:Ribosomes are among the largest folded RNAs, whose function depends on their structure. Nonetheless, in vitro studies indicate a propensity of rRNAs to misfold. We use a combination of DMS-MaPseq, structural analyses, biochemical experiments, and yeast genetics to dissect the final RNA folding steps of the small ribosomal subunit head.
