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A chemical probe based on the PreQ₁ metabolite enables transcriptome-wide mapping of binding sites.

ABSTRACT: The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ₁ riboswitch system, assessing sixteen diverse PreQ₁-derived probes for their ability to selectively modify the class-I PreQ₁ riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ₁, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ₁ aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ₁ aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ₁, which is similar in structure to guanine, interacts with human RNAs.

SUBMITTER: Balaratnam S

PROVIDER: S-EPMC8494917 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A Chemical Probe Based on the PreQ1 Metabolite Enables Transcriptome-wide Mapping of Binding Sites

Project description:We report the optimization and application of diazirine photocrosslinking probes that enable selective covalent crosslinking to the PreQ1 RNA aptamer and any other interacting RNAs in close proximity. By evaluating the impact of the linker structure and length on crosslinking efficiency we were able to shed important light on what factors have the largest impact on photocrosslinking efficiency in this model system. Additionally, the best compound (compound 11) was shown to efficiently and selectively crosslink site-specifically to the PreQ1 aptamer even in the presence of competing human cellular RNAs. Sequencing experiments were performed in human MCF-7 cell total RNA both with and without spike in of exogenous PreQ1 aptamer. In the aptamer spike-in samples, the only RNA target that was significantly enriched after differential expression analysis with the negative control (compound 17) was the PreQ1 aptamer suggesting that this compound does in fact selectively crosslink to the desired target even in the presence of other RNAs. Additionally, in the samples without spike-in of the aptamer we were able to identify 16 significantly enriched hit RNAs that may represent novel metabolite-RNA interactions. Competitive RT-qPCR experiments confirmed enrichment of a handful of select genes with 11 and showed that the free PreQ1 ligand, which lacks the diazrine crosslinking moeity, is able to compete away compound 11. This suggests that these interactions are specific.

2021-08-09 | GSE168353 | GEO

A Chemical Probe Based on the PreQ1 Metabolite Enables Transcriptome-wide Mapping of Binding Sites

Project description:A Chemical Probe Based on the PreQ1 Metabolite Enables Transcriptome-wide Mapping of Binding Sites

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Nucleobase mutants of a bacterial preQ1-II riboswitch that uncouple metabolite sensing from gene regulation.

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Structure and function of preQ1 riboswitches.

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Project description:Deciphering physiological changes that mediate transition of Mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an individual host for decades. We have combined RNA sequencing (RNA-seq) of 5' triphosphate-enriched libraries with regular RNA-seq to characterize the architecture and expression of M. tuberculosis promoters. We identified over 4,000 transcriptional start sites (TSSs). Strikingly, for 26% of the genes with a primary TSS, the site of transcriptional initiation overlapped with the annotated start codon, generating leaderless transcripts lacking a 5' UTR and, hence, the Shine-Dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria. Genes encoding proteins with active growth functions were markedly depleted from the leaderless transcriptome, and there was a significant increase in the overall representation of leaderless mRNAs in a starvation model of growth arrest. The high percentage of leaderless genes may have particular importance in the physiology of nonreplicating M. tuberculosis.

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Dual chemical labeling enables nucleotide-resolution mapping of DNA abasic sites and common alkylation damage in human mitochondrial DNA.

Project description:Mitochondrial DNA (mtDNA) modifications play an emerging role in innate immunity and inflammatory diseases. Nonetheless, relatively little is known regarding the locations of mtDNA modifications. Such information is critically important for deciphering their roles in mtDNA instability, mtDNA-mediated immune and inflammatory responses, and mitochondrial disorders. The affinity probe-based enrichment of lesion-containing DNA represents a key strategy for sequencing DNA modifications. Existing methods are limited in the enrichment specificity of abasic (AP) sites, a prevalent DNA modification and repair intermediate. Herein, we devise a novel approach, termed dual chemical labeling-assisted sequencing (DCL-seq), for mapping AP sites. DCL-seq features two designer compounds for enriching and mapping AP sites specifically at single-nucleotide resolution. For proof of principle, we mapped AP sites in mtDNA from HeLa cells under different biological conditions. The resulting AP site maps coincide with mtDNA regions with low TFAM (mitochondrial transcription factor A) coverage and with potential G-quadruplex-forming sequences. In addition, we demonstrated the broader applicability of the method in sequencing other DNA modifications in mtDNA, such as N7-methyl-2'-deoxyguanosine and N3-methyl-2'-deoxyadenosine, when coupled with a lesion-specific repair enzyme. Together, DCL-seq holds the promise to sequence multiple DNA modifications in various biological samples.

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Structure and function analysis of a type III preQ1-I riboswitch from Escherichia coli reveals direct metabolite sensing by the Shine-Dalgarno sequence.

Project description:Riboswitches are small noncoding RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine1 (preQ1)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ1, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ1-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ1 riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ1 metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ1 riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ1 over the chemically similar metabolic precursor preQ0. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ1 riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.

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