Project description:Programmable RNA-targeting tools provide the unique opportunity to study RNA regulation and control gene expression in an endogenous environment. However, the temporal control over these systems is lacking, rendering studies on the temporal control of regulation challenging. Here, we report the development of a small molecule-controllable RNA effector system to overcome this challenge.
Project description:All aspects of mRNA lifetime and function, including its stability, translation into protein, and trafficking through the cell, are tightly regulated through coordinated post-transcriptional modifications and interactions with a multitude of RNA effector proteins. Despite the increasing recognition of RNA regulation as a critical layer of mammalian gene expression control and its increasing excitement as a therapeutic target, tools to study and control RNA regulatory mechanisms with temporal precision in their endogenous environment are lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired RNA targeting system (CIRTS). The CIRTS biosensor platform is based on guide RNA (gRNA)-dependent RNA binding domains that interact with a target transcript using Watson-Crick-Franklin base pair interactions. Addition of a small molecule recruits an RNA effector to the target transcript, thereby eliciting a local effect on the transcript. In this work, we showcase that these CIRTS biosensors can trigger inducible RNA editing, degradation, or translation on target transcripts in a small molecule-dependent manner. We further go on to show that the CIRTS RNA base editor biosensor can induce RNA base editing in a small molecule-controllable manner in vivo. Collectively this work provides a new set of tools to probe the dynamics of RNA regulatory systems and control gene expression at the RNA level.
Project description:We develop a small molecule-inducible dimerization strategy of RBP of interest and adenosine deaminase domain to profile dynamic RBP-RNA interactions.
Project description:Crimean-Congo hemorrhagic fever (CCHF) caused by CCHF virus (CCHFV) is one of the epidemic-prone diseases prioritized by the World Health Organisation as public health emergency with an urgent need for accelerated research. The trajectory of host response against CCHFV is multifarious and remains unknown. Here, we reported the temporal spectrum of pathogenesis following the CCHFV infection using genome-wide blood transcriptomics analysis followed by advanced systems biology analysis, temporal immune-pathogenic alterations, and context-specific progressive and postinfection genome-scale metabolic models (GSMM) on samples collected during the acute (T0), early convalescent (T1), and convalescent-phase (T2). The interplay between the retinoic acid-inducible gene-I-like/nucleotide-binding oligomerization domain-like receptor and tumor necrosis factor signaling governed the trajectory of antiviral immune responses. The rearrangement of intracellular metabolic fluxes toward the amino acid metabolism and metabolic shift toward oxidative phosphorylation and fatty acid oxidation during acute CCHFV infection determine the pathogenicity. The upregulation of the tricarboxylic acid cycle during CCHFV infection, compared to the noninfected healthy control and between the severity groups, indicated an increased energy demand and cellular stress. The upregulation of glycolysis and pyruvate metabolism potentiated energy generation through alternative pathways associated with the severity of the infection. The downregulation of metabolic processes at the convalescent phase identified by blood cell transcriptomics and single-cell type proteomics of five immune cells (CD4+ and CD8+ T cells, CD14+ monocytes, B cells, and NK cells) potentially leads to metabolic rewiring through the recovery due to hyperactivity during the acute phase leading to post-viral fatigue syndrome.
Project description:Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems
Project description:RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.
Project description:Tuning protein expression by targeting RNA structure using small molecules is an unexplored avenue for cancer treatment. To understand whether this vulnerability could be therapeutically targeted in the most lethal form of prostate cancer, castrate-resistant prostate cancer (CRPC), we used a clinical small molecule, zotatifin, that targets the RNA helicase and translation factor eIF4A. Zotatifin repressed tumorigenesis in patient-derived and xenograft models and prolonged survival in vivo alongside hormone therapy. Genome-wide transcriptome, translatome, and proteomic analysis revealed two important translational targets: Androgen Receptor (AR), a key oncogene in CRPC, and hypoxia-inducible factor 1A (HIF1A), an essential cancer modulator in hypoxia. We solved the structure of the 5’UTRs of these oncogenic mRNAs and strikingly observed complex structural remodeling of these select mRNAs by this small molecule. Remarkably, tumors treated with zotatifin become more sensitive to radiotherapy, usually blunted by HIF1A. Therefore, ‘translatome therapy’ opens new avenues to treat the deadliest cancers.