Affecting RNA biology genome-wide by binding small molecules and chemically induced proximity

Modeling age-related neurodegenerative disorders with human stem cells is difficult due to the embryonic nature of stem cell derived neurons. We developed a chemical cocktail to induce senescence of iPSC-derived neurons to address this challenge. We first screened small molecules that induce embryonic fibroblasts to exhibit features characteristic of aged fibroblasts. We then optimized a cocktail of small molecules that induced senescence in fibroblasts and cortical neurons without causing DNA damage. The utility of the senescence cocktail was validated in motor neurons derived from ALS patient iPSCs which exhibited protein aggregation and axonal degeneration substantially earlier than those without cocktail treatment. Our senescence cocktail will likely enhance the manifestation of disease-related phenotypes in neurons derived from iPSCs, enabling the generation of reliable drug discovery platforms.

Download Full-text

miPEP858/miR858-MYB3-PSK4 module regulates growth and development in Arabidopsis

10.1101/2021.09.27.461987 ◽

2021 ◽

Author(s):

Poorwa Kamal Badola ◽

Aashish Sharma ◽

Himanshi Gautam ◽

Prabodh Kumar Trivedi

Keyword(s):

Plant Growth ◽

Small Molecules ◽

Growth And Development ◽

Wild Type ◽

Developmental Defects ◽

Over Expression ◽

Genome Wide ◽

Exogenous Treatment ◽

Regulation Of Growth ◽

Genome Wide Expression

Small molecules, peptides, and miRNAs are the crucial regulators of plant growth. Here, we show the importance of cross-talk between miPEP858a/miR858a and Phytosulfokine (PSK4) in regulating plant growth and development in Arabidopsis. Genome-wide expression analysis suggested modulated expression of PSK4 in miR858 mutant and overexpression, miR858OX, plants. The silencing of PSK4 in miR858OX plants compromised the growth, whereas over-expression of PSK4 in miR858 mutant rescued the developmental defects. The exogenous application of synthetic PSK4 further complemented the plant development in mutant plants. Exogenous treatment of synthetic miPEP858a in PSK4 mutant led to clathrin-mediated internalization of the peptide however did not enhance growth as in the case of wild-type plants. We also demonstrate that the MYB3 is an important molecular component participating in miPEP858a/miR858a-PSK4 module. Finally, our work highlights the signaling between miR858/miPEP858-MYB3-PSK4 in modulating the expression of key elements involved in auxin responses leading to the regulation of growth.

Download Full-text

The Use of Large-Scale Chemically-Induced Transcriptome Data Acquired from LINCS to Study Small Molecules

Methods in Molecular Biology - Systems Chemical Biology ◽

10.1007/978-1-4939-8891-4_11 ◽

2018 ◽

pp. 189-203 ◽

Cited By ~ 1

Author(s):

Michio Iwata ◽

Yoshihiro Yamanishi

Keyword(s):

Small Molecules ◽

Large Scale ◽

Transcriptome Data ◽

Chemically Induced

Download Full-text

Small-molecule targeted recruitment of a nuclease to cleave an oncogenic RNA in a mouse model of metastatic cancer

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914286117 ◽

2020 ◽

Vol 117 (5) ◽

pp. 2406-2411 ◽

Cited By ~ 22

Author(s):

Matthew G. Costales ◽

Haruo Aikawa ◽

Yue Li ◽

Jessica L. Childs-Disney ◽

Daniel Abegg ◽

...

Keyword(s):

Breast Cancer ◽

Mouse Model ◽

Small Molecules ◽

Small Molecule ◽

Enzymatic Degradation ◽

Metastatic Cancer ◽

Rna Degradation ◽

Oncogenic Pathways ◽

Rna Biology

As the area of small molecules interacting with RNA advances, general routes to provide bioactive compounds are needed as ligands can bind RNA avidly to sites that will not affect function. Small-molecule targeted RNA degradation will thus provide a general route to affect RNA biology. A non–oligonucleotide-containing compound was designed from sequence to target the precursor to oncogenic microRNA-21 (pre–miR-21) for enzymatic destruction with selectivity that can exceed that for protein-targeted medicines. The compound specifically binds the target and contains a heterocycle that recruits and activates a ribonuclease to pre–miR-21 to substoichiometrically effect its cleavage and subsequently impede metastasis of breast cancer to lung in a mouse model. Transcriptomic and proteomic analyses demonstrate that the compound is potent and selective, specifically modulating oncogenic pathways. Thus, small molecules can be designed from sequence to have all of the functional repertoire of oligonucleotides, including inducing enzymatic degradation, and to selectively and potently modulate RNA function in vivo.

Download Full-text

Metabolic RNA labeling for probing RNA dynamics in bacteria

Nucleic Acids Research ◽

10.1093/nar/gkaa1111 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12566-12576

Author(s):

Liying Meng ◽

Yilan Guo ◽

Qi Tang ◽

Rongbing Huang ◽

Yuchen Xie ◽

...

Keyword(s):

Global Analysis ◽

Bacterial Species ◽

Rna Degradation ◽

Metabolic Labeling ◽

Powerful Method ◽

Bacterial Rna ◽

Genome Wide ◽

Rna Labeling ◽

Rna Biology ◽

Imaging Probes

Abstract Metabolic labeling of RNAs with noncanonical nucleosides that are chemically active, followed by chemoselective conjugation with imaging probes or enrichment tags, has emerged as a powerful method for studying RNA transcription and degradation in eukaryotes. However, metabolic RNA labeling is not applicable for prokaryotes, in which the complexity and distinctness of gene regulation largely remain to be explored. Here, we report 2′-deoxy-2′-azidoguanosine (AzG) as a noncanonical nucleoside compatible with metabolic labeling of bacterial RNAs. With AzG, we develop AIR-seq (azidonucleoside-incorporated RNA sequencing), which enables genome-wide analysis of transcription upon heat stress in Escherichia coli. Furthermore, AIR-seq coupled with pulse-chase labeling allows for global analysis of bacterial RNA degradation. Finally, we demonstrate that RNAs of mouse gut microbiotas can be metabolically labeled with AzG in living animals. The AzG-enabled metabolic RNA labeling should find broad applications in studying RNA biology in various bacterial species.

Download Full-text

Generation of Mouse Haploid Somatic Cells by Small Molecules for Genome-wide Genetic Screening

Cell Reports ◽

10.1016/j.celrep.2017.07.081 ◽

2017 ◽

Vol 20 (9) ◽

pp. 2227-2237 ◽

Cited By ~ 16

Author(s):

Zheng-Quan He ◽

Bao-Long Xia ◽

Yu-Kai Wang ◽

Jing Li ◽

Gui-Hai Feng ◽

...

Keyword(s):

Small Molecules ◽

Genetic Screening ◽

Somatic Cells ◽

Genome Wide

Download Full-text

Conversion of mouse embryonic fibroblasts into neural crest cells and functional corneal endothelia by defined small molecules

Science Advances ◽

10.1126/sciadv.abg5749 ◽

2021 ◽

Vol 7 (23) ◽

pp. eabg5749

Author(s):

Shao-Hui Pan ◽

Ning Zhao ◽

Xiang Feng ◽

Ying Jie ◽

Zi-Bing Jin

Keyword(s):

Neural Crest ◽

Small Molecules ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Neural Crest Cells ◽

Corneal Opacity ◽

Defined Medium ◽

Mouse Fibroblasts ◽

Chemically Induced

Reprogramming of somatic cells into desired functional cell types by small molecules has vast potential for developing cell replacement therapy. Here, we developed a stepwise strategy to generate chemically induced neural crest cells (ciNCCs) and chemically induced corneal endothelial cells (ciCECs) from mouse fibroblasts using defined small molecules. The ciNCCs exhibited typical NCC features and could differentiate into ciCECs using another chemical combination in vitro. The resulting ciCECs showed consistent gene expression profiles and self-renewal capacity to those of primary CECs. Notably, these ciCECs could be cultured for as long as 30 passages and still retain the CEC features in defined medium. Transplantation of these ciCECs into an animal model reversed corneal opacity. Our chemical approach for direct reprogramming of mouse fibroblasts into ciNCCs and ciCECs provides an alternative cell source for regeneration of corneal endothelia and other tissues derived from neural crest.

Download Full-text

Haplotypic divergence coupled with lack of diversity at the Arabidopsis thaliana alcohol dehydrogenase locus: roles for both balancing and directional selection?

Genetics ◽

10.1093/genetics/138.3.811 ◽

1994 ◽

Vol 138 (3) ◽

pp. 811-828 ◽

Cited By ~ 3

Author(s):

U Hanfstingl ◽

A Berry ◽

E A Kellogg ◽

J T Costa ◽

W Rüdiger ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Alcohol Dehydrogenase ◽

Balancing Selection ◽

Haplotype Diversity ◽

Hypervariable Region ◽

Amino Acid Replacement ◽

Loss Of Function ◽

Closely Related Species ◽

Genome Wide ◽

Chemically Induced

Abstract We designate a region of the alcohol dehydrogenase locus (Adh) of the weedy crucifer, Arabidopsis thaliana, as "hypervariable" on the basis of a comparison of sequences from ecotypes Columbia and Landsberg. We found eight synonymous and two replacement mutations in the first 262 nucleotides of exon 4, and an additional two mutations in the contiguous region of intron 3. The rest of the sequence (2611 bp) has just three mutations, all of them confined to noncoding regions. Our survey of the hypervariable region among 37 ecotypes of A. thaliana revealed two predominant haplotypes, corresponding to the Columbia and Landsberg sequences. We identified five additional haplotypes and 4 additional segregating sites. The lack of haplotype diversity is presumably in part a function of low rates of recombination between haplotypes conferred by A. thaliana's tendency to self-fertilize. However, an analysis in 32 ecotypes of 12 genome-wide polymorphic markers distinguishing Columbia and Landsberg ecotypes indicated levels of outcrossing sufficient at least to erode linkage disequilibrium between dispersed markers. We discuss possible evolutionary explanations for the coupled observation of marked divergence within the hypervariable region and a lack of haplotype diversity among ecotypes. The sequence of the region for closely related species argues against the possibility that one allele is the product of introgression. We note (1) that several loss of function mutations (both naturally and chemically induced) map to the hypervariable region, and (2) the presence of two amino acid replacement polymorphisms, one of which causes the mobility difference between the two major classes of A. thaliana Adh electrophoretic alleles. We argue that protein polymorphism in such a functionally significant part of the molecule may be subject to balancing selection. The observed pattern of extensive divergence between the alleles is consistent with this explanation because balancing selection on a particular site maintains linked neutral polymorphisms at intermediate frequencies.

Download Full-text

Molecular Mechanisms of Cereblon-Interacting Small Molecules in Multiple Myeloma Therapy

Journal of Personalized Medicine ◽

10.3390/jpm11111185 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1185

Author(s):

Matteo Costacurta ◽

Jackson He ◽

Philip E. Thompson ◽

Jake Shortt

Keyword(s):

Multiple Myeloma ◽

Small Molecules ◽

Molecular Mechanisms ◽

Rational Drug Design ◽

Genome Wide ◽

Rational Drug ◽

Anti Cancer ◽

Cellular Machinery ◽

Endogenous Substrates ◽

New Generation

Thalidomide analogues (or immunomodulatory imide drugs, IMiDs) are cornerstones in the treatment of multiple myeloma (MM). These drugs bind Cereblon (CRBN), a receptor for the Cullin-ring 4 ubiquitin-ligase (CRL4) complex, to modify its substrate specificity. IMiDs mediate CRBN-dependent engagement and proteasomal degradation of ‘neosubstrates’, Ikaros (IKZF1) and Aiolos (IKZF3), conveying concurrent antimyeloma activity and T-cell costimulation. There is now a greater understanding of physiological CRBN functions, including endogenous substrates and chaperone activity. CRISPR Cas9-based genome-wide screening has further elucidated the complex cellular machinery implicated in IMiD sensitivity, including IKZF1/3-independent mechanisms. New-generation IMiD derivatives with more potent anti-cancer properties—the CELMoDs (Cereblon E3 ligase modulators)—are now being evaluated. Rational drug design also allows ‘hijacking’ of CRL4CRBN utilising proteolysis targeting chimeras (PROTACs) to convey entirely distinct substrate repertoires. As all these chemotypes—thalidomide, IMiDs, CELMoDs and PROTACs—engage CRBN and modify its functions, we describe them here in aggregate as ‘CRBN-interacting small molecules’ (CISMs). In this review, we provide a contemporary summary of the biological consequences of CRBN modulation by CISMs. Detailed molecular insight into CRBN–CISM interactions now provides an opportunity to more effectively target previously elusive cancer dependencies, representing a new and powerful tool for the implementation of precision medicine.

Download Full-text

Targeting translation activity at the ribosome interface with UV-active small molecules

10.1101/436311 ◽

2018 ◽

Author(s):

Divya T. Kandala ◽

Alessia Del Piano ◽

Luca Minati ◽

Massimiliano Clamer

Keyword(s):

Protein Synthesis ◽

Small Molecules ◽

Protein Interactions ◽

Rna Processing ◽

Translational Control ◽

Control Mechanisms ◽

Translation Efficiency ◽

Catalytic Core ◽

Genome Wide

ABSTRACTPuromycin is a well-known antibiotic that is used to study the mechanism of protein synthesis and to monitor translation efficiency due to its incorporation into nascent peptide chains. However, puromycin effects outside the ribo-some catalytic core remain unexplored. Here, we developed two puromycin analogues (3PB and 3PC) that can efficiently interact with several proteins involved in translation, ribosome function and RNA processing. We biochemically characterized the binding of these analogues and globally mapped the direct small molecule-protein interactions in living cells using clickable and photoreactive puromycin-like probes in combination with in-depth mass spectrometry. We identified a list of proteins that interact with ribosomes during translation (e.g. eEF1A, ENO1 and GRP78) and we addressed possible uses of the probes to sense the activity of protein synthesis and to capture associated RNA. By coupling genome-wide RNA sequencing methods with these molecules, the characterization of unexplored translational control mechanisms will be feasible.

Download Full-text