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Your search keyword '"Carthew, Richard W."' showing total 17 results
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17 results on '"Carthew, Richard W."'

8. Energy metabolism modulates the regulatory impact of activators on gene expression.

Author

Sha Qiao, Bernasek, Sebastian, Gallagher, Kevin D., O'Connell, Jessica, Shigehiro Yamada, Bagheri, Neda, Amaral, Luis A. N., and Carthew, Richard W.

Subjects
ENERGY metabolism, GENE expression, GENETIC regulation, ENERGY consumption, DROSOPHILA
Abstract

Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. Here, we explore this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. We find that, in the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. We propose that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions. [ABSTRACT FROM AUTHOR]

Published
2024
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9. Ratiometric sensing of Pnt and Yan transcription factor levels confers ultrasensitivity to photoreceptor fate transitions in Drosophila

Author

Bernasek, Sebastian M., primary, Hur, Suzy S. J., additional, Peláez-Restrepo, Nicolás, additional, Lachance, Jean-François Boisclair, additional, Bakker, Rachael, additional, Navarro, Heliodoro Tejedor, additional, Sanchez-Luege, Nicelio, additional, Amaral, Luís A. N., additional, Bagheri, Neda, additional, Rebay, Ilaria, additional, and Carthew, Richard W., additional

Published
2023
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14. Robust and heritable knockdown of gene expression using a self-cleaving ribozyme in Drosophila

Author

Nyberg, Kevin G, Navales, Fritz Gerald, Keles, Eren, Nguyen, Joseph Q, Hertz, Laura M, and Carthew, Richard W

Abstract

The current toolkit for genetic manipulation in the model animal Drosophila melanogasteris extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogasterusing the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesexusing scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3′ fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes.Techniques for genetic manipulation in Drosophila melanogasterare powerful but not without limitations. Here, the authors detail a novel approach to achieve strong RNA knockdown in Drosophilausing self-cleaving ribozymes. Ribozyme insertion via CRISPR/Cas9 into both nuclear and cytoplasmic noncoding RNAs and a male-specific splice variant of doublesexresulted in cleavage and efficient degradation of 3’ RNA fragments without observable off-target effects. This work demonstrates the potential of self-cleaving ribozymes for gene knockdown in Drosophila.

Published
2024
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15. Scaling between cell cycle duration and wing growth is regulated by Fat-Dachsous signaling in Drosophila .

Author

Liu A, O'Connell J, Wall F, and Carthew RW

Abstract

The atypical cadherins Fat and Dachsous (Ds) signal through the Hippo pathway to regulate growth of numerous organs, including the Drosophila wing. Here, we find that Ds-Fat signaling tunes a unique feature of cell proliferation found to control the rate of wing growth during the third instar larval phase. The duration of the cell cycle increases in direct proportion to the size of the wing, leading to linear-like growth during the third instar. Ds-Fat signaling enhances the rate at which the cell cycle lengthens with wing size, thus diminishing the rate of wing growth. We show that this results in a complex but stereotyped relative scaling of wing growth with body growth in Drosophila . Finally, we examine the dynamics of Fat and Ds protein distribution in the wing, observing graded distributions that change during growth. However, the significance of these dynamics is unclear since perturbations in expression have negligible impact on wing growth.

Published
2024
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16. Energy metabolism modulates the regulatory impact of activators on gene expression.

Author

Qiao S, Bernasek S, Gallagher KD, Yamada S, Bagheri N, Amaral LAN, and Carthew RW

Abstract

Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. Here, we explore this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. We find that in the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. We propose that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions., Competing Interests: COMPETING INTERESTS The authors declare no competing financial interests.

Published
2023
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17. CRISPR-/Cas9-Mediated Precise and Efficient Genome Editing in Drosophila.

Author

Nyberg KG and Carthew RW

Subjects
Animals, CRISPR-Associated Protein 9 genetics, Drosophila genetics, Recombinational DNA Repair, CRISPR-Cas Systems genetics, Gene Editing methods
Abstract

The CRISPR/Cas9 system provides the means to make precise and purposeful modifications to the genome via homology-directed repair (HDR). In Drosophila, a wide variety of tools provide flexibility to achieve these ends. Here, we detail a method to generate precise genome edits via HDR that is efficient and broadly applicable to any Drosophila stock or species. sgRNAs are first tested for their cleavage efficiency by injecting embryos with Cas9/sgRNA ribonucleoproteins using commercially available Cas9 protein. Using an empirically validated sgRNA, HDR is performed using a donor repair plasmid that carries two transformation markers. A fluorescent eye marker that can be seamlessly removed using PiggyBac transposase marks integration of the repair sequence. A counter-selection marker that produces small rough eyes via RNAi against eyes absent is used to screen against imprecise HDR events. Altogether, the enhancements implemented in this method expand the ease and scope of achieving precise CRISPR/Cas9 genome edits in Drosophila., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Published
2022
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