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Partial Sequence Identity in a 25-nucleotide Long Element is Sufficient for Transcriptional Adaptation in the caenorhabditis elegans act-5/act-3 Model

Jordan M. Welker, Vahan Serobyan, Elhamalsadat Zaker Esfahani, Didier Y. R. Stainier


Genetic robustness can be achieved via several mechanisms including transcriptional adaptation (TA), a sequence similarity-driven process whereby mutant mRNA degradation products modulate, directly or indirectly, the expression of so-called adapting genes. To identify the sequences required for this process, we utilized a transgenic approach in Caenorhabditis elegans, combining an overexpression construct for a mutant gene (act-5) and a fluorescent reporter for the corresponding adapting gene (act-3). Analyzing a series of modifications for each construct, we identified, in the 5’ regulatory region of the act-3 locus, a 25-base pair (bp) element which exhibits 60% identity with a sequence in the act-5 mRNA and which, in the context of a minimal promoter, is sufficient to induce ectopic expression of the fluorescent reporter. The 25 nucleotide (nt) element in the act-5 mRNA lies between the premature termination codon (PTC) and the next exon/exon junction, suggesting the importance of this region of the mutant mRNA for TA. Additionally, we found that single-stranded RNA injections of this 25 nt element from act-5 into the intestine of wild-type larvae led to higher levels of adapting gene (act-3) mRNA.


Under the umbrella of various mechanisms contributing to genetic robustness, or the ability of a living cell or organism to maintain homeostasis in the presence of mutations, transcriptional adaptation (TA) is of particular interest because it regulates gene expression in response to mutant mRNA degradation and not protein feedback loops [1,2]. According to the current model of TA, in the presence of mRNA destabilizing lesions, mRNA degradation products, or their derivatives, translocate from the cytosol to the nucleus where they modulate the mRNA levels of the adapting gene(s). Notably, full locus deletion alleles do not exhibit TA [1,2], indicating that mutant mRNA molecules are required. As such, TA can lead to functional compensation [37] or to more severe phenotypes [8] depending on the gene(s) whose expression becomes modulated by the mutant mRNA degradation fragments and/or their derivatives. 

Materials and method

C. elegans culture conditions and strains

All C. elegans strains were maintained on 6 cm plates with nematode growth medium agar and fed with a lawn of E. coli OP50 grown in 500 μl Luria broth [36]. All C. elegans strains used in this study are listed in Table 1. Cultures were maintained at 18–20°C. In addition, to minimize the potential for laboratory evolution of the traits, new cultures of the strains were revived annually from frozen stocks. All plates with fungal or bacterial contamination were excluded from the experiments.


Ectopic uterine RFP expression as a proxy for the transcriptional adaptation response

In order to identify the sequences in the act-3 promoter and in the act-5 mRNA that are necessary and/or sufficient for the TA response of act-3, we utilized two plasmids to generate transgenic animals. The first plasmid [act-3p(long):rfp] uses a 4 kb promoter for the adapting gene (act-3) to drive TurboRFP expression (Fig 1A) [4]. The second plasmid [eft-3p:act-5(ptc)] uses a ubiquitous promoter to overexpress the mutant gene (act-5(ptc)) (Fig 1B) [11]. By analyzing transgenic animals containing one or both of these plasmids (Fig 1C), or their derivatives, we can identify the sequence requirements by comparing the RFP expression pattern between control and experimental animals. 


Transcriptional adaptation (TA) is a widespread cellular response to mRNA destabilizing lesions, including mutations, that is driven by mRNA degradation fragments, or their derivatives [17,11]. TA could lead to changes in gene expression via different mechanisms including chromatin remodeling [1,2,11], the inhibition of antisense RNAs [1], the release of transcriptional pausing [23], and the suppression of premature transcription termination [14,9]. Here, starting with a 4 kb piece of the act-3 locus that includes the first exon and first intron as well as 2.8 kb of upstream sequence, we identified a 25 bp element 2.6 kb upstream of the transcriptional start site of the adapting gene (act-3) that is sufficient for TA in a transgenic setting. The corresponding 25 nt element in the mutant mRNA (act-5) lies between the PTC and the next exon/exon junction, suggesting the importance of this region of the mutant mRNA.


We thank Cansu Çirzi, Thomas Juan, Christopher M. Dooley, Maëlle Bellec, Samuel J. Capon, Greta Ebnicher, Mohamed El-Brolosy, and René Ketting for discussion and comments on the manuscript. We thank the genome engineering facility, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany for assistance with plasmid injections. We thank InVivo Biosystems for assistance with the generation of the COP2474 and COP2475 mutant strains.

Citation: Welker JM, Serobyan V, Zaker Esfahani E, Stainier DYR (2023) Partial sequence identity in a 25-nucleotide long element is sufficient for transcriptional adaptation in the Caenorhabditis elegans act-5/act-3 model. PLoS Genet 19(6): e1010806.

Editor: Laura Bianchi, University of Miami, UNITED STATES

Received: February 10, 2023; Accepted: June 1, 2023; Published: June 29, 2023

Copyright: © 2023 Welker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This research was supported by awards from the European Research Council (ERC) under the European Union’s research and innovation programmes (AdG 694455-ZMOD and AdG 101021349-TAaGC) to D.Y.R.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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