Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation
- University of California, Berkeley, United States
- Columbia University, United States
Abstract
Targeted selection-based-genome-editing approaches in budding yeast have enabled many fundamental discoveries and continue to be used routinely with high precision. We found, however, that replacement of DBP1 with a common selection cassette led to reduced expression and function for the adjacent gene, MRP51, despite all MRP51 coding and regulatory sequences remaining intact. Cassette-induced repression of MRP51 drove all phenotypes we detected in cells deleted for DBP1. This behavior resembled the previously observed 'neighboring gene effect' (NGE), a phenomenon of unknown mechanism whereby cassette insertion at one locus reduces the expression of a neighboring gene. Here, we leveraged strong off-target phenotypes resulting from cassette replacement of DBP1 to provide mechanistic insight into the NGE. We found that inherent bidirectionality of promoters, including those in expression cassettes, drives a divergent transcript that represses MRP51 through combined transcriptional interference and translational repression mediated by production of a long undecoded transcript isoform (LUTI). We demonstrate that divergent transcript production driving this off-target effect is general to yeast expression cassettes and occurs ubiquitously with insertion. Despite this, off-target effects are often naturally prevented by local sequence features, such as those that terminate divergent transcripts between the site of cassette insertion and the neighboring gene. Thus, cassette induced off-target effects can be eliminated by the insertion of transcription terminator sequences into the cassette, flanking the promoter. Because the driving features of this off-target effect are broadly conserved, our study suggests its consideration in the design and interpretation of experiments using integrated expression cassettes in other eukaryotes.
Data availability
mRNA sequencing and ribosome profiling data are available at NCBI GEO, with accession numbers GSE207267 and GSE207189. Mass spectrometry data are available at MassIVE, with accession number MSV000089724
-
Loss of DBP1 during meiosis in yeastNCBI Gene Expression Omnibus, GSE207267.
-
DBP1 in budding yeastNCBI Gene Expression Omnibus, GSE111255.
Article and author information
Author details
Funding
National Institutes of Health (R35GM134886)
- Gloria A Brar
National Institutes of Health (R35GM128802)
- Marko Jovanovic
National Science Foundation
- Emily N Powers
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Powers et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,558
- views
-
- 248
- downloads
-
- 9
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation
eLife 11:e81086.
https://doi.org/10.7554/eLife.81086
Further reading
-
- Chromosomes and Gene Expression
RNA interference (RNAi) is a conserved pathway that utilizes Argonaute proteins and their associated small RNAs to exert gene regulatory function on complementary transcripts. While the majority of germline-expressed RNAi proteins reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here, we find that the small RNA biogenesis machinery is spatially and temporally organized during Caenorhabditis elegans embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Curiously, coincident with the appearance of the SIMR granules, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. NRDE-3 binds ERGO-dependent 22G-RNAs in the somatic cells of larvae and adults to silence ERGO-target genes; here we further demonstrate that NRDE-3-bound, CSR-class 22G-RNAs repress transcription in oocytes. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during oogenesis to promote global transcriptional repression, and switching during embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.
-
- Chromosomes and Gene Expression
- Genetics and Genomics
A new method for mapping torsion provides insights into the ways that the genome responds to the torsion generated by RNA polymerase II.
