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Abstract Protein translation is tightly and precisely controlled by multiple mechanisms including upstream open reading frames (uORFs), but the origins of uORFs and their role in maize are largely unexplored. In this study, an active transposition event was identified during the propagation of maize inbred line B73. The transposon, which was named BTA for ‘B73 active transposable element hAT’, creates a novel dosage-dependent hypomorphic allele of the hexose transporter gene ZmSWEET4c through insertion within the coding sequence in the first exon, and results in reduced kernel size. The BTA insertion does not affect transcript abundance but reduces protein abundance of ZmSWEET4c, probably through the introduction of a uORF. Furthermore, the introduction of BTA sequence in the exon of other genes can regulate translation efficiency without affecting their mRNA levels. A transposon capture assay revealed 79 novel insertions for BTA and BTA-like elements. These insertion sites have typical euchromatin features, including low levels of DNA methylation and high levels of H3K27ac. A putative autonomous element that mobilizes BTA and BTA-like elements was identified. Together, our results suggest a transposon-based origin of uORFs and document a new role for transposable elements to influence protein abundance and phenotypic diversity by affecting the translation rate.
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Disintegration promotes protospacer integration by the Cas1-Cas2 complex
‘Disintegration’—the reversal of transposon DNA integration at a target site—is regarded as an abortive off-pathway reaction. Here, we challenge this view with a biochemical investigation of the mechanism of protospacer insertion, which is mechanistically analogous to DNA transposition, by the Streptococcus pyogenes Cas1-Cas2 complex. In supercoiled target sites, the predominant outcome is the disintegration of one-ended insertions that fail to complete the second integration event. In linear target sites, one-ended insertions far outnumber complete protospacer insertions. The second insertion event is most often accompanied by the disintegration of the first, mediated either by the 3′-hydroxyl exposed during integration or by water. One-ended integration intermediates may mature into complete spacer insertions via DNA repair pathways that are also involved in transposon mobility. We propose that disintegration-promoted integration is functionally important in the adaptive phase of CRISPR-mediated bacterial immunity, and perhaps in other analogous transposition reactions.
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- Award ID(s):
- 1949821
- PAR ID:
- 10312444
- Date Published:
- Journal Name:
- eLife
- Volume:
- 10
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Bosco, Giovanni (Ed.)Transposable elements (TE) are selfish genetic elements that can cause harmful mutations. In Drosophila , it has been estimated that half of all spontaneous visible marker phenotypes are mutations caused by TE insertions. Several factors likely limit the accumulation of exponentially amplifying TEs within genomes. First, synergistic interactions between TEs that amplify their harm with increasing copy number are proposed to limit TE copy number. However, the nature of this synergy is poorly understood. Second, because of the harm posed by TEs, eukaryotes have evolved systems of small RNA-based genome defense to limit transposition. However, as in all immune systems, there is a cost of autoimmunity and small RNA-based systems that silence TEs can inadvertently silence genes flanking TE insertions. In a screen for essential meiotic genes in Drosophila melanogaster , a truncated Doc retrotransposon within a neighboring gene was found to trigger the germline silencing of ald , the Drosophila Mps1 homolog, a gene essential for proper chromosome segregation in meiosis. A subsequent screen for suppressors of this silencing identified a new insertion of a Hobo DNA transposon in the same neighboring gene. Here we describe how the original Doc insertion triggers flanking piRNA biogenesis and local gene silencing. We show that this local gene silencing occurs in cis and is dependent on deadlock , a component of the Rhino-Deadlock-Cutoff (RDC) complex, to trigger dual-strand piRNA biogenesis at TE insertions. We further show how the additional Hobo insertion leads to de-silencing by reducing flanking piRNA biogenesis triggered by the original Doc insertion. These results support a model of TE-mediated gene silencing by piRNA biogenesis in cis that depends on local determinants of transcription. This may explain complex patterns of off-target gene silencing triggered by TEs within populations and in the laboratory. It also provides a mechanism of sign epistasis among TE insertions, illuminates the complex nature of their interactions and supports a model in which off-target gene silencing shapes the evolution of the RDC complex.more » « less
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null (Ed.)Abstract Transposable elements (TEs) are ubiquitous DNA segments capable of moving from one site to another within host genomes. The extant distributions of TEs in eukaryotic genomes have been shaped by both bona fide TE integration preferences in eukaryotic genomes and by selection following integration. Here, we compare TE target site distribution in host genomes using multiple de novo transposon insertion datasets in both plants and animals and compare them in the context of genome-wide transcriptional landscapes. We showcase two distinct types of transcription-associated TE targeting strategies that suggest a process of convergent evolution among eukaryotic TE families. The integration of two precision-targeting elements are specifically associated with initiation of RNA Polymerase II transcription of highly expressed genes, suggesting the existence of novel mechanisms of precision TE targeting in addition to passive targeting of open chromatin. We also highlight two features that can facilitate TE survival and rapid proliferation: tissue-specific transposition and minimization of negative impacts on nearby gene function due to precision targeting.more » « less
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Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance1. IS200- and IS605-family transposons undergo ‘peel-and-paste’ transposition catalysed by a TnpA transposase2, but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively3,4. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases5,6, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR–Cas adaptive immunity for antiviral defence. TnpB and IscB nucleases use transposon-encoded guide RNAs to target genomic sequences for cleavage, thereby favouring copying and spreading of transposable elements.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.65763
