An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis

Abstract

N 6 -threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon-codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.

Modulation of the Proteostasis Machinery to Overcome Stress Caused by Diminished Levels of t6A‐Modified tRNAs in Drosophila

“…Our group [12] and others [13] have observed that tcs3 mutants lacked imaginal discs and larval tissues were smaller, indicating that different cell types could have particular requirements for t 6 A-modified tRNAs; consistently Drosophila tcs3 [47] and human TCS3 /OSGEP [48] are differentially expressed in different tissues, supporting a tissue-specific demand. Accordingly, silencing the TCTC components in differentiated Drosophila photoreceptors did not cause any phenotype [13], suggesting that differentiated cells have a low demand for t 6 A-modified tRNAs; in contrast, highly proliferative cells such as the ones that the wing imaginal discs in Drosophila are composed, seem to have a high demand for these tRNAs.…”

“…Accordingly, silencing the TCTC components in differentiated Drosophila photoreceptors did not cause any phenotype [13], suggesting that differentiated cells have a low demand for t 6 A-modified tRNAs; in contrast, highly proliferative cells such as the ones that the wing imaginal discs in Drosophila are composed, seem to have a high demand for these tRNAs. There could be a cell cycle-dependent requirement of t 6 A-modified tRNAs even in the same cell type, as we observed only G 0 /G 1 cells die by apoptosis, which is consistent with the severe target of rapamycin (TOR) inhibition in tcs3 mutants [12] as its activity promotes G 1 /S progression [49].…”

“…The last is present in tRNAs that pair A-starting codons (ANN) (Figure 1) [2]. This modification is universally conserved and has a paramount role for tRNA decoding function, as has been shown in yeast [8,9], archea [10], bacteria [11], and, recently by our laboratory [12] and others [13], in Drosophila . This modification was identified over 40 years ago [14,15,16]; however, the enzymes that synthetize it were only recently identified [8,9].…”

Elongator Subunit 3 (Elp3) Is Required for Zebrafish Trunk Development

“…elp3 is expressed ubiquitously, suggesting that the modification is present in most tissues but, since the knockdown phenotype affects certain cell types, there may be differential requirement for the mcm 5 s 2 U modification. This has been shown in the case of t 6 A; mutants that disrupt this modification present cell type specific phenotypes [46,47]. In addition, in absence of Elongator, translation of several specific mRNAs is affected [48] that may explain the tissue-or cell-type specific phenotypes.…”

Biallelic variants in CTU2 cause DREAM‐PL syndrome and impair thiolation of tRNA wobble U34

“…We monitored the t6A modification at position 37 of tRNA‐Arg‐UCU and the ms2t6A modification at position 37 of tRNA‐Lys‐UUU (Figure a,b). Both the t6A and ms2t6A modifications can be monitored by primer extension assays using reverse transcriptase (RT) since the t6A modification leads to a partial block in RT extension while the ms2t6A leads to a strong block in RT (Clark, Evans, Dominissini, Zheng, & Pan, ; Lin et al, ). As expected, we detected RT pausing on the t6A modification in tRNA‐Arg‐UCU while RT was completely blocked for tRNA‐Lys‐UUU (Figure c,d).…”