Cell-Type-Specific Profiling of Alternative Translation Identifies Regulated Protein Isoform Variation in the Mouse Brain

Sapkota, Darshan; Lake, Allison M.; Yang, Wei; Yang, Chengran; Wesseling, Hendrik; Guise, Amanda J.; Uncu, Ceren; Dalal, Jasbir S.; Kraft, Andrew W.; J, Lee; Sands, Mark S.; Steen, Judith A.; Dougherty, Joseph D.

doi:10.1016/j.celrep.2018.12.077

Cited by 59 publications

(51 citation statements)

References 96 publications

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“…Of note, most N-terminal proteoforms were discovered in studies focused on single proteins. To date, there is only one widescale study that maps N-terminal proteoforms in mouse brain and their relation to functionality (Box 3 and [66]). Thus, this emerging field appears to be gaining attention.…”

Section: Outstanding Questionsmentioning

confidence: 99%

N-Terminal Proteoforms in Human Disease

Bogaert

Fernández

Gevaert

2020

Trends in Biochemical Sciences

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Section: Outstanding Questionsmentioning

confidence: 99%

N-Terminal Proteoforms in Human Disease

Bogaert

Fernández

Gevaert

2020

Trends in Biochemical Sciences

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“…CNS-specific expression of a mutated tRNA resulted in neurodegeneration in mice 43 , again verifying that components and regulation of the translation apparatus can be expressed in a tissue-specific manner. A recent study observed high rates of native stop codon readthrough in mouse brains, although differences in readthrough rates between neurons and glia were not apparent 10 . Our study suggests, in the context of premature stop codon readthrough, there may be substantial tissue-specific differences in mRNA translation.…”

Section: Discussionmentioning

confidence: 99%

“…In Drosophila , the headcase ( hdc ) gene encodes two different proteins, one of them produced by translational readthrough, and the readthrough product is necessary for Drosophila tracheal development 7 . There have also been a number of reports of readthrough in mammals, including in mouse brains 8-10 .…”

Section: Introductionmentioning

confidence: 99%

Premature termination codon readthrough inDrosophilavaries in a developmental and tissue-specific manner

Chen

Sun

et al. 2019

Preprint

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24Despite their essential function in terminating translation, readthrough of stop codons occurs 25 more frequently than previously supposed. However, little is known about the regulation of stop 26 codon readthrough by anatomical site and over the life cycle of animals. Here, we developed a 27 set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in 28 whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in 29 the reporters were recognized as premature termination codons (PTCs). We found readthrough 30 rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult 31 flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were 32 highest in fly brains, specifically neurons and not glia. This was not due to differences in 33 reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these 34 tissues. Overall, our data reveal temporal and spatial variation of PTC-mediated readthrough in 35 animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring 36 transcripts when the NMD surveillance pathway is inhibited. 37 38 39 65 codon readthrough truly represents a regulatory mechanism 13 , and there are mechanisms to 66 mitigate canonical readthrough 14 , these data suggest that stop codon readthrough in eukaryotes 67 is far more pervasive than previously appreciated. We therefore decided to measure readthrough 68 in flies using a set of novel gain-of-function reporter lines that could sensitively detect 69 translation through stop codons in animals throughout their life cycle, as well as in specific 70 tissues. Furthermore, we confirmed that the stop codons in our readthrough reporters are 71 recognized as premature termination codons (PTCs) in flies. We observed that stop codon 72 readthrough frequency in two candidate gene reporters varied widely throughout fly 73 development, and appeared to be highest in Drosophila neurons. High frequency readthrough 74 of PTCs may be an alternative rescue pathway for translation of transcripts with premature 75 termination codons in flies. 76 77 Results and Discussion 78 An in vivo gain-of-function reporter fly line can sensitively detect translational 79 readthrough 80We wished to measure stop codon readthrough in flies across developmental stages of their life 81 cycle. We initially chose to measure readthrough using a candidate gene, rab6, which had been 82 identified as undergoing moderately elevated readthrough rates in a ribosome profiling study of 83 fly embryos 12 . We decided to use a gain-of-function reporter 15,16 , since this approach would be 84 able to detect low levels of readthrough. The gene for Nanoluc luciferase -Nluc -17 , missing its 85 start codon, was cloned immediately downstream of rab6 complete with its native stop codon 86 and 3' UTR, but missing the second, in-frame, stop codon (Fig. 1a). In our reporter, translation 87 past the native UAG stop codon would re...

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“…Based on these data, there are now over 400 genes annotated in FlyBase as readthrough genes with highly conserved extensions among Drosophila species. In contrast, there are only a few dozen readthrough genes documented in the mouse/human genome (13,31,51,66,67) suggesting the Drosophila genome finds readthrough particularly useful. Curiously, the peptide extensions produced by readthrough genes in Drosophila vary considerably in length from fewer than 10 amino acids to over 800.…”

Section: Evasion Of Nmd Does Not Explain Tissue-specific Readthroughmentioning

confidence: 99%

Tissue-specific dynamic codon redefinition in Drosophila

Hudson

Loughran

Szabo

et al. 2020

Preprint

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Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3' of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue-and cell-specific dynamic stop codon redefinition in Drosophila. Stop codon readthrough | recoding | Drosophila | Kelch | central nervous system (CNS) †Correspondence: j.atkins@ucc.ie or lynn.cooley@yale.edu Hudson et al. | bioRχiv | June 22, 2020 | 1-11

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Cell-Type-Specific Profiling of Alternative Translation Identifies Regulated Protein Isoform Variation in the Mouse Brain

Cited by 59 publications

References 96 publications

N-Terminal Proteoforms in Human Disease

N-Terminal Proteoforms in Human Disease

Premature termination codon readthrough inDrosophilavaries in a developmental and tissue-specific manner

Tissue-specific dynamic codon redefinition in Drosophila

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