Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms

Top, Oguz; Milferstaedt, Stella W. L.; Gessel, Nico van; Hoernstein, Sebastian N. W.; Özdemir, Buğra; Decker, Eva L.; Reski, Ralf

doi:10.1101/2020.09.30.320721

Cited by 5 publications

(6 citation statements)

References 76 publications

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“…Mass spectrometry measurement and data analysis. MS analysis was performed on an Orbitrap Q-Exactive Plus instrument (Thermo Fisher Scientific) coupled to an UltiMate 3000 RSLCnano system (Dionex LC Packings/Thermo Fisher Scientific) as described 139 . Database search and label-free quantitation was performed using MaxQuant software V1.6.0.16 140 .…”

Section: Methodsmentioning

confidence: 99%

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis

et al. 2023

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Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms.

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Section: Methodsmentioning

confidence: 99%

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis

et al. 2023

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“…MS analysis was performed on an Orbitrap Q-Exactive Plus instrument (Thermo Fisher Scientific) coupled to an UltiMate 3000 RSLCnano system (Dionex LC Packings/Thermo Fisher Scientific) as described in Top et al (2021). Database search and label-free quantitation was performed using MaxQuant software V 1.6.0.16 (Cox & Mann, 2008) as described in Hoernstein et al (2016).…”

Section: Methodsmentioning

confidence: 99%

“…as described in Top et al (2021). Database search and label-free quantitation was performed using MaxQuant software V 1.6.0.16 (Cox & Mann, 2008) as described in .…”

Section: Co-immunoprecipitationmentioning

confidence: 99%

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis

Hoernstein

Özdemir

Gessel

et al. 2022

Preprint

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Protein oxidation results from the reaction of amino-acid side chains with reactive oxygen species (ROS) and is partly irreversible. In non-photosynthetic tissues, mitochondria are a main source of ROS, whereas plastids are the major source in photosynthetic tissues. Oxidized proteins suffer from decreased structural integrity and even loss of function, and their accumulation leads to cytotoxic aggregates. In mammals, aggregate formation correlates with aging and is linked to several age-related pathologies. Mammalian proteolytic pathways for clearance of oxidized proteins are under intensive research, while mechanistic insights into this process in plants is scarce. Acylamino acid-releasing (AARE) enzymes are ATP-independent serine proteases, presumably acting on oxidized proteins and operating in a dual exo-/endopeptidase mode. They are found in all domains of life. Here, we investigated AARE enzymes in the moss Physcomitrella and the angiosperm Arabidopsis and identified three homologous nuclear genes in Physcomitrella (PpAARE1-3) and a single nuclear gene in Arabidopsis (AtAARE). Surprisingly, we observed triple localization of the proteins AtAARE and PpAARE1 to plastids, mitochondria and the cytosol in vivo, likely conserved across the plant lineage. This represents an ATP-independent possibility for degradation of oxidized proteins in the major source organelles of ROS in plants, which is distinct to mammals. Combinatorial knockout plants and protein interaction analysis revealed specific interactions of the moss AARE isoforms and functions in progressive aging. Analysis of an AtAARE T-DNA mutant further suggests the evolutionary conservation of AARE function in age-related development.

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“…Expressing genetic sequences in a novel context, such as genes from a cytoplasmic RNA virus that never enters the nucleus as a cDNA is inevitably challenged by cryptic splicing, a situation where a cryptic intron is efficiently recognized and excised within the intended CDS. This can lead to no or low expression of the full-length protein as well as production of truncated and out-of-frame protein fragments (17, 18). Cryptic splice sites (SS) that contain a consensus motif sequence in addition to the canonical splice donor (SD) dinucleotide ‘GT’ or the canonical splice acceptor (SA) dinucleotide ‘AG’ within the CDS can be removed by codon optimization.…”

Section: Introductionmentioning

confidence: 99%

Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing

Tomberg

Antunes

Hepkema

et al. 2021

Preprint

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The natural habitat of SARS-CoV-2 is the cytoplasm of a mammalian cell where it replicates its genome and expresses its proteins. While SARS-CoV-2 genes and hence its codons are presumably well optimized for mammalian protein translation, they have not been sequence optimized for nuclear expression. The cDNA of the Spike protein harbors over a hundred predicted splice sites and produces mostly aberrant mRNA transcripts when expressed in the nucleus. While different codon optimization strategies increase the proportion of full-length mRNA, they do not directly address the underlying splicing issue with commonly detected cryptic splicing events hindering the full expression potential. Similar splicing characteristics were also observed in other transgenes. By inserting multiple short introns throughout different transgenes, significant improvement in expression was achieved, including >7-fold increase for Spike transgene. Provision of a more natural genomic landscape offers a novel way to achieve multi-fold improvement in transgene expression.

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Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms

Cited by 5 publications

References 76 publications

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis

Intronization enhances expression of S-protein and other transgenes challenged by cryptic splicing

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