Intronserter, an advanced online tool for design of intron containing transgenes

Jaeger, Daniel; Baier, Thomas; Lauersen, Kyle J.

doi:10.1016/j.algal.2019.101588

Cited by 39 publications

(44 citation statements)

References 35 publications

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“…reinhardtii [ 31 ]. Insertions are thought to mimic the host nuclear genome regulation by minimizing exon length in combination with activated IME [ 31 , 78 ]. This gene design strategy has now been frequently used for biotechnological studies aimed at metabolic engineering and recombinant protein expression in C .…”

Section: Resultsmentioning

confidence: 99%

“…We have previously demonstrated that repetitive insertions of RBCS2i1 can be used to enable robust expression of large heterologous transgenes from the nuclear genome of C. reinhardtii [31]. Insertions are thought to mimic the host nuclear genome regulation by minimizing exon length in combination with activated IME [31,78]. This gene design strategy has now been…”

Section: Intron-containing Algal Transgenes Enable Successful Geneticmentioning

confidence: 99%

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Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

et al. 2020

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Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression.

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Section: Resultsmentioning

confidence: 99%

Section: Intron-containing Algal Transgenes Enable Successful Geneticmentioning

confidence: 99%

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

et al. 2020

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“…The nucleotide sequence of C. reinhardtii Hy5 (Uniprot: A0A2K3DRN7) was optimized as previously described (Baier et al, 2018) using the Intronserter tool (Jaeger et al, 2019) (https://bibiserv.cebitec.uni-bielefeld.de/intronserter). The codon optimized, intron containing gene was chemically synthesized (GenScript, Piscataway Township, New Jersey) and cloned between Bam HI- Bg lII or Eco 32I- Eco RI in the pOpt2_mVenus_Paro vector (Wichmann et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

Ubiquitin ligase component LRS1 and transcription factor CrHy5 act as a light switch for photoprotection inChlamydomonas

Lämmermann

Wulf

Chang

et al. 2020

Preprint

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25Survival under excess light conditions requires the light-induced accumulation of protein 26 LHCSR3 and other photoprotection factors, to enable efficient energy-dependent 27 quenching in the green microalga Chlamydomonas reinhardtii. Here, we demonstrate 28 that the high light-tolerant phenotype of mutant hit1 is caused by a de-repression of 29 promoters belonging to photoprotection genes, which in turn results from an inactivation 30 of the E3 ubiquitin ligase substrate adaptor LRS1. Transcriptome analyses of hit1 31 revealed massive alterations of gene expression modulation as a consequence of 32 perturbed LRS1 function, indicating its role as a crown regulator. In conjunction with 33 random forest-based network modeling, these transcriptome analyses predicted that 34 LRS1 controls photoprotection gene expression via an algal HY5 homolog as its prime 35 transcription factor target. CrHY5 binds to T-box elements present in the promoters of 36 these genes and its inactivation in the hit1 mutant via CRISPR-Cas9 genome editing, 37 confirmed the regulatory connection between LRS1 and CrHY5, predicted by the 38 network analysis. 39 40 41 42 43 44 45 46 47 48 54 Light provides the energy driving photosynthesis and it is an important cue controlling 55 many physiological processes in phototrophic organisms. In the seed plant Arabidopsis 56 thaliana the E3 ubiquitin ligase substrate adaptor COP1/SPA is a master switch of light 57 signaling, by repressing the accumulation of various transcription factors required for the 58 induction of light-dependent processes like photomorphogenesis or flowering in 59 darkness. Repression is achieved by ubiquitination of target transcription factors and 60

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“…Moreover, the first RBCS2 intron also enhanced expression of the Renilla luciferase gene driven by the CYC6 promoter when it was placed upstream of the promoter in 3 to 5 orientation [106]. Evidence for IME in Chlamydomonas comes from the finding that the second RBCS2 intron also increased transgene expression although it lacks enhancer activity-it did not increase transgene expression when placed upstream of the R promoter [18,28,41,120].…”

Section: Determinants Of Nuclear Transgene Expression-intronsmentioning

confidence: 99%

Good News for Nuclear Transgene Expression in Chlamydomonas

Schroda

2019

Cells

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Chlamydomonas reinhardtii is a well-established model system for basic research questions ranging from photosynthesis and organelle biogenesis, to the biology of cilia and basal bodies, to channelrhodopsins and photoreceptors. More recently, Chlamydomonas has also been recognized as a suitable host for the production of high-value chemicals and high-value recombinant proteins. However, basic and applied research have suffered from the inefficient expression of nuclear transgenes. The combined efforts of the Chlamydomonas community over the past decades have provided insights into the mechanisms underlying this phenomenon and have resulted in mutant strains defective in some silencing mechanisms. Moreover, many insights have been gained into the parameters that affect nuclear transgene expression, like promoters, introns, codon usage, or terminators. Here I critically review these insights and try to integrate them into design suggestions for the construction of nuclear transgenes that are to be expressed at high levels.

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Intronserter, an advanced online tool for design of intron containing transgenes

Cited by 39 publications

References 35 publications

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

Ubiquitin ligase component LRS1 and transcription factor CrHy5 act as a light switch for photoprotection inChlamydomonas

Good News for Nuclear Transgene Expression in Chlamydomonas

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