2A Peptides Contribute to the Co-Expression of Proteins for Imaging and Genome Editing

Shibuta, Mio K.; Matsuoka, Megumi; Matsunaga, Sachihiro

doi:10.1508/cytologia.84.107

Cited by 4 publications

(4 citation statements)

References 56 publications

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“…The area enriched for Ser2P-mintbody signal represents a transcriptionally active region because RNAPII-CTD Ser2P is a hallmark of RNAPII in a transcription elongation or termination state (Heidemann et al 2013). The transgenic line expressed both Ser2P-mintbody and H2B-mRuby with IntF2A (Intain-and 2A peptide-based cleavage system) under the control of RIBOSOMAL PROTEIN 5A (RPS5a) promoter (pRPS5a:Ser2P-mintbpody-IntF2A-H2B-mRuby) (Shibuta et al 2019(Shibuta et al , 2021. Fluorescence imaging showed both Ser2P-mintbody and H2B-mRuby fluorescence signals in mature pollen, revealing that this line can monitor the RNAPII-CTD Ser2P distribution in intact vegetative chromatin and sperm chromatin without isolating nuclei from pollen (Fig.…”

Section: Resultsmentioning

confidence: 99%

Transcriptionally active regions are highly limited in the nucleus of the plant sperm cell

Shibuta,

Matsunaga

2023

CYTOLOGIA

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Flowering plants have immotile sperm cells that are delivered to the female gametes by the pollen tube cell. Although the sperm chromatin in flowering plants is highly condensed like animal sperm chromatin, it is generally accepted that the transcription status in the sperm chromatin of flowering plants is active because of the existence of active gene promoters and active histone modification status. However, the level of transcriptional activity in the sperm chromatin of flowering plants has remained unknown. In this study, we observed the distribution of C-terminal domain-phosphorylated RNA polymerase II (RNAPII), which represents a transcriptionally active status in living pollen, and fixed isolated vegetative and sperm nuclei of Arabidopsis thaliana. We found that the transcriptionally active regions were highly limited in sperm chromatin compared with those in vegetative chromatin. RNA sequencing (RNA-seq) analysis showed that the transcription of some RNAPII subunit-encoding genes was highly suppressed in sperm cells, suggesting that the amount of functional RNAPII would be small. In addition, the C-terminal domains of some RNAPII phosphatase-encoding genes, which act as a regulator of the transcription cycle, were actively transcribed in sperm cells. These findings indicate that transcriptional activity is highly suppressed in the sperm chromatin in A. thaliana due to the lack of completely assembled RNAPII and the inhibition of transcriptional elongation of RNAPII by the phosphatase activities.

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Section: Resultsmentioning

confidence: 99%

Transcriptionally active regions are highly limited in the nucleus of the plant sperm cell

Shibuta,

Matsunaga

2023

CYTOLOGIA

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“…2a ). To generate the two-component system, we focused on translation-coupled protein processing using the IntF2A-based co-expression system 18 , 19 (Fig. 2b ).…”

Section: Resultsmentioning

confidence: 99%

A live imaging system to analyze spatiotemporal dynamics of RNA polymerase II modification in Arabidopsis thaliana

et al. 2021

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Spatiotemporal changes in general transcription levels play a vital role in the dynamic regulation of various critical activities. Phosphorylation levels at Ser2 in heptad repeats within the C-terminal domain of RNA polymerase II, representing the elongation form, is an indicator of transcription. However, rapid transcriptional changes during tissue development and cellular phenomena are difficult to capture in living organisms. We introduced a genetically encoded system termed modification-specific intracellular antibody (mintbody) into Arabidopsis thaliana. We developed a protein processing- and 2A peptide-mediated two-component system for real-time quantitative measurement of endogenous modification level. This system enables quantitative tracking of the spatiotemporal dynamics of transcription. Using this method, we observed that the transcription level varies among tissues in the root and changes dynamically during the mitotic phase. The approach is effective for achieving live visualization of the transcription level in a single cell and facilitates an improved understanding of spatiotemporal transcription dynamics.

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“…Other techniques such as the self-cleaving 2A peptide system (58,61) might be used in the future for miniSOG photogenetical CCK1R activation. The 2A system provides an equal molar expression of proteins up-and down-stream of the 2A peptide sequence (62)(63)(64)(65)(66)(67), although modifying the intercistronic sequence has also been shown to significantly enhance the IRES-dependent down-stream protein expression to match that of the up-stream protein (68). It might be worthwhile in the future to examine whether ratio (from 1:10 to 10:1)-controlled alternative-splicing-based bicistronic expression (69) of min-iSOG/CCK1R might eventually lead to bicistronic photogenetical CCK1R activation with miniSOG, although this is neither necessary nor essential as photodynamic/photogenetical CCK1R activation has been achieved in this work with at least three different fusion patterns of miniSOG and CCK1R: miniSOG-CCK1R, miniSOG-(GSG) 4 -CCK1R and CCK1R-miniSOG (Fig.…”

Section: Discussionmentioning

confidence: 99%

Photodynamic Activation of the Cholecystokinin 1 Receptor with Tagged Genetically Encoded Protein Photosensitizers: Optimizing the Tagging Patterns

Cui

2022

Photochem & Photobiology

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Cholecystokinin 1 receptor (CCK1R) is activated photodynamically. For this to happen in situ, genetically encoded protein photosensitizers (GEPP) may be tagged to natively expressed CCK1R, but how to best tag GEPP has not been examined. Therefore, GEPP (miniSOG or KillerRed) was tagged to CCK1R and light-driven photodynamic CCK1R activation was monitored by Fura-2 fluorescent calcium imaging, to screen for optimized tagging patterns. Blue lightemitting diode irradiation of CHO-K1 cells expressing min-iSOG fused to N-or C-terminus of CCK1R was found to both trigger persistent calcium oscillations-a hallmark of permanent photodynamic CCK1R activation. Photodynamic CCK1R activation was accomplished also with miniSOG fused to N-terminus of CCK1R via linker (GlySerGly) 4 or 8 , but not linker (GSG) 12 or an internal ribosomal entry site insert. KillerRed fused to N-or C-terminus of CCK1R after white light irradiation resulted in similar activation of inframe CCK1R. Photodynamic CCK1R activation in miniSOG-CCK1R-CHO-K1 cells was blocked by singlet oxygen ( 1 O 2 ) quencher uric acid or Trolox C, corroborating the role of 1 O 2 as the reactive intermediate. It is concluded that photodynamic CCK1R activation can be achieved either with direct GEPP fusion to CCK1R or fusion via a short linker, fusion via long linkers might serve as the internal control.

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2A Peptides Contribute to the Co-Expression of Proteins for Imaging and Genome Editing

Cited by 4 publications

References 56 publications

Transcriptionally active regions are highly limited in the nucleus of the plant sperm cell

Transcriptionally active regions are highly limited in the nucleus of the plant sperm cell

A live imaging system to analyze spatiotemporal dynamics of RNA polymerase II modification in Arabidopsis thaliana

Photodynamic Activation of the Cholecystokinin 1 Receptor with Tagged Genetically Encoded Protein Photosensitizers: Optimizing the Tagging Patterns

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