Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation

Takahashi, Hazuki; Kozhuharova, Ana; Sharma, Harshita; Hirose, Masakazu; Ohyama, Tetsuo; Fasolo, Francesca; Yamazaki, Toshio; Cotella, Diego; Santoro, Claudio; Zucchelli, S.; Gustincich, Stefano; Carninci, Piero

doi:10.1371/journal.pone.0183229

Cited by 25 publications

(31 citation statements)

References 50 publications

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“…Similar to microRNA (miRNA) engineering approaches, where the aim was to target the translation of multiple genes without burdening the overall protein production capacity of a cell, lncRNAs are no burden on the translational machinery and could be used to control gene transcription of target genes rather than their translation, thus intervening at an even earlier stage. While miRNAs can reduce translation of their target, engineering by lncRNAs could also be used to enhance transcription of individual genes, such as the product gene, an approach that has already been shown to work at the level of mRNA translation using lncRNAs (Takahashi et al, ). Speculatively, and excitingly, one could reach a level of understanding that allows control of entire phenotypes—which are determined by transcriptome patterns—by targeting multiple genes in a pathway without interfering with their genomic sequence or context by introducing lncRNAs designed to manipulate their expression patterns.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Epigenetic regulation of gene expression in Chinese Hamster Ovary cells in response to the changing environment of a batch culture

Hernández

Dhiman

Klanert

et al. 2019

Biotech & Bioengineering

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The existence of dynamic cellular phenotypes in changing environmental conditions is of major interest for cell biologists who aim to understand the mechanism and sequence of regulation of gene expression. In the context of therapeutic protein production by Chinese Hamster Ovary (CHO) cells, a detailed temporal understanding of cell‐line behavior and control is necessary to achieve a more predictable and reliable process performance. Of particular interest are data on dynamic, temporally resolved transcriptional regulation of genes in response to altered substrate availability and culture conditions. In this study, the gene transcription dynamics throughout a 9‐day batch culture of CHO cells was examined by analyzing histone modifications and gene expression profiles in regular 12‐ and 24‐hr intervals, respectively. Three levels of regulation were observed: (a) the presence or absence of DNA methylation in the promoter region provides an ON/OFF switch; (b) a temporally resolved correlation is observed between the presence of active transcription‐ and promoter‐specific histone marks and the expression level of the respective genes; and (c) a major mechanism of gene regulation is identified by interaction of coding genes with long non‐coding RNA (lncRNA), as observed in the regulation of the expression level of both neighboring coding/lnc gene pairs and of gene pairs where the lncRNA is able to form RNA–DNA–DNA triplexes. Such triplex‐forming regions were predominantly found in the promoter or enhancer region of the targeted coding gene. Significantly, the coding genes with the highest degree of variation in expression during the batch culture are characterized by a larger number of possible triplex‐forming interactions with differentially expressed lncRNAs. This indicates a specific role of lncRNA‐triplexes in enabling rapid and large changes in transcription. A more comprehensive understanding of these regulatory mechanisms will provide an opportunity for new tools to control cellular behavior and to engineer enhanced phenotypes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Epigenetic regulation of gene expression in Chinese Hamster Ovary cells in response to the changing environment of a batch culture

Hernández

Dhiman

Klanert

et al. 2019

Biotech & Bioengineering

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“…In these cases, the transcriptional orientation of genic lncRNAs can be in sense or antisense compared to the transcriptional orientation of protein-coding genes. Genic antisense lncRNAs are transcribed from the opposite strand of the closest protein-coding gene and can overlap with a 5 head-to-head divergent orientation (SINEUPs [38][39][40][41]) or with 3 tail-to-tail convergent orientation (3 overlapping lncRNAs) ( Figure 2B). An example of intronic lncRNA is COLDAIR which is transcribed from the first intron of the coding gene Flowering Locus C (FLC) [42].…”

Section: Genic Lncrnas: Intronic and Exonicmentioning

confidence: 99%

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

Alessio

Bonadio

Buson

et al. 2020

IJMS

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In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts.Int. J. Mol. Sci. 2020, 21, 302 2 of 32 not achieved by transcription factors (TFs) alone because of their low number (approximately 1500 in mammals [4]) compared to genes to regulate (90% of the genome is transcribed in human [5]). A single TF is estimated to regulate only ten thousand genes according to Chromatin immunoprecipitation and DNA sequencing experiments (ChIp-seq) [6]. Moreover, the involvement of non-coding RNAs in gene expression regulation was evident [7,8]. The evidence that the non-protein coding component of the human genome is actively transcribed and carries out crucial functions limits the importance of coding genes in genome regulation. Non-coding transcripts become one of the stars of modern biology, especially because of their involvement in a wide range of regulatory processes and changed the association of non-coding regions to junk DNA [3].The genome of multicellular eukaryotes is mostly comprised of non-coding DNA (less than 2% of the human genome codes for proteins [9] while 90% of the human genome is transcribed). Non-coding DNA is transcribed into different classes of non-coding RNAs (ncRNAs) that include structural RNAs (rRNAs and tRNAs) and regulatory RNAs [10]. rRNAs and tRNAs are involved in mRNA translation and can be long or short in size. Regulatory RNAs are further divided into three classes: Small, medium, and long non-coding RNAs. Small non-coding RNAs are between 20 and 50 nucleotides long and include microRNAs (miRNAs) that participate in post-transcriptional regulation [11], small interfering RNAs (siRNAs) that are double-stranded RNAs involved in gene silencing through RNA interfering pathway [12], piwi interacting R...

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“…. Recent studies showed that the potential of natural SINEUPs is preserved in synthetic SINEUPs designed to specifically target various endogenous and exogenous genes 14,15 . SINEUPs have two important features: first the "binding domain" (BD) which is generally the 5ʹ end region complementary to the sequence encompassing the primary start codon of a protein-coding mRNA, and second the "effector domain" (ED) as it incorporates an inverted repeat of SINE which is a prerequisite for SINEUP function 14 (Figure 1).…”

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confidence: 99%

“…The protein induction is maintained within the normal physiological range of the cell, preventing any deleterious effect due to aberrant or excessive protein expression. SINEUPs are compatible with a wide variety of mouse, human, and hamster cultured cell lines, for instance, HEK293T/17, HepG2, HeLa, CHO, MN9D, and many more 12,13,14,15,18,19 . SINEUPs can efficiently target endogenous genes, transiently overexpressed genes, and FLAG-tagged or luciferasefusion genes, eliminating the need of gene-specific antibodies 14,18 .…”

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confidence: 99%

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Takahashi

Sharma

Carninci

2019

JoVE

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Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3ʹ end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5ʹ untranslated region (5ʹ UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein upregulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

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Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation

Cited by 25 publications

References 50 publications

Epigenetic regulation of gene expression in Chinese Hamster Ovary cells in response to the changing environment of a batch culture

Epigenetic regulation of gene expression in Chinese Hamster Ovary cells in response to the changing environment of a batch culture

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

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