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

• Published in: PloS one 2018-02, Vol.13 (2), p.e0183229-e0183229
• Main Authors: Takahashi, Hazuki, Kozhuharova, Ana, Sharma, Harshita, Hirose, Masakazu, Ohyama, Takako, Fasolo, Francesca, Yamazaki, Toshio, Cotella, Diego, Santoro, Claudio, Zucchelli, Silvia, Gustincich, Stefano, Carninci, Piero
• Format: Article
• Language: English
• Subjects: Analysis; Binding sites (Biochemistry); Biology and Life Sciences; High-throughput screening (Biochemical assaying); Physiological aspects; Research and analysis methods; RNA
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0183229

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.