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, researchers at the RIKEN Center for Life Science Technologies 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, they extensively screened variants of the BD to map features needed for optimal design. They found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, the researchers report their screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. This synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.
Optimization of SINEUP-GFP binding domain design
(A) TSS analysis of EGFP and SINEUP-GFP by CAGE. CAGE analysis was performed on RNA extracted from HEK293T/17 cells transfected with pEGFP and pcDNA3.1-SINEUP-GFP. Sequencing reads were mapped on reference EGFP (left) and SINEUP-GFP (right) transcripts. Green graph indicates TSS of EGFP and red graph indicates TSS of SINEUP-GFP. The exact position of EGFP and SINEUP-GFP TSS is numbered relative to translation initiation and SINEUP insertion, respectively. (B) Shorter variants of SINEUP-GFP BD show improved activity. Scheme of the anatomy of sense EGFP (derived from pEGFP-C2 plasmid) and SINEUP-GFP transcripts is shown on top. Details of BD sequences used for the screening are indicated. Underlined pEGFP-C2 sequence indicates AUG-Kozak sequence. HEK 293T/17 cells were transfected with pEGFP in combination with SINEUP-GFP or empty control plasmid. EGFP protein quantities were analyzed by Western Blot. Respective EGFP expressions are normalized by ACTINB (endogenous control) fold changes are normalized by control (empty vector). n = 9, ***p < 0.0005, two-tailed Student’s t-test; Error bars are STDEV. Δ: deletion.