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

Hazuki Takahashi,Piero Carninci,Masakazu Hirose,Ana Kozhuharova,Diego Cotella,Francesca Fasolo,Stefano Gustincich,Silvia Zucchelli,Toshio Yamazaki,Harshita Sharma,Claudio Santoro,Takako Ohyama,Thomas Preiss

doi:10.1371/journal.pone.0183229

Abstract

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.

Highlights

One of the key conclusions of the FANTOM project is that the majority of the genome is transcribed and the majority of transcripts are constituted by long non-coding RNAs [1]
We previously designed a synthetic SINEUP against Enhanced Green Fluorescent Protein (EGFP) that successfully acts on EGFP translation [5]
Clustering of cap analysis gene expression (CAGE) tags showed that the main peak of pEGFP was 28 nt upstream of AUG; the main SINEUP-GFP transcripts start 94 nt upstream of the SINEUP insertion site of pcDNA3.1 (Fig 1A, green is EGFP and red is SINEUP-GFP)

Summary

Introduction

One of the key conclusions of the FANTOM project is that the majority of the genome is transcribed and the majority of transcripts are constituted by long non-coding RNAs (lncRNAs) [1]. A substantial portion of lncRNA sequences are antisense to protein coding mRNAs, forming sense-antisense (S/AS) pairs [2]. S/AS pairs are very abundant, involving at least 72% of all genome-mapped transcriptional units identified in the mouse transcriptome in the FANTOM3 project [2]. Various types of regulatory functions have been generally assigned.

Methods

Results

Conclusion