SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells.

Silvia Zucchelli,Piero Carninci,Francesca Persichetti,Laura Cimatti,Stefano Gustincich,Daniele Sblattero,Roberta Russo,Hazuki Takahashi,Francesca Fasolo,Laura Patrucco,Claudio Santoro,Diego Cotella,Michael H Jones

doi:10.3389/fncel.2015.00174

Abstract

Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Highlights

Large genomic projects such as ENCODE (Djebali et al, 2012) and FANTOM (Forrest et al, 2014) have shown that the majority of the mammalian genome is transcribed, generating a previously underestimated complexity in gene regulatory networks
We propose AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense long non-coding RNA (lncRNA) that activate translation. We named these lncRNAs as SINEUPs since they take advantage of an embedded invSINEB2 element to UP-regulate translation
The remaining SINEUP sequence presents the embedded invSINEB2 element in the non-overlapping part of the transcript. Since this region has been proven essential for protein synthesis upregulation, we defined it as Effector Domain (ED) (Figure 1A)

Summary

Introduction

Large genomic projects such as ENCODE (Djebali et al, 2012) and FANTOM (Forrest et al, 2014) have shown that the majority of the mammalian genome is transcribed, generating a previously underestimated complexity in gene regulatory networks. In addition to 25000 genes encoding for proteins, at least an equal number of long non-coding RNA (lncRNA) genes have been identified so far. These generate >200 base pairs long transcripts that do not encode for proteins. About one third of annotated lncRNAs overlaps with proteincoding genes (Derrien et al, 2012). Many of these are transcribed from the opposite strand forming sense/antisense (S/AS) pairs (Katayama et al, 2005; Derrien et al, 2012)

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