SINEUPs: a novel toolbox for RNA therapeutics

Abstract

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.

Keywords: RNA therapeutics; SINEUPs; long non-coding RNA.

Figure 1. SINEUPs

(A) Schematic representation of SINEUPs functional domains. The binding domain (BD, gray) provides SINEUP specificity and it is in antisense orientation to the sense protein-coding mRNA (Target mRNA). The inverted SINEB2 element (invB2) is the effector domain (ED, green) and confers enhancement of protein synthesis. 5′ to 3′ orientation of sense and antisense RNA molecules is indicated. Structural elements of target mRNA are shown: 5′ untranslated region (5′UTR, white), coding sequence (CDS, black) and 3′ untranslated region (3′UTR, white). The scheme is not drawn in scale. (B) Mechanisms of SINEUP-mediated in trans enhancing of protein synthesis. Scheme showing S/AS 5′head-to-head divergent pairing between SINEUP and targeted mRNA.

Figure 2. Specificity of SINEUP effect

SINEUPs act on endogenous mRNA and since the BD sequence is designed to be specific only for the target mRNA, off-target activity is virtually absent. (A) SINEUP is expressed in a cell that contains the target mRNA, leading to the increase of protein levels. (B) If SINEUP is expressed in a cell that does not contain the target mRNA, there is no effect on the translation of other mRNAs.

Figure 3. Secondary structure of the invSINEB2 ED of AS Uchl1

Dimethyl sulfate (DMS) and 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) were used as methylating agents. DMS and CMCT reactive nucleotides are shaded in blue and red, respectively. Internal loops and stem-loops are labelled as ILx and SLx, respectively. Non-reactive nucleotides are only circled. The segment shaded in grey corresponds to the DNA primer hybridization site. Reproduction from Figure 1 in [73] (reused with permission).

Figure 4. SINEUPs as therapeutic strategy

SINEUPs could be used as a therapeutic approach in several pathological conditions. In particular, in haploinsufficiency disorders and multifactorial disease. Genetic diseases with the lack of one functional allele for a single (haploinsufficiency) usually present a decrease of protein levels of the target gene/s leading to a pathological phenotype. The application of SINEUP may restore the physiological levels of the target protein/s and the normal phenotype in the individual affected by the genetic disease. In multifactorial diseases, the increase of pro-survival factors and enzymes may be beneficial for the patients. However, dosage and off-target distribution activity of these factors are crucial for the efficacy of these treatments. Thus, SINEUPs may be an optimal therapeutic opportunity.