Pervasive transcription of mammalian genomes leads to a previously underestimated level of complexity in gene regulatory networks. Recently, we have identified a new functional class of natural and synthetic antisense long non-coding RNAs (lncRNA) that increases translation of partially overlapping sense mRNAs. These molecules were named SINEUPs, as they require an embedded inverted SINE B2 element for their UP-regulation of translation. Mouse AS Uchl1 is the representative member of natural SINEUPs. It was originally discovered for its role in increasing translation of Uchl1 mRNA, a gene associated with neurodegenerative diseases. Here we present the secondary structure of the SINE B2 Transposable Element (TE) embedded in AS Uchl1. We find that specific structural regions, containing a short hairpin, are required for the ability of AS Uchl1 RNA to increase translation of its target mRNA. We also provide a high-resolution structure of the relevant hairpin, based on NMR observables. Our results highlight the importance of structural determinants in embedded TEs for their activity as functional domains in lncRNAs.

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.

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.

Transposable elements (TEs) compose about half of the mammalian genome and, as embedded sequences, up to 40% of long noncoding RNA (lncRNA) transcripts. Embedded TEs may represent functional domains within lncRNAs, providing a structured RNA platform for protein interaction. Here we show the interactome profile of the mouse inverted short interspersed nuclear element (SINE) of subfamily B2 (invSINEB2) alone and embedded in antisense (AS) ubiquitin C‐terminal hydrolase L1 (Uchl1), an lncRNA that is AS to Uchl1 gene. AS Uchl1 is the representative member of a functional class of AS lncRNAs, named SINEUPs, in which the invSINEB2 acts as effector domain (ED)‐enhancing translation of sense protein‐coding mRNAs. By using RNA‐interacting domainome technology, we identify the IL enhancer‐binding factor 3 (ILF3) as a protein partner of AS Uchl1 RNA. We determine that this interaction is mediated by the RNA‐binding motif 2 of ILF3 and the invSINEB2. Furthermore, we show that ILF3 is able to bind a free right Arthrobacter luteus (Alu) monomer sequence, the embedded TE acting as ED in human SINEUPs. Bioinformatic analysis of Encyclopedia of DNA Elements‐enhanced cross‐linking immunoprecipitation data reveals that ILF3 binds transcribed human SINE sequences at transcriptome‐wide levels. We then demonstrate that the embedded TEs modulate AS Uchl1 RNA nuclear localization to an extent moderately influenced by ILF3. This work unveils the existence of a specific interaction between embedded TEs and an RNA‐binding protein, strengthening the model of TEs as functional modules in lncRNAs.—Fasolo, F., Patrucco, L., Volpe, M., Bon, C., Peano, C., Mignone, F., Carninci, P., Persichetti, F., Santoro, C., Zucchelli, S., Sblattero, D., Sanges, R., Cotella, D., Gustincich, S. The RNA‐binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs. FASEB J. 33, 13572‐13589 (2019). www.fasebj.org

Trace amine-associated receptor 5 (TAAR5) is a G protein-coupled receptor that belongs to the TAARs family (TAAR1-TAAR9). TAAR5 is expressed in the olfactory epithelium and is responsible for sensing 3-methylamine (TMA). However, recent studies showed that TAAR5 is also expressed in the limbic brain regions and is involved in the regulation of emotional behaviour and adult neurogenesis, suggesting that TAAR5 antagonism may represent a novel therapeutic strategy for anxiety and depression. We used the AtomNet^® model, the first deep learning neural network for structure-based drug discovery, to identify putative TAAR5 ligands and tested them in an in vitro BRET assay. We found two mTAAR5 antagonists with low to submicromolar activity that are able to inhibit the cAMP production induced by TMA. Moreover, these two compounds also inhibited the mTAAR5 downstream signalling, such as the phosphorylation of CREB and ERK. These two hits exhibit drug-like properties and could be used to further develop more potent TAAR5 ligands with putative anxiolytic and antidepressant activity.

Sequences in the 5′-untranslated regions of cellular and viral mRNAs can function as internal ribosome entry sites (IRESs), driving cis-acting translation of the downstream protein-coding open reading frame. Here we demonstrate that RNA sequences with either newly identified or well-characterized IRES activity can also induce trans-acting translation of an independent mRNA species through an antisense sequence. SINEUPs are antisense long non-coding RNAs that enhance the translation of overlapping sense mRNAs in trans by employing two critical domains: the invSINEB2 sequence, which up-regulates translation (effector domain), and an antisense region providing target specificity (binding domain). First, we show that the invSINEB2 from the natural SINEUP AS Uchl1 RNA acts as an IRES when functioning in cis. Next, we establish that known viral and cellular sequences with IRES activity can operate in trans as an effector domain in synthetic SINEUPs. To identify natural IRES-containing non-coding RNAs with transactivity, we found that the non-coding hsa_circ_0 085 533 (circMyc), transcribed from the c-myc locus, enhances protein expression of PX Domain Containing Serine/Threonine Kinase Like (PXK) by promoting mRNA association with polysomes through antisense sequences. These results suggest that SINEUPs and some circular RNAs are trans-acting IRESs, expanding the repertoire of molecular mechanisms to regulate translation.