SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides

Bianca Pierattini; Sabrina D'Agostino; Carlotta Bon; Omar Peruzzo; Andrej Alendar; Azzurra Codino; Gloria Ros; Francesca Persichetti; Remo Sanges; Piero Carninci; Claudio Santoro; Stefano Espinoza; Paola Valentini; Luca Pandolfini; Stefano Gustincich

doi:10.1016/j.omtn.2023.04.002

SINEUP non-coding RNA activity depends on specific N6-methyladenosine nucleotides

Mol Ther Nucleic Acids. 2023 Apr 7:32:402-414. doi: 10.1016/j.omtn.2023.04.002. eCollection 2023 Jun 13.

Authors

Bianca Pierattini^{1

2}, Sabrina D'Agostino², Carlotta Bon², Omar Peruzzo², Andrej Alendar³, Azzurra Codino², Gloria Ros⁴, Francesca Persichetti⁴, Remo Sanges^{1

2}, Piero Carninci^{5

6}, Claudio Santoro⁴, Stefano Espinoza^{2

4}, Paola Valentini², Luca Pandolfini², Stefano Gustincich²

Affiliations

¹ Area of Neuroscience, International School for Advanced Studies (SISSA), Trieste, Italy.
² Central RNA Laboratory, Istituto Italiano di Tecnologia (IIT), Genova, Italy.
³ The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
⁴ Department of Health Sciences and Research Center on Autoimmune and Allergic Diseases (CAAD), University of Piemonte Orientale (UPO), Novara, Italy.
⁵ RIKEN Center for Integrative Medical Sciences (IMS), Yokohama 230-0045, Japan.
⁶ Human Technopole, 20157 Milan, Italy.

Abstract

SINEUPs are natural and synthetic antisense long non-coding RNAs (lncRNAs) selectively enhancing target mRNAs translation by increasing their association with polysomes. This activity requires two RNA domains: an embedded inverted SINEB2 element acting as effector domain, and an antisense region, the binding domain, conferring target selectivity. SINEUP technology presents several advantages to treat genetic (haploinsufficiencies) and complex diseases restoring the physiological activity of diseased genes and of compensatory pathways. To streamline these applications to the clinic, a better understanding of the mechanism of action is needed. Here we show that natural mouse SINEUP AS Uchl1 and synthetic human miniSINEUP-DJ-1 are N⁶-methyladenosine (m⁶A) modified by METTL3 enzyme. Then, we map m⁶A-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that m⁶A removal from SINEUP RNA causes the depletion of endogenous target mRNA from actively translating polysomes, without altering SINEUP enrichment in ribosomal subunit-associated fractions. These results prove that SINEUP activity requires an m⁶A-dependent step to enhance translation of target mRNAs, providing a new mechanism for m⁶A translation regulation and strengthening our knowledge of SINEUP-specific mode of action. Altogether these new findings pave the way to a more effective therapeutic application of this well-defined class of lncRNAs.

Keywords: MT: Non-coding RNAs; N6-methyladenosine; Nanopore direct RNA sequencing; RNA therapeutics; SINEUP; lncRNA; m6A; translation.