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

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 and synthetic human miniSINEUP-DJ-1 are N-methyladenosine (mA) modified by METTL3 enzyme. Then, we map mA-modified sites along SINEUP sequence with Nanopore direct RNA sequencing and a reverse transcription assay. We report that mA 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 mA-dependent step to enhance translation of target mRNAs, providing a new mechanism for mA 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.