Systematic and Functional Identification of Small Non-Coding RNAs Associated with Excess Ammonium Stress in...

Cyanobacteria, the only prokaryotic oxygenic phototrophs, rely on sophisticated regulatory networks, including those mediated by small RNAs (sRNAs) to cope with environmental fluctuations. Here, we delineate the sRNA landscape of Synechocystis sp. PCC 6803 under short- and long-term ammonium stress, revealing a significant proportion of antisense RNAs (asRNAs). Functional characterization identified three asRNAs (sll0312-as, sll0873-as, and slr1667-as) as key regulators of ammonium stress tolerance, implicating their targets (sll0312, sll0873, and slr1667) as new players in nitrogen fluctuation acclimation. The sll0944-as and sll1515-as were also identified, revealing an additional regulatory layer targeting known carbon/nitrogen metabolism regulators. Mechanistically, we characterized the ammonium-induced asRNA ssr0692-as, demonstrating that it represses pirA translation via direct 5'UTR interaction. This finding, integrated with the known role of the nitrogen limitation-responsive sRNA NsiR4 targeting the same region, supports a synergistic model wherein these two sRNAs precisely modulate PirA protein levels-and thus the downstream nitrogen flux-across varying nitrogen availability. Together, our findings expand the functional repertoire of cyanobacterial sRNAs and elucidate a dynamic post-transcriptional mechanism to fine-tune nitrogen metabolism in response to fluctuating nutrient conditions.