NsiR3, a nitrogen stress-inducible small RNA, regulates proline oxidase expression in the cyanobacteriumNostocsp. PCC 7120

NsiR3 (nitrogen stress-inducible RNA 3) is a small noncoding RNA strongly conserved in heterocyst-forming cyanobacteria. InNostocsp. PCC 7120, transcription of NsiR3 is induced by nitrogen starvation and depends on the global nitrogen regulator NtcA. A conserved NtcA-binding site is centered around position -42.5 with respect to the transcription start site of NsiR3 homologs, and NtcA bindsin vitroto a DNA fragment containing this sequence. In the absence of combined nitrogen, NsiR3 expression is induced in all cells along theNostocfilament but much more strongly in heterocysts, differentiated cells devoted to nitrogen fixation. Co-expression analysis of transcriptomic data obtained from microarrays hybridized with RNA obtained fromNostocwild-type or mutant strains grown in the presence of ammonium or in the absence of combined nitrogen revealed that the expression profile of geneputA(proline oxidase) correlates negatively with that of NsiR3. Using a heterologous system inEscherichia coli, we show that NsiR3 binds to the 5 '-UTR ofputAmRNA, resulting in reduced expression of a reporter gene. Overexpression of NsiR3 inNostocresulted in strong reduction ofputAmRNA accumulation, further supporting the negative regulation ofputAby NsiR3. The higher expression of NsiR3 in heterocystsversusvegetative cells of the N-2-fixing filament could contribute to the previously described absence ofputAmRNA and of the catabolic pathway to produce glutamate from arginine via proline specifically in heterocysts. Post-transcriptional regulation by NsiR3 represents an indirect NtcA-operated regulatory mechanism ofputAexpression. Database Microarray data are available in GEO database under accession numbers and .