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

NsiR3, a ni ogen s ess-inducible small RNA, egula es p oline oxidase exp ession in he cyanobac e ium Nos oc sp. PCC 7120 Isid o ´ Al a ez-Esc ibano 1 , Manuel B enes- ´ Al a ez 1 , El i a Olmedo-Ve d 1 , Jens Geo g 2 , Wol gang R. Hess 2 , Agus ı´n Vioque 1 and Alicia M. Mu o-Pas o 1 1 Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Consejo Supe io de In es igaciones Cien ı´ icas and Uni e sidad de Se illa, Se illa, Spain 2 Gene ics and Expe imen al Bioin o ma ics, Facul y o Biology, Uni e si y o F eibu g, F eibu g, Ge many Keywo ds he e ocys ; N cA; pos - ansc ip ional egula ion; Pu A; egula o y RNA Co espondence A. Vioque, Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Consejo Supe io de In es igaciones Cien ı´ icas and Uni e sidad de Se illa, A da. Am´ e ico Vespucio 49, Se illa 41092, Spain Tel: +34-954489519 E-mail: [email p o ec ed] (Recei ed 14 May 2020, e ised 17 July 2020, accep ed 10 Augus 2020) doi:10.1111/ ebs.15516 NsiR3 (ni ogen s ess-inducible RNA 3) is a small noncoding RNA s ongly conse ed in he e ocys - o ming cyanobac e ia. In Nos oc sp. PCC 7120, an- sc ip ion o NsiR3 is induced by ni ogen s a a ion and depends on he global ni ogen egula o N cA. A conse ed N cA-binding si e is cen e ed a ound posi ion −42.5 wi h espec o he ansc ip ion s a si e o NsiR3 homologs, and N cA binds in i o o a DNA agmen con aining his sequence. In he absence o combined ni ogen, NsiR3 exp ession is induced in all cells along he Nos oc ilamen bu much mo e s ongly in he e ocys s, di e en ia ed cells de o ed o ni ogen ixa ion. Co-exp ession analysis o ansc ip omic da a ob ained om mic oa ays hyb idized wi h RNA ob ained om Nos oc wild- ype o mu an s ains g own in he p esence o ammonium o in he absence o combined ni ogen e ealed ha he exp ession p o ile o gene pu A (p oline oxidase) co ela es nega i ely wi h ha o NsiR3. Using a he e ologous sys em in Esche ichia coli, we show ha NsiR3 binds o he 50-UTR o pu A mRNA, esul ing in educed exp ession o a epo e gene. O e exp ession o NsiR3 in Nos oc esul ed in s ong educ ion o pu A mRNA accumula ion, u he sup- po ing he nega i e egula ion o pu A by NsiR3. The highe exp ession o NsiR3 in he e ocys s e sus ege a i e cells o he N 2 - ixing ilamen could con- ibu e o he p e iously desc ibed absence o pu A mRNA and o he ca a- bolic pa hway o p oduce glu ama e om a ginine ia p oline speci ically in he e ocys s. Pos - ansc ip ional egula ion by NsiR3 ep esen s an indi ec N cA-ope a ed egula o y mechanism o pu A exp ession. Da abase Mic oa ay da a a e a ailable in GEO da abase unde accession numbe s GSE120377 and GSE150191. In oduc ion In some ilamen ous cyanobac e ia, he acclima ion o ni ogen de iciency in ol es di e en ia ion o he e o- cys s, a cell ype specialized o he ixa ion o a mosphe ic ni ogen [1]. G ow h a he expense o N 2 in ol es comple e me abolic emodeling so ha wo di e en cell ypes wi h di e en me abolic capabili ies Abb e ia ions 2-OG, 2-oxoglu a a e; dRNASeq, di e en ial RNA sequencing; FC, old change; GEO, Gene Exp ession Omnibus; GOGAT, glu amine oxoglu a a e amino ans e ase; GS, glu amine syn he ase; GSA, glu ama e γ-semialdehyde; LB, Lu ia B o h; OAA, oxaloace a e; OAC, o ni hine–ammonium cycle; P5C, Δ 1 -py oline-5-ca boxyla e; Sm, s ep omycin; Sp, spec inomycin; sRNA, small RNA; β-Asp-A g, β- aspa yla ginine; STRR, sho andemly epea ed epe i i e; TCA, ica boxylic acid cycle. 1614 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies ( ege a i e cells and he e ocys s) coope a e o achie e g ow h o he ilamen as a whole [2]. The di e en ia- ion o he e ocys s in ol es complex ansc ip ional changes bo h in ege a i e cells and in he e ocys s no only o achie e mo phological di e en ia ion o he e- ocys s bu also o unde go me abolic adap a ions equi ed o g ow h a he expense o N 2 [3,4]. Small RNAs (sRNAs) cons i u e a ele an class o pos - ansc ip ional egula o s in ol ed in he adap a- ion o bac e ial me abolism o di e en en i onmen al si ua ions [5]. sRNAs can coo dina e changes in gene exp ession in esponse o en i onmen al s esses. Simila o he obse a ions made in o he g oups o bac e ia, cyanobac e ia exhibi abundan ansc ip ion o non- coding RNAs, including an isense RNAs and sRNAs, and ansc ip ion o many o hem is egula ed in esponse o ni ogen a ailabili y [6–9]. Se e al ni ogen- egula ed noncoding RNAs ha e been iden i ied and cha ac e ized in he ilamen ous, he e ocys - o ming cyanobac e ium Nos oc sp. PCC 7120 [3,10–14]. Fu - he mo e, he e ocys -speci ic ansc ip ion o noncoding RNAs has been desc ibed [3,11,15,16], and some o hose ansc ip s a ec he p ocess o he e ocys di e - en ia ion [17] o he egula ion o enzyma ic ac i i ies whose le els mus be adjus ed speci ically in he e ocys s e sus ege a i e cells [13]. NsiR3 is an sRNA whose exp ession is induced upon ni ogen dep i a ion and depends on N cA, he global ni ogen egula o in cyanobac e ia [9,11].I was o iginally desc ibed as conse ed in 27 he e ocys - o ming cyanobac e ia bu absen in nonhe e ocys ous s ains [11], sugges ing a possible ole o NsiR3 ela ed o he e ocys unc ion. Howe e , no egula o y a ge has so a been iden i ied o NsiR3. Compu a ional p edic ion o mRNAs egula ed by a gi en sRNA can be misleading because he in e ac ions be ween sRNAs and hei a ge s ake place h ough sho , o en discon inuous complemen a y sequences [18]. Expe imen al iden i ica ion o possible a ge s based on simple analysis o di e ences in exp ession changes upon al e a ion o he amoun o he sRNA is in many cases impeded by seconda y e ec s. Fu he - mo e, such app oaches ely on he assump ion ha he pos - ansc ip ional egula ion exe ed by he sRNA esul s in clea changes in he amoun o he a ge mRNA, which is no necessa ily ue [19]. Fo his ea- son, he combina ion o compu a ional and mo e sophis ica ed expe imen al app oaches is o en mo e p oduc i e ( e iewed in Re . [20]). In his wo k, we show ha upon ni ogen dep i a- ion, exp ession o NsiR3 is mo e s ongly induced in he e ocys s han in ege a i e cells. By using a no el app oach based on co ela ion o exp ession analysis we iden i y pu A, ha encodes p oline oxidase, as a a ge o NsiR3 because o he s ong nega i e co ela- ion wi h NsiR3. Finally, we demons a e he in e ac- ion be ween NsiR3 and he 50-UTR o he pu A mRNA, and he nega i e egula ion exe ed by NsiR3 on he exp ession o pu A in Nos oc sp. PCC 7120. Resul s NsiR3 is conse ed in he e ocys - o ming cyanobac e ia, and i s ansc ip ion is induced upon ni ogen s ess The ni ogen- egula ed ansc ip ion o NsiR3 was dis- co e ed in a p e ious dRNASeq expe imen [9]. Phylo- gene ic conse a ion o NsiR3 homologs ac oss cyanobac e ial genomes was la e desc ibed [11], and we ha e now iden i ied NsiR3 homologs in addi ional cyanobac e ia, con i ming ha NsiR3 appea s es ic ed o he e ocys - o ming s ains (Fig. 1). In Nos oc sp. PCC 7120, NsiR3 is ansc ibed down- s eam o all4558 and in he opposi e o ien a ion (Fig. 2A). The p edic ed seconda y s uc u e con ains wo possible s em–loops ac ing as ansc ip ional e - mina o s (Fig. 2B); he second one (T2) is also ound in closely ela ed cyanobac e ial s ains and is o med by STRR (sho andemly epea ed epe i i e) impe - ec 7-n epea s ha a e p esen in genomes o ila- men ous cyanobac e ia usually in in e genic egions bu in some case wi hin coding sequences. Thei unc- ion, i any, is unknown [21,22] (Fig. 2C). Exp ession o NsiR3 is induced upon ni ogen s ess (Fig. 2D,E). Induc ion upon emo al o combined ni ogen is quick, and he amoun s o NsiR3 emain high up o 24 h. T ans e om ammonium-con aining medium o ni a e-con aining medium also induces NsiR3 exp es- sion bu o lowe le els and he induc ion is ansien (Fig. 2D,E), e lec ing he ac ha ni ogen s ess in hese condi ions is quickly elie ed by de ep ession o he ni ope on which encodes ni a e educ ase and ni i e educ ase, he enzymes in ol ed in assimila ion o ni a e [23,24]. Consis en wi h he p edic ed sec- onda y s uc u e, wo RNAs, o 45 and 115 nucleo- ides, we e de ec ed by no he n hyb idiza ion wi h he NsiR3 p obe. The 45-nucleo ide RNA (NsiR3S) is o he expec ed size om he expe imen ally de e mined ansc ip ional s a si e (TSS) o NsiR3 [9] o he i s p edic ed e mina o , T1. The majo de ec ed band is 115 nucleo ides long (NsiR3L) and ma ches he calcu- la ed leng h om he TSS o he second p edic ed e - mina o , T2, u he downs eam (Fig. 2B). NsiR3L is he mos abundan o he wo species o NsiR3; he e- o e, i was used in all he expe imen s desc ibed he e. 1615The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies I. ´ Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase Fig. 1. Alignmen o NsiR3 RNA. The sequences o NsiR3S om di e en cyanobac e ia we e aligned wi h CLUSTAL OMEGA. Nucleo ide posi ions conse ed in he 70 s ains whe e NsiR3 was ound a e indica ed by as e isks. Each sequence is indica ed by he o ganism name ollowed by he GenBank accession numbe (www.ncbi.nlm.nih.go /genbank). The NsiR3 sequence om Nos oc sp. PCC 7120 is highligh ed in bold. 1616 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies sRNA egula ion o p oline oxidase I. ´ Al a ez-Esc ibano e al. N cA di ec ly egula es NsiR3 exp ession T ansc ip ion o NsiR3 in Nos oc sp. PCC 7120 depends on N cA [9,11]. Alignmen o he p omo e egion om all 70 iden i ied nsiR3 sequences shows ha a pu a i e conse ed N cA-binding sequence appea s cen e ed a ound posi ion −42.5 in 67 o hem (Fig. 3), which is compa ible wi h di ec ac i a ion o NsiR3 ansc ip ion by N cA. To e i y he in e ac ion be ween N cA and he p o- mo e egion o nsiR3, we pe o med an elec- opho e ic mobili y shi assay wi h pu i ied N cA Fig. 2. The ni ogen s ess-inducible (NsiR3). (A) Schema ic ep esen a ion o he egion encoding NsiR3 in Nos oc sp. PCC 7120. The wo lanking genes a e indica ed. The ben a ow ep esen s he ansc ip ional s a a posi ion 5452083 , and he s em–loops ep esen he wo ansc ip ional e mina o s o NsiR3. The posi ion o he N cA-binding si e is also indica ed. (B) Seconda y s uc u e model o NsiR3 om Nos oc sp. PCC 7120 based on he consensus ob ained wi h RNAali old om he alignmen s in Fig. 1and C his image. The hep anucleo ide epea s (STRR) a e amed. (C) The sequences o NsiR3 encoded in hose s ains ha could ha e a second ansc ip ional e mina o (T2) u he downs eam T1 we e aligned using CLUSTAL OMEGA. The p edic ed seed egion is highligh ed in yellow. Accession numbe s a e indica ed in Fig. 1. (D) Ni ogen- esponsi e exp ession o NsiR3 in Nos oc sp. PCC 7120. Exp ession was analyzed by no he n blo in cells g own in he p esence o ammonium and ans e ed o medium con aining no sou ce o combined ni ogen (N 2 ) o con aining ni a e (NO 3) o he numbe o hou s indica ed. The uppe panel shows hyb idiza ion o he NsiR3 p obe. The lowe panel shows hyb idiza ion o a p obe o 5S RNA used as loading and ans e con ol. Sizes (n ) a e indica ed on he le . The expe imen was epea ed ou imes wi h simila esul s. The no he n blo con aining he highes numbe o ime poin s is shown. (E) Quan i ica ion o NsiR3L in he blo shown in (D) upon ni ogen emo al (black, N 2 ) o upon ni ogen emo al ollowed by ni a e addi ion ( ed, NO 3). 1617The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies I. ´ Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase Fig. 3. Alignmen o he p omo e sequences o nsiR3. The sequences ups eam nsiR3 we e aligned wi h CLUSTAL OMEGA. Nucleo ide posi ions ully conse ed a e indica ed by as e isks. The consensus sequence is ep esen ed wi h WebLogo [49]. The likely −10 elemen s a e boxed, and wo expe imen ally de e mined TSS om Nos oc sp. PCC 7120 [9] and Nodula ia spumigena CCY9414 [50] a e ci cled in ed. Accession numbe s a e indica ed in Fig. 1. 1618 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies sRNA egula ion o p oline oxidase I. ´ Al a ez-Esc ibano e al. p o ein and a DNA agmen ex ending om posi ions −118 o +31 wi h espec o he TSS o nsiR3 ha includes he possible N cA-binding sequence. In he p esence o N cA, a e a ded complex was obse ed wi h he agmen con aining he wild- ype p omo e sequence (Fig. 4A). Howe e , he ac ion o agmen bound by N cA was s ongly (nine old) educed (Fig. 4 A,B) when he assay was pe o med wi h a DNA ag- men con aining a e sion o he nsiR3 p omo e wi h he N cA-binding si e mu a ed (GTG changed o CAC). These esul s demons a e he binding o N cA o a speci ic sequence o he nsiR3 p omo e . NsiR3 accumula es di e en ially in he e ocys s bu is no essen ial o diazo ophic g ow h I was p e iously shown ha NsiR3 ansc ip ion is dependen on N cA bu independen o He R [9,11], he mas e egula o o he e ocys di e en ia ion. The e o e, we expec ha NsiR3 ansc ip ion would be induced in all cells o he ilamen upon ni ogen s ess. To cha ac e ize he pa e n o exp ession o NsiR3, we p epa ed a plasmid con aining a ansc ip- ional usion o he NsiR3 p omo e o he g p gene (pSAM341, see Table S4). The plasmid was in oduced in Nos oc sp. PCC 7120 by conjuga ion, and exp es- sion o GFP was analyzed by con ocal luo escence mic oscopy (Fig. 5). Filamen s g owing in ammonium- con aining medium had e y low g een luo escence, while ilamen s g owing in medium lacking combined ni ogen showed luo escence in all cells, bu peaks o g een luo escence we e associa ed wi h cells ha we e di e en ia ing as he e ocys s, as indica ed by hei lowe ed au o luo escence. In e es ingly, induc ion o he nsiR3 p omo e occu s ea ly in he e ocys de elop- men because signi ican g een luo escence was al eady obse ed in imma u e he e ocys s, s ill ha ing subs an ial ed luo escence (no shown). The conse a ion o NsiR3 in he e ocys - o ming cyanobac e ia and i s N cA-dependen exp ession sug- ges s ha NsiR3 is in ol ed in some aspec o he eg- ula ion o he esponse o ni ogen s ess in hese complex cyanobac e ia ha includes di e en ia ion o specialized cells. To explo e he possible unc ion o NsiR3, we cons uc ed a Nos oc s ain lacking NsiR3 (ΔnsiR3). This s ain g ew simila ly o wild- ype in media con aining ammonium and ni a e, o lacking combined ni ogen (Fig. 6); he e o e, he di e en ia- ion o unc ional he e ocys s is no comp omised in he absence o NsiR3. NsiR3 is a egula o o pu A (al 0540) Bac e ial sRNAs impac hei a ge mRNAs o en by ep essing he ini ia ion o ansla ion ia binding o hei 50-UTRs [5]. Hence, he mRNAs do no associa e wi h ibosomes and may become mo e ulne able o iboendonucleases, leading o educed mRNA le el. To iden i y po en ial a ge s o NsiR3 whose exp ession would be nega i ely egula ed by NsiR3 in Nos oc sp. PCC 7120, we decided o sea ch o genes whose exp es- sion p o ile showed a nega i e co ela ion wi h he exp ession o NsiR3. Fo his pu pose, we ha e used a no el app oach based on co ela ion analysis o da a ob ained om hyb idiza ion o high-densi y mic oa - ays [3]. In o de o iden i y hose genes wi h nega i e co ela ion wi h NsiR3, we analyzed da a ob ained om hyb idiza ion o RNA samples ex ac ed om he ΔnsiR3 mu an g own in ammonium-con aining med- ium ( wo eplica es) o om he ΔnsiR3 mu an a e 8 h o incuba ion in medium lacking combined ni ogen ( wo eplica es). In addi ion, in o de o inc ease he Fig. 4. N cA binds o he nsiR3 p omo e . (A) Elec opho e ic mobili y shi assays showing binding o pu i ied His- agged N cA p o ein o a DNA agmen con aining he wild- ype p omo e o NsiR3 om Nos oc sp. PCC 7120 (le ) o a mu a ed e sion al e ed in he posi ions indica ed in ed ( igh ). Th ee imes mo e p obe was used in he assay wi h he mu an agmen han wi h he wild- ype agmen . A ep esen a i e expe imen is shown. (B) Quan i ica ion o he au o adiog am p esen ed in panel (A). The ac ion o p obe bound by N cA was calcula ed o he wild- ype (black) o he mu a ed ( ed) e sion. 1619The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies I. ´ Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase sensi i i y o he co ela ion analysis, p e iously a ail- able da a om hyb idiza ion o he same mic oa ay pla o m wi h o he 20 samples (bo h om he wild- ype s ain and om a he R mu an s ain ei he in he p es- ence o ammonium o a e di e en pe iods o ni ogen dep i a ion) [3] we e included in he analysis. The exp ession da a om he 24 samples (Table S1) we e analyzed as desc ibed [3], and a co ela ion able was ob ained o NsiR3 (Table S2). The p obe wi h he s onges nega i e co ela ion wi h NsiR3 (−0.943) was ha o gene al 0540 (pu A), encoding p oline oxidase. Mo eo e , ano he p obe co esponding o he 50-UTR o al 0540 had a s ong nega i e co ela ion as well (−0.884). Figu e 7A shows he exp ession p o ile o NsiR3 and pu A in he 24 samples hyb idized o he a ays. We also con i med by no he n blo he opposi e Fig. 5. Exp ession pa e n o he nsiR3 p omo e in Nos oc ilamen s. Con ocal luo escence images ( ed channel, g een channel, bo h channels me ged, and b igh - ield image) o Nos oc ilamen s ca ying he g p gene unde he con ol o he nsiR3 p omo e (plasmid pSAM341) and g owing on op o medium con aining ammonium, NHþ 4(A), o lacking any sou ce o combined ni ogen, N 2 (B). Quan i ica ion o he signals o he g een channel (GFP) is shown o he segmen s indica ed by a blue line in he co esponding b igh - ield images. All images we e acqui ed wi h he same sensi i i y se ings so ha in ensi ies can be compa ed. Scale ba s, 20 µm. Fig. 6. The ΔnsiR3 s ain can di e en ia e he e ocys s. Cells we e g own in he p esence o ni a e, washed wi h BG11 0 , and esuspended a 0.1 μg chlo ophyllmL −1 . Fi e old se ial dilu ions o liquid cul u es o wild- ype o ou independen ΔnsiR3 isola es we e p epa ed. Ten mic oli e o he cell suspension and o h ee i e old se ial dilu ions we e pla ed on BG11 0 pla es con aining ammonium (NHþ 4), ni a e (NO 3), o no sou ce o combined ni ogen (N 2 ). Pic u es we e aken a e 10 days o incuba ion a 30 °C. 1620 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies sRNA egula ion o p oline oxidase I. ´ Al a ez-Esc ibano e al. exp ession dynamics o pu A wi h NsiR3 ha was deduced om he co-exp ession analysis (Fig. 7B). The esul s desc ibed abo e s ongly sugges ha he exp ession o pu A migh be egula ed by NsiR3. In ac , we could p edic wi h In aRNA [25] an in e ac- ion be ween NsiR3 and he 50-UTR o pu A mRNA (Fig. 8A). NsiR3 would bind o posi ions −5 o−17 wi h espec o he ini ia ion codon o pu A, occluding he ibosome-binding si e. To e i y he in e ac ion be ween NsiR3 and he 50- UTR o pu A, we used a he e ologous epo e sys em [26] in which he 50-UTR plus he i s 60 n o he pu A coding sequence was ansla ionally used o he gene encoding supe olde GFP (s g p). This cons uc was co-exp essed in E. coli ei he wi h NsiR3 o wi h a con ol, un ela ed RNA. The ini ia ion codon o pu A was changed om GTG o ATG o op imal exp es- sion in E. coli. The luo escence o cells ca ying s g p usions signi ican ly dec eased when NsiR3 was co-ex- p essed, indica ing a nega i e e ec o NsiR3 on exp ession o s GFP (Fig. 8B). To e i y he in e ac ions be ween NsiR3 and he mRNA o pu A, we c ea ed a mu a ed e sion o NsiR3 al e ed in posi ion 14 (G o C, NsiR3 Mu 14) (Fig. 8A). The mu a ion in NsiR3 elimina ed he in e ac ion be ween NsiR3 and he mRNA o pu A (Fig. 8B). We designed a compensa o y mu a ion in he 50-UTR o pu A (Comp-14*17) ha would es o e he in e ac ion wi h NsiR3 (Mu 14) (Fig. 8A). When NsiR3 (Mu 14) was combined wi h he mu a ed e sion o he 50-UTR o pu A, he educ ion o luo escence was es o ed (Fig. 8B). All oge he , hese esul s e i y he in e ac- ion o NsiR3 wi h he 50-UTR o pu A a he posi ions p edic ed by In aRNA. In consequence, an inhibi o y e ec o NsiR3 on pu A in he in i o con ex o he he e ologous E. coli sys em is suppo ed. NsiR3 ep esses he exp ession o pu A in Nos oc sp. PCC 7120 To analyze he egula o y e ec o NsiR3 on pu A in Nos oc sp. PCC 7120, we gene a ed a Nos oc s ain Fig. 7. Nega i e co ela ion o pu A and nsiR3 exp ession. (A) Exp ession le el o nsiR3 ( ed) and pu A (al 0540, black), in he 24 RNA samples hyb idized o he mic oa ays used o he co-exp ession analysis. (B) pu A exp ession a e emo al o combined ni ogen. RNA was ex ac ed om Nos oc cul u es a di e en imes a e ni ogen emo al and subjec ed o no he n blo . The il e was hyb idized wi h p obes o pu A ( op), nsiR3 (middle), and npB (bo om), ha was used as loading con ol. The expe imen was epea ed h ee imes wi h simila esul s. The no he n blo con aining he highes numbe o ime poin s is shown. (C) Quan i ica ion o he blo shown in (B). The amoun o pu A mRNA (black) and NsiR3 ( ed) is exp essed as pe cen age o he maximum. Fo pu A, he ull-leng h ansc ip (a ow in B) was used in quan i ica ion. Sizes o ibosomal RNAs a e indica ed in (B). 1621The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies I. ´ Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase wi h con olled exp ession o NsiR3 by complemen ing s ain ΔnsiR3 wi h a plasmid con aining nsiR3 unde con ol o he coppe -inducible pe E p omo e (ΔnsiR3+P pe E ::nsiR3). Accumula ion o he mRNA o pu A was analyzed by no he n blo in he wild- ype, ΔnsiR3, and ΔnsiR3+P pe E ::nsiR3 s ains a di e en imes a e ni ogen emo al and coppe addi ion (Fig. 9A). A s onge educ ion in he amoun o he ull-leng h pu A mRNA was obse ed in he ΔnsiR3+P pe E ::nsiR3 s ain han in he wild- ype, in ag eemen wi h he obse a ion ha ΔnsiR3+P pe E :: nsiR3 accumula ed abou 40 imes mo e NsiR3 han he wild- ype (Fig. 9A,B). The dele ion o nsiR3 in he ΔnsiR3 s ain esul ed in le els o he pu A mRNA ha we e no signi ican ly educed a e 24 h in he absence o combined ni ogen. These esul s indica e a s ong nega i e co ela ion be ween he amoun o NsiR3 and he amoun o he pu A mRNA. As a con- ol, we hyb idized he same il e wi h a p obe o gene ag E encoding he bi unc ional enzyme a ginine dihyd olase/o ni hine cyclodeaminase ha ac s ups eam p oline oxidase in he ca abolic pa hway om a ginine o p oline [27]. The dynamic o changes in he amoun o ag E mRNA upon ni ogen emo al was di e en o ha o pu A. In he wild- ype s ain, whe eas he amoun o pu A mRNA was educed o abou 40% a e 24 h in he absence o combined ni ogen, no signi ican change was obse ed in he case o ag E. In addi ion, he le els o ag E mRNA did no co ela ed o changes in he amoun o NsiR3 (Fig. 9), as p edic ed o a mRNA ha is no a a ge o NsiR3. Discussion In his wo k, we cha ac e ize NsiR3, a highly con- se ed sRNA in he e ocys ous cyanobac e ia. NsiR3 was iden i ied in he genomes o se en y he e ocys - o ming cyanobac e ia bu was no ound in unicellu- la o ilamen ous s ains ha do no de elop he e o- cys s. This dis ibu ion o he nsiR3 gene, in con as o ha o o he N cA- egula ed sRNAs such as NsiR4, also ound in unicellula s ains [10], sugges s a ele an unc ion ela ed o he speci ic me abolic ai s o he e ocys - o ming s ains. nsiR3 is no ansc ibed in he p esence o ammonium, bu i s ansc ip ion is s ongly induced upon ans e o ni ogen- ee med- ium, sugges ing a possible unc ion in he esponse o ni ogen s ess. Fu he mo e, nsiR3 ansc ip ion is also induced upon ans e om ammonium- o ni a e-con aining medium. Howe e , in his case he induc ion is lowe and ansien . This can be explained because he ni ogen s ess is quickly elie ed upon induc ion o he ni a e assimila ion pa hway in he p esence o ni a e [23]. We show ha he global egu- la o o ni ogen assimila ion, N cA, binds di ec ly o he nsiR3 p omo e , explaining he p e ious obse a- ion ha N cA is equi ed o exp ession o NsiR3 [9,11]. By means o a usion o he g p gene, we show ha exp ession o NsiR3, al hough induced in all cells o he ilamen unde ni ogen dep i a ion, is much s onge in he e ocys s han in ege a i e cells. This obse a ion migh be explained by he highe concen- a ion o N cA p esen in he he e ocys e sus in eg- e a i e cells [28,29]. Al e na i ely, he nsiR3 p omo e Fig. 8. Ve i ica ion o NsiR3 in e ac ion wi h he 50-UTR o pu A using an in i o epo e sys em. (A) P edic ed in e ac ion be ween NsiR3 and he 50-UTR o pu A mRNA acco ding o INTARNA [25]. Nucleo ides a e numbe ed wi h espec o he s a o he coding sequence (ini ia ion codon is unde lined). The ibosome-binding si e in he pu A mRNA is highligh ed in yellow. A mu a ion in oduced in NsiR3 a posi ion 14 (G o C, Mu 14) and he co esponding compensa o y mu a ion in pu A 50-UTR posi ion −17 (C o G, Comp-14*17) a e indica ed in ed and blue, espec i ely. (B) Fluo escence measu emen s o E. coli DH5αcul u es bea ing combina ions o plasmids exp essing di e en e sions o NsiR3 and pu A::s g p usions. Plasmid pJV300 (encoding a con ol RNA) was used as con ol. The da a a e p esen ed as he mean s anda d de ia ion o cul u es om eigh independen colonies a e sub ac ion o luo escence in cells bea ing pXG-0 (***P<0.0001, S uden - es ). 1622 The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies sRNA egula ion o p oline oxidase I. ´ Al a ez-Esc ibano e al. Pseudomonas s u ze i A1501. 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(2013) Insigh s in o he physiology and ecology o he b ackish-wa e -adap ed Cyanobac e ium Nodula ia spumigena CCY9414 based on a genome- ansc ip ome analysis. PLoS One 8, e60224. Suppo ing in o ma ion Addi ional suppo ing in o ma ion may be ound online in he Suppo ing In o ma ion sec ion a he end o he a icle. Table S1. Di e en ially_exp essed_genes_all_samples. Table S2. Pea son_co ela ion_o _NsiR3_and_all_- genes. Table S3. S ains. Table S4. Plasmids. Table S5. Oligonucleo ides. Table S6. Sequences. 1629The FEBS Jou nal 288 (2021) 1614–1629 ª2020 Fede a ion o Eu opean Biochemical Socie ies I. ´ Al a ez-Esc ibano e al.sRNA egula ion o p oline oxidase