R-DeeP/TripepSVM identifies the RNA-binding OB-fold-like protein PatR as regulator of heterocyst patterning

Nucleic Acids Resea ch , 2025, 53 , gkae1247
h ps://doi.o g/10.1093/na /gkae1247
Ad ance access publica ion da e: 19 Decembe 2024
RNA and RNA-p o ein complexes
R-DeeP / T ipepSVM iden i ies he RNA-binding OB- old-like
p o ein Pa R as egula o o he e ocys pa e ning
Manuel B enes-Ál a ez
1 ,
* , Halie R. Ropp
1
, Dimi ios P apagiannidis
2
, Clemen M. Po el
2
,
F ank S ein
2
, Ing ebo g Sc holz
1
, Claudia S eglic h
1
, Mikhail M. Sa i ski
2
, Agus ín Vioque
3
,
Alicia M. Mu o-Pas o
3 and Wol gang R. Hess
1
1
Gene ics and Expe imen al Bioin o ma ics, Facul y o Biology, Uni e si y o F eibu g, Schänzles . 1, 79104 F eibu g, Ge many
2
Eu opean Molecula Biology Labo a o y (EMBL), Meye ho s . 1, 69117 Heidelbe g, Ge many
3
Ins i u o de Bioquímica Vege al y Fo osín esis, Consejo Supe io de In es igaciones Cien í icas and Uni e sidad de Se illa, A enida Amé ico
Vespucio 49, 41092 Se illa, Spain
*
To whom co espondence should be add essed. Tel: +49 761 / 203 2708 Email: [email p o ec ed]
Abs ac
RNA-binding p o eins (RBPs) a e cen al componen s o gene egula o y ne w o ks. T he di e en ia ion o he e ocys s in ilamen ous cyanobac e ia
is an example o cell di e en ia ion in p oka yo es. Al hough mul iple non-coding ansc ip s a e in ol ed in his p ocess, no RBPs ha e been
implica ed hus a . He e we used quan i a i e mass spec ome y o analyze he di e en ial ac iona ion o RNA–p o ein comple x es a e RNase
ea men in densi y g adien s yielding 333 RNA-associa ed p o eins, while a bioin o ma ic p edic ion yielded 311 RBP candida es in Nos oc sp.
PCC 7120. We alida ed in i o he RNA-binding capaci y o six RBP candida es. Some pa icipa e in essen ial physiological aspec s, such as
pho osyn hesis (Al 2890), h ylak oid biogenesis (Vipp1) o he e ocy s di e en ia ion (P pA, Pa U3), bu hei associa ion wi h RNA w as unkno wn.
Valida ed RBPs Asl3888 and Al 1700 we e no p e iously cha ac e iz ed. Al 1700 is an RBP wi h wo oligonucleo ide / oligosaccha ide-binding
(OB)- old-lik e domains ha is di e en ially exp essed in he e ocys s and in e ac s wi h non-coding egula o y RNAs. Dele ion o al 1700 led o
comple e de egula ion o he cell di e en ia ion p ocess, a s iking inc ease in he numbe o he e ocy s -lik e cells, and was ul ima ely le hal in he
absence o combined ni ogen. These obse a ions cha ac e ize his RBP as a mas e egula o o he he e ocys pa e ning and di e en ia ion
p ocess, leading us o ename Al 1700 o Pa R.
G aphical abs ac
R-DeeP
in Cyanobac e ia
In i o alida ion
(PNK assays)
Cha ac e iza ion o no el
RBPs
Wes e n-Blo Au o adiog aphy
-+ -+ UV
3xFLAG-Pa R
KDa
70
55
40
35
15
25
10
RBPs wi h
no el p o ein
domains and
a chi ec u e
6 new RBPs
alida ed in i o
Some RBPs a e
in ol ed in cell
di e en ia ion in
cyanobac e ia
333 RNA-associa ed
p o eins de ec ed
Size g adien
+ RNases
MS
No malized
p o ein
amoun
non-RBP RBP RBP
F ac ions
- RNases
In oduc ion
Filamen ous cyanobac e ia a e conside ed one o he oldes
mul icellula o ganisms on Ea h ( 1 ). Some o hese o ganisms,
such as Nos oc sp. PCC 7120 (he ea e Nos oc ), also known
as Anabaena sp. PCC 7120, ha e he abili y o di e en ia e
a specialized cell ype de o ed o ni ogen ixa ion, he he e-
Recei ed: May 31, 2024. Re ised: No embe 15, 2024. Edi o ial Decision: Decembe 2, 2024. Accep ed: Decembe 4, 2024
©The Au ho (s) 2024. Published by Ox o d Uni e si y P ess on behal o Nucleic Acids Resea ch.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License
(h ps: // c ea i ecommons.o g / licenses / by-nc / 4.0 / ), which pe mi s non-comme cial e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he
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2 Nucleic Acids Resea ch , 2025, Vol. 53, No. 3
ocys . Unde ni ogen- eple e condi ions he e a e no he e o-
cys s, bu when he ilamen s encoun e ni ogen s a a ion,
cell–cell compe i ion ollowing a one-dimensional Tu ing pa -
e n induces he gene ic p og am o cell di e en ia ion only in
some ege a i e cells. The de elopmen al p og am akes abou
24 h o comple e, p o iding an excellen expe imen al model
o he analysis o cell di e en ia ion in bac e ia. He e ocys s
a e e minally di e en ia ed, non-di iding cells dedica ed o
ni ogen ixa ion, and hei mo phology and physiology ha e
e ol ed o c ea e a mic ooxic en i onmen ha p o ec s and
suppo s ni ogenase ac i i y ( 2 ). Du ing diazo ophic g ow h,
ege a i e cells con inue o ix CO
2 h ough pho osyn hesis,
while he e ocys s ix N
2
. The e o e, bo h ypes o cells mus
exchange me aboli es o main ain he g ow h o he ilamen
as a whole, p o iding one o he simples examples o a ue
mul icellula o ganism wi h wo cell ypes coope a ing o-
ge he ( 3 ).
N cA and He R ha e been ecognized as he main ansc ip-
ional egula o s o his di e en ia ion p ocess ( 4 ). Howe e ,
in s iking pa allel o mo e complex euka yo ic o ganisms,
genome-wide analyses ha e sugges ed he in ol emen o hun-
d eds o non-coding RNAs [an isense RNAs (asRNAs) and
small RNAs (sRNAs)] in his p ocess ( 5 ,6 ). Se e al sRNAs a e
egula ed by he a ailabili y o ni ogen hemsel es and we e
designa ed n i ogen s ess-i nduced R NAs (NsiR). Some ha e
been cha ac e ized in mo e de ail ( 7–9 ) and a e in ol ed in he
eshaping o he e ocys me abolism ( 9 ) o in he co e egula-
ion o he de elopmen al p ocess ( 8 ). NsiR1 is exp essed a a
e y ea ly s age in he cells unde going di e en ia ion ( 10 ) and
inhibi s, as an asRNA, he ansla ion o he o e lapping he F
messenge RNA (mRNA) ( 11 ) encoding a p o ease c ucially
in ol ed in he e ocys di e en ia ion ( 12 ). As a ans -ac ing
sRNA, NsiR1 egula es he exp ession o al 3234 ( 8 ), encod-
ing a He P-like p o ein in ol ed in he egula ion o commi -
men o he e ocys di e en ia ion ( 13 ). Regula ion h ough
p oka yo ic non-coding RNAs equen ly depends on RNA-
binding p o eins (RBPs) such as RNA chape ons o ma ch-
make s, and ex ensi e ne wo ks o pos - ansc ip ional egu-
la ion ha e been elucida ed ( 14 ), bu li le is known abou he
in e ac ion o RBPs and hei unc ions in cyanobac e ia. In
ac , homologs o p ominen RNA chape ones in p o eobac e-
ia, such as Cs A, P oQ, FinO o H q do no exis in cyanobac-
e ia o do no bind RNA ( 15 ).
RBPs play a cen al ole in he physiology o cells. The ibo-
somes, he RnpB-RnpA (RNase P) complex, he signal ecog-
ni ion pa icle, he ans e -messenge -RNA-SmpB complex,
o he as di e si y o CRISPR sys ems a e well-known i-
bonucleop o ein complexes in p oka yo es. RBPs a e key eg-
ula o s o gene exp ession because hey de e mine he a e o
all RNA molecules in he cell ( 16 ), including he egula ion o
mRNA s abili y o he spa ial localiza ion o mRNAs in he
cell ( 17 ). Howe e , only a ew pieces o in o ma ion on he
ole o RBPs in he physiology o cyanobac e ia a e known. A
amily o RBPs con aining an RNA ecogni ion mo i (P am:
PF00076), simila o ce ain plan RBPs, was disco e ed in
cyanobac e ia almos 30 yea s ago ( 18 ). Recen ly, hey ha e
been shown o be essen ial o he a ge ing o mRNAs en-
coding pho osys em subuni s nea he hylakoid memb ane,
he si e whe e pho osyn he ic p o eins should be inse ed ( 19 ).
Se e al expe imen al sc eens o he iden i ica ion o RBPs
ha e been de eloped in ecen yea s. They ely on he ac-
iona ion o cellula ibonucleop o ein complexes using ei he
glyce ol / suc ose g adien s and ul acen i uga ion ( 20 ,21 ) o
size exclusion ch oma og aphy ( 22 ). In G ad-seq he u he
analysis o he componen s o each ac ion using RNA-seq
and mass spec ome y (MS) allows he p edic ion o RBPs
and hei RNA a ge s ( 23 ). G adR / R -DeeP ( 24 , 25 ) use an
RNase- ea ed sample o iden i y p o eins whose posi ion in
he g adien shi a e RNA emo al. I is impo an o no e
ha G adR / R-DeeP iden i ies no only RBPs, bu also p o-
eins ha a e pa o RNA–p o ein complexes, al hough hey
do no di ec ly in e ac wi h he RNA componen ( 25 ). In his
wo k, we ha e used G adR / R -DeeP ( 24 , 26 ) o iden i y new
RBPs exp essed in Nos oc cell ilamen s ha unde go he e o-
cys di e en ia ion. Since G adR and R-DeeP a e simila ap-
p oaches, only R-DeeP will be used in he ollowing. We ha e
iden i ied 333 RNA-associa ed p o eins ha may be pa o
RNA–p o ein complexes. The in i o alida ion o some o he
candida es opens a new wo ld o ibo egula ion in hese o -
ganisms, as some RBPs ha e desc ibed unc ions no ela ed o
RNA me abolism, o we e comple ely uncha ac e ized, such
as Al 1700 (Pa R), a p o ein wi h wo OB- old-like domains
ha a ec s he cell di e en ia ion p ocess.
Ma e ials and me hods
S ains and g ow h condi ions
The di e en Nos oc s ains used in his wo k
( Supplemen a y Table S1 ) we e g own pho oau o ophi-
cally wi h cons an shaking unde s anda d condi ions (30
◦C
and 50 μE illumina ion) in liquid BG11 medium ( 27 ). Fil-
amen s we e collec ed by cen i uga ion a 3270 ×g o 5
min a oom empe a u e (RT), washed and hen esuspended
in BG11
0 (medium wi hou a combined ni ogen sou ce) o
induce he e ocys di e en ia ion. Nos oc de i a i e s ains
bea ing Sm
R
Sp
R
o Nm
R
plasmids we e g own in liquid in he
p esence o 2 μg / ml s ep omycin (Sm) and spec inomycin
(Sp) each, o 5 μg / ml neomycin (Nm).
R-DeeP expe imen ; cell lysis, g adien p epa a ion
and ac iona ion
T iplica e 200 ml Nos oc liquid cul u es we e g own pho oau-
o ophically unde s anda d condi ions in BG11 medium un-
il hey eached a concen a ion o 3 μg chlo ophyll / ml. He -
e ocys di e en ia ion was induced as desc ibed abo e. A e
26 h o cul i a ion, he ilamen s we e ha es ed by cen i uga-
ion and esuspended in ice-cold lysis bu e (20 mM T is-HCl
pH 7.5, 150 mM KCl, 10 mM MgCl
2
, 1 mM di hio h ei ol)
supplemen ed wi h p o ease Inhibi o [cOmple e e hylenedi-
amine e aace ic acid (EDTA)- ee, Roche]. F om his poin
on, he samples we e kep a 4
◦C. Each eplica e was di ided
in o equal sample ubes. The con ol samples in which he
RNA should be p ese ed we e supplemen ed wi h 8 μl o
Ribolock RNase Inhibi o . To all ubes, 100 μl o a mix u e
o 0.1 and 0.5 mm glass beads we e added. Cells we e me-
chanically lysed in a p e-chilled P ecellys homogenize (Be in
Technologies) using nine cycles o 10 s shaking a 6000 pm
ollowed by a pause o 5 s. Cell deb is and glass beads we e e-
mo ed by cen i uga ion a 1500 ×g o 2 min. Nex , 1.25%
β-DM was added o he supe na an o solubilize memb ane
p o eins by o exing o 30 s. Memb anes we e pelle ed by
cen i uga ion a 21000 ×g o 15 min. Nex , 100 μl o RNase
A / T1 mix (The mo, 2 mg / ml RNase A and 5000 U / ml RNase
T1 s ock) was added o he RNase- ea ed ex ac s, while 100
μl o RNase bu e (50 mM T is-HCl pH 7.4 and 50% glyc-
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Nucleic Acids Resea ch , 2025, Vol. 53, No. 3 3
e ol) was added o he con ol ex ac s. Con ol and RNase-
ea ed samples we e incuba ed on ice o 20 min.
Linea suc ose g adien s we e p epa ed on Ul a-Clea
Beckman ubes using a G adien Mas e 108 o ming sys-
em (Biocomp) and wo solu ions o lysis bu e supplemen ed
wi h ei he 10% o 40% suc ose. A o al o 600 μl o he
con ol and RNase- ea ed o al ex ac s we e loaded ca e-
ully on o he g adien s. Mac omolecula complexes we e sep-
a a ed by ul acen i uga ion o he lysa es in a swinging-
bucke o o (Beckman SV40 Ti) o 16 h a 285000 ×g . F ac-
ions o equal olume (600 μl) we e collec ed using a Pis on
G adien F ac iona o (Biocomp). The p o ein concen a ion
o each ac ion was de e mined using he Low y p ocedu e
( 28 ).
No he n and wes e n blo analysis o ac ions
RNA was ex ac ed using ho phenol ( 29 ) wi h modi ica-
ions ( 6 ) om 300 μl o each ac ion. The RNA was ac-
iona ed on a 10% u ea-polyac ylamide (PAA) gel, s ained
wi h e hidium b omide and elec oblo ed on o a Hybond-
N + memb ane (Ame sham) a 1 mA pe cm
2 o 1 h. The
dis ibu ion o RnpB, Y 1 and NsiR8 was analyzed by no h-
e n hyb idiza ion using single-s anded adioac i ely labeled
RNA p obes ansc ibed in i o om polyme ase chain eac-
ion (PCR)-gene a ed empla es (see Supplemen a y Table S2
o p ime s). Radioac i ely labeled p obes we e gene a ed us-
ing [ α-
32
P]-UTP and he Maxisc ip T7 In i o ansc ip-
ion ki (The mo Fishe Scien i ic). The dis ibu ion o NsiR1,
4.5S and 5S along he g adien ac ions was also analyzed
by no he n hyb idiza ion using end-labeled oligonucleo ides
(see Supplemen a y Table S2 o p ime s). Oligonucleo ides
we e labeled wi h [ γ-
32
P]-ATP and polynucleo ide kinase
(PNK) (The mo). Hyb idiza ions we e pe o med as p e i-
ously desc ibed ( 30 ). The signals we e de ec ed wi h a Ty-
phoon FLA 9500 (GE Heal hca e).
Fo wes e n blo analysis, 35 μl o each ac ion we e ac-
iona ed in 15% sodium dodecyl sul a e (SDS)-PAA gels and
ans e ed o a ni ocellulose memb ane (Ame sham). As a
con ol, iden ically p epa ed gels we e s ained wi h Coomassie
B illian Blue G-250. The memb anes we e blocked in 5%
skim milk in 1% Tween in phospha e-bu e ed saline (T-PBS)
o 1 h a oom empe a u e and incuba ed wi h abbi poly-
clonal an ibody an i-RplA (#AS111738,1:5000, Ag ise a) o
mouse monoclonal an i-FLAG coupled o Ho se adish pe ox-
idase (HRP) (#A8592,1:2000, Sigma) o 16 h a 4
◦C. The
memb anes we e washed h ee imes wi h T-PBS, 5 min each.
Memb anes p e iously incuba ed wi h an i-RplA we e in-
cuba ed wi h he seconda y an ibody an i- abbi IgG-HRP
(#A8275, 1:10 000, Sigma) o 1 h a oom empe a u e and
hen washed h ee imes wi h T-PBS. Signals we e de ec ed
wi h wes e n-blo ECL sp ay (Ad an a).
Sample p epa a ion o MS and measu emen s
Samples o p o eomic analysis we e p epa ed as p e iously
desc ibed ( 31 ), wi h mino modi ica ions. A o al o 1% N-
lau oylsa cosine was added o equal olumes o samples along
he g adien ( ac ions 1 and 2 we e combined) and p o eins
we e diges ed ollowing a modi ied SP3 p o ocol ( 32 ). Speci -
ically, 20 μl samples we e incuba ed wi h 40 μl bead suspen-
sion (The mo Fische Scien i ic, Se a-Mag Speed Beads, 4515–
2105-050250, 6515–2105-050250) in 2.5% o mic acid and
50% e hanol (15 min, RT, 500 pm). Beads we e hen washed
ou imes wi h 70% e hanol and p o eins we e diges ed
o e nigh (RT, 500 pm) wi h ypsin and LysC (200 ng o
each enzyme in 5 mM chlo oace amide, 1.25 mM is(2-
ca boxye hyl)phosphine in 100 mM 4-(2-hyd oxye hyl)-1-
pipe azinee hanesul onic acid bu e pH 8, pe 5 μg p o ein).
Pep ides we e elu ed om he beads, d ied unde acuum,
and esuspended in wa e . Ten mic oli e samples (up o 5 μg
pe sample) we e labeled wi h 50 μg o TMT18plex (The mo
Fishe Scien i ic) econs i u ed in 4 μl ace oni ile. Labeling
eac ion (1 h, RT, 500 pm) was quenched wi h 4 μl 5% hy-
d oxylamine (30 min, RT, 500 pm) and samples o he same
g adien we e pooled. The samples we e hen desal ed wi h
solid phase ex ac ion by loading on o a Wa e s OASIS HLB
μElu ion Pla e (30 μm), washing wice wi h 100 μl o 0.05%
o mic acid, and elu ing wi h 100 μl o 80% ace oni ile. Sam-
ples we e hen d ied unde acuum and esuspended in 20 mM
ammonium o ma e (pH 10.0).
O line high pH e e se phase ac iona ion was pe o med
using an Agilen 1200 In ini y high-pe o mance liquid ch o-
ma og aphy sys em equipped wi h a qua e na y pump, de-
gasse , a iable wa eleng h ul a iole (UV) de ec o (se o
254 nm), Pel ie -cooled au osample and ac ion collec o
(bo h se a 10
◦C o all samples). The column was a Gem-
ini C18 column (3 μm, 110 Å, 100 ×1.0 mm, Phenomenex)
wi h a Gemini C18, 4 ×2.0 mm Secu i yGua d (Phenomenex)
ca idge as a gua d column. The sol en sys em consis ed o
20 mM ammonium o ma e (pH 10.0) (Bu e A) and 100%
ace oni ile as mobile phase (Bu e B). The sepa a ion was ac-
complished a a mobile phase low a e o 0.1 ml / min using
he ollowing linea g adien : 100% Bu e A o 2 min, om
100% Bu e A o 35% Bu e B in 59 min, o 85% Bu e B in
a u he 1 min and held a 85% Bu e B o an addi ional 15
min, be o e e u ning o 100% Bu e A and e-equilib a ion
o 13 min. Fo y-eigh ac ions we e collec ed along wi h
he LC sepa a ion and subsequen ly pooled in o 12 ac ions.
Pooled ac ions we e d ied unde acuum cen i uga ion, e-
cons i u ed in 10 μl 1% o mic acid, 4% ace oni ile p io
liquid ch oma og aphy–mass spec ome y analysis.
An Ul iMa e 3000 RSLC nano LC sys em (Dionex) i ed
wi h a apping ca idge ( μ-P ecolumn C18 PepMap 100, 5
μm, 300 μm in e nal diame e ×5 mm, 100 Å) and an ana-
ly ical column (nanoEase™ M / Z HSS T3 column 75 μm ×
250 mm C18, 1.8 μm, 100 Å, Wa e s) was used. T apping
was ca ied ou wi h a cons an low o 0.05% i luo oace ic
acid a 30 μl / min on o he apping column o 6 min. Subse-
quen ly, pep ides we e elu ed ia he analy ical column wi h a
cons an low o sol en A (3% dime hyl sul oxide (DMSO),
0.1% o mic acid in wa e ) a 0.3 μl / min wi h an inc easing
pe cen age o sol en B (3% DMSO, 0.1% o mic acid in ace-
oni ile). The ou le o he analy ical column was coupled di-
ec ly o a QExac i e plus (The mo) mass spec ome e using
he Nanosp ay Flex™ ion sou ce in posi i e ion mode.
The pep ides we e in oduced in o he QExac i e plus ia
a Pico-Tip Emi e 360 μm ou e diame e ×20 μm in e nal
diame e ; 10 μm ip (CoAnn Technologies) and an applied
sp ay ol age o 2.3 kV. The capilla y empe a u e was se
a 320
◦C. Full mass scan was acqui ed wi h a mass ange o
375–1 200 m / z in p o ile mode wi h esolu ion o 70 000. The
illing ime was se a a maximum o 250 ms wi h a limi a ion
o 3 ×106 ions. Da a-dependen acquisi ion was pe o med
wi h he esolu ion o he O bi ap se o 35000, wi h a ill
ime o 120 ms and a limi a ion o 2 ×105 ions. A no mal-
ized collision ene gy o 30 was applied. The isola ion window
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4 Nucleic Acids Resea ch , 2025, Vol. 53, No. 3
o he quad upole was se o 0.7 m / z . A dynamic exclusion
ime o 30 s was used. The pep ide ma ch algo i hm was se
o ‘p e e ed’ and cha ge exclusion ‘unassigned’, cha ge s a es
1, 5–8 we e excluded. MS2 da a was acqui ed in p o ile mode.
Bioin o ma ic analysis o R-DeeP da a
Bioin o ma ic analysis was pe o med in R. Only p o eins
wi h a leas wo iden i ied pep ides we e kep o he analysis.
In he case o small p o eins ( < 100 aa), a mo e elaxed il e
was applied and a single iden i ied pep ide was conside ed su -
icien o keep he p o ein in he analysis. Fu he mo e, only
p o eins iden i ied in a leas one pai o con ol and RNase-
ea ed g adien s we e kep . Ba ch e ec emo al and a a i-
ance s abiliza ion no maliza ion me hod we e applied o each
ac ion using he limma ( 33 ) and sn ( 34 ) packages. An in-
g adien no maliza ion was also pe o med, so ha he sum
o each p o ein along a g adien was se o 100%. A di e -
en ial exp ession-like app oach was applied o each ac ion
using he limma package. The p esence o a p o ein in mul iple
eplica es was aken in o accoun using he pa ame e weigh s
o he adjus men o he linea model h ough he lmFi unc-
ion. The alse disco e y a e ( d ) was calcula ed using he
d ools ( 35 ) package. P o eins wi h a log
2 old change ≥1
and d ≤0.05 in a leas one ac ion we e conside ed as hi s.
P o eins wi h a log
2
old change ≥0.58 and d ≤0.02 we e
conside ed candida es.
An R package wi h he Nos oc anno a ion was c ea ed
using he Anno a ionFo ge R package. Sequences and de-
sc ip ion o he Nos oc p o eome we e downloaded om
NCBI and UNIPROT. The Gene On ology (GO) e ms
we e downloaded om QuickGO ( h ps://www.ebi.ac.uk/
QuickGO/anno a ions ). The molecula weigh and isoelec-
ic poin o each p o ein we e calcula ed using he Expasy
Compu e pI / Mw ool ( h ps:// web.expasy.o g/ compu e _ pi/ ).
GO- e m en ichmen o he RNA-associa ed p o eome was
pe o med using he clus e P o ile ( 36 ) R package and a q
alue ≤0.05 was used as cu o .
In o de o g oup p o eins wi h simila sedimen a ion p o-
iles, a so clus e ing app oach was applied o he mean o
he con ol g adien s by M uzz ( 37 ). An m pa ame e o 1.14
was selec ed and a con idence > 0.85 was used as he h esh-
old o a p o ein o be conside ed in he co e o a clus e . The
log
2
old change o each ac ion o he 333 RNA-associa ed
p o eins was clus e ed using hie a chical clus e ing. Silhoue e
was used as a me ic o de e mine he bes numbe o clus e s
in bo h cases.
Bioin o ma ic p edic ion o RBPs
We used a modi ied e sion o T iPepSVM (
38 ) o p edic
RBPs in cyanobac e ia. Ins ead o using a e e ence p o-
eome and i s e olu iona y close p o eomes o he gene a-
ion o posi i e and nega i e da ase s, we selec ed p o eomes
o cyanobac e ia wi h di e se mo phologies and li es yles (see
Supplemen a y Table S3 ). In addi ion, we also included he
p o eome o wo well-s udied G am-nega i e bac e ia, Es-
c he ic hia coli K12 and Salmonella yphimu ium LT2. All p o-
eomes we e downloaded om UNIPROT. The assembly o
he posi i e and nega i e aining da ase s was pe o med ol-
lowing he T iPepSVM pipeline ( 38 ). We used he KeBABS
( 39 ) package o pe o m a 10-k- old alida ion in a g id sea ch
using he ollowing combina ion o pa ame e s: posi i e class
weigh , nega i e class weigh , k-me s and cos . The balanced
accu acy sco e was used o selec he bes combina ion o pa-
ame e s. An RBP p edic ion was pe o med using he ol-
lowing pa ame e s: posi i e class weigh = 2.7, nega i e class
weigh = 0.05, cos = 1 and k-me = 3. A h eshold o 0.25
was selec ed in he suppo ec o machine (SVM) sco es o
he p o eins o classi ica ion as po en ial RBP, esul ing in 311
po en ial RBPs.
The p edic ion o in insically diso de ed egions in he p e-
dic ed RBPs was pe o med using IUP ed2A ( 40 ). Regions
wi h an a e age diso de sco e > 0.5 we e conside ed no glob-
ula egions. A sliding window p og ammed in R was used o
coun he equency o ipep ides in globula o diso de ed e-
gions o he p o eins. A cu e was smoo hed using locally es i-
ma ed sca e plo smoo hing (LOESS) eg ession (span = 0.2)
o analyze he endency o ipep ides en iched in RBPs o lo-
calize in in insically diso de ed egions.
To in es iga e he e olu iona y conse a ion o he RNA-
binding po en ial o Nos oc p o eins, homologs we e p e-
dic ed o he en i e Nos oc genome using ‘Genome Gene Bes
Homologs’ om he IGM / MER Po al o he IMG da abase
( 41 ), 55 cyanobac e ial genomes (see Supplemen a y Table
S4 ), a minimum pe cen age o iden i y o 40% and an e- alue
o 0.05. A p edic ion o RBPs o all homologs was ca ied
ou using he pa ame e s speci ied abo e.
Cons uc ion o mu an and epo e s ains
To gene a e a pa R s ain, wo o e lapping agmen s we e
ampli ied by PCR using genomic DNA wi h oligonucleo ides
669 and 670 and oligonucleo ides 671 and 672, espec i ely
(see Supplemen a y Table S2 o oligonucleo ides). The esul -
ing p oduc s we e hen used as empla es o a hi d PCR wi h
oligonucleo ides 669 and 672 esul ing in dele ion o he se-
quences co esponding o pa R and he gene a ion o a unique
XhoI si e be ween he wo ampli ied agmen s. The agmen
was diges ed wi h BamHI a he si es p o ided by oligonu-
cleo ides 669 and 672, and cloned in o BamHI-diges ed sacB -
con aining Sm
R
Sp
R ec o pCSRO ( 42 ), yielding pMBA62
(see Supplemen a y Table S5 o plasmids). An Nm
R
casse e
was excised om pRL278 ( 43 ) as a SalI-XhoI agmen and
cloned in o he Xho si e o pMBA62, yielding pMBA66, which
was ans e ed in o Nos oc by conjuga ion ( 44 ). Genomic
DNA was ex ac ed om clones exhibi ing suc ose esis ance
and sensi i i y o neomycin. Seg ega ion o he clones was an-
alyzed by PCR using genomic DNA and oligonucleo ides 738
and 739. Clones wi hou he pa R gene and no pa R wild- ype
ch omosomal copies we e named pa R .
The plasmid pMBA79 was cons uc ed o exp ess pa R
om he npB p omo e . A PCR agmen was ampli ied using
oligonucleo ides 738 and 739, diges ed wi h NsiI and XhoI
and cloned in NsiI and XhoI-diges ed pMBA20 ( 45 ). pMBA79
o wo epo e plasmids [pSAM301 ( 46 ) and pSAM270 ( 5 )]
we e ans e ed in o pa R s ain by conjuga ion wi h selec-
ion o Sm
R
Sp
R
cells.
Two plasmids o he exp ession o p o eins used wi h
3x-FLAG we e designed (see he Supplemen a y Table S5
o he plasmid desc ip ions). F agmen s con aining sg p
used o he 3xFLAG sequence we e ampli ied by PCR us-
ing as empla e pMBA96 ( 8 ) and oligonucleo ides 32 and
34 (3xFLAG N- e minal usion) o 33 and 35 (3xFLAG
C- e minal usion). The esul ing p oduc s we e NsiI / SacI-
diges ed and cloned in o NsiI / SacI-diges ed pMBA20 ( 45 ),
yielding pMBA131 and pMBA132, espec i ely. The npB
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Nucleic Acids Resea ch , 2025, Vol. 53, No. 3 5
p omo e and s GFP o pMBA132 we e exchanged by up-
s eam sequences plus he coding sequences o he candi-
da e RBPs. DNA agmen s we e ampli ied by PCR using
as empla e genomic DNA and oligonucleo ides 129 and
130, 136 and 137, 148 and 149, 151 and 152, 156 and
157, 166 and 167, 194 and 195 o P03 and P04, esul -
ing in he ampli ica ion o ups eam sequences plus he cod-
ing sequences o al 2809 , ipp1 , pa U3 , p pA , pB , npA ,
al 2890 and asl3888 , espec i ely. F agmen s we e Ps I / XhoI-
diges ed and cloned in o Ps I / XhoI-diges ed pMBA132, yield-
ing pMBA166, pMBA170, pMBA177, pMBA179, pMBA182,
pMBA188, pMBA211 and pMB2, espec i ely. All plasmids
con ained he ups eam sequences plus he coding sequences
o he selec ed genes used o he 3xFLAG sequence and we e
designed o he in eg a ion in o he Nos oc alpha plasmid.
DNA agmen s we e ampli ied by PCR using as empla e
pMBA166, pMBA170, pMBA177, pMBA179, pMBA182,
pMBA188 o pMBA211, o wa d oligonucleo ides 131, 138,
150, 153, 158, 168, 196 and 19 as e e se oligonucleo ide,
espec i ely. These agmen s we e diges ed wi h SacI and
cloned in o SacI-diges ed pCSV3 ( 47 ), yielding pMBA167,
pMBA171, pMBA178, pMBA180, pMBA183, pMBA189 and
pMBA213, espec i ely. These plasmids con ain he ups eam
sequences plus he coding sequences o al 2809 , ipp1 , pa U3 ,
p pA , pB , npA and al 2890 used o 3xFLAG sequence
and allowed he inse ion o he agmen s a he na i e ch o-
mosomal loci exp essing he usion p o eins om hei na-
i e p omo e s. Plasmids pMBA167, pMBA171, pMBA178,
pMBA180, pMBA183, pMBA189, pMBA213 and pMB2
we e ans e ed in o Nos oc by conjuga ion wi h selec ion
o Sm
R
Sp
R
cells.
An N- e minal usion o Pa R o he 3xFLAG epi ope was
gene a ed. A agmen con aining he p omo e and 5

-UTR
o pa R was ampli ied by PCR using genomic DNA as em-
pla e and oligonucleo ides 57 and 58. The esul ing p oduc
was diges ed wi h Ps I and NsiI and cloned in o Ps I / NsiI-
diges ed pMBA131, yielding pMBA140. A agmen con ain-
ing he coding egion o pa R was ampli ied by PCR using
as empla e genomic DNA and oligonucleo ides 53 and 54.
The p oduc was diges ed wi h XhoI and SacI and cloned in o
XhoI- / SacI-diges ed pMBA140, yielding pMBA149, which
was ans e ed in o pa R by conjuga ion wi h selec ion o
Sm
R
Sp
R
cells.
In o de o gene a e a pa R p omo e epo e used wi h
g pmu 2 , a agmen con aining he p omo e egion o pa R
(posi ions 2038795 o 2039043 ) was ampli ied by PCR us-
ing genomic DNA as empla e and oligonucleo ides 978 and
979. The esul ing agmen was diges ed wi h ClaI / XhoI
and cloned in o ClaI / XhoI-diges ed pSAM270 (5), yielding
pMBA117, which was ans e ed in o Nos oc by conjuga ion
wi h selec ion o Sm
R
Sp
R
cells.
A ma ke less He R -3xFLA G s ain was gene a ed. A ag-
men con aining he p omo e , 5

-UTR and coding egion
o he R was ampli ied by PCR using as empla e genomic
DNA and oligonucleo ides 49 and 70. The esul ing agmen
was Ps I / XhoI-diges ed and cloned in o Ps I / XhoI-diges ed
pMBA132, yielding pMBA154. A agmen con aining he se-
quences downs eam o he R was also ampli ied by PCR us-
ing genomic DNA as empla e and oligonucleo ides 50 and
51. The agmen was diges ed wi h SacI a he si es p o-
ided by he oligonucleo ides and cloned in o SacI-diges ed
pMBA154, yielding pMBA157 (only clones wi h he co ec
o ien a ion o he downs eam egion we e kep ). A agmen
con aining he ull cons uc (p omo e , 5

-UTR, he R coding
egion, 3xFLAG, and downs eam egion) was inally ampli-
ied by PCR using pMBA157 as empla e and oligonucleo ides
47 and 52. The esul ing agmen was diges ed wi h BamHI
a he si e p o ided by he oligonucleo ides and cloned in o
BamHI-diges ed pCSRO ( 47 ), yielding pMBA160, which was
la e ans e ed in o he he R mu an CSSC2 ( 48 ) by conju-
ga ion. Sm
S
Sp
S
clones exhibi ing esis ance o suc ose and able
o g ow again diazo ophically we e selec ed and sequenced
o con i m he p esence o a he R copy used o he 3xFLAG
inse ed a he he R locus.
PNK assays
The PNK assays we e ca ied ou as p e iously desc ibed ( 49 )
wi h some modi ica ions. A o al o 400 ml o cul u es om e-
po e s ains exp essing 3xFLAG p o ein usions we e g own
in BG11 supplemen ed wi h he app op ia e an ibio ics un-
il hey eached a concen a ion o 2.5 μg chlo ophyll / ml.
He e ocys di e en ia ion was induced as desc ibed abo e.
A e 24 h o cul i a ion, hal o each cul u e (200 ml) was
UV- ea ed (1.6 J / cm
2
) while gen ly shaking o induce RNA–
p o ein c osslinking keeping he sample on ice ( he emaining
hal was also placed on ice). Cells we e pelle ed by cen i u-
ga ion a 3270 ×g o 5 min. The pelle s we e esuspended
in NT-P bu e (50 mM NaH
2
PO
4
, 300 mM NaCl, 0.05%
Tween, pH 8.0) supplemen ed wi h p o ease Inhibi o (cOm-
ple e EDTA- ee, Roche). Cells we e lysed as desc ibed o he
R-DeeP expe imen . Glass beads we e emo ed by cen i u-
ga ion a 1500 ×g o 2 min. Nex , memb anes we e pel-
le ed by cen i uga ion a 21 000 ×g o 30 min a 4
◦C. A
o al o 2 ml o cla i ied lysa e pe sample (c osslinked and
non-c osslinked) was incuba ed wi h 80 μl o an i-FLAG M2
magne ic beads (Sigma) p ewashed i e imes in NT-P bu e .
F om his poin on, all he washing s eps we e pe o med wi h
2 ml o bu e . The samples we e incuba ed a 4
◦C while gen-
ly o a ing o 1 h. Beads we e washed wice wi h high-sal
NT-P bu e (50 mM NaH
2
PO
4
, 1 M NaCl, 0.05% Tween20,
pH 8.0), once wi h o cold NT-P bu e and once wi h Ben-
zonase bu e (50 mM T is-HCl, 1 mM MgCl
2
, pH 8). The
beads we e esuspended in 100 μl benzonase bu e supple-
men ed wi h p o ease inhibi o and 25 U o Benzonase nucle-
ase (Sigma). The samples we e incuba ed a 37
◦C o 10 min,
while shaking a 900 pm. A e his, he beads we e washed
once wi h high-sal NT-P bu e and wice wi h Fas AP bu e
(10 mM T is-HCl, 5 mM MgCl
2
, 100 mM KCl, pH 8). The
beads we e esuspended in 150 μl o Fas AP bu e supple-
men ed wi h p o ease inhibi o and 2 U o Fas Alkaline Phos-
pha ase (The mo). Samples we e incuba ed a 37
◦C o 30
min, shaking a 900 pm. The beads we e washed once wi h
high-sal NT-P bu e and wice wi h PNK bu e (50 mM T is-
HCl, 10 mM MgCl
2
, 0.1 mM spe midine, pH 8). The mag-
ne ic beads we e esuspended in 100 μl o PNK bu e supple-
men ed wi h p o ease inhibi o , 10 μCi o [ γ-
32
P]-ATP and 1
μl o PNK (The mo). Samples we e incuba ed a 37
◦C o 30
min, while shaking a 900 pm. The beads we e washed once
wi h high-sal NT-P bu e and wice wi h NT-P bu e . Fo
he elu ion o he p o ein–RNA complexes, he beads we e e-
suspended in 35 μl o T is-bu e ed saline (TBS) (50 mM T is-
HCl, 150 mM NaCl, pH 7.5) supplemen ed wi h 0.5 μg / μl
FLAG pep ide (Sigma) and incuba ed a 4
◦C o 15 min, while
gen ly shaking a 900 pm. A e sepa a ion o beads om he
supe na an using a magne ic ack, p o ein loading dye was
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6 Nucleic Acids Resea ch , 2025, Vol. 53, No. 3
added o supe na an (5 ×concen a ed loading dye: 25 mM
T is-HCl pH 6.8, 25% glyce ol, 10% SDS, 50 mM di hio h e-
i ol and 0.05% b omophenol blue). A e 5 min o dena u a-
ion a 95
◦C, he samples we e sepa a ed on 15% SDS-PAA
gels and elec oblo ed on o ni ocellulose memb anes (Ame -
sham). Radioac i e ink was added o he co ne s o he mem-
b ane o acili a e he o e lay o he size ma ke s. Radioac i e
signals we e de ec ed using a Typhoon FLA 9500 (GE Heal h-
ca e). A e de ec ion o he adioac i e signal, he memb anes
we e blocked in 5% skim milk in T-PBS o 1 h and subse-
quen ly used o wes e n blo ing using an i-FLAG an ibodies
as desc ibed abo e.
CLIPseq expe imen
Th ee biological eplica es o wild ype (WT) and 3xFLAG-
Pa R s ain we e cul i a ed in 200 ml o BG11 supplemen ed
wi h he app op ia e an ibio ics o a concen a ion o 2.5 μg
chlo ophyll / ml. He e ocys di e en ia ion was induced as de-
sc ibed abo e. A e 24 h o cul i a ion, cells we e i adia ed
wi h UV ligh (1.6 J / cm
2
) o c osslink he p o eins o hei
in e ac ing RNA. The pu i ica ion o he RNA–p o ein com-
plexes was pe o med as explained abo e o he PNK assay
wi h some modi ica ion. No Ribolock RNase Inhibi o was
used du ing he lysis s eps o allow he endogenous RNases
o pe o m a mild diges ion o he RNAs no p o ec ed by he
RBP. A e incuba ion o he clea lysa es wi h 100 μl o an i-
FLAG M2 magne ic beads (Sigma), he beads we e washed
h ee imes wi h high sal NT-P bu e and wice wi h cold
NT-P bu e . To alida e he p og ess, 20 μl o beads we e an-
alyzed ollowing he PNK assay p o ocol. The o he 80 μl o
beads we e esuspended in 50 μl o TBS bu e supplemen ed
wi h 0.5 μg / μl FLAG pep ide (Sigma) and 2 μl o Ribolock
RNase Inhibi o o elu ion o he RNA–p o ein complexes.
Beads we e incuba ed a 4
◦C o 15 min, while shaking a 900
pm. P o eins we e diges ed in 400 μl p o einase K bu e (50
mM T is-HCl, pH 6, 5 mM EDTA and 0.5% SDS) wi h 400
μg o p o einase K o 30 min a 37
◦C. Ano he 400 μl o
p o einase K bu e + 9 M u ea was added o an addi ional
incuba ion o 30 min a 37
◦C. RNA agmen s we e isola ed
using phenol-chlo o o m and chlo o o m ex ac ions. RNA
was la e ea ed wi h 2 U o TURBO DNase (Li e Technolo-
gies) o elimina e DNA aces, ollowed by RNA cleanup using
RNA Clean & Concen a o columns (Zymo Resea ch).
Syn hesis o complemen a y DNA (cDNA) was acili a ed
by SMARTSc ibe e e se ansc ip ase (TaKaRa), which adds
he loop adap e a he 3

-end in he i s -s and syn hesis
s ep and he SMART oligo a he 5

-end in he subsequen
empla e swi ching and ex ension s ep. The loop adap e
( Supplemen a y Table S2 ) was p epa ed as desc ibed ( 50 ), and
annealing o he loop adap e o RNA was achie ed by incu-
ba ing bo h a 72
◦C o 3 min and 42
◦C o 2 min. All compo-
nen s we e added o his mix u e, including he SMART oligo
( Supplemen a y Table S2 ), 100 U SMARTSc ibe e e se an-
sc ip ase (TaKaRa) and 40 U o Ribolock RNase inhibi o ,
and he eac ion was incuba ed a 42
◦C o 90 min and 70
◦C
o 10 min, ollowed by cooling o 12
◦C. A e cDNA syn-
hesis, he samples we e excised om 3% Nusie e aga ose
gels and pu i ied wi h he NucleoSpin Gel and PCR Clean-up
Ki (Mache ey-Nagel), using NTC bu e o solubilize he gel
slices. The cDNA was ampli ied in 22 PCR cycles wi h Phu-
sion high- ideli y DNA polyme ase (New England BioLabs) as
desc ibed in ( 50 ) using Illumina 8-n index p ime s o bo h
sides, and PCR samples we e pu i ied using he Agencou
AMPu e XP sys em (Beckman Coul e ). All indi idual ampli-
ied cDNA lib a ies we e pooled in equimola a ios and size
selec ed be ween 100 and 320 bp using he Pippin P ep sys em
(Sage Sys em) and subsequen ly pu i ied using he Agencou
AMPu e XP sys em (Beckman Coul e ). The quali y o RNA
and DNA was analyzed on a F agmen Analyze pa allel cap-
illa y elec opho esis sys em (Agilen ). Sequencing o lib a ies
was pe o med on a Nex Seq 2000 sequence wi h a 100 bp
single-end ead mode a he Co e Uni Sys ems Medicine o
he Uni e si y o Wü zbu g.
Quali y con ol analysis o he eads was ca ied ou us-
ing FASTQC ( h ps://www.bioin o ma ics.bab aham.ac.uk/
p ojec s/ as qc/ ). Adap e p ime s we e emo ed using
cu adap ( h ps:// cu adap . ead hedocs.io/ en/ s able/ ). Reads
we e mapped o he Nos oc genome using bow ie2 in mode
‘ e y-sensi i e-local’ ( 51 ). Bam iles con aining only aligned
eads we e used o peak calling using PEAKachu ( h ps:
// gi hub.com/ bischle / PEAKachu ). Only peaks wi h a log
2
old change > 2.3 and d < 0.0001 we e used o down-
s eam analysis. Fo isualiza ion pu poses, he numbe o
eads aligning o a posi ion o he genome was no malized
using he numbe o mapped eads o each lib a y. Bedg aph
iles we e gene a ed by BamCo e age ( 52 ).
G ow h assay and ime-cou se exp ession o
3xFLAG-Pa R
Cells om liquid Nos oc cul u es, pa R and pa R+ we e
g own in BG11 medium unde s anda d condi ions as ex-
plained abo e. A e 5 days, hey we e collec ed and esus-
pended in BG11
0 a OD
750
= 0.1. Fi e- old se ial dilu ions
we e p epa ed and 10 μl o each dilu ion was pla ed on
BG11
0 pla es con aining ammonium (2.5 mM NH
4
+
and 12
mM N- is (hyd oxyme hyl) me hyl-2-aminoe hanesul onic
acid-NaOH bu e (pH 7.5), ni a e (17 mM NO
3
−) o lack-
ing combined ni ogen (N
2
). G ow h was analyzed a e 13
days o incuba ion a 30
◦C. To analyze he e ocys di e en ia-
ion by op ical mic oscopy, liquid cul u es o Nos oc , pa R
and pa R + g owing pho oau o ophically in BG11 unde
s anda d condi ions we e ha es ed, washed and esuspended
in media wi hou combined ni ogen sou ce (BG11
0
) as de-
sc ibed abo e. A e 24 h o cul i a ion, samples o pa R
we e aken and he e ocys s we e s ained wi h alcian blue as
p e iously desc ibed ( 53 ). A e 7 days o cul i a ion, isual-
iza ion o he e ocys s was no possible in pa R because his
s ain did no g ow diazo ophically.
Liquid cul u es o he s ain 3xFLAG-Pa R we e g own in
BG11 unde s anda d condi ions, cells we e ha es ed by cen-
i uga ion, and he e ocys di e en ia ion was induced as ex-
plained abo e. Samples we e aken a di e en ime poin s a -
e combined ni ogen emo al. Cells we e mechanically lysed
as explained abo e and 30 μg o o al soluble p o ein was sep-
a a ed on a 15% PAA-SDS gel and ans e ed o a ni ocellu-
lose memb ane. The memb ane was s ained wi h Ponceau Red
[0.1% ( w / ) in 5% ace ic acid] as loading con ol, blocked
in 5% skim milk in T-PBS o 1 h and subsequen ly used o
wes e n blo ing using an i-FLAG an ise a as desc ibed abo e.
Mic oscopy and image analysis
Filamen s s ained wi h alcian blue we e isualized using an
Olympus BX60 mic oscope. Fluo escence o Nos oc ilamen s
ca ying plasmid pMBA118 g owing o 5 days on op o so-
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Nucleic Acids Resea ch , 2025, Vol. 53, No. 3 7
lidi ied BG11 o BG11
0
(ni ogen- ee) medium supplemen ed
wi h app op ia e an ibio ics was analyzed by a Nikon CFI
Plan apoch oma VC 60 ×1.4 oil imme sion objec i e and
DIC N2 condense a ached o a Nikon Eclipse T1 con ocal
mic oscope. The samples we e exci ed a 488 nm and he luo-
escen emission was il e ed by a HQ il e se (g een luo es-
cen p o ein (GFP), 500–550 nm window) and a Cy-5 il e
se (pho osyn he ic pigmen au o luo escence, 663–738 nm
window). Fo quan i ica ion pu poses, all images we e aken
wi h he same se ings. Fluo escence o WT and Δpa R il-
amen s ca ying plasmids pSAM301 o pSAM270 g owing
o 5 days on op o solidi ied BG11 o BG11
0 (ni ogen-
ee) medium we e analyzed using a Leica HCX PLAN-APO
63 ×1.4 NA oil imme sion objec i e a ached o a Leica TCS
SP2 lase -scanning con ocal mic oscope. Samples we e exci ed
a 488 nm by an a gon-ion lase and he luo escen emis-
sion was moni o ed by collec ion ac oss windows o 500–538
nm (GFP) and 630–700 nm (pho osyn he ic pigmen au o lu-
o escence). Fo quan i ica ion pu poses, he WT and Δpa R
s ains we e g own in he sample pla e and images we e aken
wi h he same se ings. Images we e analyzed wi h Fiji ( 54 ).
Phylogene ic analysis o Pa R and conse a ion o
pa R p omo e sequences
A BLASTP sea ch was pe o med using de aul pa ame e s
and he Nos oc Pa R p o ein sequence (WP044521042.1) as a
que y. Because 1658 homologs we e ound, we decided o use
only 107 homologs om genomes associa ed wi h e e ence
cyanobac e ia ( 55 ) o he downs eam analysis. A mul iple
alignmen was pe o med using Clus alW. The bes dis ance
model was selec ed using MEGAX ( 56 ). The e olu iona y his-
o y was in e ed by using he Maximum Likelihood me hod
and Jones e al . w / eq. model ( 57 ). A o al o 1000 i e a ions
o boo s apping we e ca ied ou o in e he con idence o
he b anches. Ini ial ee(s) o he heu is ic sea ch we e ob-
ained au oma ically by applying Neighbo -Joining and BioNJ
algo i hms o a ma ix o pai wise dis ances es ima ed using
he JTT model, and hen selec ing he opology wi h he highe
log likelihood alue. A disc e e Gamma dis ibu ion was used
o model e olu iona y a e di e ences be ween si es ( i e ca -
ego ies; +G, pa ame e = 1.1415). The a e a ia ion model
allowed o some si es o be e olu iona ily in a iable ([+I],
1.40% si es).
S uc u al analysis
Alpha old p edic ion o Pa R (AF-Q8YWB5-F1), Sl 1122
(AF-P72645-F1) and PMM0486 (AF-Q7V2J0-F1) we e
downloaded om he AlphaFold P o ein S uc u e Da abase.
Sequences o he conse ed domains o Pa R ( esidues 70–
158 and esidues 198–279, espec i ely) we e olded by he
ESMFold me hod and compa ed wi h he s uc u e o o he
p o eins h ough he Foldseek webse e ( 58 ). The isualiza-
ion o he Pa R s uc u e, he calcula ion o he Coulombic
elec os a ic po en ial (ESP) and he supe imposi ion o s uc-
u al homologs we e pe o med by Chime aX ( 59 ).
Resul s
Nos oc R-DeeP o ac iona e mul ime ic complexes
o a memb ane- ich cyanobac e ium
T iplica e cul u es o Nos oc we e g own o 26 h in medium
wi hou a combined ni ogen sou ce o ob ain ilamen s con-
aining ege a i e cells, p o-he e ocys s, and ma u e he e o-
cys s o R-DeeP / G adR analysis ( 24 ,26 ). In his echnique,
all mac omolecula complexes in he cell a e ac iona ed in
a densi y g adien ollowed by high- esolu ion p o eomics o
19 ac ions aken om each g adien . G adien s o RNase-
ea ed samples we e compa ed agains hose o un ea ed
samples in iplica es. I a p o ein was pa o an RNA–p o ein
complex, he complex should be disassembled a e RNase
ea men and a shi o he p o ein posi ion in he g adien
should be obse ed (RNA-associa ed p o eins) (Figu e 1 A).
A c i ical s ep in he success o he app oach is he p ese -
a ion o he na i e p o ein–p o ein and RNA–p o ein com-
plexes. Since cyanobac e ia, as pho osyn he ic o ganisms, a e
ich in pigmen s, he ac iona ion o hei mac omolecula
complexes esul ed in colo ul g adien s e lec ing he abun-
dance o la ge pho osyn he ic complexes (Figu e 1 B). We used
10–40% suc ose g adien s, which ha e a esolu ion be ween
0 and 900 KDa. I he Nos oc p o eome was comple ely disas-
sembled in o monome ic p o eins, no p o eins should be de-
ec ed in ac ions beyond ac ion 5 ( Supplemen a y Figu e
S1 A). Howe e , p o eins we e measu ed in highe ac ions
( Supplemen a y Figu e S1 B) and SDS-PAA gels s ained wi h
Coomassie blue showed highly exp essed p o eins, such as Ru-
BisCO o glu amine syn he ase, appea ing in highe ac ions,
consis en wi h hei associa ion in o mul isubuni complexes.
Fu he mo e, he o e all dis ibu ion o p o eins did no ap-
pea o be a ec ed by RNase ea men , excluding unspeci ic
e ec s o ea men ( Supplemen a y Figu e S1 C and D). In o -
de o es he in eg i y o he RNA in he un ea ed samples,
RNA was ex ac ed om he di e en ac ions and sepa a ed
in dena u ing u ea-PAA gels. Se e al in ac highly exp essed
RNAs we e di ec ly iden i ied (Figu e 1 C). We analyzed by
no he n blo hyb idiza ion he dis ibu ion o six p e iously
known non-coding RNAs; 5S ibosomal RNA ( RNA), RnpB,
4.5S, Y 1 ( 7 ), NsiR8 ( 6 ) and NsiR1 ( 8 ). The posi ion o an
RNA in he g adien s did no depend on i s size, bu a he on
i s in e ac ion wi h la ge p o ein complexes. This ac can be
obse ed in he di e en dis ibu ions o he app oxima ely
equal-sized RNAs 4.5S (signal ecogni ion pa icle RNA) and
5S RNA (Figu e 1 D). The de ec ion o NsiR1 and NsiR8, wo
sRNAs exp essed only in he e ocys s, con i med he capac-
i y o de ec ing pu a i e he e ocys -speci ic ibonucleop o ein
complexes. This was a ele an aspec ega ding he sensi i i y
o he analysis, because a e he e ocys di e en ia ion, he e is
on a e age only one he e ocys pe 15 ege a i e cells. Finally,
as a posi i e con ol, he change in he posi ion o he iboso-
mal RBP RplA a e RNase ea men was es ed by wes e n
blo (Figu e 1 E). As a nega i e con ol, we gene a ed a s ain
exp essing he mas e ansc ip ion ac o o he e ocys di -
e en ia ion He R ( 60 ) used o a 3xFLAG epi ope ag unde
con ol o i s na i e p omo e . We p epa ed g adien s wi h his
s ain ollowing he same p ocedu e as o he WT. The ab-
sence o a shi o He R -3xFLA G con i med he speci ici y o
ou app oach (Figu e 1 E).
Tandem mass ags ( 61 ) we e used o he pa allel de ec ion
and ela i e quan i ica ion o p o eins by MS. We pooled ac-
ions 1 and 2; hus, 18 samples we e measu ed pe g adien .
These measu emen s iden i ied 2638 p o eins in a leas one
pai o con ol-RNase- ea ed g adien s, which ep esen 44%
o he Nos oc p o eome. Ou o 139 p o eins p e iously de-
ined as being exp essed exclusi ely in he e ocys s ( 5 ), 40 we e
de ec ed in his s udy (29%).The o e all in-g adien dis ibu-
ion o p o ein p o iles showed co ela ion sco es o 0.92–
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8 Nucleic Acids Resea ch , 2025, Vol. 53, No. 3
2
CA
B
D
G adien
Con ol
+ RNases
MS
No malized
p o ein
amoun
Con ol
+ RNases
F ac ions
26h N
Size (n )
RplA snoi ca Fsnoi ca F
F ac ions
F ac ions
123456 78910111213141516171819
700
600
500
400
300
200
100
RnpB
4.5S
RNAs
5S
1234 56 78 910111213141516171819
5S
RnpB
4.5S
Y 1
NsiR8
NsiR1
He R-3xFLAG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
40
35
25
40
35
25
Con ol
+ RNases
40
35
40
35
EKDa KDa
non-RBP RBP RBP
F ac ions
Figu e 1. Nos oc R-DeeP expe imen . (A) O e iew: Lysa es om he e ocys -con aining ilamen s a e ac iona ed in suc ose densi y g adien s ollo w ed
by MS p o eomics. G adien s om RNase- ea ed and un ea ed samples a e compa ed. An RNA–p o ein complex should dissocia e a e RNase
ea men and a shi in he posi ion o he espec i e p o eins in he g adien should be obse ed. (B) G adien ubes a e cen i uga ion and ac ions
a e elu ion in collec ion ubes. A ep esen a i e pai o g adien s is shown. The di e en colo s co espond o abundan pigmen -p o ein complexes,
con aining o ange ca o enoids, blue ph y cobilins o g een chlo ophylls. (C) Sepa a ion o RNA om 19 ac ions on a 10% u ea-PAA gel. RNA was
ex ac ed om 300 μl o each ac ion o one un ea ed g adien ou o h ee. All ex ac ed RNA was loaded on o he gel. The posi ion o abundan RNA
species is indica ed o o ien a ion. The posi ion o size ma ke s is indica ed on he le . (D) Dis ibu ion o selec ed non-coding RNAs analyzed by
no he n blo . RNA was ex ac ed om 300 μl o each ac ion o a ep esen a i e un ea ed g adien , sepa a ed on a 10% u ea-PAA gel and a e
blo ing h yb idiz ed wi h adiolabeled p obes o he indica ed non-coding RNAs. (E) Dis ibu ion o RplA and He R-3xFLAG along un ea ed and ea ed
ep esen a i e g adien s. Wes e n blo analyses we e pe o med using 35 μl o each ac ion sepa a ed on a 15% SDS-PAA gel and an ise a agains RplA
o he FLAG epi ope. The posi ion o size ma ke s (in kilodal ons) is indica ed on he igh . The esul s om one o h ee g adien pai s a e displayed.
0.94 be ween he eplica es, indica ing good ep oducibili y
( Supplemen a y Figu e S2 ). To compa e he dis ibu ion o
p o eins wi h di e en exp ession le els, an in-g adien no -
maliza ion was pe o med, so ha he sum o each p o ein
along a g adien was se o 100%. The o e all quali y o he
app oach can be obse ed in he co-sedimen a ion o p o eins
belonging o essen ial ibonucleop o ein complexes such as
50S and 30S ibosomal subuni s, RNA polyme ase o one o
he Nos oc CRISPR sys ems ( 62 ) (Figu e 2 A–D). In addi ion
o he housekeeping sigma ac o SigA, h ee al e na i e sigma
ac o s SigE, SigD and SigI we e de ec ed (Figu e 2 D). SigA
showed a clea co-sedimen a ion pa e n wi h he RNA poly-
me ase co e while he al e na i e sigma ac o s did no . Only
a small subpopula ion o he al e na i e sigma ac o s may in-
e ac wi h he RNA polyme ase co e. In he case o SigE, a
he e ocys -speci ic ac o ( 63 ), only 2% o he o e all p o ein
amoun was de ec ed in each o he ac ions we e he RNA
polyme ase co e co-sedimen ed ( Supplemen a y Da a S2 ).
To solubilize memb ane p o eins, we used n-dodecyl β-D-
mal oside, a mild de e gen commonly used o s udy pho o-
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Nucleic Acids Resea ch , 2025, Vol. 53, No. 3 9
RplA (Al 5301)
RplB (All4212)
RplC (All4215)
RplD (All4214)
RplE (All4203)
RplF (All4201)
RplI (All0579)
RplJ (Al 5302)
RplK (Al 5300)
RplL (Al 5303)
RplM (All4188)
RplN (All4205)
RplO (All4198)
RplQ (All4190)
RplR (All4200)
RplS (Al 5297)
RplT (Al 3428)
RplV (All4210)
RplW (All4213)
RplX (Asl4204)
RplY (Al 4785)
RpmA (Asl0146)
RpmB (Asl2630)
RpmC (Asl4207)
RpmG (Asl4452)
RpmI (As 3427)
RpmJ (Asl4194)
A
080
% P o ein
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
Rps1_1 (Al 1078)
Rps1_2 (All0136)
RpsB (All4792)
RpsC (All4209)
RpsD (Al 2737)
RpsE (All4199)
RpsF (All4802)
RpsG (All4339)
RpsH (All4202)
RpsI (All4187)
RpsJ (All4336)
RpsK (All4192)
RpsL (All4340)
RpsM (All4193)
RpsN (All3969)
RpsO (Asl0749)
RpsP (As 1953)
RpsQ (Asl4206)
RpsR (Asl4451)
RpsS (Asl4211)
RpsT (As 1592)
RpsU1 (As 0742)
B
040
% P o ein
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
C
030
% P o ein
RpoA (All4191)
RpoB (Al 1594)
RpoC1 (Al 1595)
RpoC2 (Al 1596)
RpoZ (As 4648)
SigA (All5263)
SigD (Al 3810)
SigE (Al 4249)
SigI (All2193)
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
D
030
% P o ein
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
All1472
All1474
Csx7 (All1475)
Csx10 (All1477)
Cas10 (All1479)
PecA (Al 0524)
PecB (Al 0523)
PecC (Al 0525)
ApcA (Al 0021)
ApcB (Al 0022)
ApcC (As 0023)
ApcF (All2327)
ApcE (Al 0020)
CpcA (Al 0529)
CpcB (Al 0528)
CpcC (Al 0530)
CpcD (As 0531)
CpcG2 (Al 0535)
CpcL (Al 0536)
CpcG1 (Al 0534)
CpcG4 (Al 0537)
ApcD (All3653)
PsbA1 (Al 4866)
PsbA3 (Al 4592)
PsbB (All0138)
PsbC (Al 4291)
PsbD (Al 4290)
PsbE (As 3845)
PsbH (Asl0846)
PsbL (As 3847)
PsbO (All3854)
PsbP (All3076)
PsbQ (All1355)
PsbU (Al 1216)
PsbW (All0801)
PsbY (As 1025)
Psb27(All1258)
Psb29 (All0646)
PsaA (Al 5154)
PsaB (Al 5155)
PsaC (As 3463)
PsaD (All0329)
PsaF (All0109)
PsaJ (Asl0108)
PsaE (Asl4319)
PsaK2 (As 5289)
PsaM (As 4657)
PsaL1 (All0107)
E
050
% P o ein
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
NdhA (Al 0023)
NdhB (All4883)
NdhD (Al 3957)
NdhF (Al 3956)
NdhG (Al 0225)
NdhH (Al 3335)
NdhI (Al 0024)
NdhJ (All3840)
NdhK (All3841)
NdhM (All1732)
NdhN (Al 4216)
NdhO (As 4321)
NdhQ
NdhS (As 0654)
NdhV (Al 3297)
H
030
% P o ein
K
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
CoxA1 (Al 0951)
CoxB1 (Al 0950)
CoxA2 (Al 2515)
030
% P o ein
SdhA (All2970)
SdhB (All0945)
SdhC (All3341)
I
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
030
% P o ein
J
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
Pe A (All2452)
Pe B (Al 3421)
Pe C (All2453)
025
% P o ein
L
040
% P o ein
A pA (All0005)
A pB (All5039)
A pC (All0004)
A pE (All5038)
A pD (All0006)
A pF (All0007)
A pG (All0008)
A pI (All0010)
A pH (Asl0009)
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
035
% P o ein
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
U eB (Al 3668)
U eC (Al 3670)
G
F
025
% P o ein
Ni H (All1455)
Ni D (All1454)
Ni K (All1440)
19
18
17
16
15
14
13
12
10
11
9
8
7
6
5
4
3
1_2
F ac ions
Figu e 2. In-g adien dis ibu ion o p o eins ha a e pa o la ge mac omolecula comple x es in Nos oc . Hea map ep esen a ion o he mean
no malized abundance o each de ec ed p o ein in h ee un ea ed g adien s. The sum o a p o ein abundance along he g adien is no malized o 100%.
P o eins ha a e pa o RNA–p o ein complexes a e shown as ollows: (A) 50S ibosomal subuni , (B) 30S ibosomal subuni , (C) RNA polyme ase, (D)
Sub ype III-D CRISPR complex. P o eins ha belong o mul ip o ein complexes in ol ed in pho osyn hesis, ni ogen me abolism o he espi a o y chain
a e also shown: (E) Pho osyn he ic complexes, (F) Ni ogenase, (G) U ease, (H) NADH dehyd ogenase, (I) Succina e dehyd ogenase, (J) Cy och ome
b
6
, (K) Te minal espi a o y oxidases, (L) ATP syn hase. Componen s o each complex wi h simila dis ibu ions a e amed in ed. In panel (E), a
di e en colo pale e o he ames is used and a pho o o he g adien s is displayed abo e he hea map o show he colo s o he di e en ac ions.
Ph y cobilisome componen s con aining ph y coe y h ocy anin, alloph y cocy anin o ph y cocy anin a e amed in o ange, black and blue, espec i ely.
Co-sedimen a ion o he PSII and PSI co e is also highligh ed in da k g een and ligh g een, espec i ely.
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16 Nucleic Acids Resea ch , 2025, Vol. 53, No. 3
A
D
C
WT
Δpa R
Δpa R+
NH4
+
NO3
-
NO3
-
N2
WT
Δpa R
Δpa R+
WT
Δpa R
Δpa R+
B
Δpa R N2 (24h)
WT
Δpa R
Δpa R+
Δpa R N2
(7 days)
-10 10
ESP
Δpa R + PnsiR1
N2
E
NO3
-
NO3
-N2
TWTWΔpa R
PnsiR1Phe R
Δpa R
Figu e 6. P a R egula es he e ocy s di e en ia ion. (A) G o w h on solid media. Cells om liquid cul u es o he indica ed s ains g o wn in he p esence
o ni a e we e collec ed and esuspended in BG11
0
a an OD
750
= 0.1. Fi e old se ial dilu ions we e p epa ed and 10 μl o each dilu ion pla ed on BG11
0
pla es con aining ammonium (NH
4
+
), ni a e (NO
3
−) o lacking combined ni ogen (N
2
). Pic u es we e aken a e 13 da y s o incuba ion a 30
◦C. (B)
P esence o he e ocys -like cells in pa R . Uppe panel: b igh - ield image o ilamen s o pa R g owing in liquid medium 24 h a e emo al o
combined ni ogen. He e ocys s and he e ocys -like cells a e s ained wi h alcian blue. Bo om panel: G ow h in liquid media o indica ed s ains 7 days
a e combined ni ogen emo al. (C) Con ocal luo escence images o ilamen s om WT and pa R s ains ca ying nsiR1 and he R cell- ype speci ic
p omo e usions o g p. Filamen s we e g own on op o medium con aining ni a e (NO
3
−) o wi hou a combined ni ogen sou ce (N
2
). G een channel
(GFP luo escence) and magen a channel (au o luo escence) a e shown. Highe magni ica ion (D) . Scale ba s, 25 μm. (E) Pa R s uc u e p edic ed by
Alpha old (AF-Q8YWB5-F1). Le panel: Coulombic ESP o Pa R s uc u e calcula ed by Chime aX. Posi i ely cha ged su aces (blue) and nega i ely
cha ged su aces ( ed) a e shown. Righ panel: co e o he Pa R p o ein s uc u e. The wo andem conse ed OB- old-like domains a e highligh ed;
domain 1 in ed ( esidues 70–158) and domain 2 in blue ( esidues 198–279).
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Nucleic Acids Resea ch , 2025, Vol. 53, No. 3 17
A
WT 3xFLAG-Pa R
KDa
70
55
40
35
15
25
10
WB
WT 3xFLAG-Pa R
PNK
B
0
1000
2000
3000
4000
5000
Ax 1500 la
000750006500055
Pa R_1
Pa R_3
Pa R_2
WT1
WT2
WT3
Pa R_1
Pa R_3
Pa R_2
WT1
WT2
WT3
y 1
Genomic posi ion
Genomic posi ion
No malized eads
C
4271500 4272500 4273500
50
40
30
20
10
0
No malized eads
134567891112
he F
Pa R_1
Pa R_3
Pa R_2
WT1
WT2
WT3
Genomic posi ion
2232500 2233500
600
500
400
300
200
100
0
D
No malized eads
all1871
al 1870
E
1368000 1369000
all1164
s
asl1165
250
200
150
100
50
0
No malized eads
Genomic posi ion
Pa R_1
Pa R_3
Pa R_2
WT1
WT2
WT3
201
nsiR1
Figu e 7. RNA a ge ome o Pa R. (A) PNK assay o 3xFLAG-Pa R (posi i e con ol) and WT (nega i e con ol) samples used o CLIP-seq. Wes e n blo
analysis (WB) and au o adiog aphy (PNK) a e shown. Size ma ke s a e indica ed on he le . ( B –E ) Dis ibu ion o eads mapped in ele an egions o
RNA ob ained om h ee biological eplica es o WT (blue) o 3xFLAG-Pa R (g een). The genomic egions o y 1 (B) , nsiR1 a ay (C) , all1871 (D) and
s (E) a e shown. Genes a e ep esen ed by a ows oge he wi h he gene name o ID. Genomic coo dina es deno e he posi ion on he Nos oc
ch omosome. The scale indica es he numbe o mapped eads pe nucleo ide posi ion no malized by he o al numbe o eads mapped in each lib a y.
The indi idual nsiR1 ins ances a e displayed in panel (C) wi h numbe s 1–12.
seem s iking, i has also been obse ed in he G adR / R-DeeP
analyses o Salmonella o human samples ( 24 ,25 ). One possi-
ble explana ion is he p ecipi a ion o a p o ein when i s RNA
coun e pa is no p esen . In he he e ologous pu i ica ion o
some RBPs, such as CRISPR p o eins, i was obse ed ha he
coexp ession o hei a ge RNA was equi ed o s abilize he
p o eins in soluble o m ( 81 ). Howe e , he igh -shi could
also e lec a egula o y mechanism (see he discussion below
abou Vipp1).
The p edic ion o RBPs usually elies on he sea ch o con-
se ed RNA-binding domains. Howe e , he abili y o bind
RNA may ely on mo e basic biochemical p ope ies. As an
example, p e iously known RBPs a e usually en iched in pos-
i i ely cha ged esidues ha may acili a e hei in e ac ions
wi h he nega i ely cha ged RNAs, a endency ha is obse ed
in Figu e 3 B. In o de o conside sub le pa e ns, we used
a modi ied e sion o T iPepSVM ( 38 ), an algo i hm ha is
ained wi h known RBPs o non-RBPs sequences, acco ding
o he da abases, and conside s hei ipep ide con en o
RBP p edic ion. The ad an age o his me hod is he abili y
o p edic new RBPs whose RNA-binding si es could be lo-
ca ed in in insically diso de ed egions. In ac , we obse ed
a sligh endency o he cha ac e is ic RBP ipep ides o be
loca ed in diso de ed egions in Nos oc (Figu e 3 C). This en-
dency was p e iously epo ed o he human p o eome, bu
no o he p o eomes o E. coli and S. yphimu ium ( 38 ). The
impo ance o diso de ed egions in RBPs is a ecen ield o
esea ch ( 82 ) since some RBPs a e assembled in liquid-liquid
phase sepa a ion d ople s ( 83 ), including an RNA helicase in
cyanobac e ia ( 84 ).
The e was only a pa ial o e lap be ween he shi ing p o-
eins and he p edic ion o T iPepSVM (Figu e 3 D), indica -
ing complemen a i y be ween he wo me hods. The shi ing
p o eins no p edic ed as RBP could be pa o RNA–p o ein
complexes bu may no bind RNA hemsel es. This ac could
be suppo ed by he low pI o he p o eins om his g oup
(Figu e 3 B). The p esence o p e iously known RBP wi h no
appa en signi ican shi has also been epo ed ( 24 ). Weak
and ansien RNA–p o ein in e ac ions o he R-DeeP esolu-
ion limi s ( Supplemen a y Figu e S5 ) could be he easons o
hese alse nega i es. Because he use o size exclusion ch o-
ma og aphy in SEC-seq has an imp o ed esolu ion ( 22 ), i
may be a good app oach o u u e esea ch on RBPs appea -
ing in he low-molecula -weigh ac ions.
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18 Nucleic Acids Resea ch , 2025, Vol. 53, No. 3
Some o he p o eins no signi ican ly shi ing bu p edic ed
as RBPs migh be ue RBPs. Two me abolic enzymes ela ed
o ni ogen me abolism, CphA1, cyanophycin syn he ase ( 85 ),
and U eB, an u ease subuni ( 86 ), had a isible sligh shi in
ou g adien s jus below ou s ic cu o , bu in con as had
e y good SVM sco es no only in Nos oc bu in all cyanobac-
e ial p o eins es ed (Figu e 3 E and F). They could be alse
posi i es p edic ed by T iPepSVM, bu a ecen epo ound
bo h p o eins as in e ac ing pa ne s o YlxR, an RBP in Syne-
chocys is ha in e ac s wi h he RNA subuni o RNase P
( 87 ) . The de ec ion o new RNA-binding capaci ies in many
me abolic enzymes has also been epo ed in S. yphimu ium
( 80 ), and may ep esen a di ec link be ween esponses o nu-
ien a ailabili y and pos - ansc ip ional gene egula ion.
All ou p o eins wi h signi ican shi s and high SVM sco es
(Al 2890, Vipp1, P pA and Asl3888) had he abili y o in e -
ac wi h RNA in i o (Figu e 4 C–E), con i ming he speci ici y
o ou combined app oach. Howe e , we also ob ained e y
clea ly labeled bands o Pa U3 and Pa R (Figu e 4 I and J),
wo p o eins wi h low SVM sco es in mos o he cyanobac-
e ial p o eomes. This esul sugges s ha some o he 282
p o eins ha shi ed bu we e no p edic ed o be RBPs (Fig-
u e 3 D) could be ue RBPs. I is impo an o no e ha SVMs
a e ained wi h cu en knowledge and, he e o e, o ally new
domains o in e ac ion pa e ns could be missed.
The newly alida ed RBPs ha e p e iously anno a ed unc-
ions ela ed o essen ial physiological p ocesses such as pho-
osyn hesis and he e ocys di e en ia ion. The Al 2890 ho-
mologs a e s ic ly conse ed in cyanobac e ia and plan
chlo oplas s. Al hough Al 2890 con ains a pu a i e S4 RNA-
binding domain and was he e o e agged as ‘RNA-binding’
in he UNIPROT da abase, Sll1252, i s close homolog in Syne-
chocys is , was ound in PSII p epa a ions, is necessa y o he
p ope ac i i y o PSII ( 69 ) and is in ol ed in he coo dina-
ion o elec on anspo be ween plas oquinone and he cy-
och ome b
6
complex ( 88 ). The au ho s hypo hesized a eg-
ula o y unc ion o Sll1252 ( 69 ), bu he exac mechanism
has emained unknown. He e, we con i m ha he Nos oc o -
holog Al 2890 is an RBP, sugges ing ha i is bi unc ional.
The case o Vipp1 (All2342) is e en mo e s iking, since i
was he only alida ed RBP ha had a shi owa d highe
ac ions a e RNase ea men (Figu e 4 F). Oligome s o
Vipp1 a e essen ial o main aining he in eg i y o he hy-
lakoid memb ane ( 70 ) and Vipp1 was ecognized as a bac e-
ial ances o o he euka yo ic ESCRT-III memb ane emod-
eling sys em ( 89 ). In cyanobac e ia, he p o ein con inuously
mo es be ween a di used ac ion and disc e e oci a he
hylakoid memb anes ( 90 ), which a e mo e abundan a e
high ligh s ess ( 91 ) and consis o Vipp1 oligome s ( 70 ). The
igge ha causes he polyme iza ion o he p o ein is un-
known. Based on ou obse a ion ha Vipp1 mig a ed in ou
g adien s a e RNase ea men o mul iple ac ions co e-
sponding o highe molecula weigh , i is emp ing o specu-
la e ha i is he p esence o an RNA ha seques e s Vipp1
in monome ic o m. A e he emo al o his hypo he ical
RNA (o deg ada ion in ou ea men ), Vipp1 is able o o m
oligome s.
P o eins in ol ed in he e ocys di e en ia ion we e also
alida ed as new RBPs. P pA was i s desc ibed as a phos-
pha ase ha a ec s he e ocys di e en ia ion ( 71 ). He e, we
ha e demons a ed he in i o RNA-binding capaci y o P pA,
which i s he epo ha a closely ela ed homolog in T icho -
mus a iabilis ATCC29413 is pa o an RNA epai sys em
( 92 ). In con as , no link o RNA has been epo ed o Pa U3,
a p o ein exp essed speci ically in he e ocys s ( 93 ) ha is in-
ol ed in he egula ion o he e ocys o ma ion ( 12 ) and cell
di ision ( 12 , 94 , 95 ).
Ou mu a ion o pa R highligh ed he undamen al ole ha
his RBP plays in he he e ocys di e en ia ion p ocess (Fig-
u e 6 ). The absence o pa R caused an al e a ion o he e o-
cys pa e ning and di e en ia ion (Figu e 6 ). pa R ilamen s
had a highe endency o di e en ia e he e ocys s, since e en
g owing in pla es wi h ni a e, a condi ion in which he di -
e en ia ion p ocess should no occu , he s ain had a highe
exp ession o nsiR1 and he R (Figu e 6 C). A e combined
ni ogen emo al, his endency o cell di e en ia ion gene -
a ed an unna u ally high numbe o he e ocys -like cells (Fig-
u e 6 C), some imes con ol o he pa e ning is al e ed and
gene a es double s and iple s o he e ocys -like cells (Fig-
u e 6 D). I is in iguing ha his pheno ype o Mch was e-
po ed o cells lacking Pa U3 ( 93 ), also cha ac e ized as RBP
he e. This highe equency o he e ocys -like cells did no im-
p o e diazo ophic g ow h, bu a he esul ed in an inabili y
o g ow diazo ophically a all. An explana ion o his ob-
se a ion is he inabili y o he he e ocys -like cells o each
he inal s eps o ma u a ion, including he assembly o unc-
ional ni ogenase. This explana ion is suppo ed by he ab-
sence o pola g anules, an indica o o ma u e unc ional
he e ocys s.
The CLIP-seq da a p o ide insigh in o he possible egu-
la o y mechanism o Pa R, al hough he p esence o mul iple
RNA a ge s complica es he in e p e a ion. Pa R seems o be
an impo an hub o he in e ac ion wi h se e al di e en sR-
NAs ( Supplemen a y Da a S10 ). Among hem, Y 1 was he
s a is ically mos en iched sRNA (Figu e 7 B). Y 1 is highly
exp essed in all cells o he ilamen s i espec i e o he ni-
ogen s a us and, in Nos oc, egula es he ansla ion o mR-
NAs encoding enzymes o he syn hesis and emodeling o
he cell wall ( 7 ). In ac , an al e ed exp ession o y 1 gen-
e a es agmen ed ilamen s ha canno suppo he low o
ni ogen compounds be ween ege a i e cells and he e ocys s
( 7 ), a agmen a ion ha is also obse ed in he pa R s ain
a longe ime poin s a e combined ni ogen emo al. The in-
e ac ion be ween Y 1 and Pa R is also suppo ed by he p es-
ence o Y 2 in he Pa R a ge ome ( Supplemen a y Da a S10 ),
a ni ogen- egula ed sRNA ha aps Y 1 and p e en s i s
egula o y e ec s ( 7 ,96 ).
The iden i ica ion o he he e ocys -speci ic sRNA NsiR1
(Figu e 7 C) among he in e ac ing pa ne s o Pa R may ex-
plain he speci ic unc ion o Pa R in he egula ion o he he -
e ocys di e en ia ion p ocess. nsiR1 is speci ically exp essed
in young he e ocys s, which could be cohe en wi h a highe
exp ession o pa R in hese cells (Figu e 5 D) and, gi en ha
his sRNA modula es he e ocys di e en ia ion h ough i s in-
e ac ion wi h impo an nodes o he he e ocys egula o y
ne wo k ( 8 ,11 ), i is emp ing o specula e ha al e ed ex-
p ession o NsiR1 may gene a e a de egula ion o he whole
cell di e en ia ion p ocess. The in e ac ion be ween Y 1 o
NsiR1 and Pa R de ec ed by CLIP-seq is suppo ed by di -
e en expe imen al da a. Fi s , he e is a clea co ela ion in
he dis ibu ion o he RBP (Figu e 4 J) and hese wo sRNAs
(Figu e 1 D) in ou densi y g adien s. Second, he small shi o
Pa R a e RNase ea men (Figu e 4 J) sugges s an in e ac ion
wi h small RNAs. Fu he mo e, hese in e ac ions seem o be
speci ic because o he highly ansc ibed non-coding RNAs
wi h simila dis ibu ions in he g adien s, such as he 4.5S
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Nucleic Acids Resea ch , 2025, Vol. 53, No. 3 19
RNA (Figu e 1 D), we e no en iched in ou CLIP-seq da a
(Figu e 7 E).
The s ong pheno ypic e ec s sugges Pa R as a key eg-
ula o o he e ocys ma u a ion. In e es ingly, he e is also a
pa allel o He R, he ansc ip ional mas e egula o o he e-
ocys di e en ia ion. The he R gene is ansc ibed om ou
disce nible s a si es ( 97 ). Appa en ly, pa R exp ession de-
c eased a e combined ni ogen emo al (Figu e 5 A and B),
bu ou luo escence con ocal analysis o single cells e ealed
a mo e complex pic u e (Figu e 5 C). Exp ession o his gene
appea ed o be ep essed om ea ly s ages o he e ocys di -
e en ia ion only in cells ha we e no unde going he cell di -
e en ia ion p ocess (Figu e 5 D). Al hough we analyzed he
ansc ip ion om he i s ansc ip ional s a si e (TSS) o
pa R (Figu e 5 A), ou mo e ansc ip ional s a si es we e
iden i ied o his gene ( 72 ) ( Supplemen a y Figu e S9 ). The
p esence o mul iple ansc ip ional s a si es, some imes wi h
pa adoxical egula ions ha c ea e complex exp ession pa -
e ns along he ilamen , has also been obse ed o o he
di e en ia ion- ele an genes in Nos oc ( 98 ) . In he case o
pa R ansc ip ional s a si es, he usual pa e n o TSS us-
age in he e ocys s, i.e induc ion in he WT a e ni ogen limi-
a ion bu no in he he R mu an , is no clea ly de ec ed ( 72 ).
The e o e, he educed ansc ip ion in he ege a i e cells
may be achie ed by ep ession o he same TSSs ha a e used
in he he e ocys , bu by a speci ic ep esso ha is exp essed
only in he ege a i e cells a e ni ogen limi a ion.
The wide phylogene ic conse a ion o Pa R sugges s ha
i is also impo an o non-he e ocys ous cyanobac e ia. This
esul is cohe en wi h a mo e gene al unc ion ha may in-
ol e i s in e ac ion wi h Y 1, a uni e sally conse ed sRNA
in cyanobac e ia ( 6 ). The p o ein has a s uc u al a chi ec-
u e ha con ains wo OB- old-like domains combined in an-
dem (Figu e 6 E). A simila a angemen has been desc ibed
o a ssDNA binding p o ein in Deinococcus adiodu ans
( 99 ). Al hough he p esence o Pa R homologs in unicellu-
la cyanobac e ia could be pa adoxical conside ing i s e ec s
on he e ocys di e en ia ion, homologs o o he p o eins es-
sen ial o he e ocys di e en ia ion, such as He R, also exis
in non-he e ocys - o ming cyanobac e ia ( 100 ). Pa R and i s
homologs p obably ha e some essen ial unc ion ela ed o
he physiology o all cyanobac e ia, bu he inse ion o wo
addi ional α-helices in he homologs o he e ocys - o ming
cyanobac e ia ( Supplemen a y Figu es S7 and S8 ) may ha e
allowed he pa icula egula o y ole o Pa R in he e ocys
di e en ia ion. The exac egula o y mechanism(s) in ol ing
Pa R and i s a ge s a e highly in e es ing opics o u u e
wo k.
Da a a ailabili y
Sou ce da a a e p o ided as a Sou ce Da a ile. MS aw
da a is deposi ed a he P o eomeXchange Conso ium ( h p:
//p o eomecen al.p o eomexchange.o g ) ia he PRIDE pa -
ne eposi o y( 101 ) unde he iden i ie : PXD050404. CLIP-
seq da a is deposi ed in GEO unde he iden i ie GSE280056.
All code in ol ing he bioin o ma ics wo k low and he shiny
app is a ailable on Gi hub ( h ps:// gi hub.com/ manb eal / R-
DeeP-Nos oc ) and Zenodo ( h ps:// doi.o g/ 10.5281/ zenodo.
14265455 ). A shiny app was p og ammed o acili a e he
accessibili y and isualiza ion o he da a: h ps://sunshine.
biologie.uni- eibu g.de/R- DeeP- Nos oc/.
Supplemen a y da a
Supplemen a y Da a a e a ailable a NAR Online.
A c kno wledg emen s
The au ho s hank P o . En ique Flo es and P o . An onia
He e o o sha ing he Δhe R CSSC2 s ain. We also hank
Ma c B oghamme o help in cloning he Asl3888-3xFLAG
usion, Ka s en Voig and Edi h Ams o help in he implemen-
a ion o he shiny app on he webse e , Panagio a A ampa zi
and Tho s en Bischle o sequencing he CLIP-seq RNA li-
b a ies and Ma cus Ziemann o p o iding assis ance in he
isual ep esen a ion o CLIP-seq da a.
Au ho con ibu ions : M.B.A. designed he p ojec . M.B.A.,
A.M.M-P ., M.M.S. and W .R.H. secu ed unding. The R-DeeP
app oach, PNK assays, phylogene ic analysis and cons uc ion
o he mu an s and epo e s ains was pe o med by M.B.A.
D.P ., C.P ., F .S. and M.M.S. pe o med and in e p e ed he p o-
eomic analysis. Bioin o ma ic analysis o R-DeeP da a was
pe o med by F .S., D.P . and M.B.A. The modi ied e sion o
T iPepSVM was implemen ed by H.R.R. and M.B.A. In e -
p e a ion o he R-DeeP da a and SVM ou pu was pe o med
by M.B.A. and W.R.H. G ow h analysis and mic oscopy ex-
pe imen s o pa R we e pe o med and in e p e ed by A.V.,
A.M.M-P. and M.B.A. CLIP-seq expe imen was ca ied ou
by I.S., C.S. and M.B.A., and analyzed by M.B.A. M.B.A. and
W.R.H. w o e he manusc ip wi h he inpu o all au ho s.
Funding
Alexande on Humbold -S i ung ( o M.B.A.); Deu sche
Fo schungsgemeinscha [HE 2544 / 20-1 o W.R.H.];
Paul G. Allen F on ie s G oup ( o M.M.S.); Agen-
cia Es a al de In es igación [PID2022-138128NB-I00,
MCIN / AEI / 10.13039 / 501100011033, FEDER, Eu opean
Regional De elopmen Fund, UE o A.M.M-P]. We acknowl-
edge suppo by he Open Access Publica ion Fund o he
Uni e si y o F eibu g.
Con lic o in e es s a emen
None decla ed.
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