Ci a ion: O mancey, M.; Thuleau, P.;
Combie , J.-P.; Plaza, S. The Essen ials
on mic oRNA-Encoded Pep ides
om Plan s o Animals. Biomolecules
2023,13, 206. h ps://doi.o g/
10.3390/biom13020206
Academic Edi o s: William W. Du
and Feiya Li
Recei ed: 16 Decembe 2022
Re ised: 6 Janua y 2023
Accep ed: 8 Janua y 2023
Published: 19 Janua y 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
biomolecules
Re iew
The Essen ials on mic oRNA-Encoded Pep ides
om Plan s o Animals
Mélanie O mancey 1,2,* , Pa ice Thuleau 1, Jean-Philippe Combie 1and Se ge Plaza 1,*
1Labo a oi e de Reche che en Sciences Végé ales, CNRS/UPS/INPT, 31320 Auze ille-Tolosane, F ance
2Epigene ics and Plan De elopmen , Cen e o Resea ch in Ag icul u al Genomics (CRAG), UAB,
Ce danyola del Vallès, 08193 Ba celona, Spain
*Co espondence: [email p o ec ed] (M.O.); [email p o ec ed] (S.P.)
Abs ac :
P ima y ansc ip s o mic oRNAs (p i-miRNAs) we e ini ially de ined as long non-coding
RNAs ha hos miRNAs u he p ocessed by he mic oRNA p ocesso complex. A ew yea s ago,
howe e , i was disco e ed in plan s ha p i-miRNAs ac ually con ain unc ional open eading
ames (sORFs) ha ansla e in o small pep ides called miPEPs, o
mi
c oRNA-encoded
pep
ides.
Ini ially de ec ed in A abidopsis haliana and Medicago unca ula, ecen s udies ha e e ealed he
p esence o miPEPs in o he p i-miRNAs as well as in o he species anging om a ious plan species
o animals. This sugges s ha miPEP numbe s emain la gely unde es ima ed and ha hey could be
a common signa u e o p i-miRNAs. He e we p esen he mos ecen ad ances in miPEPs esea ch
and discuss how hei disco e y has b oadened ou ision o he egula ion o gene exp ession by
miRNAs, and how miPEPs could be in e es ing ools in sus ainable ag icul u e o he ea men o
ce ain human diseases.
Keywo ds: mic oRNA; sORF; pep ide; plan miPEP; animal miPEP
1. In oduc ion
The adap a ion o all li ing o ganisms o hei en i onmen equi es he s ic con ol o
a ious biological p ocesses ha a e essen ial o hei g ow h, de elopmen , ep oduc ion,
and esponses o s esses. This con ol is achie ed h ough he modula ion o signaling
pa hways by egula o y molecules ha , in ine, ac i a e o ep ess downs eam a ge genes.
In his con ex , mic oRNAs (miRNAs) play pi o al oles in g ow h, de elopmen , and
s ess esponses.
MiRNAs a e small endogenous single-s anded RNAs (20 o 22 nucleo ides) ha a e
in ol ed in pos - ansc ip ional gene silencing in euka yo es. They allow he down egu-
la ion o a ge genes by speci ically igge ing he deg ada ion o hei messenge RNAs
(mRNAs) o by inhibi ing hei ansla ion [
1
–
4
]. Mos plan species ha e se e al hund ed
anno a ed miRNA genes. Fo example, he miRNA da abase miRbase (www.mi base.o g)
con ains 326 known miRNAs (accessed on 1 Decembe 2022) in A abidopsis haliana, 604
in O yza sa i a, 247 in Physcomi ella pa ens, 594 in Picea abies, 2654 in Homo sapiens, 1978
in Mus musculus, 437 in Caeno habdi is elegans, and 469 in D osophila melanogas e . These
genes a e g ouped in amilies ep esen ed by a numbe in e e ence o simila i ies be ween
hei ma u e miRNA sequences. The a ious homologs wi hin one mul igene miRNA
amily a e dis inguished wi h di e en le e s. Unlike animal miRNAs, which equen ly
a ge hund eds o genes, plan miRNAs usually ha e ewe han 10 a ge s, ypically key
egula o s such as ansc ip ion ac o s (TFs), ho monal ecep o s, and nu ien senso s [
5
].
By down egula ing key egula o s, which in u n modi y he exp ession o se e al genes,
miRNAs hus ac as de elopmen al swi ches capable o modula ing en i e signaling ne -
wo ks. Since miRNA ac i i y g ea ly in luences physiological esponses o de elopmen al
and en i onmen al cues, i is ob ious ha any e en ha egula es miRNA ac i i y could
ha e d as ic consequences on plan physiology and pheno ypes.
Biomolecules 2023,13, 206. h ps://doi.o g/10.3390/biom13020206 h ps://www.mdpi.com/jou nal/biomolecules
Biomolecules 2023,13, 206 2 o 14
Fo a long ime, plan p ima y ansc ip s o miRNAs (p i-miRNAs) we e anno a ed
as long non-coding RNAs (lncRNAs). Howe e , pionee ing wo k on plan s has high-
ligh ed he p esence o small open ames (ORFs) in he 5’a m o hese p i-miRNAs [
6
].
These ORFs can encode pu a i e miRNA-encoded pep ides (miPEPs) [
6
]. MiPEPs we e
u he iden i ied in animals [
7
]. He e, we highligh he mos impo an published indings
ega ding miPEPs, om hei disco e y o hei biological unc ions in bo h plan s and
animals, and we p esen he mos ecen da a ega ding he molecula mechanisms unde -
lying miPEP ac i i y. Finally, we discuss he po en ial applica ion o miPEPs in ag onomy
and human he apeu ics.
2. MiPEP Disco e y
Pep ides a e known o be in ol ed in many p ocesses including de elopmen al egu-
la ion, acclima ion o abio ic s ess, and de ense agains pa hogens [
8
–
12
] (Figu e 1). The
majo i y o known egula o y pep ides in plan s a e de i ed om p ecu so p o eins [
13
].
Howe e , pep ides ha a e di ec ly ansla ed om sORFs ha e also been epo ed [
8
,
10
].
Among hem, hose loca ed in he 5’ egion o p i-miRNAs, e med miPEPs, ha e ecen ly
ecei ed mo e a en ion [
14
–
16
]. Indeed, based on in-house and exis ing RACE-PCR-based
anno a ions o p i-miRNAs o M. unca ula and A. haliana, Lau esse gues and colleagues
(2015) pe o med an in silico analysis e ealing he p esence o a leas one pu a i e sORF
in he 5’ egion o M miR171b and A miR165a p i-miRNAs [
6
]. The unc ionali y o hese
sORFs was alida ed o he i s ime in his s udy using A. haliana and M. unca ula as
model plan s. Indeed, in bo h cases, he p esence o endogenously exp essed miPEPs was
isualized by wes e n blo and/o immuno luo escence using speci ic an ibodies [6].
Biomolecules 2023, 13, x FOR PEER REVIEW 5 o 15
miPEPs ha e been shown o ac as an ac i a o o hei cogna e miRNA exp ession con-
as ing wi h animals whe e only e ec s o sORF we e de ec ed [30,37,40].
3. MiPEP Func ions
3.1. In Plan s
Se e al pieces o e idence sugges ha miPEPs ac i a e he exp ession o hei
miRNA genes. Indeed, he o e exp ession o A miPEP165a in a he e ologous species (Ni-
co iana ben hamiana), o he applica ion o i s syn he ic e sion, inc eased he exp ession o
bo h i s co esponding p i-miRNA and he ma u e miRNA, and co ela i ely dec eased
he exp ession o miRNA a ge genes in A. haliana. Simila ly, he M. unca ula
miPEP171b was able o inc ease i s M p i-miR171b exp ession, sugges ing ha he unc-
ion o miPEPs is conse ed and no limi ed o a ew species [6]. The posi i e e ec o
miPEPs on he accumula ion o hei espec i e p i-miRNAs was inhibi ed by co dycepin,
a ansc ip ion inhibi o , sugges ing ha miPEPs induce his accumula ion by inc easing
he ansc ip ion o hei co esponding miRNA genes [6].
Due o he posi i e eedback ha miPEPs exe on hei co esponding p i-miRNAs
in plan s, miPEPs can be expec ed o exhibi di e se biological unc ions anging om
plan de elopmen o bene icial plan -mic obial in e ac ions o s ess esis ance, and
could hus be conside ed as a na u al al e na i e o pes icides and chemical e ilize s
(Figu e 1a).
Figu e 1. Mic oRNA-encoded pep ides (miPEPs) egula e many biological unc ions bo h in plan s
and animals. (a) The abili y o plan miPEPs o posi i ely egula e he exp ession o hei espec i e
p i-miRNAs is desc ibed o se e al miPEPs and plan species. (b) Con e sely, in animals, he eg-
ula ion o p i-miRNAs by miPEPs is less clea . MiPEPs equen ly ac independen ly.
A s udy pe o med on g ape ine was ecen ly published in his con ex [23].
MiRNA171 amily membe s a e known o a ge genes in ol ed in he o ma ion and de-
elopmen o oo s in di e en plan s [6,41]. Chen and colleagues ound ha V iMIR171
gene membe s we e speci ically exp essed du ing he o ma ion and de elopmen o
g ape ine (Vi is ini e a) ad en i ious oo s [23]. When V imiR171d was o e exp essed in
A. haliana, he plan s displayed sho e p ima y oo s, highe la e al oo densi y, and
ea lie ad en i ious oo de elopmen compa ed o wild- ype (WT) plan s. An in silico
analysis p edic ed h ee pu a i e sORFs in he 5’ egion o V ip i-miRNA171d. Thei e-
spec i e ansien o e exp ession in g ape issue cul u e plan le s showed ha only he
i s p i-miRNA sORF enabled an inc ease in V imiR171d exp ession. In addi ion, when
a cons uc con aining he egion om he V imiR171d p omo e o he ATG s a si e o
his sORF used o he GUS gene was exp essed in N. ben hamiana lea es o g ape issue
cul u e plan le s, GUS ac i i y was obse ed. These da a demons a e ha his sORF en-
codes a pep ide, which was named V imiPEP171d1. Simila o wha was p e iously de-
sc ibed, when g ape issue cul u e plan le s we e ea ed wi h syn he ic V imiPEP171d1,
V imiR171d exp ession speci ically inc eased while he exp ession o miRNA a ge genes
co ela i ely dec eased. In addi ion, when g ape plan le s we e g own on a medium
(a)
(b)
Figu e 1.
Mic oRNA-encoded pep ides (miPEPs) egula e many biological unc ions bo h in plan s
and animals. (
a
) The abili y o plan miPEPs o posi i ely egula e he exp ession o hei espec i e
p i-miRNAs is desc ibed o se e al miPEPs and plan species. (
b
) Con e sely, in animals, he
egula ion o p i-miRNAs by miPEPs is less clea . MiPEPs equen ly ac independen ly.
Since hei disco e y, he exis ence o miPEPs has been ex ended o a ious p i-
miRNAs in se e al plan species as lis ed in Table 1[17–28].
Biomolecules 2023,13, 206 3 o 14
Table 1.
Lis o miPEPs (and hei embedded miRs) desc ibed in he li e a u e (and miRbase) bo h in
plan s and animals.
O ganism MiPEP
(miR) MiPEP Size In Vi o miPEP
De ec ion
E ec on he
Co esponding
P i-miRNA
Regula ion o
miRNA
Ta ge s
Regula ed Biological
Func ions Re
Plan s
A abidospsis
haliana
A miPEP165a
(a h-miR165a) 18 GUS epo e
gene exp ession
and wb Up egula ion
Down egula ion
o HD-ZIP III
PHAVOLUTA,
PHABOLUSA,
REVOLUTA
S imula ion o main
oo g ow h;
Accele a ion o he
in lo escence s em
appea ance and o he
lowe ing ime;
Inhibi o y e ec on o al
oo g ow h
[6,17,18]
A abidopsis
haliana
A miPEP858a
(a h-amiR858a) 44 GUS epo e
gene exp ession
and wb Up egula ion
Down egula ion
o MYB
ansc ip ion
ac o A MYB12
Fla onoid biosyn hesis
and plan de elopmen [19]
A abidopsis
haliana
A miPEP164b
(a h-miR164b) 29 N/A Up egula ion
Down egula ion
o NAC1,NAC4,
NAC5,CUC1
and CUC2
Inhibi o y e ec on o al
oo g ow h [18]
A abidopsis
haliana
A miPEP397a
(a h-miR397a) 7 N/A Up egula ion Down egula ion
o LAC2,LAC4
and LAC17
S imula ion o o al oo
g ow h [18]
Dimoca us
Longan Lou N/A 50 N/A Up egula ion Down egula ion
o HD-ZIP
IIIATHB15
Emb yogenesis [20]
Glycine max GmmiPEP172c
(gma-miR172c) 16 N/A Up egula ion
Down egula ion
o AP2
ansc ip ion
ac o NODULE
NUMBER
CONTROL 1
Inc ease in nodule
numbe [21]
Lo us japonicus LjmiPEP171b
(lja-miR171b) 22 N/A Up egula ion N/A Inc ease in
myco hiza ion a e [22]
Medicago
unca ula
M miPEP171b
(m -miR171b) 20 GUS epo e
gene exp ession
and wb Up egula ion
Up egula ion o
GRAS
ansc ip ion
ac o LOST
MERISTEMS 1
(LOM1)
Reduc ion o la e al oo
de elopmen and
inc ease in
myco hiza ion a e
[6,22]
Medicago
unca ula
M miPEP171a
(m -miR171a) 10 N/A N/A Down egula ion
o LOM1
Dec ease in
myco hiza ion a e [22]
Medicago
unca ula
M miPEP171c
(m -miR171c) 7 N/A N/A Down egula ion
o LOM1
Dec ease in
myco hiza ion a e [22]
Medicago
unca ula
M miPEP171d
(m -miR171d) 6 N/A N/A Down egula ion
o LOM1
Dec ease in
myco hiza ion a e [22]
Medicago
unca ula
M miPEP171e
(m -miR171e) 23 N/A N/A Down egula ion
o LOM1
Dec ease in
myco hiza ion a e [22]
Medicago
unca ula
M miPEP171
(m -miR171 ) 5 N/A N/A Down egula ion
o LOM1
Dec ease in
myco hiza ion a e [22]
O yza sa i a OsmiPEP171i
(osa-miR171i) 31 N/A Up egula ion N/A Inc ease in
myco hiza ion a e [22]
Solanum
lycope sicum
SlmiPEP171e
(slymiR171e) 19 N/A Up egula ion N/A Inc ease in
myco hiza ion a e [22]
Vi iis ini e a V imiPEP171d1
( i-MIR171d1 *) 7GUS epo e
gene exp ession Up egula ion Down egula ion
o sca ec ow-like
V SCL27
Ad en i ious oo
o ma ion [23]
Vi is ini e a V imiPEP164c
( i-miR164c) 16 N/A Up egula ion
Down egula ion
o V MYBPA1
g ape ine
ansc ip ion
ac o
Inhibi ion o
p oan hocyanidin
syn hesis and
s imula es an hocyanin
accumula ion
[24]
Vi is ini e a V imiPEP172b
( i-miR172b) 16 N/A Up egula ion Down egula ion
o V RAP2-7-1
Inc ease in cold
ole ance in g ape ine [25]
Vi is ini e a V imiPEP3635b
( i-MIR3635b *) 11 N/A Up egula ion Down egula ion
o V ENT3
Inc ease in cold
ole ance in g ape ine [25]
Biomolecules 2023,13, 206 4 o 14
Table 1. Con .
O ganism MiPEP
(miR) MiPEP Size In Vi o miPEP
De ec ion
E ec on he
Co esponding
P i-miRNA
Regula ion o
miRNA
Ta ge s
Regula ed Biological
Func ions Re
Plan s
Ba ba ea ulga is B miPEP164b
(b -miR164b *) 8 N/A Up egula ion
Down egula ion
o NAC1,NAC4,
NAC5,CUC1
and CUC2
Inhibi o y e ec on
main oo g ow h and
olia su ace [18]
B assica oleace a BomiPEP397a
(bo-miR397a *) 10 N/A Up egula ion Down egula ion
o LAC2,LAC4
and LAC17
S imula ion o main
oo g ow h and olia
su ace [18]
B assica apa B miPEP156a
(b -miR156a) 33 TAMRA-
labeled pep ide Up egula ion N/A Mode a e s imula ion o
main oo g ow h [26]
Animals
Human miPEP200a
(hsa-miR-200a) 187
wb; HA used
pep ide
o e -exp essed
in cells
No egula ion
Inhibi he
exp ession o
imen in in
cance cells
Inhibi ion o he
mig a ion o p os a e
cance cells [29,30]
Human miPEP200b
(hsa-miR-200b) 54
wb; HA used
pep ide
o e -exp essed
in cells
N/A
Inhibi he
exp ession o
imen in in
cance cells
Inhibi ion o he
mig a ion o p os a e
cance cells [29]
Human miPEP155
(hsa-miR-155) 17 EGFP- used ORF No egula ion No egula ion
Supp ession o
au oimmune
in lamma ion by
modula ing an igen
p esen a ion
[30,31]
Human miPEP497
(hsa-miR-497) 21 N/A No egula ion No egula ion N/A [30]
Human miPEP22
(hsa-miR-22) 57 wb N/A N/A Tumo supp esso [32]
Human miPEP133
(hsa-miR-34a) 133 wb Up- egula ion N/A
Inc ease in p53
ansc ip ional ac i i y
by dis up ing
mi ochond ial unc ion
[33]
Human MISTRAV
o MOCCI
(hsa-miR-147b) 83
Wb; Immuno-
luo escence o
o e -exp essed
pep ide
No egula ion N/A Vi al s ess esponse,
in lamma ion and
immuni y [34,35]
D osophila
melanogas e
MSAmiP
(dme-miR-iab-8) 9 o 20 EGFP- used ORF No egula ion N/A In ol ed in spe m
compe i ion [36]
D osophila
melanogas e
DmmiPEP8
(dme-miR- 8) 71 wb No egula ion No egula ion Wing size educ ion [37]
Mus musculus MmmiPEP31
(mmu-miR-31) 44 EGFP- used ORF
and wb down- egula ion N/A
Supp ession o EAE by
p omo ing he
di e en ia ion o T eg
cells
[38]
*: miR no p esen in miRbase.
A he same ime, he ques ion o whe he miPEPs exis in animals has a isen. The
i s desc ip ion came om Razooky and co-wo ke s (2017), who iden i ied a miPEP
called C17o 91 exp essed om he p i-miRNA22 hos gene [
32
]. MiPEP C17o 91 was
up egula ed upon i al in ec ion bu no associa ed unc ion was epo ed. La e , se e al
p i-miRNAs encoding miR34a, miR31, miR155, miR147b in mammals and miR8 and iab8
in D osophila we e desc ibed as capable o exp essing miPEPs [31,33–38].
While i emains o be cla i ied in animals, se e al s udies pe o med in plan s on
di e en miRNA genes ha e epo ed ha he i s ORF a e he ansc ip ion s a si e is
p e e en ially ansla ed in o a miPEP [
6
,
19
,
23
,
39
]. No common signa u e has been ound
among hese di e en sORF-encoded pep ides. Howe e , so a , in plan s, all es ed miPEPs
ha e been shown o ac as an ac i a o o hei cogna e miRNA exp ession con as ing wi h
animals whe e only e ec s o sORF we e de ec ed [30,37,40].
Biomolecules 2023,13, 206 5 o 14
3. MiPEP Func ions
3.1. In Plan s
Se e al pieces o e idence sugges ha miPEPs ac i a e he exp ession o hei miRNA
genes. Indeed, he o e exp ession o A miPEP165a in a he e ologous species (Nico iana
ben hamiana), o he applica ion o i s syn he ic e sion, inc eased he exp ession o bo h
i s co esponding p i-miRNA and he ma u e miRNA, and co ela i ely dec eased he
exp ession o miRNA a ge genes in A. haliana. Simila ly, he M. unca ula miPEP171b
was able o inc ease i s M p i-miR171b exp ession, sugges ing ha he unc ion o miPEPs
is conse ed and no limi ed o a ew species [
6
]. The posi i e e ec o miPEPs on he
accumula ion o hei espec i e p i-miRNAs was inhibi ed by co dycepin, a ansc ip ion
inhibi o , sugges ing ha miPEPs induce his accumula ion by inc easing he ansc ip ion
o hei co esponding miRNA genes [6].
Due o he posi i e eedback ha miPEPs exe on hei co esponding p i-miRNAs in
plan s, miPEPs can be expec ed o exhibi di e se biological unc ions anging om plan
de elopmen o bene icial plan -mic obial in e ac ions o s ess esis ance, and could hus
be conside ed as a na u al al e na i e o pes icides and chemical e ilize s (Figu e 1a).
A s udy pe o med on g ape ine was ecen ly published in his con ex [
23
]. MiRNA171
amily membe s a e known o a ge genes in ol ed in he o ma ion and de elopmen
o oo s in di e en plan s [
6
,
41
]. Chen and colleagues ound ha V iMIR171 gene mem-
be s we e speci ically exp essed du ing he o ma ion and de elopmen o g ape ine
(Vi is ini e a) ad en i ious oo s [
23
]. When V imiR171d was o e exp essed in A. haliana,
he plan s displayed sho e p ima y oo s, highe la e al oo densi y, and ea lie ad en i-
ious oo de elopmen compa ed o wild- ype (WT) plan s. An in silico analysis p edic ed
h ee pu a i e sORFs in he 5’ egion o V ip i-miRNA171d. Thei espec i e ansien
o e exp ession in g ape issue cul u e plan le s showed ha only he i s p i-miRNA sORF
enabled an inc ease in V imiR171d exp ession. In addi ion, when a cons uc con aining he
egion om he V imiR171d p omo e o he ATG s a si e o his sORF used o he GUS
gene was exp essed in N. ben hamiana lea es o g ape issue cul u e plan le s, GUS ac i i y
was obse ed. These da a demons a e ha his sORF encodes a pep ide, which was named
V imiPEP171d1. Simila o wha was p e iously desc ibed, when g ape issue cul u e
plan le s we e ea ed wi h syn he ic V imiPEP171d1, V imiR171d exp ession speci ically
inc eased while he exp ession o miRNA a ge genes co ela i ely dec eased. In addi ion,
when g ape plan le s we e g own on a medium con aining syn he ic V imiPEP171d1, he
numbe o ad en i ious oo s signi ican ly inc eased, indica ing ha he miPEP is able o
egula e he o ma ion and de elopmen o g ape ine ad en i ious oo s. This p ope y
appea s speci ic o g ape ines since V imiPEP171d1 had no e ec on A. haliana oo s.
Mo e ecen ly, he same g oup cha ac e ized he unc ion o wo o he miPEPs in
g ape ines, namely V imiPEP172b and V imiPEP3635b [
25
]. Fi s , he au ho s iden i ied
V imiRNAs in g ape issue cul u e plan le s, whose exp essions we e modi ied du ing
cold s ess (4
◦
C). They hen selec ed V imiR172b and V imiR3635b o u he analysis.
Using an in silico app oach, hey iden i ied six and ou pu a i e sORFs, espec i ely, in
he 5’ egion o he co esponding p e-miRNAs. They ansien ly exp essed hese sORFs
in issue cul u e plan le s independen ly and ound ha one ORF om each p e-miRNA
was biologically ac i e as i was able o inc ease he exp ession o i s nascen p i-miRNA.
They syn hesized he co esponding miPEPs and, in e es ingly, hei ex e nal applica ion
on g ape issue cul u e plan le s imp o ed hei ole ance o cold.
Ano he example illus a ing he po en iali y o miPEPs came om he s udy o
he e ec o A miPEP858a on A abidopsis de elopmen [
19
]. A miR858 had p e iously
been shown o down egula e he exp ession o di e en ansc ip ion ac o s such as
A MYB11, A MYB12, and A MYB11, which egula e he phenylp opanoid pa hway ha
sou ces he me aboli es equi ed o he biosyn hesis o lignin and he p oduc ion o
many o he impo an compounds such as la onoids, couma ins, and lignans [
42
]. In
addi ion, A miR858 modi ies plan de elopmen by inc easing oo g ow h and accele a ing
lowe ing. By analyzing he egion ups eam o A p e-miR858a, he au ho s ound h ee

Biomolecules 2023,13, 206 6 o 14
pu a i e sORFs, o which one was shown o be ansla ed in plan a using epo e gene
usion assays and wes e n blo expe imen s. This pep ide, named A miPEP858a, inc eased
he exp ession o bo h A p i-miR858a and ma u e A miR858 when exogenously applied
o A abidopsis seedlings; his also co ela ed wi h a down egula ion o he exp ession o
A MYB12 and i s a ge genes, and pheno ypically wi h an inc ease in oo leng h. The
e ec o A miPEP858a was hen con i med ia gene ic app oaches using bo h ansgenic
A abidopsis plan s o e exp essing he miPEP and Cas9-edi ed A miPEP858a mu an plan s.
Thus, A miPEP858a-o e exp essing plan s exhibi ed longe main oo s han WT plan s,
while edi ed mu an lines showed an in e ed pheno ype. In e es ingly, he exogenous
ea men o A miPEP858a-edi ed mu an plan s wi h A miPEP858a complemen ed his
pheno ype. Compa ed o WT plan s, A miPEP858a-o e exp essing plan s exhibi ed a
educ ion in an hocyanin accumula ion as well as an inc ease in lignin con en , oge he
wi h enhanced exp ession o lignin biosyn hesis genes. The ecip ocal pheno ype was
obse ed in A miPEP858a-edi ed plan s [
19
]. Ve y ecen ly, he same g oup showed ha a
disul a ed pen apep ide, named Phy osul okine4 (PSK4), plays a key ole in he g ow h
and de elopmen o A miR858-dependen A abidopsis, h ough auxin [
28
]. In e es ingly,
A miPEP858a posi i ely egula es he exp ession o PSK4 ia A miR858a. The exp ession
o A miR858a and PSK4 is also posi i ely egula ed by he A MYB3 ansc ip ion ac o
h ough he di ec binding o A MYB3 o i s a ge p omo e s. A MYB3, whose exp ession is
egula ed by A miPEP858a/A miR858a, is a key componen in A miPEP858a/A miR858a-
PSK4-dependen plan g ow h and de elopmen [
28
]. Concomi an ly o his s udy, he
same au ho s showed ha ligh di ec ly egula es A miPEP858a accumula ion in A abidopsis
and is necessa y o A miPEP858a ac ion. This ligh -dependen miPEP egula ion equi es
he shoo - o- oo mobile, ligh -media ed ansc ip ion ac o , A HY5 [
43
]. O e all, he da a
place A miPEP858a a he c oss oads o se e al biological p ocesses, mos likely h ough
he egula ion o i s co esponding miRNA.
MiPEPs can also modula e hizosphe ic plan -mic oo ganism in e ac ions. Fo in-
s ance, an exogenous applica ion o GmmiPEP172c speci ically inc eases nodule numbe s in
soybean (Glycine max) when inocula ed wi h B ady hizobium diazoe iciens and leads o an in-
c ease in GmmiR172c ansc ip s [
21
]. These esul s a e in ag eemen wi h hose p e iously
obse ed by Wang e al., (2014), which show ha GmmiR172c o e exp ession posi i ely
egula es nodula ion in soybean h ough he ep ession o i s a ge gene— he Ape ala
2 (GmAP2) ansc ip ion ac o Nodule Numbe Con ol 1 (GmNNC1)—which di ec ly
binds o he p omo e o Ea ly Nodulin 40 (GmENOD40) o ep ess i s ansc ip ion [
44
].
Ano he example is he ole played by M miPEP171b in a buscula myco hizal symbiosis
in M. unca ula [
22
]. Unlike o he membe s o he M miPEP171 amily, M miPEP171b
s imula es a buscula myco hizal symbiosis and posi i ely egula es he exp ession o
i s co esponding M miR171b as well as he exp ession o M miR171b a ge M LOM1
(Los Me is ems 1). M miR171b is speci ically exp essed in oo cells con aining a bus-
cules and p o ec s M LOM1 om being silenced by o he M miR171 membe s h ough i s
misma ched clea age si e [22].
3.2. In Animals
Wi h he miPEP desc ip ion wi hin miR34a, miR31, miR155, and miR147b genes in
mammals and miR8 and iab8 genes in D osophila (see abo e), i is now well es ablished ha
p i-miRNAs can encode miPEPs in animal cells and, o some o hem, hei unc ion and
biology ha e e en been documen ed. Howe e , whe he and how miPEPs egula e hei
co esponding p i-miRNA exp ession emains con adic o y. To da e, he only example
in he animal li e a u e desc ibing a posi i e e ec o a miPEP on he exp ession o i s
co esponding p i-miRNA is ha o HsmiPEP133. HsmiPEP133 is a 133 amino acid pep ide
encoded by Hsp i-miR34a. HsmiPEP133 induces he exp ession o Hsp i-miR34a/miR34a
which leads o he down egula ion o HsmiR34a- a ge ed genes [
33
]. HsmiPEP133 is
exp essed in a ious heal hy issues bu is down egula ed in cance cell lines and umo s.
The o e exp ession o HsmiPEP133 indica es ha he pep ide ac s as a human umo
Biomolecules 2023,13, 206 7 o 14
supp esso in cellulo and
in i o
by inducing apop osis and inhibi ing he mig a ion and
in asion o cance cells. Howe e , HsMiPEP133 is mainly localized in mi ochond ia and
no in nuclei as epo ed o plan miPEPs. I modula es a ye - o-be-de ined signaling
cascade ha inc eases p53 ansc ip ional ac i i y by dis up ing mi ochond ial unc ion.
Since miR34a is a di ec a ge gene o he ansc ip ion ac o p53, he la e up egula es
bo h HsmiPEP133 and i s co esponding HsmiR34a, mos likely among a ple ho a o o he
p53 a ge genes. In addi ion, he au ho s showed ha he posi i e eedback egula ion o
HsmiR34a by HsmiPEP133 can occu in bo h a p53-dependen and -independen manne ,
sugges ing ha miPEP133 can ac h ough o he molecula playe s [
33
]. Mo e ecen ly,
Zhou and colleagues (2022) showed, in mice (Mus musculus), ha MmmiPEP31 p omo es
he di e en ia ion o egula o y T cells by ep essing he exp ession o MmmiR31 in a
sequence-dependen manne [
38
]. In e es ingly, he au ho s showed ha miPEP31 en e s
cells spon aneously and localizes o nuclei. The au ho s also demons a e ha miPEP31
nega i ely con ols he exp ession o miR31, p o iding he i s e idence ha a miPEP can
nega i ely con ol he exp ession o a miRNA gene. Howe e , he mechanism in ol ed
seems di e en om ha o miPEP133. Indeed, MmmiPEP31 binds o he Mmp i-miR31
p omo e , induces he deace yla ion o his one H3K27 (likely h ough he ec ui men o a
co ac o ), and compe es o he binding o an unknown ansc ip ion ac o [38].
Al hough hese wo examples show ha mammalian miPEPs a e able o egula e
hei co esponding p i-miRNAs, ei he posi i ely o nega i ely, animal miPEPs likely
play o he unc ions, which emain o be iden i ied. The mechanism desc ibed in plan s is
p obably no a gene al mechanism conse ed in animal p i-miRNAs. Indeed, HsmiPEP200a,
HsmiPEP155, HsmiPEP497, HsMOCCI/MISTRAV, DmmiPEP8 and DmMSAmiP do no
e eal any e ec on hei co esponding p i-miRNA [
30
,
31
,
34
–
37
]. Fu he mo e, hese
miPEPs exhibi egula o y and biological unc ions uncoupled om hei miRNA ac i i y,
ac ing ei he an agonis ically o [
31
], in pa allel wi h [
37
], o independen ly o [
36
], he
miRNA pa hway.
To conclude his pa , he s udies desc ibed abo e show ha while posi i e eedback
egula ion has been ound in all plan miRNA genes s udied so a , di e se miPEP e ec s
ha e been epo ed in di e en animal model sys ems (Figu e 1b), indica ing ha miPEP-
dependen posi i e eedback egula ion o miRNA genes is no a gene al mechanism ha
can be ex ended o all o ganisms.
4. Wha Fea u es Unde lie miPEP Ac i i y?
4.1. MiPEP En y in o Cells
Gi en ha an exogenous plan ea men wi h miPEPs induces signi ican pheno-
ypic e ec s, one ques ion emains unanswe ed: how do miPEPs en e plan s? Recen ly,
luo escein-labeled A miPEP165a was shown o be in e nalized in A. haliana oo s by
bo h endocy osis and passi e di usion [
17
]. Ne e heless, his miPEP did no en e he
cen al cylinde and was no subsequen ly anspo ed sys emically. I s pene a ion was
limi ed o he oo pe iphe al zone, indica ing ha miPEPs migh only ac in a localized
way. Acco dingly, he applica ion o miPEPs on lea es did no induce pheno ypic changes
o oo s and, ecip ocally, he applica ion o miPEPs on oo s did no induce changes o
lea es. The en y o miPEPs h ough cla h in-media ed endocy osis was also ecen ly
epo ed by Badola and co-wo ke s (2022) who used luo escein-labeled A miPEP858a
in A abidopsis [
28
]. Howe e , ano he s udy consis ing o an exogenous applica ion o a
TAMRA-labeled miPEP, B miPEP156a, o B assica apa seedlings indica es ha his luo es-
cen pep ide en e s h ough he oo sys em and accumula es p edominan ly in lea es [
26
].
This sugges s ha signi ican di e ences can be obse ed om one miPEP o ano he ,
pe haps due o hei di e en physical/chemical p ope ies.
In human, he cellula up ake o miPEPs has also been s udied and i was shown ha
FITC-labeled miPEP155 e icien ly en e ed in o HEK293T cells and co-localized wi h en-
dogenous miPEP155 (called P155) [
31
]. Ano he s udy in mouse showed ha MmmiPEP31
beha es as a
C
ell
P
ene a ing
P
ep ide (CPP) bo h
in i o
and
in i o
[
37
]. FAM-labeled
Biomolecules 2023,13, 206 8 o 14
MmmiPEP31 en e s ac oss he cell memb ane in an ene gy-independen manne hanks o
i s posi i ely cha ged esidues (5 A g and 4 Lys on a 44-amino acid pep ide), a common
ea u e wi h o he CPPs. This is howe e con a y o i s nuclea anspo , which appea s
o be ene gy-dependen . As obse ed in CPPs and ansc ip ion ac o s, a MmmiPEP31
s uc u e p edic ion highligh ed an α-helix s uc u e [38].
Thus, hese esul s show ha miPEPs can beha e as CPPs and cons i u e molecules ac -
ing in a non-cell-au onomous manne . The ex en o which hese pep ides a e endogenously
sec e ed and able o unc ion as long- ange signaling molecules emains o be in es iga ed.
4.2. MiRNA Genes Exp ess He e ogeneous Popula ions o T ansc ip s in Plan s
P i-miRNAs a e p edic ed o be localized o nuclei whe e he p ocessing occu s. The
p ocessing is ca ied ou by DCL1 in plan s and D osha/Pa cha in animals. This howe e
aises he ques ion o how miPEP ORFs a e ansla ed.
In human, a s udy pe o med on he p i-miRNAs o genes encoding exonic miRNAs
showed ha some spliced p i-miRNA ansc ip s exhibi a cy oplasmic localiza ion, con-
sis en wi h a possible ansla ion [
45
]. Mo eo e , as obse ed in plan s, long non-coding
miRNA hos genes (p i-miRNAs), exhibi a complex gene s uc u e, and a e exp essed as
mul iple ansc ip a ian s due o al e na i e p omo e usage and/o al e na i e splic-
ing [
46
]. This shows ha miRNA genes p oduce many di e en ansc ip s, some o which
lack he miRNA s em loop (Figu e 2a).
Figu e 2.
Plan p i-miRNAs a e p ocessed in o a he e ogeneous popula ion o ansc ip s. (
a
) P i-
miRNAs a e p oduced om al e na i e splicing (
b
) o al e na i e ansc ip ional e mina ion si es.
(
c
) Mos sho and al e na i ely spliced (AS) p i-miRNA ansc ip s a e localized in he cy oplasm
whe e hey in e ac wi h he 60S ibosomal p o ein L18 (RPL18), sugges ing ha hey a e loaded
in o ibosomes o ansla ion. P i-miRNA ansc ip s con aining he miRNA s em loop a e en iched
wi hin nuclei whe e hey can be used as empla es o gene a e ma u e miRNAs (adap ed om [
39
]).
Biomolecules 2023,13, 206 9 o 14
Using Iso-Seq, RNA-Seq, and RACE-PCR da a in A. haliana, Lau esse gues and
co-wo ke s (2022) ecen ly showed ha plan miRNA genes also exp ess a he e ogeneous
popula ion o ansc ip s in almos all s udied cases: long canonical ansc ip s con aining
ull-leng h sequences wi h he en i e p e-miRNA (miRNA and miPEP sequences), and
sho e ansc ip s, o , al e na i ely, spliced (AS) ansc ip s ha only possess he miPEP
sequence bu no he en i e p e-miRNA s em-loop sequence, i.e., he miRNA and miRNA*
sequences (Figu e 2b) [
39
]. Mos sho and AS ansc ip s appea ed o be associa ed wi h
he 60S ibosomal p o ein L18 (RPL18), sugges ing ha hey a e loaded in o ibosomes. This
is also he case o a ew long p i-miRNA ansc ip s. These long ansc ip s a e en iched
wi hin he nuclei, p obably o gene a e ma u e miRNAs, compa ed o he cy oplasm whe e
hey a e unde ep esen ed. Sho and AS ansc ip s, which a e mainly associa ed wi h
ibosomes and ound in he cy oplasm, a e mos likely o gene a e miPEPs (Figu e 2c).
In i o
ansc ip ion/ ansla ion in whea ge m ex ac s ein o ced he hypo hesis ha
sho ansc ip s a e e icien ly ansla ed and cons i u e he main sou ce o miPEPs [39].
4.3. Molecula Bases o miPEP Speci ici y in Plan s
Al hough plan miPEPs appea o be poo ly conse ed ac oss species, in iguingly,
hey ul ill an appa en ly e y speci ic unc ion by modi ying he exp ession o hei co e-
sponding p i-miRNAs only, i.e., wi hou dis up ing he exp ession o o he p i-miRNAs,
e en wi hin he same miRNA amily [
6
,
39
]. In his con ex , i is legi ima e o wonde wha
molecula mechanisms unde lie miPEP speci ici y, and mo e gene ally hei unc ions.
Recen da a ha e highligh ed ha he sORF-encoding miPEP (miORF) i sel plays a pi o al
ole in miPEP esponses and speci ici y [
39
]. Thus, i s dele ion leads o an absence o
p i-miRNA induc ion by he co esponding miPEP. Mo eo e , swapping he sequence
o M. unca ula miORF171b wi h ha o A. haliana miORF319a in M p i-miRNA171b p e-
en s M miPEP171b ac i i y, whe eas A miPEP319a becomes ac i e o posi i ely modula e
he exp ession o M p i-miR171b. Simila ly, he inse ion o an a i icial miPEP sequence
(no p esen in he plan genome) in M p i-miR171b makes his p i-miRNA ac i a able by
he co esponding a i icial miPEP. Fu he mo e, miORF localiza ion appea s impo an o
miPEP-induced ac i a ion. Indeed, when he ORF is placed on he 3’ a m o he mic oRNA
wi hin he p i-miRNA, no miPEP-induced ac i a ion could be obse ed. Howe e , dupli-
ca ing he ORF in mul iple copies on he 5’ a m inc eased he miPEP-induced esponse.
These obse a ions sugges an in e play be ween he miPEP and i s co esponding ORF
ha is impo an o he miPEP esponse. Consis en ly, FRET–FLIM (Fö s e esonance
ene gy ans e – luo escence li e ime imaging mic oscopy) and ITC (iso he mal i a ion
chemis y) app oaches ocusing on M miPEP171b indica e ha he miPEP is indeed a
p oximi y o p obably in e ac s wi h i s p i-miRNA ia he miORF [39].
Taken oge he , hese da a shed ligh on how non-conse ed miPEPs can pe o m
speci ic egula o y unc ions in hei hos species only.
4.4. MiPEP Conse a ion
Unlike miRNAs, which a e highly conse ed among plan species, miPEP sequences
appea o be much mo e a iable and do no possess any common signa u e [
22
,
39
]. Wi h
he excep ion o A miPEP156a, A miPEP164a, and A miPEP165a which exhibi some con-
se a ion, miPEP sequences a e gene ally no conse ed wi hin B assicaceae [
6
,
39
,
47
]. An
exogenous applica ion o A miPEP156a and A miPEP167a, highly (
≈
90% iden i y) and
poo ly (
≈
50–70% iden i y) conse ed miPEPs among B assicaceae, espec i ely, e ealed
ha only A miPEP156a posi i ely up egula ed i s p i-miRNA in all plan s es ed—i.e.,
A. haliana,B assica apa and B assica ole acea [
39
]. In he same way, Chen and collabo a o s
(2020) ha e shown ha an exogenous applica ion o an o e exp ession o V imiPEP171d1
om g ape ines does no a ec oo de elopmen in A. haliana [
23
]. Simila ly, an applica-
ion o A miPEP171c, he A. haliana miPEP o holog o V imiPEP171d1, does no induce
any pheno ype change in g ape ines, whils i does p omo e he g ow h o A. haliana
la e al and ad en i ious oo s. These miPEPs a e he e o e only ac i e on hei plan o