Available online at www.sciencedirect.
com
ScienceDirect
Emerging roles of RNA-binding proteins
in plant development
Hyunwoo Cho1, Hyun Seob Cho2 and Ildoo Hwang2
RNA-binding proteins (RBPs) influence the fate of target RNAs
via direct interactions. During transcription, RBPs and
interacting partners are recruited to and modify transcripts,
after which they may also participate in critical steps to
generate functional RNA. RBP–RNA interactions govern post-
transcriptional processing of RNA, consequently regulating
gene expression in a spatio-temporal manner. In plants, an
increasing number of proteins have been classified as RBPs,
many of which have been shown to function as key players in
diverse developmental processes. However, a comprehensive
understanding of how RBPs function, which RNAs are targeted,
and where RBP–RNA interactions occur within plant cells is
lacking. Here, we discuss recent findings in the field and newly
defined roles for RBPs in plant growth and development. We
also describe the mechanistic effects of RBPs on target RNA
metabolism and translation.
Addresses
1
Department of Industrial Plant Science and Technology, College of
Agricultural, Life and Environmental Sciences, Chungbuk National
University, Cheongju 2864, Republic of Korea
2
Department of Life Sciences, POSTECH Biotech Center, Pohang
University of Science and Technology, Pohang 37673, Republic of Korea
Corresponding author: Hwang, Ildoo (ihwang@postech.ac.kr)
Current Opinion in Plant Biology 2019, 51:51–57
This review comes from a themed issue on Cell signalling and gene
regulation
Edited by Ildoo Hwang and Masaaki Umeda
For a complete overview see the Issue and the Editorial
Available online 6th May 2019
https://doi.org/10.1016/j.pbi.2019.03.016
1369-5266/ã 2019 Elsevier Ltd. All rights reserved.
Introduction
Advancements in our understanding of plant
development and growth have largely relied on studies
of the cellular functions of genes and their hierarchical
signaling networks, with a strong focus on the regulation
of gene expression. Forward and reverse genetic screens,
in combination with transcriptome sequencing and
protein–DNA interactome analyses, have led to an
improved understanding of the interactions and func-
tions of various DNA-binding proteins (DBPs) as key
regulators of gene expression during plant development.
www.sciencedirect.com
However, recent studies coupled with large-scale tran-
scriptome and translatome datasets in plants [1–4] have
newly emphasized importance of RNA-binding proteins
(RBPs), a diverse class of proteins defined by their ability
to interact with single-stranded or double-stranded
RNA, in RNA metabolism.
These studies have revealed that up to 60% of messen-
ger RNA (mRNA) molecules in Arabidopsis thaliana
have at least two isoforms and that many genes display
different translational efficiencies in different tissue
types and at different developmental stages, suggesting
that RBP-mediated regulation is a key aspect of RNA
metabolism and translation [1,3,5,6]. During RNA syn-
thesis (transcription), maturation (post-transcriptional
processing, including capping, splicing, and polyade-
nylation), export to the cytosol, and translation, all
RNAs are tightly bound and controlled by diverse
RBPs, allowing these proteins to serve as master reg-
ulators of RNA fate [7–12]. In the Arabidopsis genome,
more than 800 RBPs have been identified based on
computational prediction of RNA-binding domains
(RBDs) and sequence homology to known RBPs in
other eukaryotes [13,14]. Among the Arabidopsis RBPs,
197 proteins contains an RNA recognition motif (RRM)
and 28 with a K homology (KH) domain [13,14]. In
addition, 26 pumilio (PUM) domain proteins,
58 DEAD-box helicase proteins, 5 proteins with cold
shock domains (CSDs), and numerous proteins contain-
ing pentatricopeptide repeat (PPR) domains have been
characterized as proteins with at least one RBD [15–18].
Moreover, recent proteomic studies have identified
mRNA-interacting partners, providing unprecedented
insight into the complexity of RBDs, many of which
were not previously implicated in RNA metabolism and
translation in plants [19–21].
Although these studies dramatically expanded the reper-
toire of potential RBPs to more than 1800 in Arabidopsis,
which is nearly double the number predicted by in silico
techniques, current knowledge on the roles of RBPs in
plant development and efforts to determine their mech-
anisms of action are limited compared to other organisms,
including viruses, fungi, and animals. Here, we highlight
the roles of RBPs in plant development, discuss the
molecular mechanisms regulating RNA targets, and con-
sider how technological advances will improve our under-
standing of RBP function. For more detailed information
on the general roles of RBPs in post-transcriptional
processes, including splicing and translation, and
Current Opinion in Plant Biology 2019, 51:51–57
52 Cell signalling and gene regulation

approaches to identify RBPs and their target RNAs, we hypocotyl elongation, lateral root development, and
refer readers to other reviews [22–25]. root hair formation (Figure 1).

Roles of RBPs in plant development and Vegetative–reproductive phase transition:

molecular action on target RNAs splicing and stability regulation by RBPs
The plasticity of plant growth and development Splicing regulators, which consist of several uridine-rich
enables sessile plants to adapt continuously to environ- small nuclear ribonucleoproteins (U snRNPs), serine/argi-
mental changes. Recent studies in Arabidopsis reported nine-rich (SR) proteins, and heterogeneous nuclear
widespread post-transcriptional and -translational reg- ribonucleoproteins (hnRNPs) are components of the spli-
ulation in diverse developmental stages and signaling ceosome [25]. Compared with constitutive splicing, alterna-
modules that serve as critical regulatory elements in tive splicing increases the potential for regulatory and
maintaining plasticity. Here, we summarize multiple proteomic diversity during plant development [3]. Alterna-
tiers of gene regulation mediated by plant RBPs, par- tive splicing of pre-mRNA requires different activities of the
ticularly focusing on RNA targets and the physiological spliceosome and specific binding site recognition of pre-
consequences of RBP–RNA interactions in the context mRNAs, both of which may depend on RBPs or protein
of specific developmental processes, including flower- subunits of the spliceosome. For example, glycine-rich
ing, stem cell maintenance, vascular development, RNA-binding proteins (GRPs) and RZ-1A-C, members of

Figure 1

(a) vegetative-reproductive phase transition (b) stem cell maintenance (c) vascular development
hnRNA-like normal condition stress condition AAAAAA
La1 La1

La1
alternative splicing of FLC I
alternative polyadenylation of FLC LG
P JU FIP3
BP PA
B
PA P
BP B
F3

PA FIP37 PA SMXL4/5 mRNA

F3
eI

FCA FPA FY HLP1 CPSF100

eI

eIF4G eIF4G JULGI

m6A methylation
La1
SMXL4/5 of unknown targets
m6A demethylation of FT, SPL3/7 La1
WUS mRNA

ALKBH10B phloem cambium xylem

cap-dependent translation IRES-mediated translation
FCA FY La1 JUL1
FPA HLP1

(d) hypocotyl elongation (e) root development

EIN2 air EIN2 ethylene nodule & LR primordia other tissues root hair
SE
low phosphate
C
-E

NSR1
ND
N

ND
D

A
AA
C-E

C-E

AA ENOD40 A RNA
GRP8 AA
ASCO-RNAs AA degradation
WRKY75 mRNA machinery

auxin related genes auxin related genes WRKY75

PHT1 CAX4 MOR1
ND

AAAAA AAAAA PKL

C-E

EIL1 NSR1
EIN3 alternative
splicing splicing
P-body
EIN3/
ethylene phosphate uptake root hair
EIL1
response AAAAA AAAAA termination

EIN2 NSR1 GRP8 SE

RBPs RNA binding proteins RRM WD HTH ZF Non-canonical

Current Opinion in Plant Biology

Examples of RBP-mediated regulatory mechanisms in plant growth and development. (a) Suppression of FLC by alternative splicing by GRPs and
RZ-1A-C and alternative polyadenylation by FCA, FPA, FY, HLP1 and AtCPSF100 triggers vegetative-reproductive phase transition. In addition,
stabilization of FT and SPL3/7 mRNA by m6A demethylase promotes the timing of flowering. (b) In SAM, La1 promotes cap-independent
translation of WUS under stress condition through redistribution to cytosol and direct recognition of IRES element. Moreover, FIP37 participates
WUS mRNA destabilization through m6A (blue dot). (c) Translational regulation of SMXL4/5 through RNA secondary structure and JULGI
interaction involves in phloem differentiation. FIP3 controls m6A methylation of unknown targets involved in xylem differentiation. (d) C-terminal
end (CEND) of EIN2 cleaved under ethylene directly binds to EBF1/2 mRNA, sequesters, and suppresses their translation to inhibit hypocotyl
elongation. (e) NSR1 controls auxin response in root developments by alternative splicing of auxin related genes. ENOD40 and ASCO-RNAs
compete with auxin related genes in terms of NSR1 binding and affect their alternative splicing. GRP8 controls phosphate uptake in hair cell,
presumably through direct interaction of WRKY75 mRNA, and SERRATE participates non-hair cell fate by regulating CAX4, MOR1, and PKL
mRNA stability independent of its miRNA pathway. Blue circles and orange lines indicate RBPs and target RNAs, respectively. The RNA-binding
domain of individual RBP is described in the corresponding figure. Orange, green, blue and red indicate RNA recognition motif (RRM), tryptophan-
aspartic acid repeat domain (WD), helix-turn-helix domain (HTH) and zinc-finger domain (ZF), respectively.

Current Opinion in Plant Biology 2019, 51:51–57 www.sciencedirect.com

 Emerging roles of RNA-binding proteins in plant development Cho, Cho and Hwang 53
the hnRNP-like RBPs, interact with SR proteins to promote
pre-mRNA splicing of diverse floral transition regulators,
including FLOWERING LOCUS C (FLC), MADS-box tran-
scription factor, a key repressor of flowering time [26,27].
Additionally, two plant-specific RRM domain-containing
RBPs, FLOWERING CONTROL LOCUS A (FCA)
and FPA, were characterized as flowering time regulators
[28,29]. Both FCA and FPA promote flowering by decreas-
ing FLC mRNA abundance through alternative polyadeny-
lation of its pre-mRNA or antisense-transcript (COOLAIR)
[30–32]. In addition, diverse polyadenylation factors, includ-
ing FY, hnRNP A1-like protein 1 (HLP1), and AtCPSF100,
were reported to control FCA pre-mRNA processing by
alternative polyadenylation on the distal and proximal sites,
consequently regulating flowering time [33–35].
In combination with pre-mRNA processing, a major
epitranscriptomic modification, N6-methyladenosine
(m6A), is important to regulate flowering time by control-
ling transcript stability [36]. The m6A demethylase
ALKBH10B removes m6A modifications on mRNAs
through specific RNA recognition and enzymatic activity
at the m6A site. Plants deficient in ALKBH10B activity
showed a significant delay in flowering time, whereas
overexpression of ALKBH10B resulted in early flowering
[37]. The direct binding of ALKBH10B to the mRNA of
FLOWERING LOCUS T (FT) [38], a key floral inducer,
and SQUAMOUSA PROMOTER BINDING PROTEIN-
LIKE 3/7 (SPL3/7) [39], key age-dependent flowering
time regulators, induced demethylation of these
transcripts and ultimately affected flowering time in each
loss-of-function or gain-of-function mutant [37]. Thus,
the dynamic effects of RBPs on RNA processing and
stability play important regulatory roles during
development and in response to changing environmental
conditions.
Stem cell maintenance: cap-independent
translation regulation and RBP-mediated RNA
modification
Recent studies have addressed gene-specific translation
regulation in the context of specific developmental
processes. The predominant mode of translation in
eukaryotes is 50 cap-dependent; translation initiation
begins when cap binding proteins, eukaryotic transla-
tion initiation factor 4E (eIF4E) recognize 7-methyl
guanosine (m7G) of mRNA [22]. Interestingly, under
environmental stress conditions when cap-dependent
translation is generally suppressed, internal ribosomal
entry site (IRES)-mediated translation initiation [40]
occurs for WUSCHEL (WUS) mRNA, a key
transcriptional regulator that controls stem cell activity
in the shoot apical meristem (SAM) [41] through the
action of the AtLa1 protein [42]. La proteins typically
contain an N-terminal La motif (LAM) and two RRM
domains [43], and they contribute to RNA processing
under normal conditions and to IRES-dependent
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translation during stress [44,45]. In Arabidopsis, AtLa1
moves from the nucleus to the cytosol in response to
stress, where it directly binds the IRES in the 50 UTR of
WUS mRNA [42], thus promoting WUS protein syn-
thesis. This translational regulation mechanism may
allow plants to maintain stem cell populations under
stress conditions through AtLa1-mediated gene-
specific control of translation.
Considering the role of AtLa1 in stem cell homeostasis
during stress, a genome-wide identification of its target
mRNAs, especially those exhibiting differential transla-
tion in stem cell populations, would allow us to expand
our understanding of how stem cells integrate external
cues to balance proliferation and differentiation. In
addition to translational control of WUS, the epitran-
scriptomic m6A marker [36] also plays a pivotal role by
affecting WUS mRNA stability [46]. Among the diverse
group of RBPs involved in m6A modification and
response, FKBP-interacting protein 37 (FIP37), a homo-
log of animal m6A modification component Wilm’s
tumor association protein (WTAP) [47], was recently
shown to function as an m6A modifier of WUS mRNA in
plants [48]. A mutant lacking FIP37 activity has an
enlarged stem cell population in the SAM, and the
m6A profile in the fip37 mutant showed strong reduction
of this modification, especially in key genes involved in
SAM function such as WUS [48]. A recent analysis of in
vivo WTAP targets identified AGGACU as an RNA-
binding motif for deposition of m6A [49], paving the way
for future studies exploring the role of this RNA modifi-
cation in developmental processes and how it contrib-
utes to the other roles of RBPs in RNA metabolism.
Vascular development: RNA
G-quadruplex-guided translational
regulation by RBPs
Similar to the structural importance of the IRES ele-
ment in translational regulation, an RNA
G-quadruplex, a secondary structure assembled from
Hoogsteen-bonded G-quartet stacks, was shown to
suppress translation in a mammalian system [50],
but the mechanistic bases of translational inhibition
and secondary structure formation have yet to be
determined. A recent study revealed that a novel
vascular plant-specific RBP, JULGI, contains three
RanBP2 (RAN-binding protein 2)-type zinc finger
domains, possesses single-strand RNA binding activ-
ity [51], and plays a crucial role in translational
regulation during plant development. Deficiency of
JULGI caused increased phloem formation, and a
combinational analysis of the phloem-specific tran-
scriptome and the intrinsic specificity of JULGI on
RNA sequences revealed that JULGI directly binds to
consecutive repeats of guanines and induces the for-
mation of an RNA G-quadruplex in the 50 -UTRs of
SUPPRESSOR OF MAX2-LIKE1-4/5 (SMXL4/5). The
Current Opinion in Plant Biology 2019, 51:51–57
54 Cell signalling and gene regulation
study also found that the JULGI-induced RNA G-
quadruplex in the 50 UTRs of SMXL4/5, key positive
regulators of phloem formation [52], suppressed trans-
lation presumably by attenuating the assembly of a
translational initiation complex, and consequently
inhibited phloem development. This work
provided the first mechanistic evidence on transla-
tional regulation via the RNA G-quadruplex through
RBP binding and its impact on the folding/unfolding
status of the complex, as well as its functional role in
phloem development. Interestingly, sucrose, the main
sugar transported in phloem, strongly attenuates
JULGI expression [51]. This implies that sucrose
plays an important role in phloem development
through direct regulation of translation, at least for
SMXL4/5. Furthermore, this observation suggests that
a sucrose-responsive RBP-SMXL4/5 module may func-
tion as a signaling integrator at the translational level
and relay information about the supply of cellular
energy in specific tissues of the plant. A transcrip-
tome-wide identification of JULGI targets and their
functional characterization would allow us to better
understand diverse vascular plant-specific post-
transcriptional regulatory mechanisms. In addition to
RBP-mediated translational regulation of phloem
development, one RBP required for the m6A modifi-
cation complex, FIP3, is also involved in vascular
pattern formation in protoxylem [47]. Deficiency of
RBP components of this complex led to strong defects
in protoxylem formation, suggesting that m6A modi-
fications of uncharacterized targets and subsequent
changes in RNA metabolism have important functions
in vascular cell development.
Hypocotyl elongation: control of local mRNA
distribution and translational regulation by
RBPs
Among the well-studied plant hormonal pathways, the
membrane-anchored ethylene signaling component
ETHYLENE INSENSITIVE 2 (EIN2), which plays
a critical role in the perception and signaling response
between the endoplasmic reticulum and nucleus
[53,54], was surprisingly found to be a non-canonical
RBP [55,56]. Upon perception of ethylene, the
C-terminal part of EIN2 is released from the mem-
brane, after which it acts as a translational regulator of
EBF (EIN3-BINDING F-BOX PROTEIN) 1/2 [57]
through direct recognition of 30 UTR poly-uridylate
(poly-U) motifs and sequestration of mRNA in
processing bodies (P-bodies) [55,56]. This suppres-
sive effect of ethylene on hypocotyl elongation
mediated by the RBP function of EIN2 (i.e.
sequestration of mRNA in P-bodies and translational
regulation), coupled with nuclear-localized EIN2
function, allows plants to respond to ethylene more
dynamically and reversibly depending on changes in
its concentration during hypocotyl growth.
Current Opinion in Plant Biology 2019, 51:51–57
Root development: impact of long non-coding
RNAs and RBPs on alternative splicing and
RNA stability
Another well-characterized RBP is the C-terminal RRM
domain-containing nuclear speckle RNA-binding protein
(NSR) [58]. Medicago truncatula NSR1 (MtNSR1) was
found to directly interact with a highly structured
RNA, EARLY NODULIN 40 (ENOD40), which encodes
short open-reading-frame-derived peptides and long non-
coding functions that are required for the formation of
nitrogen-fixing root nodules [59,60]. MtNSR1 is redis-
tributed from the nucleus to the cytoplasm, where
ENOD40 is expressed in nodule primordia and lateral
roots [61]. Two homologs of MtNSR1 in Arabidopsis,
AtNSRa and AtNSRb, interact with mRNA targets and
the long non-coding RNAs (lncRNAs) ENOD40 and
lnc351 [58]. AtNSR deficiency and ectopic expression of
alternative splicing competitor long non-coding RNAs
(ASCO-RNAs) resulted in differential auxin sensitivity
during lateral root development and changes in the
alternative splicing pattern of the auxin-responsive
transcriptome [58].
RNA-immunoprecipitation and other biochemical
techniques to assess splicing patterns in Atnsra/b
revealed that interactions between the ASCO-RNA
and AtNSR mediate an alternative splicing regulatory
module that controls lateral root development through
post-transcriptional remodeling of auxin-responsive
genes. Additionally, a recent global analysis of RNA–
protein interactions based on footprinting of RNases
(protein interaction profile seq) [62] using Arabidopsis
root hair and non-hair cell nuclei identified distinct
patterns of RNA secondary structure formation and
RBP binding profiles in these cell types [63]. Differ-
ential RNA secondary structure profiling enabled the
authors to identify tissue-specific RBPs and target sites,
such as GGN repeat motif binding of SERRATE and
TG-rich motif binding of GRP7/8 in hair cell nuclei.
Additional functional studies on potential targets of
these RBPs demonstrated that the mRNA abundance
of CATION EXCHANGER 4 (CAX4), MICROTUBULE
ORGANIZATION 1 (MOR1), and PICKLE (PKL) are
controlled by SERRATE independently of its micro-
RNA-pathway, and that mRNAs encoding phosphate
transporters are controlled by GRP8, presumably to
promote phosphate uptake in hair cells [63]. These
studies successfully identified novel RBPs using RNA
secondary structure-driven interaction screening
methods, providing new avenues to investigate biolog-
ically relevant RBPs with unique RNA structures/
sequences and explore novel RBPs involved in diverse
developmental processes in plants.
Concluding remarks
Despite recent advances in our understanding of the
roles of RBPs in development, few RBPs have been
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 Emerging roles of RNA-binding proteins in plant development Cho, Cho and Hwang 55
functionally explored in plants. By taking advantage of
high-throughput approaches, combined with affinity
capture of RNA–protein interactions, we are beginning
to understand the diversity of RBPs and their functions
[11,19–21,23,24,64]. A growing body of evidence sup-
ports multifaceted roles for RBPs, which not only bind to
and regulate target RNAs, but also possess enzymatic,
transport, and signaling activities [65]. In addition,
remarkable differences in RBP accumulation occur
under different physiological conditions, which implies
that post-transcriptional regulation and translational con-
trols are tightly associated with cell type and/or devel-
opmental stage [24]. Because of the dynamic nature and
spatio-temporal specificity of RBP–RNA interactions, a
more detailed determination of RBP targets and their
expression patterns in different developmental stages
will improve our understanding of RBPs and their under-
lying molecular mechanisms. Applications using cell
type-specific isolation and subcellular fractionation have
the potential to reveal the full spectrum of plant RBPs
and their roles in targeting RNA, and also to determine
the precise functions of RBP in the nucleus, cytosol, and
other organelles. Functional characterization of newly
identified RBPs and their RNA targets within a
physiological framework will pave the way for better
understanding the complexity and flexibility of post-
transcriptional and -translational regulation of plant
growth and development.
Conflict of interest statement
Nothing declared.
Acknowledgements
This work was supported by grants to I.H. from Basic Science Research
Program (2017R1A2A1A17069734) through the National Research
Foundation (NRF) funded by the Ministry of Science, ICT and Future
Planning and from the Cooperative Research Program for Agriculture
Science and Technology Development (PJ010953022018) funded by Rural
Development Administration, Korea.
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