MicroProteins

Small size-big impact

Credit
Seminar
M. Raghavendra
2013BS09D

Date: 17.10.2015
Time: 3:00 PM

MicroProteins (miPs)
Most biological processes require the formation of protein
complexes that are established through protein-protein
interaction (PPI) domains.
The formation, stability, and disruption of protein complexes are
dependent on various cellular factors, such as regulatory
proteins and post-translational modifications.
One important means by which complex formation is regulated
is by microProteins (miPs) mediated inhibition of complex
formation.

Eguen et al. 2015

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MicroProteins (miPs)
MiPs are short, usually single-domain proteins interact
(heterodimerize) with target and exert a dominant-negative
effect.
Although some miPs are not small but they are named so
-because the results of their actions are analogous to microRNAs
(miRNAs), which are negative regulators of mRNAs.
MiPs thus behave as post-translational regulators by forming
homotypic dimers with their targets, and act through the
dominantnegative suppression of protein complex function

Eguen et al. 2015

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 The first miP identified is that disrupt a functional transcription factor

complexinhibitor of DNA binding (Id).
It was isolated about two decades ago in a search for genes encoding
basic helixloophelix (bHLH) transcription factors from a murine
erythro leukaemia cell cDNA library.

Id protein

Id: Inhibitor of DNA binding

bHLH: basic helix loop helix

Id is missing the basic region adjacent to the HLH domain that is essential for
specific DNA binding in another HLH protein, MyoD.
In vitro studies shown that Id can bind with atleast three HLH TFs (MyoD,
E12 and E47)
This interaction attenuate the ability to bind DNA as homodimeric or
heterodimeric complexes Ex: Id prevents MyoD from binding to DNA.
Researchers assumed that Id acts in a negative manner to fine-tune muscle
development.
Later studies showed that Id has a higher affinity for the more ubiquitously
expressed E-type bHLH transcriptional regulators.
Suggesting that Id probably regulates the abundance of MyoD/E-type
heterodimers and thus allows the MyoD homodimers to exert their action in a
tissue-specific manner.

Model of miP interference

Targets of miPs are often
transcriptional regulators that
bind to DNA as active
homodimers.
miPs interfere with their targets
by forming nonfunctional
heterodimeric complexes that
cannot bind to DNA.

DBD: DNAbinding domain

PPI: proteinprotein interaction domain

Staudt & Wenkel 2011

Homotypic and heterotypic miP inhibition

MiPs can form inhibitory
complexes by interacting with
proteins that have protein
protein interaction (PPI)
domains
Homotypic miP: similar PPI (A)
Heterotypic miP: non-identical
compatible PPI (B)

Eguen et al. 2015

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MiPs inhibition are reported in

plants

HLH miP inhibition in plants

Arabidopsis
(Arabidopsis
thaliana)
brassinosteroid-sensitive
HLH,
PACLOBUTRAZOL RESISTANT1 (PRE1; 11 kDa) regulates petiole length.
In rice (Oryza sativa) homolog INCREASED LAMINA INCLINATION1
(ILI1; 11 kDa) lamina joint inclination (an erect leaf).

Staudt & Wenkel 2011

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PRE1 and IBH1 antagonistically regulate cell elongation

Overexpression of PRE1 and ILI1 increased
BR-induced cell elongation in both
Arabidopsis and rice.
Overexpression of IL1 BINDING bHLH
PROTEIN1 (IBH1) leads to dwarfism in
Arabidopsis, and this phenotype is suppressed
by overexpression of PRE1, indicating that
PRE1 promotes cell elongation by inactivating
IBH1.

PRE1
suppresses the
effects of IBH1

Col:
Arabidopsis
thaliana
Columbia wildtype control.
IBH1-Ox: Overexpression of IBH1
PRE1-Ox: Overexpression of PRE1
Bai et al. 2012
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The basic domain is not required for IBH1 function

Various degrees of dwarf phenotype observed among T1 plants

overexpressing wild-type IBH1.
Number and percentage of transgenic plants showing each
category of phenotype severity observed among populations of
transgenic plants overexpressing wild-type IBH1 or mutant IBH1
containing deletion of the basic domain (IBH1B) or of the AS
domain (IBH1AS).
Bai et al. 2012
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IBH1 Interacts with HBI1 in vitro and in vivo

By Arabidopsis interactome database It was found that IBH1 interact with bHLH
proteins BEE2, At2g18300 (HBI1 ), BPE,CIB1, At5g48560
By yeast two-hybrid assays it is confirmed the interactions.
Both HBI1 and BEE2 interact with IBH1 in yeast, and this interaction needs the IBH1 AS
domain but not the basic domain.
IBH1 interacts with PRE1 through its HLH domain and does not require the AS domain or basic
domain

HBI1:-

HOMOLOG OF
BEE2 INTERACTING WITH
IBH1.

BEE2:

BR ENHANCED
EXPRESSION2
CIB1: CYTOCHROME
INTERACTING BASIC HLH

Bai et al. 2012

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HBI1 promotes cell elongation

Representative plants of the wild type and HBI1 cosuppression (CS) and overexpression (#1 and 2#)
lines were grown in soil for 4 weeks.
Quantitative RT-PCR analyses of HBI1 expression in
plants

Zhang et al. 2009

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IBH1 and PRE1 antagonistically regulate the activity of HBI1

Overexpression of IBH1 or knockdown the expression of PREs

suppresses the phenotype ofHBI1-Ox. Plants and detached leaves.

Bai et al. 2012

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PRE-IBH1-HBI1 regulatory chain controls cell

elongation
Overexpression of PRE1 and ILI1 leads to increased petiole length
and lamina joint inclination in Arabidopsis and rice, respectively,
and increased sensitivity to brassinosteroids (BR).
Conversely, IBH1 overexpression leads to reduced sensitivity to BR,
smaller petioles in Arabidopsis and reduced lamina inclination in
rice
Overexpression of HBI1 results in longer petioles and longer
hypocotyls in Arabidopsis.
The antagonistic effect of miPs on their target proteins is a common
trait in miPs.

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R3-MYB miP

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Single repeat R3-MYB transcription factor

1. TRIPTYCHON
(TRY)
2. CAPRICE (CPC)
3. ENHANCER OF
TRY AND CPC1
(ETC1)
4. ETC2
5. ETC3/CAPRICELIKE MYB3
(CPL3)
6. TRICHOMELESS1
(TCL1)
7. TCL2/CPL4

Sequence signature [D/E]Lx2[R/K]x3Lx6Lx3R required for the interaction between

R3 MYBs and R/B-like bHLH transcription factors.
R3 MYBs contain a sequence motif WxM required for its cell-to-cell movement
R3 MYBs follow a pattern of Mx 18Wx18W, instead of [F/I]x18Wx18W that is typically
found in the R3 MYB repeat of R2R3-MYB proteins and MYBL2.
A single MYB repeat, R3 MYBs lack the activation domain that is typically present
in most transcription factors
Wang & Chen 2014

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 The few functions of R3-MYB are

Trichome development, Root hair development, anthocyanin
accumulation and stomatal formation.
The etc3/cpl3 mutant was shown to flower earlier with fewer leaves than
the wild-type.
GL3, EGL3, and TTG1 of the activator complex involved in the
regulation of seed coat mucilage and anthocyanin production(also
trichome and root hair formation).
CPC was shown to be a negative regulator of anthocyanin biosynthesis
and positive regulator of stomatal formation in the hypocotyl.
TRY is also expressed in inflorescence and siliques (Narrow elongated
seed capsule peculiar to the family Cruciferae).

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Promoter activity
The differences in tissue/organ expression patterns of R3 MYBs
determine the first level of specificities.
Among all seven R3 MYB genes, ETC2 and TCL1, TCL2/CPL4 was also not
detected or at very low level in the root . All of these three genes tested were
able to partially rescue the root hair phenotype of cpc mutant when their
expression was driven by CPC promoter.

Regulation of expression of R3 MYB genes is important for their

functioning .
The constructs (pETC3:ETC3, pTRY:ETC3, and pCPC:ETC3) were used to
complement triple mutants containing etc3.
pETC3:ETC3 construct rescued the cpc try etc3 mutant to the same extent
pTRY:ETC3and pCPC:ETC3 displayed an over-rescued phenotype resembling that of try.

Promoters of R3-MYBs are interchangeable (single mutant rescue)

When ETC3 was expressed under the promoter of ETC3, TRY or CPC, it could
equally rescue the trichome phenotype of etc3 mutant.

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Function of R3-MYBs in trichome development

Trichomes are hair cells produced by

the outward growth of epidermal cells.
Trichome initiation is promoted by an
activator complex consisting of a
WD40-repeat protein,
TRANSPARENT TESTA GLABRA1
(TTG1), an R2R3 MYB-type
transcription factor, GLABRA1
(GL1), and a bHLH transcription
factor, GLABRA3 (GL3) or
ENHANCER OF GLABRA3 (EGL3)

Fig: R3 MYB transcription factors function redundantly to control trichome

formation in Arabidopsis. In Col wild type plant (left), trichomes were observed on
leaves, lower part of stems and flower organs. Intry cpc etc1 tcl1quadruple mutant
(right), trichomes were observed in almost all aerial parts of the plants.

Wang & Chen 2014

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Modes of action of R3-MYBs

TTG1, GL3/EGL3, and GL1 form an activator
complex.
The activator complex regulate the transcription
of GL2 and R3 MYB genes.
GLABRA2 (GL2) is required for trichome
initiation.
R3 MYBs can move from a trichome precursor cell
(left) to its neighboring cell (right) to compete with
GL1 for binding

This results in decreased expression of GL2

In addition, some R3 MYBs, such as TCL1 and
TCL2, can suppress the transcription of GL1
These events create differences between cells and
ultimately result in a pattern of trichome and nontrichome cells

Wang et al.
2007

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Little Zipper proteins (ZPR)

are miPs

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ZPR proteins are structurally similar to ZIP motif

Phylogenetic analysis revealed that they can be classified into two
distinct groups:
1.
2.

ZPR1 and ZPR2

ZPR3 and ZPR4

ZPR1 and ZPR2 have N-terminal extensions which play a role in DNA
binding.
ZIP motif is located
1.
2.

C-terminal regions of ZPR1

and ZPR2.
N-terminal regions of ZPR3
and ZPR4.

ZPR Protein contain a single leucine zipper (ZIP) domain that

enables them to bind and regulate class III Homeodomain.
ZPR3 and ZPR4 play role in SAM Development, ZPR1 and ZPR2
linked to patterning of leaf polarity
Wenkel et al.
2007

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Regulatory modules LITTLE ZIPPER (ZPRs)

ZPR Protein bind and inhibit class III Homeodomain-leucine zipper
(HD-ZIPIII) proteins activation.
HD ZIP III are conserved plant proteins that act as potent regulators
of ad/abaxial polarity in Arabidosis.

Fig: ZPR proteins inhibit HD-ZIPIII

DNA binding activity by forming
heterodimers with them

Kim et al. 2008

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MINI ZINC FINGERs (MIFs)

miPs

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MINI ZINC FINGER (MIF)

Three Arabidopsis genes, MIF1, MIF2 andMIF3, encode
small zinc finger proteins that are highly conserved in
plants.
MIF1 constitutive overexpression inhibited cell division
and elongation
reduction in size of organs
morphological alteration of cotyledons and flowers and
ectopic root hairs on cotyledons.

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Protein structure of MIF and zinc finger

The MIF1 gene encodes protein of 101
aminoacid residues.
It is similar to N-terminal region of
ZF-HD proteins
The putative ZF domain contains eight
cysteine and/or histidine residues
(CX3HX11CX1226CX2CXCHX3H)
Full length aminoacid sequence
of MIF1

Hu & Ma 2006
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Expression pattern of MIF genes in Arabidopsis

A representative
example of three RTPCR examinations of
MIF1, MIF2, and MIF3;
APT1is used as the
internal control; the
cycle numbers of the
PCR reaction are
indicated at the right
of the images.
Hu & Ma 2006

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Phenotypic recovery of transgenic plants overexpressing ZF-HD5 by

MIF1 co expression
The MIF1 or ZF-HD5 gene
overexpressed under control
of CaMV 35 S promotor.
Expression of MIF1 and ZFHD5 genes in transgenic
plants (transcript levels of
qRT-PCR).

Hong et al.
2011
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MIF1 act as dominant negative regulators of ZF-HD5 transcription factors

Working model of
MIF1. The MIF1 protein
inhibits nuclear
localization of the ZHD5
protein by forming
MIF1-ZHD5
heterodimers in the
cytoplasm. In the
nucleus, the MIF1-ZHD5
heterodimers have a
lower DNA binding
activity compared with
that of the ZHD5
homodimers.

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There are 20 published bonafide Arabidopsis miP

Eguen et al.

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What is not miP?

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Proteins with sequence similarity to TFs but lack a DBD

The authors identified 438 TF-related proteins and referred to these as

putative miPs.
The absence of a DBD domain does not necessarily lead to an
inhibitory effect
But these does not fit into defined miP, such as size and dominantnegative mode of action.
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LITTLE SIPPER (SPPR)

52-kDa multidomain protein that
regulates HD-Zip IV FLOWERING
WAGENINGEN (FWA).
SPPR contained sequence similarity
to FWA interacted with it.
SPPR was not shown to sequester its
target FWA into a nonfunctional
complex and neither was it related to
the late flowering knockout
phenotype of fwa.

Mangnani et al.
2014
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Testing the effect of SPPR on

FWA
Plants carry thefwa-1mutation
show a late flowering phenotype.
Overexpressed SPPR using
constitutive 35S promoter in wildtype or fwa-1backgrounds
Seven of eight independent
transgenic lines overexpressing
SPPR in thefwa-1background
flowered significantly later
thanfwa-1plants.

Thus it was concluded that SPPR is a miP that functions synergistically

with its target; however, most known miPs are known for their antagonistic
effect on wild type alleles.
Mangnani et al.
2014

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RESPONSE TO ABSCISIC ACID AND SALT1 (RAS1)

RAS1 (26kDa), predicted to be involved in salt response related to

TGACG SEQUENCE-SPECIFIC BINDING PROTEIN

(TGA) a defense related TFs .
This protein also failed to live up to the promise of true miP.
RAS1 overexpression lines lacked any observable dominant
negative effect on TGA function.
SPPR and RAS1 fit the classification of modulatory proteins
which are capable of acting synergistically or
antagonistically to regulate the targets.

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 The putative list of 483 potential miPs that were published ranged in
size from 3 to 125 kDa.
Approximately 70% of which were above 20kDa size.
This list contains some known miPs and probably some undiscovered
miPs.
Most of the proteins in list are better classified as modulatory proteins
due to their size

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MiP biogenesis

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How did miPs arise?

Most miPs are trans-miPs

Mangnani et al.
2014
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Cis miPs
MADS box box TF genes FLOWERING LOCUS M (FLM) and
SHORT VEGETATIVE PHASE (SVP) have key roles.
FLM is subjected to temperature dependent alternative splicing
(FLM- and FLM-).
FLM- (at low temperature) and FLM- (at higher temperaturedominant negative activator) compete for interaction with the floral
repressor SVP.
Low ambient temperature favours the formation of SVPSVP and
SVPFLM- complexes that actively repress flowering.
These results show a new mechanism that controls the timing of the
floral transition in response to changes in ambient temperature.
Pose et al. 2013
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ZPR genes are phylogenetically derived from the C3HDZ genes.

ZPR evolution: degeneration of an ancient C3HDZ duplicate gene
(euphyllophyte: ferns). HD-ZIPIII acquire stop codon that resulted
in truncation (encoded the HD and ZIP domain). This truncation
was followed by loss of C-terminal genomic sequence and
sequential mutation of HD.
In Arabidopsis ZPR (ZPR1,2,3,4) are resulted from gene
duplication).

Floyd et al. (2014)

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Literature cited
Bai, M., Fan, M., Oh, E. & Wang, Z. (2012) A triple helix-loop-helix/ basic helixloop-helix cascade controls cell elongation downstream of multiple hormonal and
environmental signalling pathways in Arabidopsis. The Plant Cell, 24, 49174929.
Eguen, T., Straub, D., Graeff, M. & Wenkel, S. (2015) MicroProteins: small sizebig impact. Cell Press, 20, 477-482.
Floyd, S. K., Ryan, J. G., Conway, S .J., Brenner, E., Burris, K. P, Burris, J. N.,
Chen, T., Edger, P. P., Graham, S. W., Leebens-Mack, J. H., Pires, J. C., Rothfels,
C .J., Sigel, E. M., Stevenson, D. W., Stewart Jr, C.N, Wong, G. K. & Bowman, J.
L. (2014) Origin of a novel regulatory module by duplication and degeneration of
an ancient plant transcription factor. Molecular Phylogenetics and Evolution, 81,
159-173.
Hong, S., Kim, O., Kim, S., Yang, M. & Park, C. (2011) Nuclear import and DNA
binding of the ZHD5 transcription factor is modulated by a competitive peptide
inhibitor in Arabidopsis. The Journal of Biological Chemistry, 286, 1659-1668.

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 Hu, W. & Ma, H. (2006) Characterisation of a novel putative zinc finger

gene MIF1: involvement in multiple hormonal regulation of Arabidopsis
development. The Plant Journal, 45, 399-422.
Kim, Y., Kim, S., Lee, M., Lee, I., Park, H., Seo, P., Jung, J., Kwon, E.,
Suh, S. W., Paek, K. & Park, C. (2008) HD-ZIP III activity is modulated
by competitive inhibitors via a feedback loop in Arabidopsis shoot apical
meristem development. The Plant Cell, 20, 920-933.
Mangnani, E., Klein, N., Nam, H., Kim, J., Pham, K., Fiume, E.,
Mudgett, M.B. & Rhee, S.Y. (2014) A comprehensive analysis of
microproteins reveals their potentially widespread mechanism of
transcriptional regulation. Plant Physiology, 165, 149-159.
Pose, D., Verhage, L., Ott, F., Yant, L., Mathieu, J., Angenent, G. C.,
Immink, R. G. H. & Schmid, M. (2013) Temparature- dependent
regulation of flowering by antagonistic FLM variants. Nature, 000, 1-17.
doi:10.1038/nature12633.

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 Staudt, A., & Wenkel, S.(2011) Regulation of protein function by microproteins.

EMBO Reports, 12, 35-42.
Wang, S. & Chen, J. (2014) Regulation of cell fate determination by single repeat
R3-MYB transcription factors in Arabidopsis. Frontiers in Plant Science, 5 (133),
1-11.
Wang, S., Kwak, S., Zeng, Q., Ellis, B.E., Chen, X., Schiefelben, J. & Chen, J.
(2007) TRICHOMELESS1 regulates trichome patterning by suppressing
GLABRA1 in Arabidopsis. Development, 134, 3873-3882.
Wenkel, S., Emery, J., Hou, B., Evans, M. M. S. & Barton, M.K. (2007) A feedback
regulatory module formed by LITTLE ZIPPER and HD-ZIPIII genes. The Plant
Cell, 19, 3379-3390.
Zhang, L., Bai, M., Wu, J., Zhu, J., Wang, H., Zhang, Z., Wang, W., Sun, Y., Zhao,
J., Sun, X., Yang, H., Xu, Y., Kim, S., Fujioka, S., Lin, W., Chong, K., Lu, T. &
Wang, Z. (2009) Antagonistic HLH/bHLH transcription factors mediate
brassinosteroid regulation of cell elongation and plant development in Rice and
Arabidopsis. The Plant Cell, 21, 3767-3780.

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CONCLUSION
Microproteins (miPs) are small proteins that disrupt functional complexes.
MiPs have a dominant-negative mode of action.
All known bona fide Arabidopsis miPs are less than 20kDa.
Transcription factor-like proteins lacking dominant-negative effects are
not mips.
Microproteins (miPs) are short, usually single-domain proteins that, in
analogy to miRNAs, heterodimerize with their targets and exert a
dominant-negative effect.
Modulatory proteins misrepresented as miPs do not qualify as true miPs

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