PRL1, an RNA-Binding Protein, Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis
Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis. PLoS
Genet 10(12): e1004841. doi:10.1371/journal.pgen.1004841
PRL1, an RNA-Binding Protein, Positively Regulates the Accumulation of miRNAs and siRNAs in Arabidopsis
Shuxin Zhang 0
Yuhui Liu 0
Bin Yu 0
Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America
0 1 Center for Plant Science Innovation & School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America, 2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences & Key Laboratory of Agricultural Genomics, Ministry of Agriculture , Beijing , China
The evolutionary conserved WD-40 protein PRL1 plays important roles in immunity and development. Here we show that PRL1 is required for the accumulation of microRNAs (miRNAs) and small interfering RNAs (siRNAs). PRL1 positively influences the processing of miRNA primary transcripts (pri-miRNAs) and double-stranded RNAs (dsRNAs). Furthermore, PRL1 interacts with the pri-miRNA processor, DCL1, and the dsRNA processors (DCL3 and DCL4). These results suggest that PRL1 may function as a general factor to promote the production of miRNAs and siRNAs. We also show that PRL1 is an RNA-binding protein and associates with pri-miRNAs in vivo. In addition, prl1 reduces pri-miRNA levels without affecting pri-miRNA transcription. These results suggest that PRL1 may stabilize pri-miRNAs and function as a co-factor to enhance DCL1 activity. We further reveal the genetic interaction of PRL1 with CDC5, which interacts with PRL1 and regulates transcription and processing of pri-miRNAs. Both miRNA and pri-miRNA levels are lower in cdc5 prl1 than those in either cdc5 or prl1. However, the processing efficiency of pri-miRNAs in cdc5 prl1 is similar to that in cdc5 and slightly lower than that in prl1. Based on these results, we propose that CDC5 and PRL1 cooperatively regulate pri-miRNA levels, which results in their synergistic effects on miRNA accumulation, while they function together as a complex to enhance DCL1 activity.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This work was supported by National Science Foundation Grants MCB-1121193 (to BY). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
In plants and animals, microRNAs (miRNAs), ,2025
nucleotides (nt) in size, regulate gene expression in various
biological processes such as development and metabolism .
They are produced as duplexes through precise excision from
imperfect fold-back primary transcripts (pri-miRNAs) . In the
miRNA duplex, the miRNA strand is loaded into ARGONAUTE
(AGO) proteins to repress the expression of target genes containing
its complementary sequences while the other strand (passenger
strand; miRNA*) is degraded . Plants and animals also use
small interfering RNAs (siRNAs) to repress gene expression.
siRNAs are chemically identical to miRNAs . However they are
produced from long double stranded RNAs. The two major classes
of plant siRNAs are siRNAs derived from repeated DNAs
(rasiRNAs) and trans-acting siRNAs (ta-siRNAs) [4,5].
In plants, most pri-miRNAs are transcribed by DNA-dependent
RNA polymerase II (Pol II) from endogenous miRNA encoding
genes (MIR) [1,2]. The mediator complex and Negative on TATA
less2 (NOT2; a transcription factor) regulate the transcription of
MIR [6,7]. After generation, pri-miRNAs are proposed to be
stabilized by DAWDLE (DDL), an RNA binding protein .
PrimiRNAs are then processed to stem-loop precursors (pre-miRNAs)
and finally to the miRNA/miRNA* duplex by Dicer-LIKE 1
(DCL1; an RNAse III enzyme) in the nucleus in plants [9,10]. The
C2H2 zinc-finger protein SERRATE (SE) and the RNA binding
proteins HYPONASTIC LEAVES 1 (HYL1) and TOUGH
(TGH) form a complex with DCL1 to ensure efficient and
accurate process of pri-miRNAs [9,1117]. To ensure its proper
function, HYL1 needs to be dephosphorylated during pri-miRNA
processing . Several other proteins including DDL,
CapBinding Protein 20 (CBP20), CBP80, RACK1 and NOT2 are
associated with the DCL1 complex to facilitate miRNA
maturation [7,8,1921]. NOT2 and MODIFIER OF SNC1, 2 (MOS2;
an RNA binding protein) have been shown to guide the correct
localization of the DCL1 complex [7,22]. SICKLE (SIC; a proline
rich protein) is shown to regulate the accumulation of some
miRNAs . Besides protein factors, the structure of pri-miRNAs
plays essential roles in regulating DCL1 activity . For
instance, an imperfectly paired lower stem of ,15 bp below the
miRNA:miRNA* duplex is crucial for the initial pri-miRNA
We previously showed that Cell Division Cycle 5 (CDC5), a
DNA-binding protein, positively regulates miRNA biogenesis in
Arabidopsis . CDC5 interacts with Pol II and MIR promoters
. Lack of CDC5 reduces the occupancy of Pol II at MIR
promoters and pri-miRNA levels, suggesting that CDC5 is a
positive transcription factor of MIR . Besides acting as a
transcription factor, CDC5 functions as a co-factor of the DCL1
complex to participate pri-miRNA processing . CDC5 is a
PRL1, a conserved WD-40 protein, is required for plant
development and immune responses. However, its
functional mechanisms are not well understood. Here,
we show the positive impact of PRL1 on the
accumulation of miRNAs and siRNAs, which are key regulators of
plant growth and immunity. PRL1 interacts with multiple
DCLs (the processors of miRNAs and siRNAs) and is
required for their optimal activities, suggesting that PRL1
acts as a general factor to facilitate the production of
miRNAs and siRNAs. In addition, PRL1 is an RNA-binding
protein, binds pri-miRNAs in vivo and positively
influences the levels of pri-miRNAs levels without affecting the
promoter activities of genes encoding pri-miRNAs. These
results suggest that PRL1 may also stabilize pri-miRNAs.
We further show that RPL1 and its interactor CDC5 (a
DNA-binding protein) synergistically regulate pri-miRNA
levels, resulting in enhanced effects on miRNA
accumulation, although they function together as a complex to
facilitate DCL1 activity.
component of the conserved MOS4-associated complex (MAC).
MAC was first identified as a suppressor of snc1, which carries a
gain-of-function mutation in the SNC gene and show constitutive
resistance to a wide spectrum of pathogens . Loss-of-function
mutations in the MAC complex reduce plant immunity to
bacterial infections and cause multiple developmental defects such
as reduced fertility and delayed growth . The counterparts of
MAC in yeast and Human associate with spliceosome and
function in splicing . Other components of MAC include
MOS4 (a coil-coil domain containing protein), PRL1 (a WD-40
protein), MAC3A and MAC3B (two functionally redundant
Ubox E3 ubiquitin ligases). Among these proteins, PRL1 and MOS4
have been shown to interact with CDC5 directly .
In this study, we show that PRL1 but not MOS4 plays
important roles in the accumulation of miRNAs and siRNAs. Lack
of PRL1 in prl1 reduces miRNA accumulation and pri-miRNA
processing efficiency. In addition, PRL1 interacts with the DCL1
complex, suggesting it may function as co-factor of DCL1 to
promote miRNA maturation. Pri-miRNA levels are reduced in
prl1 relative to wild-type plants. However, MIR promoter activity
is not affected by prl1, despite of the association of PRL1 with Pol
II. Based on the facts that PRL1 is an RNA-binding protein and
binds pri-miRNAs in vivo, we propose that PRL1 may stabilize
pri-miRNAs. Furthermore, the levels of both miRNAs and
primiRNAs are further reduced in cdc5 prl1 relative to either cdc5 or
prl1. However, CDC5 and PRL1 do not show additive effects on
the processing of pri-miRNAs. These results suggest that CDC5
and PRL1 may synergistically influence pri-miRNAs levels and act
together as a complex to promote miRNA maturation. PRL1 also
interacts with DCL3 and DCL4, which produces siRNAs, and is
required for their optimal activities, suggesting that PRL1 may be
a general accessory factor for the production of small RNAs.
The accumulation of miRNAs and siRNAs is reduced in
Given the role of CDC5 in miRNA biogenesis, it is possible that
other components of the MAC complex may also be required for
miRNA accumulation. Therefore, we examined the effect of the
mutants mac3b (SALK_050811), mos4 (SALK_0090851C) and
prl1-2 on miRNA abundance using Northern blot. We also
included snc1 (SALK_047058C) in the analysis since SNC1 is
related to the MAC complex and snc1 causes development defects.
These mutants are likely null since the transcripts of corresponding
genes could not be detected by RT-PCR (Figure S1A). Like in
cdc51, the abundance of all four tested miRNAs (miR167, miR171,
miR172 and miR173) was decreased in prl1-2 compared to Col
(wild-type control). In contrast, miRNA levels in mos4, mac3b and
snc1 were comparable with those in Col (Fig. 1A). We examined the
accumulation of additional miRNAs in prl1-2 and found that all
these miRNAs were reduced in abundance in prl1-2 relative to Col
(Fig. 1B). In addition, expression a wild-type copy of PRL1 fused
with a YFP tag under the control of its native promoter
(pPRL1::PRL1-YFP) fully recovered miRNA levels in prl1-2
(Fig. 1B). These results demonstrated that PRL1 but not MOS4
and MAC3b is required for miRNA accumulation. We next
analyzed the transcript levels of several miRNA targets (ARF3,
CUC1, MYB33, MYB65 and PHV) in prl1-2 and Col by
quantitative RT-PCR (qRT-PCR) in order to test the effect of
prl1-2 on miRNA function. The transcription levels of these targets
were slightly increased in prl1-2 relative to Col (Figure. S1B). The
PRL1 transgene fully recovered miRNA function in prl1 (Figure
S1B). We also asked if PRL1 has a role in siRNA biogenesis. The
levels of nine examined siRNAs (three ta-siRNAs and six
rasiRNAs) were reduced compared to those in Col (Fig. 1B and 1C),
which was complemented by the expression of
pPRL1::PRL1YFP. These results revealed that like CDC5, PRL1 positively
regulates the levels of miRNAs and siRNAs in Arabidopsis.
PRL1 associates with Pol II and DCL1
The PRL1-CDC5 interaction suggests that similar to CDC5,
PRL1 may act as a co-factor of Pol II and DCL1 to regulate
miRNA accumulation. To test these two possibilities, we first
examined the PRL1-Pol II association using
co-immunoprecipitation (co-IP) assay. In this experiment, anti-YFP and anti-RPB2
that detects the second largest subunit of Pol II (RPB2)  were
used to capture the PRL1-YFP and Pol II complex, respectively,
from the protein extracts of prl1-2 complementation line
expressing the pPRL1::PRL1-YFP transgene. After IP,
PRL1YFP was detected in the Pol II precipitates whereas RPB2 existed
in the PRL1-YFP complex (Fig. 2A and 2B). In contrast, no
interaction was detected in the control reactions (Fig. 2A and 2B),
demonstrating the PRL1-Pol II association.
We next tested the association of PRL1 with the components of
DCL1 complex using a bimolecular fluorescence complementation
(BiFC). In the BiFC assay, transient co-expression of PRL1 fused
with C-terminal fragment of cyan fluorescent protein (cCFP) with
DCL1, SE, HYL1 or CDC5 fused with the N-terminal fragment of
Venus (nVenus) produced yellow fluorescence signals (Fig. 2C),
suggesting that PRL1 might associate with the DCL1 complex. To
verify this result, we tested co-IP of PRL1 with DCL1 and SE. After
PRL1-YFP or YFP was transiently co-expressed with DCL1-MYC
and SE-MYC fusion proteins in N. benthamiana, respectively, IPs
were performed with anti-YFP or anti-MYC antibodies. Western
blots detected PRL1-YFP in the DCL1-MYC and SE-MYC
complexes and DCL1-MYC/SE-MYC in the PRL1-YFP
precipitates, suggesting that PRL1-YFP and DCL1/SE reciprocally pulled
down each other (Fig. 2D, 2E, 2F and 2G). As a control, YFP did
not show interaction with either DCL1 or SE. These results
demonstrated the association of PRL1 with DCL1 and SE.
PRL1 positively influences pri-miRNA levels without
affecting MIR promoter activity
The interaction of PRL1 with Pol II suggests that PRL1 may
positively regulate MIR transcription. If so, lack of PRL1 will
Figure 1. PRL1 is required for the accumulation of miRNAs. (A) The effect of various MAC components on the abundance of miRNAs (B) The
levels of miRNAs are reduced in prl1-2. (C) The levels of siRNAs are reduced in prl1-2. Col: wild-type control. For miR159/319: upper band, miR159;
lower band, miR319. Northern blot was used to detect small RNAs and the radioactive signals were quantified with ImageQuant (V5.2). To determine
the amount of miRNAs/siRNAs in various mutants relative to that in Col, the radioactive signals of miRNAs/siRNAs were normalized to U6 RNA and
compared with that in Col (set as 1). The numbers indicate the average value of three repeats (P,0.05).
impair MIR transcription, resulting in reduced levels of
primiRNAs. To test this, we compared the pri-miRNA levels in
prl12 with those in Col using qRT-PCR. In fact, the levels of all seven
examined pri-miRNAs in prl1-2 were less than those in Col
(Fig. 3A), which were recovered in the complementation line of
prl1-2 (Fig. 3A). To test whether the reduction of pri-miRNA
levels is due to impaired MIR promoter activity, we introduced the
prl1-2 mutation into a Col transgenic line containing a single cope
of GUS transgene driven by MIR167a promoter
(pMIR167a::GUS), which was previously used to test the function of the
mediator complex in regulating MIR transcription . However,
GUS staining and qRT-PCR analysis showed a similar GUS
expression level in PRL1+ (PRL1/PRL1 or PRL1/prl1 genotype)
and prl1-2 containing the pMIR167a::GUS transgene (Fig. 3B
and 3C). This result demonstrated that PRL1 does not affect MIR
promoter activity. Consistent with this notion, prl1 did not show
obvious effect on MIR172b promoter activity (Figure S2A).
We next evaluated the effect of prl1-2 on the half-lives of
primiRNAs using cordycepin as a transcriptional inhibitor .
Twoweek old plants were transferred to medium containing cordycepin.
After incubation was stopped at various time points, we measured
the levels of pri-miR164a and pri-miR167a using qRT-PCR. The
results showed that the degradation rate of pri-miR164a and
primiR167a in prl1-2 is similar to that in Col (Figure S2B).
PRL1 functions in miRNA maturation
We next asked whether PRL1 has a role in processing of miRNA
precursors through an in vitro processing assay [13,31] since it is
associated with DCL1 and SE. In this experiment, a portion of
primiR162b that contains the stem-loop of miR162b with 6-nt arms at
each end (MIR162b; Fig. 4A) and pre-miR162b (Fig. 4B) were first
produced through in vitro transcription in the presence of [a-32P]
UTP. [32P]-labeled MIR162b and pre-miR162b were then processed
in the protein extracts of young flower buds of prl1-2 or Col. The
production of miR162b from both MIR162b and pre-miR162b in
prl1-2 at various time points was less than that in Col (Fig. 4C and
4D). The processing of MIR162b and pre-miRNA162b was
recovered in the PRL1 complementation line (Figure S3) The levels
of miR162 produced from MIR162b and pre-miRNA162b in prl1 at
80 min were ,40% of those produced in Col (Fig. 4E and 4F). These
results suggested that PRL1 might have a role in promoting miRNA
The role of PRL1 in siRNA biognesis
We next asked the role of PRL1 in siRNA biogenesis, as prl1-2
reduces the accumulation of siRNAs. By analog, we examined the
interaction of PRL1 with DCL3 and DCL4 and the effect of
prl12 on dsRNA processing. To test the PRL1-DCL3/DCL4
interaction, we expressed a recombined PRL1 fused with a
maltose-binding protein at its N-terminus (MBP-PRL1) and MBP
in E.coli. The protein extracts containing MBP-PRL1 or MBP
were mixed with protein extracts containing DCL3-YFP or
DCL4-YFP, which were transiently expressed in N. benthamiana.
Then the DCL3-YFP or DCL4-YFP complex was IPed with
antiYFP antibodies. MBP-PRL1, but not MBP, was co-IPed with
DCL3-YFP and DCL4-YFP (Fig. 5A). In addition, YFP did not
interact with MBP or MBP-PRL1. These results demonstrated
that PRL1 interacts with DCL3 and DCL4 (Fig. 5A).
To test the effect of prl1 on dsRNA processing, we generated
,460 bp dsRNAs through in vitro transcription of a DNA fragment
(59 portion of UBIQUITIN 5) containing the T7 promoter at the
end of each strand under the presence [a-32P] UTP. The
radioactive labeled dsRNA then incubated with prl1 or Col protein
extracts. The production of both 21 nt and 24 nt small RNAs was
impaired in prl1 compared with that in Col and that in the
complementation line (Fig. 5B). This result indicated that multiple
DCL activities are impaired by prl1-2, because DCL3 is responsible
for the production of 24 nt small RNAs and DCL1/DCL4 is
involved in the production of 21 nt small RNAs from dsRNAs.
PRL1 and CDC5 synergistically regulate miRNA
CDC5 and PRL1 have been shown to directly interact with each
other. Both CDC5 and PRL1 interact with DCL1 and positively
Figure 2. PRL1 associates with the Pol II and DCL1 complexes. (A) and (B) Co-immunoprecipitation (Co-IP) between PRL1 and Pol II. Protein
extracts from transgenic plants containing PRL1-YFP were incubated with Anti-YFP or anti-RPB2 antibodies to precipitate PRL1-YFP or Pol II. PRL1-YFP
and RBP2 were detected with western blot and labeled on the left side of the picture. Ten percent of input proteins were used for IP and one percent
of input proteins were used for Co-IP. (C) BiFC analysis of PRL1 with DCL1, HYL1, SE, AGO1 and CDC5. Paired cCFP- and nVenus-fusion proteins were
co-infiltrated into N. benthamiana leaves. The BiFC signal (Yellow fluorescence) was detected at 48 h after infiltration by confocal microscopy,
assigned as green color and marked with arrow. 30 nuclei were examined for each pair and an image is shown. Red: auto fluorescence of chlorophyll.
(D) and (E) Co-immunoprecipitation between PRL1 and DCL1. (F) and (G) Co-immunoprecipitation between PRL1 and SE. PRL1-YFP or YFP were
coexpressed with DCL1-MYC and SE-MYC in N. benthamiana, respectively. Anti-YFP and anti-MYC (MBL) antibodies were used to detect YFP- and
MYCfused proteins, respectively. The protein pairs in the protein extracts were indicated on the on tope of the picture and proteins detected by western
blot were indicated on the left side of the picture. Ten percent of input proteins were used for IP and one percent of inputs proteins were used for
regulate miRNA processing. These results raise a possibility that
CDC5 and PRL1 may act as a complex to regulate DCL1 activity. In
addition, CDC5 regulates MIR promoter activity while PRL1 does
not. These suggest that PRL1 and CDC5 might act additionally in
miRNA pathway. To test these two possibilities, we constructed a
cdc5-1 prl1-2 double mutant by crossing prl1-2 into cdc5-1 and
compared miRNA levels in cdc5-1 prl1-2 with those in cdc5-1 and
prl1-2, respectively. The cdc5-1 prl1-2 double mutant displayed more
severe developmental defects than either cdc5-1 or prl1-2, suggesting
that PRL1 and CDC5 function additionally in regulating
development (Fig. 6A). Northern blot analyses showed that the levels of
several examined miRNAs in cdc5-1 prl1-2 were lower than those in
either prl1-2 or cdc5-1 (Fig. 6B), indicating that PRL1 and CDC5
function synergistically in miRNA pathway.
There are at least two possible explanations for the further
reduction of miRNA levels in cdc5-1 pr1l-2 relative to either
cdc51 or prl1-2 based on the fact that both PRL1 and CDC5 positively
regulate pri-miRNA levels and miRNA maturation. One is that
pri-miRNA levels might be further reduced in cdc5-1 prl1-2. The
other is that the processing efficiency of miRNA precursors might
be lower than either cdc5-1 or prl1-2. To test these two
possibilities, we first determined the pri-miRNA levels in cdc5-1
prl1-2, cdc5-1 and prl1-2 through qRT-PCR. The levels of
several pri-miRNAs were decreased in cdc5-1 prl1-2 when
compared with those in either cdc5-1 or prl1-2 (Fig. 6C),
demonstrating that CDC5 and PRL1 indeed act synergistically
in regulating pri-miRNA levels. Next, we evaluated the in vitro
processing of pre-miR162b in cdc5-1 prl1-2. The amount of
miR162b produced in cdc5-1 prl1-2 was similar to that in cdc5-1
and slightly lower than that in prl1-2 (Fig. 6D), suggesting that
PRL1 and CDC5 may not act additionally in promoting miRNA
PRL1 binds pri-miRNAs in vitro and in vivo
Given the role of PRL1 in RNA metabolism, it is reasonable to
speculate that PRL1 might have an RNA-binding activity. We
therefore performed an in vitro RNA pull-down assay to test this
possibility. In this assay, recombinant PRL1 fused with a
maltosebinding protein at its N-terminus (MBP-PRL1) and MBP were
expressed in E.coli and purified with amylose resin (Fig. 7A).
MBP-PRL1 and MBP were then incubated with [32P]-labeled
MIR162b or pre-miR162b, respectively. MBP-PRL1 but not MBP
bound MIR162b and pre-miR162b after incubation. In addition,
when excess amount of unlabeled MIR162b or pre-miR162b was
added in the reaction, radioactive labeled MIR162b or
premiR162b could not be retained in the MBP-PRL1 complex. These
results suggested that PRL1 binds RNAs in vitro. We next tested
the binding ability of MBP-PRL1 to dsRNA, ssRNA and DNA
using the in vitro RNA pull-down assay described above.
MBPPRL1 was able to bind a ,100-nt RNA corresponding to a
portion of the 59 end of the UBIQUITIN 5 (UBQ5) , but not
an in vitro synthesized ,50 bp DNA fragment  and a dsRNA
generated through in vitro transcription vitro transcription of a
DNA fragment (59 portion of UBQ5, ,460 bp) containing the T7
promoter at end of each strand  (Fig. 7B).
Next, we performed an RNA immunoprecipitation (RIP) assay
to test whether PRL1 binds pri-miRNAs in vivo . Seedlings of
the prl1-2 complementation line expressing pPRL1::PRL1-YFP
transgene and the control plants harboring YFP were used for
RIP. After PRL1-YFP or YFP complex were precipitated with
anti-YFP antibody, pri-miRNAs were detected with RT-PCR.
Several tested pri-miRNAs (pri-miR159a, pri-miR167a,
primiR171 and pri-miR172a) existed in the PRL1-YFP complex
but not in the YFP complex and no Anti-body (NoAb) controls
(Fig. 7C). These results suggested that PRL1 associates with
primiRNAs in vivo.
In this study, we identify PRL1, a WD-40 protein, as an
important regulator of miRNA accumulation. Several evidences
including reduced accumulation of pri-miRNAs and miRNAs in
prl1, PRL1-DCL1 interaction and PRL1-pri-miRNA association
demonstrate that PRL1 positively impacts miRNA biogenesis. It
has been suggested that PRL1 influences plant immunity and
development through its impacts on RNA processing [29,33].
Given the essential roles of miRNAs in plant immunity and
development, it is possible that reduced miRNA levels in prl1 may
partially contribute to the observed phenotypes.
PRL1 likely has a role in promoting miRNA maturation, as lack
of PRL1 reduces processing of MIR162b and pre-miR162b. PRL1
interacts with the DCL1 complex and does not positively regulate
the transcription of genes involved in miRNA biogenesis (Figure
S4), suggesting that PRL1 may act as a co-factor to regulate DCL1
activity. CDC5, a direct interactor of PRL1 also regulates the
DCL1 activity through its interaction with the helicase and
dsRNA binding domains of DCL1. The effect of PRL1 on
primiRNA processing appears to be weaker than that of CDC5. The
processing efficiency of MIR162b and pre-miR162b in cdc5-1
prl1-2 is similar to that in cdc5-1 and slightly lower than that in
prl1-2. This result suggests that PRL1 and CDC5 may act
together as a complex to regulate DCL1 activity. Furthermore, gel
filtration analysis suggests that PRL1 may not affect DCL1-CDC5
association (Figure S5). Thus, it is possible that PRL1 may act as
accessory factor to facilitate CDC5 function.
PRL1 also positively regulates the pri-miRNA levels since prl1
reduces the accumulation of pri-miRNAs. We previously showed
that CDC5 interacts with Pol II and positively regulate MIR
transcription . Since PRL1 associates with Pol II as well, it is
possible that PRL1 acts as a component of the CDC5 complex to
regulate MIR promoter activity. However this seems not to be the
Figure 4. PRL1 is required for miRNA maturation in vitro. (A) and (B) A schematic diagram of the MIR162b (A) and pre-miR162b (B) used in vitro
processing assay. (C) and (D) The amount of miR162b produced from MIR162b and pre-miR162b were reduced in prl1-2. Proteins were isolated from
inflorescences of prl1-2 and Col and incubated with MIR162b or pre-miR162b. The reactions were stopped at various time points as indicated in the
picture. (E) and (F) Quantification of miR162b production in prl1-2 compared to that in Col. Quantification analysis was performed at 80 min. The
radioactive signal of miR162 were normalized to input and compared with that of Col. The amount of miR162 produced in Col was set as 1. The value
represents mean of three repeats (*** P,0.001; t-test).
case, as loss-of-function of PRL1 does not affect the GUS levels
driven by the MIR167a promoter. Consistent with this notion, the
levels of pri-miRNAs are further reduced in cdc5-1 prl1-2
compared with cdc5-1 or prl1-2. Given the fact that PRL1 binds
pri-miRNAs in vitro and vivo, we propose that PRL1 may stabilize
pri-miRNAs. Indeed, the fact that the half-life of pri-miR164a and
pri-miR167a in prl1 is similar to that in Col suggests that the
degradation of pri-miRNAs may be increased in prl1, because less
efficient processing may lead to increased abundance of
primiRNAs in prl1. However, we cannot rule out the possibility that
PRL1 acts in MIR transcription after initiation, as it associates
with Pol II.
In summary, we reveal that PRL1 positively regulates miRNA
levels through its impacts on pri-miRNA levels and processing.
PRL1 functions additively with its interactor CDC5 as miRNA
abundance is lower in cdc5-1 prl1-2 than in cdc5-1 or prl1-2. The
Figure 5. The role of PRL1 in siRNA biogenesis. (A) PRL1 interacts with DCL3 and DCL4. Co-IP was performed to detect the interaction of PRL1
with DCL3 or DCL4. MBP and MBP-PRL1 fused protein were expressed in E.coli. YFP, DCL3-YFP and DCL4-YFP were expressed in N. benthamiana
leaves. Anti-YFP was used for IP. For loading, ten percent and one percent of input proteins were used for IP and Co-IP, respectively. (B) prl1-2 impairs
siRNA production from double-stranded RNAs (dsRNAs). Protein extracts isolated from inflorescences of Col, prl1-2 and prl1-2 containing a PRL1-YFP
transgene were incubated dsRNAs for 120 min. dsRNAs were synthesized through in vitro transcription of a DNA fragment (59 portion of UBQ5 gene,
,460 bp) under the presence of [a-32P] UTP.
Figure 6. PRL1 and CDC5 synergistically regulate miRNA accumulation. (A) Morphological phenotypes of Col, cdc5-1, prl1-2 and cdc5-1
prl12. (B) The abundance of miRNAs is lower in cdc5-1 prl1-2 than that in cdc5-1 or prl1-2. Small RNAs were detected by Northern Blot. To determine the
amount of miRNAs, radioactive signals of miRNAs were normalized to U6 RNA. The number represents the relative abundance compared to Col (set
as 1) quantified by three repeats (P,0.05). (C) The abundance of pri-miRNAs is reduced in cdc5-1 prl1-2. The levels of pri-miRNAs in various mutants
were determined by qRT-PCR, normalized to UBQUITIN5 (UBQ5) and compared with those of Col (set as 1). Standard deviation of three technical
replications was shown as error bars. **: P,0.01. (D) miR162b production from pre-miR162b in Col, cdc5-1 prl1-2, cdc5-1 and prl1-2. The reaction was
stopped at 120 min. The radioactive signals of miR162b were normalized to input. The number represents the relative production in various
genotypes compared to Col (set as 1) quantified by three repeats (P,0.05).
synergistic effect of CDC5 and PRL1 on miRNA levels can be
explained by their different roles in controlling pri-miRNA levels
rather than their function in promoting miRNA maturation.
Besides CDC5 and PRL1, the core components MAC complex
includes MOS4, MAC3A and MAC3B . We show that MOS4
and MAC3b have no impact on miRNA levels. However, whether
MAC3B has a role in miRNA accumulation needs to be further
explored since it acts redundantly with MAC3A . The MAC
complex appears to have a role in siRNA biogenesis. Both CDC5
and PRL1 promote the accumulation of siRNA  while MOS4
is required for the accumulation of ra-siRNAs . How does
MAC participate in siRNA biogenesis? We have showed both
PRL1 and CDC5 interact with the DCL1 complex and regulate its
activity. By analogy, it is possible that the MAC complex associates
with the DCL3 complex to regulate its activity. In fact, DCL3
interacts with PRL1. prl1 also reduces the abundance of
tasiRNAs, whose production requires DCL4 and DCL1-dependent
miRNAs. Since PRL1 interacts with DCL4 and is required for the
accumulation of DCL1-dependent miRNAs, it may promote
tasiRNA production through facilitating DCL4 function and
miRNA production. The MAC complex is an evolutionarily
conserved complex . As many aspects of small RNA pathway
are conserved, it is tempting to propose that the counterparts of
MAC play some roles in small RNA pathways in other organisms.
Materials and Methods
The mac3b (SALK_050811), mos4 (SALK_0090851C), prl1-2
(Salk_008466), snc1 (SALK_047058C) and cdc5-1 (SAIL_207_F03)
mutants were ordered from Arabidopsis Biological Resources Center
(ABRC). All of them are in the Columbia-0 genetic background.
Transgenic line containing a single copy of pMIR167a::GUS was
crossed to prl1-2. In the F2 population, PRL1/PRL1, PRL1/prl1-2
and prl1-2/prl1-2 harboring pMIR167a::GUS were identified
through PCR genotyping for prl1-2 and GUS.
PRL1 genomic DNA was amplified from Col genome and
cloned to pMDC204 binary vector to generate
pPRL1::PRL1YFP construct. The construct was transformed to prl1-2. The
fulllength PRL1 cDNA was amplified by RT-PCR and ligated to
pMAL-c5x (NEB) to generate MBP-PRL1. To generate the
cCFPPRL1 fusion vector, the PRL1 cDNA was first cloned into the
pSAT4-cCFP-C vector . The DNA fragment containing
cCFP-PRL1 was released by I-SecI restriction enzyme and
subsequently cloned into the pPZP-RCS2-ocs-bar vector. All the
primers are listed in Table S1.
In the PRL1-PoII co-IP experiment, proteins were extracted
from the transgenic plants harboring the pPRL1::PRL1-YFP
transgene and incubated with anti-YFP (Clontech) or anti-RPB2
antibodies coupled to protein G agarose beads (Clontech) for 4 h
at 4uC. After the beads were washed five times with protein
extraction buffer, proteins were resolved by SDS/PAGE.
AntiYFP and anti-RPB2 antibodies were then used to detect
PRL1YFP and RPB2, respectively. To test the interaction of PRL1 with
components of the DCL1 complex, PRL1-YFP (YFP) was
coexpressed with DCL1-MYC or SE-MYC in N. benthamiana.
Protein extracts were then incubated with anti-YFP or anti-MYC
antibodies coupled to protein G agarose beads. Anti-YFP and
antiMYC (MBL) antibodies were used to detect PRL1-YFP/YFP and
Dicer activity assay
In vitro dicer activity assay was performed according to Qi et al
and Ren et al [13,31]. MIR162b and pre-miR162b RNAs were
produced by in vitro transcription under the presence of [a-32P]
UTP. In the dicer activity assay, protein extractions were incubated
with [32P] labeled MIR162b or pre-miR162b in reaction buffer
containing 100 mM NaCl, 1 mM ATP, 0.2 mM GTP, 1.2 mM
MgCl2, 25 mM creatine phosphate, 30 mg/ml creatine kinase and 4
U RNase inhibitor at 25uC. After the reactions were stopped at 40,
80 or 120 mins, respectively, RNAs were extracted and resolved on
PAGE gel. Radioactive signals were detected with a
PhosphorImager and quantified by ImageQuant version 5.2.
Paired cCFP and nVenus constructs were co-infiltrated in N.
benthamiana leaves for 40 h. YFP signals were then detected with a
confocal microscopy (Fluoview 500 workstation; Olympus) at 488 nm
with a narrow barrier (505525 nm, BA505525; Olympus).
Northern blot was used to detect small RNA abundance as
described . qRT- PCR was performed to detect the levels of
pri-miRNAs, transcripts of miRNA targets and GUS using cDNA
templates reverse transcribed by the SuperScript III (Invitrogen)
and oligo dT18 primer. qRT-PCR was run on an iCycler
apparatus (Bio-Rad). RNA pull-down were performed according
to Ren et al . MBP and MBP-PRL1 were expressed in E.coli.
MIR162b, pre-miR162b, dsRNAs and ssRNA were produced by
in vitro transcription with T7 RNA polymerase at the presence
[a-32P] UTP whereas DNA was synthesized at IDT and labeled
with T4 PNK at the presence [c-32P] ATP. [32P]-labeled probes
are incubated with amylose resin beads combined MBP or
MBPPRL1 at 4uC for 1 hour. After 4 times wash with washing buffer,
DNA or RNA are extracted and resolved on PAGE gel.
Radioactive signals were detected with a PhosphorImager and
quantified by ImageQuant version 5.2. RIP was performed
according to [13,36]. Seedlings of transgenic plants harboring
the pPRL1::PRL1-YFP transgene or YFP were used to examine
the RNA binding activity of PRL1 in vivo. All the primers are
listed in Table S1.
RNA half-life assay was performed according to Lidder et al
. Two-week-old Col and prl1-2 seedlings were transferred to
flask with incubation buffer (1/2 MS medium), respectively. After
30 min incubation, 39-deoxyadenosine (Cordycepin, Sigma) was
added to final concentration of 0.6 mM (time 0). Seedlings were
collected at various time points (0, 15, 30, 60, 90, 120 and
240 min). qRT- PCR then was performed to detect the transcript
levels of pri-miRNAs and DDL. For quantification, the transcript
levels of pri-miRNAs and DDL at various time points were
normalized to that of eIF4a, respectively. Value of time 0 was set
to 1. Error bars indicate standard deviation of three technical
replications. Three biological repeats were performed and similar
results were obtained.
Gel filtration analysis
The gel filtration was performed on an HPLC system and a
HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare) at a
rate of 0.5 ml/min, and 0.5 ml fractions were collected every
minute. Fractions were separated by 8% SDSPAGE and
analyzed by Western blotting using antibodies recognizing CDC
or YFP. The protein standards (Bio-Rad, http://www.bio-rad.
com/) were used to calibrate the column contain five size
Figure S1 (A) The expression of MAC3b, MOS4, PRL1 and
SNC1 in four null mutants detected by RT-PCR. The T-DNA
line of mac3b (SALK_050811), mos4 (SALK_090851C), prl1-2
(SALK_039427), snc1 (SALK_047058C) are all in Columbia-0
genetic background. (B) The transcript levels of several small RNA
targets in prl1-2, Col and complementation line. The amount of
target transcripts in prl1-2 and complementation line were
normalized with UBQUITIN5 (UBQ5) and compared with that
of Col (set as 1). Error bars indicate standard deviations of three
technical replications. *:P,0.05; **:P,0.01.
Figure S2 (A) The levels of GUS mRNA l in PRL1+ and prl1-2
harboring pMIR172a::GUS. GUS mRNA levels were determined
by qRT-PCR and normalized to UBQ5. Value of PRL1+ was set
to 1. Standard deviation of three technical replications was shown
as error bars. (B) Pri-miR164a, pri-miR167a and DDL mRNA
decay in the half-life assay. Two-week-old Col and prl1-2 seedlings
were treated with 0.6 mM 39-deoxyadenosine (Cordycepin,
Sigma) at various times (0, 15, 30, 60, 90, 120 and 240 min).
qRT-PCR was performed to detect pri-miRNA, and DDL
Figure S3 The PRL1-YFP transgene restores in vitro processing
of MIR162b and pre-miR162b in prl1-2. Protein extracts isolated
from inflorescences of Col, prl1-2 and prl1-2 containing a
PRL1YFP transgene were incubated with MIR162b and pre-miR162b
for 120 min.
Figure S4 The effects of prl1-2 on the expression of several
genes involved in miRNA biogenesis. (A) The transcript levels of
several genes involved in miRNA biogenesis determined by
qRTPCR. UBQ5 was used as a control. Standard deviation of three
technical replications was shown as error bar. (B) DCL1, (C)
HYL1 and (D) CDC5 protein levels in various genotypes detected
by western blot. Controls were dcl1-9 containing a truncated
DCL1 protein, hyl1-2 lacking of HYL1 and cdc5-1 lacking CDC5.
Figure S5 Gel filtration analysis of CDC5 and DCL1. Col and
prl1-2 protein extracts from inflorescences were separated by
HPLC. Eluted fractions were separated by SDSPAGE and
detected by Western blotting using anti-CDC5 or anti-DCL1
antibodies. Elution times of protein standards are shown on the
top of the blots.
Primers used in this study.
We thank Dr. Xin Li from University of British Columbia, Vancouver, BC,
Canada for providing the CDC5 antibody and Dr. Shengjun Li from the
University of Nebraska-Lincoln for critical reading of the manuscript.
Conceived and designed the experiments: SZ YL BY. Performed the
experiments: SZ. Analyzed the data: SZ YL BY. Contributed reagents/
materials/analysis tools: SZ YL BY. Wrote the paper: SZ YL BY.
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