STV1, a ribosomal protein, binds primary microRNA
transcripts to promote their interaction with the
processing complex in Arabidopsis
Shengjun Lia, Kan Liua, Shuxin Zhanga,b, Xiaoyan Wangc, Kestrel Rogersd, Guodong Renc, Chi Zhanga, and Bin Yua,1
a
Center for Plant Science Innovation & School of Biological Sciences, University of Nebraska, Lincoln, NE 68588–0660; bState Key Laboratory of Crop Biology,
College of Life Sciences, Shandong Agricultural University, Tai’an 271018, China; cState Key Laboratory of Genetic Engineering, Collaborative Innovation
Center for Genetics and Development, School of Life Sciences, Fudan University, Shanghai 200438, China; and dDepartment of Botany and Plant Sciences,
Institute of Integrative Genome Biology, University of California, Riverside, CA 92521
Edited by Richard Scott Poethig, University of Pennsylvania, Philadelphia, PA, and approved December 27, 2016 (received for review August 5, 2016)
MicroRNAs (miRNAs) are key regulators of gene expression. They
are processed from primary miRNA transcripts (pri-miRNAs), most of
which are transcribed by DNA-dependent polymerase II (Pol II).
miRNA levels are precisely controlled to maintain various biological
processes. Here, we report that SHORT VALVE 1 (STV1), a conserved
ribosomal protein, acts in miRNA biogenesis in Arabidopsis. A por-
tion of STV1 localizes in the nucleus and binds pri-miRNAs. Using pri-
miR172b as a reporter, we show that STV1 binds the stem-loop
flanked by a short 5′ arm within pri-miRNAs. Lack of STV1 reduces
the association of pri-miRNAs with their processing complex. These
data suggest that STV1 promotes miRNA biogenesis through facili-
tating the recruitment of pri-miRNAs to their processing complex.
Furthermore, we show that STV1 indirectly involves in the occu-
pancy of Pol II at the promoters of miRNA coding genes (MIR) and
influences MIR promoter activities. Based on these results, we pro-
pose that STV1 refines the accumulation of miRNAs through its
combined effects on pri-miRNA processing and transcription. This
study uncovers an extraribosomal function of STV1.
miRNA biogenesis | Arabidopsis | STV1 RNA binding | pri-miRNAs
M icroRNAs (miRNAs) are noncoding RNAs that regulate
multiple biological processes by inhibiting gene expression
at posttranscriptional levels (1–3). The majority of miRNA-coding
genes (MIR) are transcribed by the DNA-dependent RNA poly-
merase II (Pol II) to produce primary miRNA transcripts (pri-
miRNAs) (4). In Arabidopsis, a complex containing DICER-
LIKE1 (DCL1, an RNase III enzyme), HYPONASTIC LEAVES
1 (HYL1, a double-stranded RNA-binding protein), SERRATE
(SE, a Zinc-finger protein), and TOUGH (TGH, an RNA-binding
protein) process the imperfect stem-loops residing in the pri-
miRNAs to release the miRNA/miRNA* (passenger strand) du-
plex in the nucleus (3, 5–9). The miRNA duplex is then methylated
by HUA1 ENHANCER1 (HEN1) to prevent uridylation and
degradation (3). Following biogenesis, miRNAs are loaded into the
effector protein called ARGONAUTE 1 (AGO1), which requires
HSP90 and the Cyclophilin 40 (CYP-40), to repress the expression
of genes containing their homolog sequences through mRNA
cleavage and translational inhibition in Arabidopsis (10–13).
Plant miRNA biogenesis is regulated through pri-miRNA tran-
scription, stability, and processing (14). In Arabidopsis, the tran-
scription factors, including the mediator complex, the elongator
complex, Negative on TATA less 2 (NOT2), and CELL DIVISION
CYCLE 5 (CDC5) have been shown to positively regulate MIR
transcription through recruiting Pol II to MIR promoters (15–17).
The transcription of some MIRs, such as MIR172 and MIR156, is
also subject to physiological, temporal, or spatial regulation by spe-
cific transcription factors (18, 19). The RNA-binding proteins
DAWDLE (DDL) and PLEIOTROPIC REGULATORY LOCUS
1 (PRL1) are proposed to stabilize pri-miRNAs following their
transcription (14, 20). Several protein factors, including DDL,
CDC5, PRL1, and CBP20/80 (two cap-binding proteins), associate
1424–1429 | PNAS | February 7, 2017 | vol. 114 | no. 6
with the DCL1 complex to promote pri-miRNA processing (14, 17,
20–22), whereas the elongator complex, NOT2 and MODIFIER OF
SNC1, 2 (MOS2, an RNA-binding protein), are required for the correct
assembly of the DCL1 complex (16, 23). Proteins including SICKLE
(SIC, a proline-rich protein), RECEPTOR FOR ACTIVATED
C KINASE 1 (RACK1), STABILIZED1 (STA1, a pre-mRNA
processing factor 6 homolog), HOS5 and GRP7 (a glycine-rich
RNA-binding protein), act outside of the DCL1 complex to
participate in pri-miRNA processing (24–29). Protein modifications
also regulate the activity of the DCL1 complex. For example, HYL1
activity is controlled by phosphorylation and dephosphorylation (30,
31). Furthermore, the expression levels of the DCL1 complex are
regulated at transcriptional and posttranscriptional levels to influ-
ence miRNA accumulation (32–34). The structure and length of
the stem-loop also impact pri-miRNA processing. For example, the
presence of an internal loop following a ∼15-bp stem below the
miRNA/miRNA* is required for efficient processing of some pri-
miRNAs (35–39). Despite these progresses, it is still enigmatic how
the DCL1 complex recognizes pri-miRNAs, because the stem loops
of pri-miRNAs are diversified in structures, lengths, and sequences.
Ribosomal proteins are ribosome components responsible for
ribosome assembly and protein translation. Because of their ability
to interact with RNAs and proteins, their extraribosomal functions
have been proposed (40). However, most extraribosomal functions
of ribosomal proteins are still related to the ribosome or its syn-
thesis. Thus, the real extraribosomal functions of ribosomal pro-
teins still remain to be uncovered (40). The ribosomal protein L24
(RPL24) exists in archaebacteric and eukaryotic, but not pro-
karyotic, ribosomes (41); It facilitates the interaction between the
ribosomal large subunit and the small subunit in archaebacteria (41).
Significance
SHORT VALVE 1 (STV1), a ribosomal protein, is required for the
development of Arabidopsis. However, its functional mechanism
remains to be identified. This research shows that STV1 binds the
stem-loop flanked by a short 5′ arm within primary miRNAs and
facilitates the recruitment of primary miRNAs to the DICER-LIKE1
complex. Consequently, this study provides insights into the
mechanisms controlling miRNA production and identifies an
extraribosomal function of STV1. Because STV1 is a conserved
protein in eukaryotes, the results may produce a broader impact.
Author contributions: S.L. and B.Y. designed research; S.L., K.L., S.Z., X.W., K.R., G.R., and
C.Z. performed research; K.L. and C.Z. analyzed data; and S.L. and B.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The data reported in this paper have been deposited in the Gene Ex-
pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE91013).
1
To whom correspondence should be addressed. Email: byu3@unl.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1613069114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1613069114
However, in yeast, RPL24 is not essential for cell viability (42). In
Arabidopsis, SHORT VALVE 1 (STV1), an RPL24 homolog, is
required for the translation initiation of some proteins including
ETT and MP, two auxin response factors, involved in gynoecium
patterning (43). However, whether it functions outside ribosome is
not known.
Here, we report that STV1 is required for miRNA accumulation
in Arabidopsis. Lack of STV1 in stv1-1 alters the occupancy of Pol
II at MIR promoters and influence MIR transcription. However,
STV1 does not interact with Pol II and MIR promoters, indicating
that STV1 may indirectly regulate MIR transcription. A portion of
STV1 localizes in the nucleus and directly binds pri-miRNAs
in vivo. STV1 interacts with the stem-loop flanked by a short 5′ arm
within pri-miRNAs. In addition, both the stem-loop and the short
5′ arm are required for STV1 binding. In stv1, the association of
pri-miRNAs with HYL1 is reduced. These results suggest that
STV1 directly acts in miRNA biogenesis through facilitating the
recruitment of pri-miRNA to the processing complex.

Results
STV1 Is Required for Proper miRNA Accumulation. Besides abnormal
vascular patterning and gynoecium structures (43), the stv1 mu-
tants displayed other developmental defects, such as delayed

growth, smaller plant size, clustered inflorescence, shorter si-
liques, and reduced fertility (Fig. S1 A and B). We suspected that
the developmental defects of stv1 could result from the impaired
translation at global levels, because STV1 is a ribosomal protein.
To test this possibility, we compared the polysomes profile be-
tween Columbia (Col) and stv1-3 (Salk_045401, in Col genetic
background) (Fig. S1C). Both 80S monosomes and polysomes
were largely intact in stv1-3, although their relative abundance
was slightly different with that in Col (Fig. S1C). This result in-
dicates that like in other organisms, STV1 may not play essential
roles in protein translation in Arabidopsis.
We next tested miRNA accumulation in inflorescences of stv1-1
because impaired miRNA biogenesis is known to cause pleiotro-
pic developmental defects. Indeed, quantitative RT-PCR (qRT-
PCR) analyses showed that several examined miRNAs displayed
increased or decreased abundance in stv1-1 [Wassilewskija (WS)
genetic background] relative to WS (WT) (Fig. 1 A and B).
Northern blot analyses further confirmed reduced accumulation
of miR167 and miR169 and elevated abundance of miR173 in
stv1-1 relative to WS (Fig. 1C). To demonstrate that loss-of-
function of STV1 is responsible for the alteration of miRNA ac-
cumulation in stv1, we introduced an STV1 genomic fragment
under the control of its native promoter (pSTV1::STV1-MYC) into
stv1-1. The expression of STV1-MYC rescued the developmental
defects of stv1-1 and fully recovered miRNA levels (Fig. S2 A–E).
Next, we analyzed the miRNA profile of inflorescences from WS
and stv1-1 through deep sequencing. The abundance of most
miRNAs was altered in stv1-1 relative to WS in two biological
replicates (Fig. S2F and Dataset S1). We further asked whether
STV1 could affect the accumulation of transacting siRNAs (ta-
siRNAs). We found that siR255 and 1511, two examined ta-siRNAs,
were increased in abundance in stv1-1 relative to WS (Fig. 1 B and
C). However, the increased levels of siR255 and 1511 are likely
caused by elevated abundance of miR173, which triggers the
production of siR255 and 1511.
We then examined the transcript levels of miRNA targets in
stv1-1 and WS using qRT-PCR. The result showed that ARF6/8,
ARPN, CKB3, CUC1, HAP2b, PHO2, SCL6, SPL9/10, SUVH6,
and TCP3/24, which are the target of miR167, miR408, miR397,
miR164, miR168, miR399, miR171, miR156, miR778, and miR319
(reduced levels in stv1-1), respectively, were increased in abun-
dance (Fig. 1D). In contrast, the accumulation of ARLPK2, BOP1,
and CMT3, which are the targets of miR863, miR836 and miR823
(elevated levels in stv1-1), was reduced (Fig. 1D). These results
PLANT BIOLOGY
Fig. 1. stv1-1 influences the accumulation of miRNAs and ta-siRNAs. (A and
B) miRNA level in stv1-1 and WS detected by qRT-PCR. miRNA levels in stv1-1
were normalized to those of U6 RNA and compared with WS.Values of WS
were set as 1. Error bars indicate SEs (SD) of three replicates (**P < 0.01).
(C) The expression levels of small RNAs in WS and stv1-1 detected by Northern
blot. U6 RNA was used for the loading control. The relative abundance of small
RNAs is shown below the picture and represents the mean of three repeats
(*P < 0.05). For miR159/319, the upper band was miR159 and the lower band
showed miR319. (D) The expression levels of miRNA target genes in WS and
stv1-1 detected by qRT-PCR. The expression levels of miRNA targets were
normalized to those of UBQ5 and compared with WS. Error bars indicate SEs
(SD) of three replicates (*P < 0.05; **P < 0.01).
suggest that alterations of miRNA abundance may influence the
expression levels of some targets.
A Portion of STV1 Localizes in the Nucleus. Because plant miRNA
biogenesis occurs in the nucleus, STV1 should localize in the nucleus
if it had a direct role in miRNA generation. To test this possibility,
we expressed GFP-STV1 and STV1-GFP driven by the native STV1
promoter in stv1-1, respectively. However, both transgenes could not
complement the defection caused by stv1, which is likely because the
GFP protein (∼26 kDa) interfered the function of STV1 (∼19 kDa).
Thus, to determine STV1 localization, we prepared proteins from
nuclear and cytoplasmic fractions of three independent comple-
mentation lines harboring pSTV1::STV1-MYC and examined the
presence of STV1-MYC in each fraction by Western blot. As
expected, the majority of STV1 localized in the cytoplasm (Fig. 2A
and Fig. S2G). However, a portion of STV1 did exist in the nucleus
(Fig. 2A and Fig. S2G). The presence of STV1 in the nucleus was
unlikely to be caused by the contamination of cytoplasmic proteins,
because phosphoenolpyruvate carboxylase (PEPC), a cytoplasmic
marker protein, could not be detected in the nuclear fraction (Fig.
2A and Fig. S2G). These results suggest that STV1 may function in
the nucleus and participate in miRNA biogenesis.
STV1 Indirectly Influences MIR Transcription. There are at least two
possible ways by which STV1 acts in miRNA biogenesis. It may
regulate the transcription of pri-miRNAs or participate in pri-
miRNA processing. We first evaluated pri-miRNA levels in stv1-1

Li et al. PNAS | February 7, 2017 | vol. 114 | no. 6 | 1425

Fig. 2. STV1 influences the transcription of genes encoding miRNAs. (A) The STV1 protein localizes at the cytoplasm and nucleus. MYC-STV1 and PEPC from
nuclear and cytoplasmic fractions were detected with anti-MYC and anti-PEPC antibodies, respectively, and indicated on the right. (B and C) stv1-1 alters the
expression levels of pri-miRNAs. The expression levels of pri-miRNAs in stv1-1 were analyzed by qRT-PCR, normalized to those of those of UBQ5, and com-
pared with WS (values were set as 1). Error bars indicate SEs (SD) of three replicates (*P < 0.05; **P < 0.01). (D) GUS transcript levels in STV1+ and stv1-1
harboring the pMIR167a:GUS transgene detected by qRT-PCR. GUS transcript levels were normalized to those of UBQ5 and compared with WS (values were
set as 1). Error bars indicate SEs (SD) of three replicates (**P < 0.01). STV+: STV1/STV1 or STV1/stv1-1. (E) The occupancy of Pol II at MIR promoters in WS and
stv1-1 detected by ChIP with anti-RPB2 antibodies. The intergenic region between At2g17470 and At2g17460 (Pol II C1) serves as a negative control for Pol II
occupancy. Means and standard derivation of three technical repeats are presented. (**P < 0.01). (F) STV1 does not associate with RPB2 by co-IP. MYC-STV1
was immunoprecipitated with anti-MYC antibodies. MYC-STV1 and RPB2 were detected with anti-MYC and anti-RPB2 antibodies, respectively. (G) STV1 does
not associate with MIR promoters detected by a ChIP assay. Pol II C1 was used as a control.

using qRT-PCR. Among 25 examined pri-miRNAs, 21 pri-miR-
NAs were either decreased or increased in abundance in stv1-1
compared with those in WS, whereas the remaining 4 were not
affected by stv1 (Fig. 2 B and C and Fig. S3A). Because tran-
scription is one of several factors affecting pri-miRNA levels, we
examined the effect of STV1 on MIR promoter activity using a GUS
reporter gene driven by the MIR167a promoter (pMIR167a::GUS),
which has been used to study the functions of mediator complex
and PRL1 in miRNA biogenesis (15, 39). We crossed the
transgenic line harboring pMIR167a::GUS to stv1-1. In the F2
generation, we obtained the STV1+(STV1/STV1 or STV1/stv1)
and stv1-1 genotypes containing the pMIR167a::GUS transgene,
respectively. The GUS mRNA level was ∼40% lower in the stv1-1
mutant than that in STV+ background (Fig. 2D), indicating that
STV1 might affect miRNA promoter activity. To validate this
result, we checked the occupancy of Pol II on MIR promoters
using ChIP assay with anti-RNA polermerase II (RPB2, the sec-
ond largest subunit of Pol II) antibodies. As expected, the ChIP
signals of MIR promoters were enriched in Pol II immunopre-
cipitates relative to “no-antibody” controls in WS (Fig. 2E). Com-
pared with WS, stv1-1 reduced Pol II occupancy at the promoters of
MIR159A, MIR167A, and MIR171A, but increased Pol II occupancy
at the promoters of MIR166A, MIR173 and MIR863 (Fig. 2E).
These results were consistent with reduced levels of pri-miR159a,
pri-miR167a, and pri-miR171a and increased levels of pri-miR166a,
pri-miR173, and pri-miR863, which support the potential role of
STV1 in regulating MIR transcription.
Next, we asked if STV1 has a direct role in promoting MIR
transcription. First, the association of Pol II with STV1 in the stv1
complementation line was examined using a coimmunoprecipita-
tion (co-IP) assay. However, STV1 did not coprecipitate with RPB2
(Fig. 2F). Next, we tested if STV1 occupied at MIR promoter using
ChIP assay. In the stv1 transgenic line harboring pSTV1::STV1-
MYC, the occupancy of STV1 in the promoters of MIR159A,
MIR66A, MIR167A, MIR171A, MIR173, and MIR863 was not de-
tected (Fig. 2G). These results indicate that STV1 may indirectly
regulate MIR transcription.
STV1 Does Not Associate with the DCL1 Complex. Next we asked if
STV1 has a role in pri-miRNA processing. We tested if STV1
affected the expression levels of genes that are involved in pri-
miRNA processing using qRT-PCR. stv1-1 did not significantly
change the transcript levels of DCL1, HYL1, HEN1, and CPB80,
although it slightly increased the expression levels of DDL, SE,
and CPB20 (Fig. S4A). Consistent with these results, stv1-1 did
not show effect on the protein levels of HYL1, SE, and DCL1
(Fig. S4B). We next examined if STV1 acted as a component of
the DCL1 complex to participate in pri-miRNA processing be-
cause its homologs in other organisms can function through
protein–protein interactions. To test the interaction of STV1
with DCL1, HYL1, SE, DDL, and CDC5, we coexpressed MYC-
STV1 with DCL1-YFP, SE-YFP, HYL1-YFP, YFP-DDL, or YFP-
CDC5 in Nicotiana benthamiana and performed co-IPs assay
using anti-MYC antibodies. STV1 did not interact with DCL1,
HYL1, SE, DDL, and CDC5 (Fig. 3A and Fig. S4 C and D),
indicating that STV1 may not associate with the DCL1 complex.
STV1 Binds pri-miRNAs in Vivo. The facts that STV1 homologs as-
sociate with ribosome RNAs (41), and that a portion of STV1 exists
in the nucleus, prompted us to test if STV1 binds pri-miRNAs
in vivo. RNA immunoprecipitation assay (RIP) was performed on
the seedlings of stv1-1 harboring the pSTV1::STV1-MYC transgene.
After cross-linking, nuclear isolation, and IP, RT-PCR analyses
showed that pri-miR167a, pri-miR172b, pri-miR173, pri-miR408,
and pri-miR863 were enriched in the MYC-STV1 immunoprecip-
itates, but not in the no-antibody control or IPs from the control
plants (Fig. 3B). In addition, the control UBIQUITIN 5 (UBQ5)

1426 | www.pnas.org/cgi/doi/10.1073/pnas.1613069114 Li et al.


Fig. 3. STV1 associates with pri-miRNAs but not the DCL1 complex. (A) STV1
does not interact with DCL1, HYL1, and SE. MYC-STV1 was coexpressed with
DCL1-YFP, SE-YFP, or HYL1-YFP in N. benthamiana. After protein extraction,
MYC-STV1 was immunoprecipitated with anti-MYC antibodies. MYC-STV1 and
YFP-tagged proteins were detected with anti-MYC and anti-YFP antibodies,
respectively, and indicated on the left. The interaction of SE and CDC5 is shown
as a positive control. Numbers on top of the pictures indicate protein extracts
containing: 1, YFP and MYC-STV1; 2, DCL1-YFP and MYC-STV1; 3, YFP and
MYC-STV1; 4, SE-YFP and MYC-STV1; 5, YFP and MYC-STV1; 6, HYL1-YFP and
MYC-STV1; 7, YFP and MYC-SE (Middle); 8, YFP-CDC5 and MYC-SE (Top).
(B) STV1 binds pri-miRNAs in vivo detected by RIP. Inflorescences from WS and
stv1-1 harboring MYC-STV1 were used for RIP assay with anti-MYC antibodies.
No antibody (No Ab.) was used as a negative control. Ten-percent of IPs and
2% input proteins were used for Western blot. Five percent RNAs were used as
input RNA. STV1 on the top of the picture indicates MYC-STV1.
mRNA was not detected in the STV1 complex (Fig. 3B). These
results show that STV1 binds pri-miRNAs in vivo.
STV1 Binds a pri-miR172b Fragment Containing the Stem-Loop
Flanked by a 39-nt 5′ Arm. Next, we sought to identify the poten-
tial STV1-binding region within pri-miRNAs. Because the
recombinant STV1 protein could not be expressed in Escherichia
coli, an in vivo RNA-binding assay was used to examine the
Fig. 4. The interaction of STV1 with MIR172b. (A) Diagram of the stem-loop and flanking sequences of MIR172b. (B) Diagrams of various MIR172b constructs
used for the STV1-binding assay. Gray box indicate the sequences from vectors. miR172 is shown in red; miR172b* is shown in dark blue. (C–J) The interaction
of STV1 with full-length and truncated MIR172b RNAs. MYC-STV1 and MIR172b were transiently coexpressed in N. benthamiana. IP was performed with anti-
MYC antibodies. No Ab: IP without antibody. No RT was performed with primers recognizing MIR172b. Input RNA was 5%.
Li et al.
STV1–pri-miRNA interaction. In this assay, we coexpressed
35S::MYC-STV1, which complements the defects of stv1 (Fig. S5A)
with 35S::MIR172b in N. benthamiana and checked the STV1-
MIR172b interaction by RIP. Transcription of 35S::MIR172b gen-
erated a transcript containing an ∼120-nt upstream vector se-
quence, MIR172b that composes of the stem-loop flanked by an
∼287-nt upstream arm and a 417-nt downstream arm, and an ∼354-nt
downstream vector sequence (MIR172bFL) (Fig. 4 A and B).
MIR172bFL, but not NtEF1A that is an endogenous mRNA from
N. benthamiana, was enriched in the MYC-STV1 IPs relative to the
“no antibody” control (Fig. 4C). As a negative control, we coex-
pressed MIR172b with an unrelated RNA-binding protein (URP,
At5g59390). This protein did not pull down MIR172bFL. These
results suggest that this assay could be used to determine the STV1-
binding position within MIR172b. We first examined the interaction
of STV1 with the 5′ arm (MIR172bF1; ∼287nt), a truncated
MIR172b fragment (MIR172bF2) containing a 5′ arm (∼287 nt) and
a short 3′ arm (6 nt; MIR172bF2), and the 3′ arm (MIR172bF3;
∼417 nt) (Fig. 4B). STV1 bound MIR172bF2, but not MIR172bF1
and MIR172bF3 (Fig. 4 D–F), indicating that STV1 may bind the
stem-loop of MIR172b. To confirm this result, we tested the asso-
ciation of STV1 with the stem-loop of MIR172b with the 6-nt 3′ arm
(MIR172bF4) (Fig. 4B). However, STV1 did not bind MIR172bF4
PLANT BIOLOGY
(Fig. 4G). These results suggest that STV1 requires both the 5′ arm
and stem-loop to bind pri-miR172b. To further determine sequence
features of the 5′ arm required for STV1 binding, we checked the
effect of 5′ arm truncation (MIR172bF5-F7) on the interaction of
STV1 with MIR172b. STV1 bound MIR172bF5 (containing a 39-nt
5′ arm) (Fig. 4 B and H), but not MIR172bF6 (containing a 6-nt
5′ arm) (Fig. 4 B and I) and MIR172bF7 (containing a 10-nt 5′ arm)
(Fig. 4 B and J). To rule out the effect of the vector sequence on
STV1-binding, we in vitro-transcribed MIR172bF5 and MIR172bF7
and tested their interactions with IPed MYC-STV1 using an RNA
pull-down assay (7). MYC-STV1 binds MIR172bF5, but not
PNAS | February 7, 2017 | vol. 114 | no. 6 | 1427
MIR172bF7 (Fig. S5 B and C). These results show that STV1 bind
the stem-loop of pri-miR172b with the 39-nt 5′ arm and the se-
quence between 39 nt and 10 nt upstream of the stem loop is re-
quired for STV1-binding.
stv1-1 Reduces the Interaction of pri-miRNAs with HYL1. In yeast,
RPL24 acts in the 60S subunit-joining step during translation
initiation (42). By analog, we suspected that STV1-binding might
enhance the recruitment of pri-miRNAs to the DCL1 complex.
If so, we would expect a reduced association of HYL1 with pri-
miRNAs in stv1. We examined the HYL1–pri-miRNA in-
teraction in WS and stv1-1 by RIP using antibodies against
HYL1. The amount of HYL1 protein in the IPs from stv1-1 was
similar to that in WS (Fig. 5A). RT-PCR and qRT-PCR analyses
showed that the levels of several examined pri-miRNAs
(MIR167a, MIR172b, MIR173, MIR408, and MIR863) in the
HYL1 IPs were lower in stv1-1 than those in WS (Fig. 5 B and C
and Fig. S5D), demonstrating that STV1 contributes to the
loading of pri-miRNAs to the DCL1 complex.
Fig. 5. STV1 affects the interaction between HYL1 and pri-miRNAs.
(A) Detection of HYL1. IP was performed with the anti-HYL1 antibodies. Ten
percent of IPs and 2% input proteins were detected by Western blot. (B and
C) The association of HYL1 with pri-miRNAs is reduced in stv1-1. Pri-miRNAs
associated with HYL1 were examined by qRT-PCR and normalized to the
input. UBQ5 serves as a negative control. *P < 0.05; **P < 0.01.
1428 | www.pnas.org/cgi/doi/10.1073/pnas.1613069114
Discussion
In conclusion, we show here that STV1, a ribosomal protein, is
required for miRNA biogenesis. This finding is supported by the
association of STV1 with pri-miRNAs and the altered accumulation
of miRNAs and pri-miRNAs in stv1. Altered miRNA accumulation
may partially contribute to the pleiotropic developmental defects of
stv1, given the essential roles of miRNAs in regulating development.
However, STV1 must have other functions, as its effect on miRNA
accumulation is less than DCL1, HYL1, and SE. It is still possible
that STV1 influences the translation of some proteins. In fact, STV1
modulates the translation initiation of ETTIN, an auxin response
factor (43), and influences the activity of the cauliflower mosaic
virus transactivator, TAV, which controls the translation reinitiation
of polycistronic RNAs encoded by virus (44). Furthermore, some
ribosomal proteins are involved in the formation of the preribosome
in the nucleus (45). It is reasonable to speculate that STV1 may
participate in the process.
In miRNA biogenesis, STV1 may facilitate the interaction of
pri-miRNAs with the processing complex, which is supported by
the association of STV1 with pri-miRNAs and the reduced
amount of pri-miRNAs in the HYL1 complex. Because STV1
does not interact with the DCL1 complex, we propose that STV1
may bind pri-miRNAs before the loading of the DCL1 complex to
pri-miRNAs. STV1 also affects the transcription of many pri-
miRNAs, based on the observations that the occupancy of Pol II
at the MIR promoters and the levels of many pri-miRNAs are
altered in stv1. However, this effect maybe indirect, as STV1 itself
does not interact with Pol II and MIR promoters. How does STV1
indirectly regulate activities of these MIR promoters? We suspect
that STV1 may indirectly impact the expression of transcription
factors of MIRs. For example, stv1 reduces the transcript levels of
AGL15 and AGL18 (Fig. S3B), which encode positive transcrip-
tion factors of MIR156 (46), but increases the abundance of SPL9
and 10 (Fig. 2B), which are targets of miR156 and promote the
expression of MIR172B (47, 48). These results are consistent with
decreased pri-miR156 levels and elevated pri-miR172b abundance
in stv1 (Fig. 2B and Fig. S3A). How STV1 affects MIR transcrip-
tion factors is not clear, but may partially attribute to its effect on
miRNA levels. Additionally, STV1 may influence the translation
of some proteins that may play roles in regulating the transcription
of MIR transcription factors. STV1 may also affect pri-miRNA
stability, which clearly needs further investigation.
The assembly of RNA–protein complex often involves the con-
formation changes of RNAs (41). Many ribosomal proteins bind
rRNAs to alter their structures for ribosome assembly (41). Thus,
STV1 may alter the structure of pri-miRNAs to enable them more
accessible for the processing complex. STV1 binds the stem-loop
with a short 5′ arm with MIR172b, and this binding requires the
presence of both stem-loop and the 39-nt 5′ arm. Because the
presence of 120-nt vector sequence in various forms of MIR172b
transcripts, it is unlikely the length of 5′ arm is required for STV1
binding. Rather, the sequence or structure features within the 39-nt
5′ arm sequence together with the stem-loop enables STV1 binding.
In summary, we discovered a previously unknown extra-
ribosomal activity of STV1. STV1 binds pri-miRNAs and promotes
miRNA biogenesis through facilitating the interaction of pri-
miRNAs with the processing complex. Besides this function, STV1
may indirectly modulate pri-miRNA accumulation through nega-
tively or positively influencing the levels of transcription factors
that regulate MIR transcription. Thus, the accumulation of miR-
NAs in stv1 reflects the effect of STV1 on pri-miRNA loading and
transcription. Through these combined effects, STV1 may be able
to fine-tune the levels of miRNAs to ensure normal development
and physiology of Arabidopsis. STV1 is a conserved protein in
eukaryotes. It is possible that STV1 homologs act in miRNA bio-
genesis in other organisms. Indeed, lack of STV1 homolog in mice
Li et al.
causes lethality, consistent with the crucial role of miRNA in de-
velopment (49).
Materials and Methods
Plant materials, plasmid construction, polysome profile analysis, deep se-
quencing, and RNA analysis are described in SI Materials and Methods. Data
generated from deep sequencing of small RNA libraries were deposited in the
National Center for Biotechnology Information (accession no. GSE91013).
Nuclear-Cytoplasmic Fractionation. Inflorescences of stv1-1 harboring the
pSTV1::STV1-MYC transgene were used to perform nuclear-cytopasmic fractionation
according to Park et al. (50). The isolated nuclear pellets were further sonicated in
the nuclear lysis buffer for 1 min on ice, as described previously (51). Following this
step, the supernatants were collected as nuclear extracts through centrifugation
(51). Anti-PEPC antibody was used to detect the cytoplasmic fraction.
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Board Award 16R-05-3/3 #1706 (to B.Y.); National Science Foundation Award
OIA-1557417 (to B.Y.); and National Natural Science Foundation of China
Award 31471221 (to G.R.).
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