Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

Accepted Manuscript Identification of Nucleosome Assembly Protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription Ora Son, Sunghan Kim, Yun-jeong Shin, Woo-Young Kim, Hee-Jong Koh, Choong-Ill Cheon PII:

S0006-291X(15)30380-6

DOI:

10.1016/j.bbrc.2015.07.150

Reference:

YBBRC 34362

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 29 July 2015 Accepted Date: 29 July 2015

Please cite this article as: O. Son, S. Kim, Y.-j. Shin, W.-Y. Kim, H.-J. Koh, C.-I. Cheon, Identification of Nucleosome Assembly Protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.07.150. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Identification of Nucleosome Assembly Protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6)

Ora Son

a*,1

, Sunghan Kim

a

c

b

, Yun-jeong Shin , Woo-Young Kim , Hee-Jong Koh ,

a

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and Choong-Ill Cheon

a,b*

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and a positive regulator of rDNA transcription

a

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Department of Biological Science, Sookmyung Women’s University, Seoul 140-742, b

Korea; Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul c

151-921, Korea; College of Pharmacy, Sookmyung Women’s University, Seoul 140-

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742, Korea

*

These authors contributed equally to this work

1

The present address: Department of Biomedical Science, Research Institute of

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Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea

Authors for correspondence: Choong-Ill Cheon Department of Biological Science, Sookmyung Women’s University, 52 Hyochangwongil, Yongsan-gu, Seoul 140-742, Korea Phone: +82-2-710-9396.

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Fax:

+82-2-2077-7322.

E-mail address: [email protected].

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Hee-Jong Koh Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea

+82-2-873-2056.

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E-mail address: [email protected].

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Fax:

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Phone: +82-2-880-4541.

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ABSTRACT The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated

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by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-

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specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, Nucleosome Assembly Protein

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1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in

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concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly

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through derepression of the negative effect of the RPS6-AtHD2B complex.

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Keywords: Ribosomal protein S6; Histone deacetylase 2B; Nucleosome assembly protein 1; TOR; rDNA transcription

Abbreviations: RPS6, Ribosomal protein S6; HD2B, Histone deacetylase 2B; NAP1, Nucleosome assembly protein 1; BiFC, bimolecular fluorescent complementation; TOR, target of rapamycin.

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1. Introduction Target of rapamycin (TOR) is a serine/threonine kinase that controls various

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cellular processes such as protein synthesis, autophagy, ribosome biogenesis, lipid synthesis in response to environmental cues [1,2,3]. One of the well-documented examples of TOR pathway is the control of translation via S6 kinase, which phosphorylates the ribosomal protein S6 (RPS6) [4,5,6]. RPS6 phosphorylation by S6

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kinase 1 (S6K1) can increase the translation of diverse proteins containing 5’-terminal oligopyrimidine tract (5’-TOP) mRNA, suggesting that RPS6 contributes to the control

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of ribosome biogenesis [7]. RPS6 was found to form a complex with a histone deacetylase AtHD2B and to bind to rDNA promoter in Arabidopsis [8]. Protoplasts overexpressing both RPS6 and AtHD2B respectively showed decrease in rDNA transcription.

Nucleosome Assembly Protein 1 (NAP1) represents a highly conserved histone

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chaperone from yeast to human, which functions in assisting the traffic of histones, directly impacting nucleosome dynamics [9,10,11,12,13]. Among the NAP1 family members in Arabidopsis, AtNAP1;1, AtNAP1;2, and AtNAP1;3 show high sequence

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homology to each other except for AtNAP1;4. They function in genome transcription, nucleotide excision repair and somatic homologous recombination [14,15,16]. NAP1

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homologs were localized to mainly cytoplasm, but can shuttle between nucleus and cytoplasm [15,17,18]. NAP1 has been known to be able to bind diverse proteins that are located in several intracellular regions and considered to have numerous functions involved in nucleosome assembly/disassembly, transcription, RNA processing/protein synthesis, cell wall biosynthesis, lysine biosynthesis, protein degradation, and energy usage [17,19,20], but the details of their mechanism still remain under study. In the previous study, we identified putative interacting partners of RPS6 in Arabidopsis by GST-pull-down followed by LC/MS [8]. Among them are AtNAP1;1,

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AtNAP1;2, and AtNAP1;3. Here we confirmed the interactions among RPS6, AtNAP1, and AtHD2B. AtS6K1 overexpression lessened the negative effect of RPS6 on rDNA transcription. The transcription of rDNA was increased upon AtNAP1;1 overexpression,

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which appeared to counteract the effect of RPS6 and AtHD2B on the rDNA transcription. Consistently, the specific down-regulation of AtNAP1 homologs by artifitial miRNAs led to the decrease of rDNA transcription. These results imply that NAP1 may affect positively rDNA transcription, acting against the effect of AtHD2B in

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the protein complex involving RPS6, which suggests that the regulation of rDNA

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transcription may be controlled by multiple pathways, including the TOR pathway.

2. Materials and methods

2.1 Bimolecular fluorescence complementation (BiFC) assay

The BiFC assay was performed as described [21]. cDNAs encoding C-terminal region

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of RPS6, the full-length cDNAs of AtHD2B and AtNAP1;1, and RPS6 C-terminal region fused to AtHD2B were cloned into p326YFPN and p326YFPC vectors [21]. The full-length cDNAs of ATHB12 and pFAγ were cloned into p326YFPN to be used as

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controls. The full-length cDNA of AtNucL1 was subcloned into 326-CFP vector [21] to be used as localization marker. For transient expression, equal amounts of different

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combinations of vectors were mixed and transfected into Arabidopsis (ecotype Columbia) protoplasts [22]. The transfected protoplasts were observed by confocal microscopy after 16 h. MitoTracker Red CMXRos (Molecular Probes) was used as mitochondrial marker.

2.2 Protein pull-down assay His-tagged AtNAP1;1 was purified from E. coli (BL21) cell lysates and incubated with total protein extracts from Arabidopsis expressing 3HA-RPS6 for 16 h. After

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incubation, His-AtNAP1;1 and 3HA-RPS6 were washed and resolved by SDS-PAGE.

2.3 Real-time RT-PCR

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Total RNA was isolated from Arabidopsis protoplasts using RNA isolation kit (QIAGEN). Approximately 1 µg of total RNA prepared was used in each reverse transcription (RT) reaction using M-MLV reverse transcriptase (Promega). Real-time RT-PCR was set up with Power SYBR Green PCR Master mix using AB7500 (Applied

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Biosystems). All reactions were normalized using ACTIN2 and EF1α as internal

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controls. Primers used were shown in Supplementary Table S1.

2.4 Cloning of 35S::amiRNA- AtNAP1

A suitable target site of the microRNA for gene silencing was identified by using the information

at

http://wmd3.weigelworld.org.

Primers

used

were

shown

in

Supplementary Table S1. Constructs for 35S::amiRNA-AtNAP1 and -AtNAP1;1,

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respectively, were made by modification of pRS300 plasmid, and cloned into the pUC19 vector [23]. The resulting constructs were used in transfecting Arabidopsis

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protoplast.

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3. Results and Discussions 3.1. Interaction of NAP1 with RPS6

Several proteins were co-purified with the Arabidopsis ribosomal protein S6,

including a unique plant histone deacetylase (AtHD2B) and isotypes of the nucleosome assembly protein 1 (AtNAP1;1, AtNAP1;2, and AtNAP1;3), from GST-pull-down followed by LC/MS analysis in our previous study [8]. In the study, AtHD2B was shown to negatively regulate rDNA transcription as a protein complexed with the RPS6.

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In order to examine if NAP1 is also involved in regulation of rDNA transcription as part of the RPS6-AtHD2B protein complex, we first confirmed its interaction with RPS6 using bimolecular fluorescence complementation (BiFC) and pull-down assays.

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Corroborating our original finding which was made through pulling down the cellular AtNAP1s by GST-fused RPS6, it was possible to pull down 3HA-tagged RPS6 expressed in transgenic Arabidopsis by His-tagged AtNAP1;1 purified from E.coli, reaffirming specific interaction between the two proteins (Fig. 1A). For the BiFC assay,

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the C-terminal region of RPS6 [24] and the full-length ORF of AtNAP1;1 were fused to N- and C-terminal halves of yellow fluorescent protein (YFP), generating S6-CT-YFPN

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and AtNAP1;1-YFPC, respectively, and the two constructs were transiently co-expressed in Arabidopsis protoplasts. The YFP fluorescence signal was detected in these cells confirming specific interaction between AtNAP1;1 and RPS6 in vivo, and the primary site of their interaction appeared to be nucleus or nucleolus, as the signal was colocalized to the fluorescence signal of GFP-fused Arabidopsis Nucleolin Like

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1(AtNucL1), which was included in the analysis as a nucleolar marker (Fig. 1B). Despite its general function as a histone chaperone responsible for the nuclear import of histone H2A/H2B for chromatin assembly [9], the association of AtNAP1;1 with RPS6

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was specific, considering that it did not show any interaction with a homeodomain transcription factor ATHB12 in the same analysis (Fig. 1B, lower panel). The data also

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confirmed earlier works of other groups, who have reported the interaction of NAP1 with RPS6A [25,26], a different isoform of Arabidopsis RPS6 than the one that has been the subject of our study, RPS6B. Therefore, we determined to obtain a functional insight into the implication of the interaction between NAP1 and RPS6 in the present study.

3.2. Characterization of the RPS6-protein complex involving NAP1 and HD2B One of the main agenda of this study was to determine if NAP1 is a component of the RPS6-HD2B complex, an alternative scenario of which would be NAP1 and

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HD2B are competing with each other for binding to RPS6. To distinguish these two possibilities, we tested the interaction between NAP1 and HD2B via BiFC assay after transfecting Arabidopsis protoplasts with two constructs harboring full-length ORFs of

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the AtNAP1;1 and AtHD2B fused to each half of YFP, respectively (AtNAP1;1-YFPN AtHD2B-YFPC) (Fig. 2, top panel). A specific fluorescence signal, which was absent in the negative control, was detected in the cells expressing the two constructs indicating that the two proteins interact with each other in vivo either directly or indirectly.

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Therefore, it is likely that, rather than acting as mutually exclusive components, these proteins are present together in the RPS6 protein complex regulating the rDNA

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transcription.

We also tested interaction between the AtNAP1;1 and a chimeric construct, S6CT-AtHD2B which was made by fusing the full-length AtHD2B ORF to the C-terminal fragment of RPS6. According to our hypothesis regarding the regulation of rDNA transcription by the RPS6-AtHD2B complex, the repressor function is provided by

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AtHD2B while RPS6 allows binding of the complex to the rDNA promoter. We also speculate that dissociation of the complex, perhaps through phosphorylation of the RPS6 by AtS6K1, would lead to derepression of the rDNA transcription. Thus, such a

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chimeric fusion protein consisting of non-dissociable RPS6 and AtHD2B would be instrumental in assessing roles of each component of the RPS6-complex and the

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AtS6K1 in the regulation of rDNA transcription, as shown in later sections. Our result showed that AtNAP1;1 also interacted with the RPS6-AtHD2B fusion protein in vivo and localized to the nucleus/nucleolus, corroborating our conclusion about the interaction dynamics between AtNAP1 and AtHD2B drawn from the BiFC data between AtNAP1;1 and AtHD2B. One peculiar observation made from both occasions was that the interacting complexes were also located outside of nucleus, the distribution pattern of which appeared similar to those of mitochondria. These locations indeed turned out to be

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mitochondria as they overlap with the mitochondrial signals generated by MitoTracker Red (Fig. 2). Although AtNAP1 has been reported to be implicated in a variety of cellular

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functions interacting with a number of different proteins in various intracellular locations including mitochondria and nucleolus [17,18], which was also confirmed in our own observation (Fig. S1), whether mitochondrial localization of these complexes is mere artifact or the one actually reflecting another novel function involving the AtNAP1

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remains to be resolved.

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3.3. Effect of S6 Kinase 1 overexpression on rDNA transcription

In our pervious study, we predicted that the repression of rDNA transcription by RPS6-HD2B complex could be lifted by the activity of S6K1 [8], as RPS6 has been known as the primary substrate of S6K1 which may be able to result in dissociation of the complex through phosphorylation. In order to test this possibility, we generated

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transgenic Arabidopsis lines that overexpress the AtS6K1 upon induction by dexamethasone (DEX) treatment. Protoplasts were prepared from these plants and their levels of rDNA transcription were measured after transiently expressing the RPS6 C-

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terminus (RPS6) or the chimeric fusion of the fragment with AtHD2B (RPS6-AtHD2B). As with our previous publication, the rDNA transcription was measured by real-time

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RT-PCR amplification of the internal intergenic region of pre-18S rRNA in order to minimize a possible confounding effect arising from the long half-life of the rRNA in the cell [8]. As expected, transient overexpression of the RPS6 in these cells under uninduced condition for the AtS6K1 transgene resulted in a dramatic repression of rDNA transcription (Fig. 3). Upon induction of the transgenic AtS6K1 expression by DEX treatment, however, these cells showed a significant increase in the rDNA transcription even far beyond the level of mere derepression, judging from in comparison with the cells expressing the GFP as control. Therefore the result is in

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support of our hypothesis that envisions S6K1 as a positive regulator of rDNA transcription through modulation of the RPS6-AtHD2B repression complex integrity. Moreover, a greater repression of rDNA transcription was observed with the expression

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of the RPS6-AtHD2B fusion chimera, demonstrating that a non-dissociable RPS6AtHD2B complex could function as a more potent negative regulator of rDNA transcription, which was also in line with our hypothesis regarding the mode of the regulation by the RPS6-AtHD2B complex. Indeed, unlike the cells transfected with

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RPS6 alone, induction of AtS6K1 by DEX in these cells did not restore the rDNA level very much, and perhaps even the observed small increase in rDNA transcript was

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mostly attributable to the effect exerted on endogenous, native RPS6-AtHD2B complex.

3.4. Effect of NAP1 on rDNA transcription

To explore the possible role played by NAP1 in regulating rDNA transcription as an interacting partner of RPS6, which can co-exist with HD2B in the complex, we

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measured rDNA transcription by real-time RT-PCR after transfecting Arabidopsis protoplasts with an overexpressing construct of AtNAP1;1 along with the RPS6 or AtHD2B. The result showed that the inhibition of rDNA transcription brought about by

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the ectopic expression of either RPS6 or AtHD2B was de-repressed by co-expression of the AtNAP1, suggesting that NAP1 may function as an antagonizing effector for the

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HD2B in the complex (Fig. 4A). Consistent with the finding, expression of artificial micro RNAs (amiRNA) against AtNAP1 in these cells resulted in a greater magnitude of repression in the rDNA transcription when the cells were co-transfected with RPS6 or AtHD2B (Fig. 4B,C). Two different types of amiRNAs were utilized; one is targeted against all three paralogues of the AtNAP1 (amiR-AtNAP1), and another for specifically targeting only AtNAP1;1 (amiR-AtNAP1;1) (Fig. S2). About 20 ~ 30 % more repression was observed with the broader target amiR-AtNAP1, as compared with the one obtained from the AtNAP1;1-specific amiR-AtNAP1;1 expression, suggesting that this positive

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regulatory role for rDNA transcription may be shared by all three isoforms of the AtNAP1. As with the positive effect of S6K1 on rDNA transcription we showed earlier, the positive effect of NAP1 could only be discernible on rDNA level and had no effect

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on other Pol II- driven transcription such as RPS27 or RPL7. In short, the results of our present work provide solid evidence for AtNAP1 acting as a positive regulator of rDNA transcription, suppressing the negative effect of the RPS6-AtHD2B protein complex. As it turned out that AtNAP1 can co-exist with

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AtHD2B within the complex, contrary to our initial speculation, the balance of dynamics between negative and positive effect of the RPS6-protein complex on rDNA

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transcription involving the functions of AtHD2B and AtNAP1, respectively, could then be primarily determined by temporal adjustment of transcription for AtNAP1, for which a cell cycle-dependent transcriptional induction pattern has been reported earlier [14]. The dual mode of positive regulatory effects on rDNA transcription identified in this study, one provided by AtS6K1 and another from AtNAP1, appears to reflect the

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complexity of the ribosome biogenesis regulatory circuits in the cell. Thus not only the TOR pathway which controls the rDNA transcription through S6K1, additional pathway

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may be involved in the regulation through NAP1.

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Acknowledgments

This work was supported by National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No. 2011-0030074) and by Next-Generation Biogreen 21 Program (PJ01102401), RDA, Republic of Korea. We thank Dr. Detlef Weigel and Addgene for providing the pRS300 plasmid vector.

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Figure legends Fig. 1. The interaction between RPS6 and AtNAP1. A, Interaction between RPS6-CT

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and AtNAP1;1 by a pulldown assay with His-tag followed by a western blot with anti3HA antiserum. Protein extracts from plants expressing 3HA-RPS6 were incubated with different amounts (150 µg and 300 µg) of His-GFP or His-AtNAP1 fusion proteins expressed in E.coli, respectively. B, Bimolecular fluorescence complementation (BiFC)

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analyses of interaction between RPS6-CT and AtNAP1;1: coexpression of P35S-S6-CTYFPN and P35S-AtNAP1-YFPC (top panels); coexpression of P35S-ATHB12-YFPN and In both cases, P35S-AtNucL1-CFP was

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P35S-AtNAP1-YFPC (bottom panels).

coexpressed for use as a nuclear marker. AtNucL1, Arabidopsis Nucleolin Like 1. These experiments were replicated three times with similar results. Bar = 10 m.

Fig. 2. The interaction between AtHD2B or RPS6-AtHD2B and AtNAP1. BiFC

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analysis of interaction between AtHD2B and AtNAP1;1 or RPS6 C-terminal region fused to AtHD2B and AtNAP1;1: coexpression of P35S-AtHD2B-YFPC and P35S AtNAP1-YFPN (top panels); coexpression of P35S-S6-CT-AtHD2B-YFPC and P35S-

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AtNAP1-YFPN (second panels); coexpression of P35S-S6-CT-AtHD2B-YFPC and P35SATHB12-YFPN (third panels); coexpression of P35S-S6-CT-AtHD2B-YFPC and P35S-

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pFA -YFPN (bottom panels). P35S-AtNucL1-CFP was coexpressed for use as a nuclear marker and MitoTracker Red used for mitochondrial localization. These experiments were replicated three times with similar results. Bar = 10 m.

Fig. 3. rDNA transcription regulated by AtS6K1. Arabidopsis protoplasts were isolated from DEX-treated transgenic plants with AtS6K1 in the DEX-inducible pTA7002 vector (GVG-AtS6K1). Protoplasts were transfected with control plasmid (P35S-GFP), or RPS6expressing plasmid (P35S-RPS6), or a fusion of RPS6-AtHD2B-expressing plasmid (P35S-

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RPS6-AtHD2B). Expression of pre-18S rRNA, RPS27, and RPL7B was examined by real time RT-PCR and the expression level of Actin2 and EF was used as an internal control. Statistically significant difference as evaluated by Student s t-test: *, P < 0.05;

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**, P < 0.01. Error bars represent standard deviations (n=3).

Fig. 4. rDNA transcription regulated by AtNAP1. A, Protoplasts were transfected with control plasmid (P35S-GFP), or AtHD2B-expressing plasmid (P35S-AtHD2B), or RPS6-

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expressing plasmid (P35S-RPS6), respectively, or each together with AtNAP1;1expressing plasmid (P35S-AtNAP1;1). B, Protoplasts were transfected with control

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plasmid (P35S-GFP), or AtHD2B-expressing plasmid (P35S-AtHD2B), or RPS6expressing plasmid (P35S-RPS6), respectively, or each together with amiRNA-AtNAP1expressing plasmid (P35S-amiRNA-AtNAP1). C. Protoplasts were transfected with control plasmid (P35S-GFP), or AtHD2B-expressing plasmid (P35S-AtHD2B), or RPS6expressing plasmid (P35S-RPS6), respectively, or each together with amiRNA-

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AtNAP1;1-expressing plasmid (P35S-amiRNA-AtNAP1;1). Expression of pre-18S rRNA, RPS27, and RPL7B was examined by real time RT-PCR and the expression level of Actin2 and EF was used as an internal control. Statistically significant difference as

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evaluated by Student s t-test: *, P < 0.05; **, P < 0.01. Error bars represent standard

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deviations (n=3).

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Son et al. Fig.1

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Son et al. Fig.2

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Son et al. Fig.3

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Son et al. Fig.4

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Supplementary materials Supplementary Fig. S1. Subcellular localization of AtNAP1;1. Protoplasts were

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transfected with a fusion of GFP to the full-length cDNA of AtNAP1;1 (GFPAtNAP1;1). P35S-AtNucL1-CFP was coexpressed for use as a nuclear marker.

Supplementary Fig. S2. Sequence of amiRNAs targeting AtNAP1. A. Sequence

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alignment of AtNAP1;1, AtNAP1;2, and AtNAP1;3 with amiRNA-AtNAP1. Asterisks indicate the target sequences of amiR-AtNAP1. B. Sequence alignment of AtNAP1;1,

sequences of amiR-AtNAP1;1.

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AtNAP1;2, and AtNAP1;3 with amiRNA-AtNAP1;1. Asterisks indicate the target

Supplementary Fig. S3. Effect of amiR-AtNAP1 and amiR-AtNAP1;1 on the expression of AtNAP1s. A. Protoplasts were transfected with control plasmid (P35S-

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GFP), or co-transfected with control plasmid (P35S-GFP), AtHD2B-expressing plasmid (P35S-AtHD2B), RPS6-expressing plasmid (P35S-RPS6), together with amiRNAAtNAP1-expressing plasmid (P35S-amiRNA-AtNAP1), respectively. B. Protoplasts were

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transfected with control plasmid (P35S-GFP), or co-transfected with control plasmid (P35S-GFP), AtHD2B-expressing plasmid (P35S-AtHD2B), RPS6-expressing plasmid

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(P35S-RPS6), together with amiRNA-AtNAP1;1-expressing plasmid (P35S-amiRNAAtNAP1;1), respectively. Expression of AtNAP1s was examined by real time RT-PCR and the expression level of Actin2 and EF was used as an internal control. Statistically significant difference as evaluated by Student s t-test: *, P < 0.05; **, P < 0.01. Error bars represent standard deviations (n=3).

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Son et al. Supplementary Table S1

PCR primers used in this work Sequence (5’-> 3’)

ACTIN2-F

GAAAAGATCTGGCATCACACTTTATA

ACTIN2-R

ACATACATAGCGGGAGAGTTAAAGGT

EF1a-F

TGAGCACGCTCTTCTTGCTTTCA

EF1a-R

GGTGGTGGCATCCATCTTGTTACA

18S-pre-rRNA-F

GCGTTTGAGAGGATGTGGCGGGGAAT

18S-pre-rRNA-R

TAAATGCGTCCCTTCCATAAGTCGGG

AtHD2B-F

TCTCTTCTCTCTTCCTCGTTCAACAACA

AtHD2B-R

TCATCATCCGAACTAGTGAAGTCATCCT

RPS6B-F

CTGTTGTAGCAGCAGTGTCTATCGGA

RPS6B-R

CAATGACCAAGTTAAGAACAGACAGGTCA

RPS27B-F

TTAGCTTCTTGCGAAGATGGTTCTTCAA

RPS27B-R RPL7B-F

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RPL7B-R

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qRT-PCRs

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Primer name

AGAATTGTCTGGCAGTTTCCGCACACCA CTCCACAGAGGATTCGGAAATGGTTGAG

ACTCCTTCTCCTTCTCGGCATATTCCTT GATAGTAAACCATGATACGGCACTCTCTCTTTTGTATTCC

NAP1-amiR1-R1

GAGTGCCGTATCATGGTTTACTATCAAAGAGAATCAATGA

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NAP1-amiR1-F1

NAP1-amiR1-F2

GAGTACCGTATCATGCTTTACTTTCACAGGTCGTGATATG

NAP1-amiR1-R2

GAAAGTAAAGCATGATACGGTACTCTACATATATATTCCT

NAP1-1-amiR1-F1

GATTTTTATGTCTGGTAGGGCGTTCTCTCTTTTGTATTCC-

NAP1-1-amiR1-R1

GAACGCCCTACCAGACATAAAAATCAAAGAGAATCAATGA

NAP1-1-amiR1-F2

GAACACCCTACCAGAGATAAAATTCACAGGTCGTGATATG

NAP1-1-amiR1-R2

GAATTTTATCTCTGGTAGGGTGTTCTACATATATATTCCT

amiRNA

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Son et al. Supplementary Fig. S1

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Son et al. Supplementary Fig. S2

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Son et al. Supplementary Fig. S3

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• Nucleosome Assembly Protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1.

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• Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription.