bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 NRF1 and NRF3 complementarily maintain a basal proteasome activity in cancer cells

2 through CPEB3-mediated translational repression

3

4 Running title: NRF3-CPEB3-NRF1 axis for proteasome regulation

5 6 Tsuyoshi Waku1 † , Hiroyuki Katayama2 † , Miyako Hiraoka2, Atsushi Hatanaka2, 3, Nanami

7 Nakamura2, Yuya Tanaka1, Natsuko Tamura2, Akira Watanabe4, Akira Kobayashi1,2*

8

9 †Equal contribution

10

11 1Laboratory for Genetic Code, Department of Medical Life Systems, Faculty of Life and Medical

12 Sciences, Doshisha University, Kyotanabe, Kyoto, Japan

13 2Laboratory for Genetic Code, Graduate School of Life and Medical Sciences, Doshisha

14 University, Kyotanabe, Kyoto, Japan

15 3Research Fellow of Japan Society for the Promotion of Science, Tokyo, Japan

16 4Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto

17 University, Kyoto, Japan

18

19

20

21

22

23 *To whom correspondence should be addressed:

24 Akira Kobayashi

25 1-3 Tatara Miyakodani, Kyotanabe 610-0394, Japan

26 Phone: +81-774-65-6273

27 Fax: +81-774-65-6274

28 E-mail: [email protected]

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29 Abstract

30 Proteasomes are protease complexes essential for cellular homeostasis, and their activity is crucial

31 for cancer cell growth. However, the mechanism of how proteasome activity is maintained in

32 cancer cells has remained unclear. The CNC family transcription factor NRF1 induces the

33 expression of almost all proteasome-related under proteasome inhibition. NRF1 and its

34 phylogenetically closest homolog NRF3 are both highly expressed in several types of cancers,

35 such as colorectal cancer. Herein, we demonstrate that NRF1 and NRF3 complementarily

36 maintain basal proteasome activity in cancer cells. A double knockdown of NRF1 and NRF3

37 impaired the basal proteasome activity in cancer cells and the cancer cell resistance to a

38 proteasome inhibitor anticancer drug bortezomib by significantly reducing basal expression of

39 seven proteasome-related genes, including PSMB3, PSMB7, PSMC2, PSMD3, PSMG2, PSMG3,

40 and POMP. Interestingly, the molecular basis behind these cellular consequences was that NRF3

41 repressed NRF1 translation by the induction of translational regulator CPEB3, which binds

42 to NRF1-3′UTR and decreases polysome formation on NRF1 mRNA. Consistent results were

43 obtained from clinical analysis, wherein patients with cancer having higher CPEB3/NRF3-

44 expressing tumors exhibit poor prognosis. These results provide the novel regulatory mechanism

45 of basal proteasome activity in cancer cells through an NRF3-CPEB3-NRF1 translational

46 repression axis.

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

47 Introduction

48 Cancer cell survival and growth is dependent on the activity of a proteasome, which is a large

49 protease complex that catalyzes the precise and rapid degradation of involved in

50 antitumorigenic events, such as cell-cycle arrest and apoptosis induction1,2. One of the proteasome

51 regulators in mammals is the cap'n'collar (CNC) family transcription factor, NRF1 (NFE2L1).

52 NRF1 induces the expression of almost all proteasome-related genes under proteasome

53 inhibition3–6, and the brain-specific knockout actually impairs proteasome function and causes

54 neurodegeneration in mice7. However, the transcriptional regulation of proteasome activity in

55 cancer cells has remained unclear.

56 Phylogenetically, NRF3 (NFE2L3) has been identified as the closest homolog of NRF1

57 in the CNC family8. Recently, NRF3 gene amplification has been reported in patients with

58 colorectal cancer9. In the Gene Expression Profiling Interactive Analysis (GEPIA) web server10,

59 the NRF3 gene is highly expressed in many types of tumors, such as testicular germ cell tumors,

60 pancreatic adenocarcinoma, colon adenocarcinoma (COAD), and rectum adenocarcinoma

61 (READ), whereas the NRF1 gene is highly expressed in almost all types of normal and tumor

62 tissues. These insights imply the functional correlation between NRF1 and NRF3 with respect to

63 the maintenance of a basal proteasome activity in cancer cells.

64 In this study, we show that both NRF1 and NRF3 are required to maintain a basal

65 proteasome activity in cancer cells through the induction of several proteasome-related genes,

66 including PSMB3, PSMB7, PSMC2, PSMD3, PSMG2, PSMG3, and POMP. Interestingly, NRF3

67 represses NRF1 translation by inhibiting polysome formation on NRF1 mRNA. We identify a

68 translational regulator CPEB3 (cytoplasmic polyadenylation element-binding 3) as an

69 NRF3-target gene that is involved in the repression of NRF1 translation. A functional CPEB

70 recognition motif is also identified in the NRF1-3′UTR. Furthermore, we validate that CPEB3 is

71 a key factor for not only the complementary maintenance of a basal proteasome activity by NRF1

72 and NRF3 but also the poor prognosis of colorectal cancer patients with tumors highly expressing

73 NRF3, but not NRF1. In conclusion, we demonstrate the novel regulatory mechanism of basal

74 proteasome activity in cancer cells where NRF1 and NRF3 complementarily, but not

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

75 simultaneously, maintain the basal expression of proteasome-related genes through CPEB3-

76 mediated translational repression.

77

78 Results

79 Both NRF1 and NRF3 are required to maintain a basal proteasome activity in cancer cells

80 Initially, we investigated the biological relevance of NRF1 and NRF3 for proteasome activity at

81 the basal level in living cancer cells. Using human colorectal carcinoma HCT116 cells, we

82 generated cells which stably expressed the ZsProSensor-1 fusion protein, a proteasome-sensitive

83 fluorescent reporter (Supplementary Fig. 1A). Once the proteasome in this stable cell line was

84 inhibited by proteasome inhibitor MG-132, green fluorescence derived from the reporter protein

85 was detected using a flow cytometer (Supplementary Fig. 1B). We found that a double

86 knockdown of NRF1 and NRF3 significantly decreased basal proteasome activity in living cancer

87 cells (Fig. 1A). Consistent results were obtained by in vitro proteasome activity assays

88 (Supplementary Fig. 1C). The double knockdown also impaired the cancer cell resistance to a

89 proteasome inhibitor bortezomib (BTZ), which is clinically used as an anticancer drug11,12 (Fig.

90 1B).

91 Next, we investigated the impact of NRF1 and NRF3 on the expression of proteasome-

92 related genes. To address this issue, we performed DNA microarray analysis using NRF1- or

93 NRF3-knockdown HCT116 cells, and found 42 proteasome-related genes with a decrease in

94 expression of less than 1.4-fold (Supplementary Table 1). Gene Set Enrichment Analysis (GSEA)

95 using these array datasets showed reduced expression of 17 common core genes in both NRF1

96 and NRF3 knockdown cells (Supplementary Fig. 1D and E, Supplementary Table 2). Therefore,

97 using RT-qPCR we showed that the mRNA levels of PSMB3, PSMB7, PSMC2, PSMD3, PSMG2,

98 PSMG3, and POMP were significantly decreased by the double knockdown of NRF1 and NRF3

99 (Fig. 1C). These results indicate that both NRF1 and NRF3 are required to maintain the basal

100 expression of several proteasome-related genes.

101 NRF1 induces proteasome-related genes by directly binding antioxidant response

102 elements (ARE) in their gene promoters3–6. NRF3 also binds to ARE sequence in vitro8, although

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103 it has not been reported whether NRF3 binds to the ARE in the promoters of proteasome-related

104 genes in cells. To address this issue, we analyzed our chromatin immunoprecipitation (ChIP)

105 sequencing datasets in the presence of proteasome inhibitor MG-132, which stabilizes both NRF1

106 and NRF3 proteins (manuscript in preparation) and found the positive ChIP peak of NRF1 and

107 NRF3 proteins on the promoters of PSMB7, PSMC2, PSMD3, and POMP genes (Fig. 1D),

108 suggesting that NRF3 as well as NRF1 directly induces the basal expression of several

109 proteasome-related genes.

110

111 NRF3 represses NRF1 translation by inhibiting polysome formation on NRF1 mRNA

112 To clarify the molecular mechanism behind the maintenance of a basal proteasome activity in

113 cancer cells by both NRF1 and NRF3, we then investigated the relationship between NRF1 and

114 NRF3 expression in HCT116 cells. Interestingly, NRF1 protein levels were increased by NRF3

115 knockdown, while NRF3 protein levels were not changed by NRF1 knockdown (Fig. 2A). Similar

116 results were obtained in other cancer cell lines, including T98G (human glioblastoma multiforme),

117 and MCF-7 (human breast cancer) (Supplementary Fig. 2A). The levels of NRF1 mRNA were

118 unchanged by NRF3 knockdown (Fig. 2B, Supplementary Fig. 2B). We also obtained consistent

119 results in cells in which NRF3 was overexpressed (Supplementary Fig. 2C and D). These results

120 indicate that NRF3 decreases NRF1 protein levels without altering NRF1 gene expression,

121 suggesting the possible effect of NRF3 on the protein stability or translation of NRF1. To

122 investigate the former effect, we performed a cycloheximide (CHX) chase experiment and found

123 that NRF3 knockdown did not stabilize NRF1 proteins (Fig. 2C). Meanwhile, we investigated the

124 latter effect by a polysome profiling analysis and showed that NRF3 knockdown increased the

125 amount of NRF1 mRNA in polysomes between fractions #11 and #20 (Fig. 2D), although rRNA

126 distribution and global protein synthesis remained unchanged (Supplementary Fig. 2E and F).

127 These results indicate that NRF3 decreases polysome formation on NRF1 mRNA, thereby

128 repressing NRF1 translation.

129

130 NRF3 directly induces the gene expression of translational regulator CPEB3

5 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

131 To identify an NRF3-target gene which represses NRF1 translation, we performed DNA

132 microarray analysis using NRF3-knockdown HCT116 cells and NRF3-overexpression H1299

133 (human non-small cell lung cancer) cells and identified 146 genes whose expression was

134 positively associated with NRF3 gene expression (Supplementary Fig. 3A). Among these genes,

135 we found CPEB3 to be a candidate gene related to translational regulation, using

136 analysis (Supplementary Fig. 3B) and validated this result using RT-qPCR (Fig. 3A). We then

137 confirmed that NRF3 proteins bound to one of two ARE regions within the CPEB3 promoter (Fig.

138 3B and C), indicating that NRF3 directly induces CPEB3 gene expression. We investigated

139 whether CPEB3 is related to the NRF3-mediated regulation of NRF1 translation and found that

140 CPEB3 knockdown increased NRF1 protein levels and the amount of NRF1 mRNA in polysomes,

141 while rRNA distribution and global protein synthesis remained unchanged (Fig. 3D and E,

142 Supplementary Fig. 3C–E). We further confirmed that NRF1 protein levels were decreased by

143 CPEB3 overexpression (Fig. 3F). These results clearly demonstrate that NRF3 specifically

144 represses NRF1 translation in a CPEB3-dependent manner. We also found evidence of CPEB3

145 gene induction by NRF1 (Supplementary Fig. 3F), suggesting the presence of negative feedback

146 regulation of NRF1 through CPEB3-mediated translation repression. We discuss the biological

147 implications of this finding in the Discussion section.

148 The CPEB family as RNA-binding proteins are essential regulators of post-

149 transcriptional gene expression, with functions including the polyadenylation of 3′UTRs and

150 ribosome recruitment onto mRNA13. CPEB proteins recognize a pentanucleotide RNA sequence

151 (5′-UUUUA-3′) in the 3′UTR of a target gene14. Five CPEB recognition motifs (CPEs) are found

152 in the NRF1-3′UTR (numbers highlighted in red in Supplementary Fig. 3G). To identify the

153 functional CPE, we initially checked by an RNA immunoprecipitation (RIP) assay that NRF1

154 mRNA was precipitated with transiently expressed 3×Flag-CPEB3 proteins (Fig. 3G). We next

155 confirmed that NRF3 knockdown induced the translation via NRF1-3′UTR (Fig. 3H) using a

156 translational assay with a luciferase reporter and also obtained results consistent with those of the

157 CPEB3 knockdown (Fig. 3I). We finally performed deletion and mutation analysis using this

158 translational assay (Fig. 3J). The deletion of the region containing CPE#2–#5, but not deletion of

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159 the region containing CPE#3–#5, induced translation via NRF1-3′UTR in comparison with the

160 full length (WT-Full vs. WT-D#2–#5 or WT-Full vs. WT-D#3–#5). The adenine mutation of the

161 CPE#2 (UUUUA->AAAAA) also induced the translation via the full length, and #3–#5 deleted

162 NRF1-3′UTR (WT-Full vs. mCPE#2-Full, or WT-D#3–#5 vs. mCPE#2-D#3–#5). These results

163 clearly demonstrate that CPEB3 and CPE#2 in NRF1-3′UTR function as the trans regulator and

164 its cis-element that are involved in NRF3-mediated repression of NRF1 translation.

165

166 CPEB3 is a key factor for the maintenance of a basal proteasome activity and the poor

167 prognosis of colorectal cancer patients

168 We investigated the impact of CPEB3 overexpression on the basal expression of proteasome-

169 related genes, the basal proteasome activity, and BTZ resistance. The basal mRNA levels of

170 PSMB3, PSMB7, PSMC2, PSMG2, and POMP were not changed under a single knockdown of

171 NRF3 (Fig. 4A, Emp + siCont vs. Emp + siNRF3). Meanwhile, CPEB3 overexpression

172 significantly reduced these mRNA levels under a single knockdown of NRF3 (Fig. 4A, CPEB3 +

173 siCont vs. CPEB3 + siNRF3). Furthermore, CPEB3 overexpression under NRF3 single

174 knockdown impaired the basal proteasome activity and the cancer cell resistance to BTZ (Fig. 4B

175 and C). Finally, we validated the clinical relevance of the current findings in colorectal cancer

176 where both NRF genes are highly expressed. Using the datasets archived at The Cancer Genome

177 Atlas, we confirmed that colorectal cancer patients with higher CPEB3/NRF3-expressing tumors

178 exhibit shorter overall survival rates, but higher CPEB3/NRF1 expression is not associated with

179 this prognosis (Fig. 4D and Supplementary Fig. 4A). These results clearly show that CPEB3 acts

180 as a key factor for not only the complemental maintenance of basal proteasome activity in cancer

181 cells by NRF1 and NRF3, but also the poor prognosis of colorectal cancer patients with high

182 expression levels of NRF3, but not NRF1.

183

184 Discussion

185 Here, we demonstrated the complementary, but not simultaneous, regulatory mechanism of basal

186 proteasome activity in cancer cells through the NRF3-CPEB3-NRF1 translational repression axis

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187 (Fig. 4E); In cancer cells, NRF3 induces the basal expression of proteasome-related genes in

188 parallel with NRF1 translational repression by inducing CPEB3 gene expression (left panel). If

189 NRF3 gene expression is reduced, NRF1 escapes from the CPEB3-mediated translational

190 repression and complementarily plays a transcriptional role for the robust maintenance of basal

191 proteasome activity in cancer cells (right panel). We also found that NRF1 induces CPEB3 gene

192 expression (Supplementary Fig. 3F) and that CPEB3 represses NRF1 translation (Fig. 3D and F).

193 Although we have not confirmed yet whether NRF1 binds to the CPEB3 promoter, we identified

194 an NRF3-bound ARE in the CPEB3 promoter (Fig. 3B). Considering that NRF1 and NRF3 bind

195 to the ARE sequence, these results suggest the possibility of negative feedback regulation of

196 NRF1 through CPEB3-mediated translational repression (Supplementary Fig. 4B).

197 NRF1 is ubiquitously expressed in normal tissues, and mice in which it is knocked out

198 suffer embryonic lethality15. Meanwhile, NRF3 expression levels are low except in several tissues

199 such as the placenta8, and Nrf3 knockout mice do not exhibit any apparent abnormalities under

200 normal physiological conditions16–18. However, NRF3 is highly expressed in many types of cancer

201 cells, implying that the proteasome in cancer or normal cells is maintained through the NRF3-

202 CPEB3-NRF1 translational repression axis or the negative feedback regulation of NRF1. Indeed,

203 we revealed a clinical association of higher CPEB3/NRF3 expression, but not higher

204 CPEB3/NRF1 expression, with poor prognosis of cancer patients (Fig. 4D and Supplementary

205 Fig. 4A). Therefore, our findings suggest the possibility that while NRF1 maintains a basal

206 proteasome activity within an appropriate range for normal development, NRF3 adjusts the

207 proteasome in the quality and quantity for cancer development.

208 We guess another biological meaning of our findings in cancer. The functional

209 difference between NRF3 and NRF1 is not in the regulation of the proteasome activity rather in

210 the expression of cancer-related genes such as VEGFA and DUX4 that other groups have recently

211 identified as NRF3-specific target genes19,20. Meanwhile, cancer cells have to keep NRF1 as a

212 backup for proteasome regulation, because the proteasome activity is crucial for more rapid

213 proliferation of cancer cells21. Thus, the NRF3-CPEB3-NRF1 translational repression axis

214 enables cancer cells to grow more rapidly through NRF3-mediated signals and to maintain the

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215 proteasome activity through NRF1 immediately in case NRF3 does not work. Further study is

216 needed to confirm this point.

217 Unexpectedly, NRF1 knockdown did not affect NRF3 protein levels (Fig. 2A,

218 Supplementary Fig. 2A), although NRF1 knockdown reduced CPEB3 mRNA levels

219 (Supplementary Fig. 3F) and NRF3 gene possessed several CPEs in its 3′UTR (blue highlighted

220 numbers in Supplementary Fig. 3G). CPEB3 belongs to the CPEB family consisting of four

221 paralogs, CPEB1, CPEB2, CPEB3 and CPEB422. According to the binding RNA sequence, the

222 CPEB family is functionally categorized into two groups, CPEB1 and CPEB2-423, implying that

223 CPEB2 and/or CPEB4 compensates for the reduction of the CPEB3 gene by NRF1 depletion. An

224 alternative possibility is a conversion of CPEB function in a cellular context-dependent manner13.

225 In maturing mouse and Xenopus oocytes, CPEB acts as the translational activator through

226 phosphorylation by aurora kinase24,25. Therefore, there is an unknown mechanism of cross-talking

227 between NRF and CPEB protein families.

228

229 Materials and Methods

230 Cell lines

231 HCT116 (human colorectal carcinoma), T98G (human glioblastoma multiforme), and MCF-7

232 (human breast cancer) cells were cultured in DMEM/high glucose (Wako) supplemented with

233 10% FBS (Nichirei), 40 µg/ml streptomycin, and 40 units/ml penicillin (Life Technology). H1299

234 (human non-small cell lung cancer) cells were cultured in RPMI-1640 (Nacalai Tesque)

235 supplemented with 10% FBS (Nichirei), 40 µg/ml streptomycin, and 40 units/ml penicillin (Life

236 Technology). To generate the cells stably expressing a ZsProSensor reporter (ZsPS cells),

237 HCT116 cells were transfected with the proteasome sensor vector (Clontech). To generate NRF3-

238 and GFP-overexpression cells, H1299 cells were transfected with the p3×FLAG-CMV 10 vector

239 (Sigma-Aldrich) containing human full-length NRF3 or GFP. The transfected cells were selected

240 with G-418.

241

242 Plasmid construction and mutagenesis

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243 The 3×Flag-CPEB3 plasmid was generated by subcloning the PCR-amplified human CPEB3

244 cDNA into the p3×FLAG-CMV 10 vector (Sigma-Aldrich). The human CPEB3 cDNA was

245 synthesized using the indicated primers. Forward: 5′-TTTGAATTCAATGCAGGATGATTTAC-

246 3′, Reverse: 5′-AAAGGATCCTCAGCTCCAGCGGAAC-3′. The luciferase reporter driven by

247 the NRF1-3′UTR-Full was generated by PCR amplification of human genomic DNA using the

248 indicated primers (Forward: 5′-GGCCGGCCGCCTGGGAAGAAGGGGGTT-3′, Reverse: 5′-

249 GGCCGGCCTTTTTTTTTTTTTTTTTTACAATGAGTCCA-3′), and cloned into pGL3-Control

250 Vector (Promega). The deletion of NRF1-3′UTR (NRF1-3′UTR-D#2-#5 or D#3-#5) was

251 performed by a PCR-based method using the indicated primers. Forward: 5′-

252 TTTTTGGCCGGCCCCTGGGGAAGAAG-3′, Reverse: 5′-

253 TTTTTGGCCGGCCGACATTGTAGTCC-3′ (NRF1-3′UTR- Δ #2-#5), or 5′-

254 TTTTTGGCCGGCCGCACTCCCAGGTT-3′ (NRF1-3′UTR-Δ#3-#5). The adenine mutation of

255 CPE#2 in NRF1-3′UTR were introduced by site-directed mutagenesis using PCR. The primer

256 sequence was as follows (altered nucleotides are underlined): 5′-

257 ACAATGTCTTTATAAAAAACTGTTTGCCA-3′. All constructs were confirmed by sequencing.

258

259 Antibody

260 The antibodies utilized in the current immunoblot analysis were anti-α-tubulin (DM1A; Sigma-

261 Aldrich), anti-NRF1 (D5B10; Cell Signaling Technology), anti-NRF3 (#9408), and anti-FLAG

262 (M2; Sigma-Aldrich). A monoclonal NRF3 antibody (#9408) raised against human NRF3 (amino

263 acids 364-415) was generated as described previously26.

264

265 Transfection

266 The transfection of the plasmid DNA and short interfering RNA (siRNA) was performed using

267 polyethylenimine (PEI) and RNAiMAX (Invitrogen), respectively. The sequences of the siRNA

268 duplexes are listed in Supplementary Table 3.

269

270 RNA extraction and real-time quantitative PCR (RT-qPCR)

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271 Total RNA was extracted and purified using ISOGEN II (NIPPON GENE) according to the

272 manufacturer’s instructions. Aliquots of total RNA (1 µg) were reverse transcribed using pd(N)6

273 random primer (Takara Bio) and Moloney murine leukemia virus (M-MLV) reverse transcriptase

274 (Invitrogen) with 250 µM deoxy nucleoside triphosphate (dNTP, Takara Bio) concentration,

275 according to the manufacturer’s instructions. RT-qPCR was performed with SYBR Premix Ex

276 Taq II (Takara Bio) and primers for genes using a Thermal Cycler Dice Real Time System (Takara

277 Bio). Each gene expression level in human cells was normalized to the mRNA levels of human

278 β-actin gene. qPCR primer sequences are described at Supplementary Table 3.

279

280 Immunoblot analysis

281 To prepare whole cell extracts, the cells were lysed with SDS sample buffer (50 mM Tris-HCl

282 [pH 6.8], 10% glycerol and 1% SDS). The protein quantities in cell extracts were measured with

283 a BCA kit (ThermoFisher Scientific). The proteins were separated by sodium dodecyl sulfate-

284 polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes

285 (Immobilon-P transfer membrane, EMD Millipore corporation). After blocking the membranes

286 with Blocking One (Nacalai Tesque) at 4°C for overnight, the membranes were incubated with a

287 primary antibody, washed with TBS-T (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% Tween20)

288 and were incubated with a horseradish peroxidase-conjugated secondary antibody (Invitrogen).

289 The blots were washed with TBS-T and developed with enhanced chemiluminescence (GE

290 Healthcare).

291

292 Cycloheximide chase experiments

293 HCT116 cells were transfected with the indicated siRNA. At 48 hours after transfection, the cells

294 were treated with 20 µg/ml cycloheximide (CHX), and the whole cell extracts were prepared at

295 the indicated time points. The immunoblot analysis was conducted with the indicated antibodies.

296

297 Protein synthesis assays

298 The protein synthesis assay was conducted using Click-iT Plus OPP Protein Synthesis Assay Kit

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299 (Invitrogen) according to the manufacturer’s protocol. HCT116 cells were transfected with the

300 indicated siRNA. At 48 hours after transfection, the cells were treated with O-propargyl-

301 puromycin (OPP; 20 µM) for 1 hour at 37°C and washed twice with PBS. The cells were fixed

302 by using 4% paraformaldehyde for 15 min at room temperature, followed by washing them twice

303 with PBS (containing 0.5% BSA) and then permeabilizing with PBSTx (0.5% TritonX-100/PBS)

304 for 15 min at room temperature. The cells were washed twice with PBS (containing 0.5% BSA).

305 After their treatment with Click-iT Reaction Mixtures for 30 min at room temperature in a dark

306 place, the cells were then washed with Click-iT Reaction Rise Buffer. Finally, the cells were

307 washed with PBS and subjected to the protein synthesis assay using BD FACS Aria-II (Becton

308 Dickinson).

309

310 Polysome fractionation assays

311 HCT116 cells were treated with 100 µg/ml cycloheximide (CHX) in culture medium for 5 min at

312 37°C. The cells were washed twice with PBS containing CHX (100 µg/ml) and lysed with lysis

313 buffer (15 mM Tris-HCl [pH 7.5], 300 mM NaCl, 25 mM EDTA, 1% Triton X-100, 0.5 mg/ml

314 heparin, 100 µg/ml CHX) with 1× EDTA-free protease inhibitor cocktail (Nacalai Tesque). The

315 lysate was centrifuged at 9,300 × g for 10 min at 4°C and loaded onto a linear 20–50% sucrose

316 gradient buffer in 15 mM Tris-HCl [pH 8.0], 25 mM EDTA and 300 mM NaCl. Centrifugation

317 was conducted at 40,000 rpm for 2 hours at 4°C in a SW-41 Ti rotor (Beckman Coulter), and

318 fractions were collected from the top of the gradient (#1 to #20). The RNA from each fraction

319 was subjected to RT-qPCR analysis. The quantified mRNA was normalized by the input. qPCR

320 primer sequences are described at Supplementary Table 3.

321

322 DNA microarray analysis

323 Total RNA was processed with the Ambion WT Expression Kit (Affymetrix) according to the

324 manufacturer’s instructions. cRNA was then fragmented, labelled, and hybridized to the

325 Affymetrix Human Gene 1.0 ST Arrays using the Gene Chip WT Terminal Labeling and

326 Hybridization Kit (Affymetrix). GeneChip fluidics station 450 was used for processing of the

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327 arrays and fluorescent signals were detected with the GeneChip scanner 3000-7 G. Images were

328 analyzed with the GeneChip operating software (Affymetrix).

329 Expression console and Transcription analysis console (Affymetrix) were used to

330 analyze the data. PANTHER Classification System with the theme “Molecular function” was used

331 for gene ontology analysis of the genes whose expression was reduced upon siRNA-mediated

332 NRF3-knockdown HCT116 cells (fold change ≧ 1.4) and increased by NRF3-stable

333 overexpression H1299 cells (fold change ≧ 1.4). The expression data of all genes in siRNA-

334 mediated NRF3- or NRF1-knockdown HCT116 cells were subjected to GSEA using open source

335 software v3.027 and the gene set containing 42 proteasome-related genes in Supplementary Table

336 1.

337

338 Chromatin immunoprecipitation (ChIP) qPCR

339 The cells were fixed with 1% formaldehyde for 10 min at room temperature, and then glycine

340 was added to a final concentration of 0.125 M. The cells were lysed with cell lysis buffer (5 mM

341 Tris–HCl [pH 8.0], 85 mM KCl, 0.5% NP-40) with protease inhibitors (Nacalai Tesque), and then

342 centrifuged at 2,000 rpm at 4°C for 3 min. The pellets were further lysed with nuclei lysis buffer

343 (50 mM Tris–HCl [pH 8.0], 10 mM EDTA, 1% SDS) with protease inhibitors (Nacalai Tesque),

344 and the lysates were sonicated. After centrifugation at 15,000 rpm at 8°C for 10 min, the

345 supernatants were collected. The supernatants were diluted in ChIP dilution buffer (16.7 mM

346 Tris–HCl [pH 8.0], 167 mM NaCl, 1.2 mM EDTA, 1.1% TritonX-100, 0.01% SDS). The diluted

347 samples were precleared with 20 µl of Dynabeads Protein G (ThermoFisher Scientific), and then

348 the supernatants (used as an input sample) were incubated with 2 µg of anti-NRF3 antibody. The

349 immunocomplexes were collected by incubation with 20 µl of Dynabeads Protein G

350 (ThermoFisher Scientific), and then washed with the following buffers: low salt wash buffer (20

351 mM Tris–HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high salt

352 wash buffer (20 mM Tris–HCl [pH 8.0], 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1%

353 SDS), and LiCl wash buffer (10 mM Tris–HCl [pH 8.0], 250 mM LiCl, 1 mM EDTA, 1% sodium

354 deoxycholate, 1% NP-40). Finally, the beads were washed twice with 1 ml of TE buffer (10 mM

13 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

355 Tris–HCl [pH 8.0], 1 mM EDTA). The immunocomplexes were then eluted by adding 200 µl of

356 elution buffer (50 mM NaHCO3, 1% SDS). After the reverse cross-linking by adding 200 mM

357 NaCl, the remaining proteins were digested by adding proteinase K. For quantification of NRF3-

358 binding to the target regions, RT-qPCR was performed using the purified DNA with the primers

359 described at Supplementary Table 3.

360 For ChIP-sequencing, the libraries were prepared from 500 pg of immunoprecipitated

361 DNA fragments using the KAPA Hyper Prep Kit (KAPA Biosystems). The libraries were applied

362 to Single-end sequencing for 93 cycles on HiSeq2500 (Illumina). All sequence reads were

363 extracted in FASTQ format using BCL2FASTQ Conversion Software 1.8.4 in the CASAVA 1.8.2

364 pipeline. Mapping was performed by BWA (version 0.5.9rc1) using the reference ,

365 NCBI build 37 (hg19), and peak call was conducted using MACS (version 1.4.2).

366

367 RNA immunoprecipitation (RIP)

368 HCT116 cells were transfected with empty vector or 3×Flag-CPEB3 plasmid. At 48 hours after

369 transfection, cells were lysed in cell lysis buffer containing protease and RNase inhibitors. Lysates

370 were sonicated for 10 min at low intensity and then precleared. Samples were immunoprecipitated

371 using anti-FLAG antibody coupled to Dynabeads Protein A (Invitrogen) for 3 hours at 4℃. RNA

372 bound the FLAG-beads was purified by a phenol/chloroform extraction, and was subjected to RT-

373 qPCR analysis. qPCR primer sequences are described at Supplementary Table 3.

374

375 Luciferase reporter assays

376 Cells expressing the reporters indicated in the legends for Fig. 3H-J were lysed, and the luciferase

377 activities were measured with the PicaGene luciferase assay system (Toyo Ink) and a microplate

378 reader (Synergy HTX, Bio Tek Instruments).

379

380 Proteasome activity analysis using a ZsProSensor reporter

381 The ZsProSensor-1 protein is a fusion of the green fluorescent protein ZsGreen and mouse

382 ornithine decarboxylase (MODC), and can be degraded by the proteasome without being

14 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

383 ubiquitinated. ZsPS cells were transfected with the indicated siRNA or plasmid. At 48 hours after

384 transfection, the cells were collected, followed by washing them twice with STM (2% FBS in

385 cold PBS). 500 µl of STM containing 2 µg/ml of propidium idodide (PI) was added to each sample. 386 After their treatment at 4℃ in a dark place, samples were subjected to the proteasome activity

387 analysis using BD FACS Aria- II (Becton Dickinson).

388

389 in vitro proteasome activity assays

390 in vitro proteasome activity assays were based on a glycerol density gradient centrifugation and

391 fluorogenic peptidase assays as described previously28. After centrifugation at 26,000 rpm for 22

392 hours in a Beckman SW40Ti swing rotor, the gradient was manually separated into 20 fractions

393 of 500 µl each. Since the 26S proteasome, which is made up of a 20S proteasome and one or two

394 19S-RPs, is contained in fraction #13 and #14, the average of the fluorescent intensity was

395 calculated as the proteasome activity as follows: 30 µl of each fraction sample was transferred to

396 a 96-well BD Falcon microtiter plate (BD Biosciences), and mixed with 2 mM ATP and 0.1 mM

397 fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC (Peptide Institute). Fluorescence (380

398 nm excitation, 460 nm emission) was monitored on a microplate fluorometer (Synergy HTX,

399 BioTek Instruments) every 5 min for 1 hour29.

400

401 Cell viability assays using Trypan Blue staining

402 HCT116 cells were transfected with siRNA and/or plasmid DNA under the conditions indicated

403 in the legends for Figs. 1B and 4C. After the treatment with 5 nM or 10 nM bortezomib (BTZ,

404 Peptide Institute), the cells were stained by Trypan Blue and automatically counted using a TC20

405 Automated Cell Counter (Bio-Rad Laboratories).

406

407 Statistics and human cancer database

408 The unpaired Student’s t-test was used to compare the two groups, and one-way analysis of

409 variance followed by Tukey’s post-hoc test was used to compare multiple groups. The GEPIA, a

410 web server for cancer and normal gene expression profiling and interactive analyses10 was used

15 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

411 for Kaplan–Meier analyses.

412

413 Conflict of Interest: The authors declare that no competing interests exist.

414

415 Acknowledgments

416 We thank Hiderori Watanabe, Mika Matsumoto and Junko Naritomi for experimental supports.

417 This work was supported in part by grant-in-aid for Young Scientists (B) (17K18234 to T.W.);

418 grant-in-aid for Scientific Research (C) (19K07650 to T.W.); The Uehara Memorial Foundation

419 (to T.W.); grant-in-aid for Scientific Research (B) (16H03265 to A.K.); grant-in-aid for

420 Challenging Research (Exploratory) (19K22826 to A.K.); grant-in-aid for JSPS Fellows

421 (18J20672 to A.H.).

422

423 Contributions

424 T.W., H.K., and Y. T. conceived the project; T.W. and H.K. designed experiments; T.W., H.K.,

425 M.H., A.H., N.N., Y.T., N.T. and A.W. performed experiments and analyzed data; T.W. and A.K.

426 wrote the manuscript; T.W. and A.K. provided funding; A.K. provided supervision.

427

428 References 429 1 Adams J. The proteasome: Structure, function, and role in the cell. Cancer Treat Rev 430 2003; 29: 3–9. 431 2 Luo J, Solimini NL, Elledge SJ. Principles of Cancer Therapy: Oncogene and Non- 432 oncogene Addiction. Cell 2009; 136: 823–837. 433 3 Radhakrishnan SK et al. Transcription Factor Nrf1 Mediates the Proteasome Recovery 434 Pathway after Proteasome Inhibition in Mammalian Cells. Mol Cell 2010; 38: 17–28. 435 4 Tsuchiya Y et al. The Casein Kinase 2-Nrf1 Axis Controls the Clearance of 436 Ubiquitinated Proteins by Regulating Proteasome Gene Expression. Mol Cell Biol 2013; 437 33: 3461–3472. 438 5 Sha Z, Goldberg AL. Proteasome-mediated processing of Nrf1 is essential for coordinate 439 induction of all proteasome subunits and p97. Curr Biol 2014; 24: 1573–1583. 440 6 Steffen J, Seeger M, Koch A, Krüger E. Proteasomal degradation is transcriptionally

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441 controlled by TCF11 via an ERAD-dependent feedback loop. Mol Cell 2010; 40: 147– 442 158. 443 7 Lee CS et al. Loss of nuclear factor E2-related factor 1 in the brain leads to 444 dysregulation of proteasome gene expression and neurodegeneration. Proc Natl Acad Sci 445 U S A 2011; 108: 8408–8413. 446 8 Kobayashi A et al. Molecular cloning and functional characterization of a new Cap’n’ 447 collar family transcription factor Nrf3. J Biol Chem 1999; 274: 6443–6452. 448 9 Liu Y et al. Comparative Molecular Analysis of Gastrointestinal Adenocarcinomas. 449 Cancer Cell 2018; 33: 721–735. 450 10 Tang Z et al. GEPIA: A web server for cancer and normal gene expression profiling and 451 interactive analyses. Nucleic Acids Res 2017; 45: W98–W102. 452 11 Adams J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 453 2004; 5: 417–421. 454 12 Manasanch EE, Orlowski RZ. Proteasome inhibitors in cancer therapy. Nat Rev Clin 455 Oncol. 2017; 14: 417–433. 456 13 Fernández-Miranda G, Méndez R. The CPEB-family of proteins, translational control in 457 senescence and cancer. Ageing Res Rev 2012; 11: 460–472. 458 14 Afroz T et al. A fly trap mechanism provides sequence-specific RNA recognition by 459 CPEB proteins. Genes Dev 2014; 28: 1498–1514. 460 15 Chan JY et al. Targeted disruption of the ubiquitous CNC-bZIP transcription factor, Nrf- 461 1, results in anemia and embryonic lethality in mice. EMBO J 1998; 17: 1779–1787. 462 16 Derjuga A et al. Complexity of CNC transcription factors as revealed by gene targeting 463 of the Nrf3 . Mol Cell Biol 2004; 24: 3286–3294. 464 17 Kobayashi A, Ohta T, Yamamoto M. Unique Function of the Nrf2-Keap1 Pathway in the 465 Inducible Expression of Antioxidant and Detoxifying Enzymes. Methods Enzymol 2004; 466 378: 273–286. 467 18 Chevillard G, Nouhi Z, Anna D, Paquet M, Blank V. Nrf3-deficient mice are not 468 protected against acute lung and adipose tissue damages induced by butylated 469 hydroxytoluene. FEBS Lett 2010; 584: 923–928. 470 19 Wang H et al. Elevated expression of NFE2L3 predicts the poor prognosis of pancreatic 471 cancer patients. Cell Cycle 2018; 17: 2164–2174. 472 20 Bury M et al. NFE2L3 Controls Colon Cancer Cell Growth through Regulation of 473 DUX4, a CDK1 Inhibitor. Cell Rep 2019; 29; 1469–1481.

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474 21 Loda M et al. Increased proteasome-dependent degradation of the cyclin-dependent 475 kinase inhibitor p27 in aggressive colorectal carcinomas. Nat Med 1997; 3: 231–234. 476 22 Mendez R, Richter JD. Translational control by CPEB: A means to the end. Nat Rev Mol 477 Cell Biol 2001; 2: 521–529. 478 23 Huang YS, Kan MC, Lin CL, Richter JD. CPEB3 and CPEB4 in neurons: Analysis of 479 RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA. 480 EMBO J 2006; 25: 4865–4876. 481 24 Hodgman R, Tay J, Mendez R, Richter JD. CPEB phosphorylation and cytoplasmic 482 polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes. 483 Development 2001; 128: 2815–2822. 484 25 Mendez R et al. Phosphorylation of CPE binding factor by Eg2 regulates translation of 485 c- mos mRNA. Nature 2000; 404: 302–307. 486 26 Chowdhury AMMA et al. Multiple regulatory mechanisms of the biological function of 487 NRF3 (NFE2L3) control cancer cell proliferation. Sci Rep 2017; 7: 12494 488 27 Subramanian A et al. Gene set enrichment analysis: a knowledge-based approach for 489 interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 2005; 102: 490 15545–15550. 491 28 Murata S, et al. Immunoproteasome assembly and antigen presentation in mice lacking 492 both PA28α and PA28β. EMBO J 2001; 20: 5898–5907. 493 29 Tanahashi N et al. Hybrid Proteasomes. J Biol Chem 2000; 275: 14336–14345. 494 495 Figure legends

496 Figure 1. NRF1 and NRF3 complementarily regulate proteasome activity and proteasome subunit

497 gene expression at a basal level. (A) Impact of NRF knockdown on a basal proteasome activity.

498 At 24 hours after siRNA transfection into ZsPS cells, the fluorescence intensity derived from a

499 ZsProSensor-1 reporter was measured using flow cytometry. The cell populations in Q1 enclosed

500 by a red line are those with “low proteasome activity.” siNRF1/3 is a double knockdown of NRF1

501 and NRF3. Control siRNA (siCont) was used as a control. ***p < 0.005; n.s., not significant (n =

502 3, mean ± SD, ANOVA followed by Tukey’s test). (B) Impact of NRF knockdown on the BTZ

503 resistance. At 24 hours after siRNA transfection to HCT116 cells, the cells were treated with 5

504 nM BTZ and further incubated for 48 hours. Then, the cells were subjected to cell viability assay

18 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

505 using trypan blue staining. siNRF1/3 represents a double knockdown of NRF1 and NRF3. Control

506 siRNA (siCont) and DMSO were used as controls. Cell viability was determined by the number

507 of living cells with BTZ treatment normalized by that with DMSO. *p < 0.05; n.s., not significant

508 (n = 3, mean ± SD, ANOVA followed by Tukey’s test) (C) Impact of NRF knockdown on mRNA

509 levels of 17 common core genes with a “Yes” value in the core enrichment in both NRF1 and

510 NRF3 knockdown cells (Supplementary Table 2). At 48 h after siRNA transfection into HCT116

511 cells, the cells were analyzed by RT-qPCR. siNRF1/3 represents a double knockdown of NRF1

512 and NRF3. Control siRNA (siCont) was used as a control. mRNA levels of each proteasome

513 subunit were normalized according to levels of β-actin mRNA. *p < 0.05; **p < 0.01; ***p <

514 0.005 (n = 3, mean ± SD, ANOVA followed by Tukey’s test, *siCont vs. siNRF1; †siCont vs.

515 siNRF3; §siCont vs. siNRF1/3) (D) ChIP peaks of NRF1 and NRF3 in the promoters of

516 proteasome-related genes. ChIP sequencing of endogenous NRF1 or exogenous NRF3 was

517 performed using wild-type HCT116 cells or NRF3-overexpression H1299 cells treated with 1 µM 518 MG-132 for 24 hours.

519

520 Figure 2. NRF3 represses NRF1 translation by inhibiting polysome formation on NRF1 mRNA.

521 (A and B) Impact of NRF knockdown on protein and mRNA levels of another NRF in HCT116

522 (colorectal carcinoma) cells. At 48 hours after siRNA transfection, the cells were analyzed by

523 immunoblotting (A) and RT-qPCR (B). Protein or mRNA levels of each NRF were normalized

524 with reference to α-tubulin protein or β-actin mRNA, respectively. Control siRNA (siCont) was

525 used as a control. *p < 0.05; n.s. not significant (n = 3, mean ± SD, t-test in A, ANOVA followed

526 by Tukey’s test in B) (C) Impact of NRF knockdown on protein degradation of another NRF. At

527 48 hours after siRNA transfection, the cells were treated with CHX, and analyzed by

528 immunoblotting at the times indicated. Protein levels were normalized by α-tubulin. Control

529 siRNA (siCont) was used as a control. (n = 3, mean ± SD) (D) Distribution of NRF mRNA in

530 HCT116 transfected with indicated siRNA (n = 2). At 48 hours after siRNA transfection, the cells

531 were analyzed by sucrose gradient centrifugation followed by RT-qPCR. mRNA levels in each

532 fraction were normalized against those in the input. Mean and individual values are represented

19 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

533 as a line and mark, respectively. Control siRNA (siCont) was used as a control.

534

535 Figure 3. CPEB3 is an NRF3-target gene that negatively regulates NRF1 translation. (A) Impact

536 of NRF3 knockdown on mRNA levels of CPEB3 in HCT116 cells (left pane) and NRF3-

537 overexpression H1299 cells (right panel). Control siRNA (siCont) or GFP-overexpression H1299

538 cells were used as controls. mRNA levels of CPEB3 were normalized according to β-actin mRNA.

539 *p < 0.05 (n = 3, mean ± SD, t-test) (B and C) The recruitment of NRF3 on CPEB3 promoters in

540 NRF3-overexpression H1299 cells. GFP-overexpression H1299 cells were used as controls. In

541 (B), the genome locus of the CPEB3 promoter and multiple sequences of two candidate AREs in

542 indicated species are shown. TSS; Transcription start site. **p < 0.01; n.s., not significant (n = 3,

543 mean ± SD, t-test) (D) Impact of CPEB3 knockdown on NRF1 protein levels. Each siRNA was

544 transfected into HCT116 cells. After 48 hours, the cells were analyzed by immunoblotting.

545 Representative images of immunoblotting are shown in the left panel, and the protein levels were

546 normalized by α-tubulin in the right panel. Control siRNA (siCont) was used as a control. *p <

547 0.05 (n = 3, mean ± SD, ANOVA followed by Tukey’s test) (E) Impact of CPEB3 knockdown on

548 the amount of NRF1 mRNA on polysomes. At 48 hours after siRNA transfection, the cells were

549 subjected to sucrose gradient centrifugation. The fractions from #2 to #8 and from #11 to #19

550 shown in Supplementary Fig. 3D were collected as the non-polysomal and polysomal fractions,

551 respectively. Each fraction was analyzed by RT-qPCR. mRNA levels of NRF1 in each fraction

552 were normalized against those in the input. NRF1 mRNA levels in the polysomal fractions were

553 then divided by NRF1 mRNA levels in the non-polysomal fractions. Control siRNA (siCont) and

554 siNRF3 were used as controls. Mean ± SD and three independent values are represented as bars

555 and indicated colored marks, respectively (n = 3). (F) Impact of CPEB3 overexpression on NRF1

556 protein levels. At 48 hours after 3×Flag-CPEB3 plasmid transfection into HCT116 cells, the cells

557 were analyzed by immunoblotting. Empty vector (Emp) was used as a control. (G) Interactions

558 between CPEB3 protein and NRF1 mRNA. At 24 hours after 3×Flag-CPEB3 plasmid transfection,

559 cells were analyzed by RIP assay followed by RT-qPCR. RIPed mRNA levels of NRF1 were

560 normalized against the input values. Empty vector (Emp) was used as a control. *p < 0.05 (n = 3,

20 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

561 mean ± SD, t-test) (H and I) Impact of NRF3 or CPEB3 knockdown on NRF1 translation via its

562 3′UTR. At 24 hours after the transfection of siNRF3 (H) or siCPEB3 (I), a luciferase reporter

563 vector fused with NRF1-3′UTR was transfected and culture for 24 hours. Control siRNA (siCont)

564 was used as a control. Luciferase activity was normalized by mRNA levels of a luciferase gene.

565 *p < 0.05 (n = 3, mean ± SD, t-tests) (J) Impact of NRF1-3′UTR deletion or mutation on

566 translation. The luciferase reporter vector fused with the indicated NRF1-3′UTR was transfected

567 into HCT116 cells, and the cells were analyzed after 24 hours. Luciferase activity was normalized

568 according to the mRNA levels of a luciferase gene. Red and blue rectangles represent wild-type

569 (WT) CPEs, which are highlighted in Supplementary Fig. 3G, and an adenine mutated CPE#2

570 (mCPE#2), respectively. **p < 0.01; n.s., not significant (n = 3, mean ± SD, ANOVA followed

571 by Tukey’s test)

572

573 Figure 4. NRF3 and CPEB3 contribute to maintain proteasome activity in cancer cells and the

574 prognosis of colorectal cancer patients. (A–C) Impact of CPEB3 overexpression under NRF3

575 knockdown on the expression of the proteasome-related genes (A), a proteasome activity (B), and

576 the BTZ resistance (C). HCT116 or ZsPS cells were used in (A and C) or in (B), respectively. At

577 24 hours after NRF3 siRNA transfection, the cells were additionally transfected with the 3×Flag-

578 CPEB3 plasmid and cultured for 24 hours. Control siRNA (siCont) and empty vector (Emp) were

579 used as controls. In (A), the cells were subject to RT-qPCR using the primers for seven

580 proteasome-related genes analyzed in Fig. 1C. mRNA levels of each proteasome subunit were

581 normalized according to levels of β-actin mRNA. In (B), the fluorescence intensity derived from

582 a ZsProSensor-1 reporter was measured using flow cytometry. The cell populations in Q1

583 enclosed by a red line are those with “low proteasome activity.” In (C), the cells were treated with

584 10 nM BTZ and further incubated for 24 hours. Then, the cells were subjected to cell viability

585 assay using trypan blue staining. Cell viability was determined by the number of living cells with

586 BTZ treatment normalized by that with DMSO. * p < 0.05, **p < 0.001, ***p < 0.005; n.s., not

587 significant (n = 3, mean ± SD, t-test). (D) Kaplan–Meier analysis comparing overall survival

588 between higher and lower CPEB3/NRF3 groups. The hazard ratio (HR) was calculated based on

21 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

589 Cox’s proportional hazards model. Patients having colorectal adenocarcinoma (COAD) or rectal

590 adenocarcinoma (READ) from The Cancer Genome Atlas. (E) Schematic model of the cross-talk

591 mechanism between NRF1 and NRF3 to complementarily maintain a basal proteasome gene

592 expression and activity in cancer cells through CPEB3-mediated translational repression.

593

594 Supplementary Figure 1. NRF1 and NRF3 function on the basal gene induction of proteasome-

595 related factors. (A) mRNA levels of the ZsProSensor reporter gene. Parental and ZsPS cells were

596 analyzed using RT-qPCR. mRNA levels of the reporter gene were normalized according to levels

597 of β-actin mRNA. ***p < 0.005 (n = 3, mean ± SD, t-test) (B) The in vitro activity of the 26S

598 proteasome fractionated from NRF knockdown cells. At 24 hours after indicated siRNA

599 transfection into HCT116 cells, the cell extracts were manually fractionated into 20 fractions

600 using a 10%–40% glycerol gradient centrifugation and the average of the fluorescent intensity in

601 fraction #13 and #14 was calculated as the 26S proteasome activity. siNRF1/3 is a double

602 knockdown of NRF1 and NRF3. Control siRNA (siCont) was used as a control. *p < 0.05; n.s.,

603 not significant (n = 3, mean ± SD, t-test) (C) Impact of MG-132 treatment of proteasome activity

604 in ZsPS cells. At 24 hours after treatment with 1 µM MG-132, the cells were analyzed using flow

605 cytometry. DMSO was used as control. The cell populations in Q1 enclosed by a red line are

606 shown as “low proteasome activity.” ***p < 0.005 (n = 3, mean ± SD, t-test) (D and E)

607 Enrichment plots for the gene sets involved in a proteasome. DNA microarray data using NRF1-

608 or NRF3-knockdown HCT116 cells are shown in (C) and (D), respectively. Control siRNA

609 (siCont) was used as a control.

610

611 Supplementary Figure 2. NRF3 does not change polysome formation or global translation as

612 well as protein degradation of NRF1. (A and B) Impact of NRF knockdown on protein and mRNA

613 levels of NRF1 or NRF3 in T98G (glioblastoma multiforme) and MCF-7 (breast cancer) cells. At

614 48 hours after siRNA transfection, cells were analyzed by immunoblotting (A) and RT-qPCR (B).

615 Control siRNA (siCont) was used as a control. mRNA levels of each NRF were normalized using

616 β-actin mRNA. n.s., not significant (n = 3, mean ± SD, ANOVA followed by Tukey’s test in B)

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

617 (C and D) Impact of NRF3 overexpression on protein and mRNA levels of NRF1. NRF3-

618 overexpression H1299 cells were analyzed by immunoblotting (C) and RT-qPCR (D). GFP-

619 overexpression H1299 cells were used as controls. mRNA levels of NRF1 were normalized by β-

620 actin mRNA. n.s., not significant (n = 3, mean ± SD, t-test in D) (E) Distribution of ribosomal

621 RNAs in HCT116 cells transfected with NRF1 or NRF3 siRNA. At 48 hours after siRNA

622 transfection, cells were analyzed using sucrose gradient centrifugation followed by agarose gel

623 electrophoresis. Control siRNA (siCont) was used as a control. (F) Impact of NRF knockdown on

624 a global translation. At 48 hours after siRNA transfection, the cells were stained using an OPP-

625 based protein synthesis assay followed by flow cytometry. Control siRNA (siCont) and CHX were

626 used as controls. n.s., not significant (n = 3, mean ± SD, ANOVA followed by Tukey’s test).

627

628 Supplementary Figure 3. The translational regulator CPEB3 does not affect global protein

629 synthesis. (A) Venn diagram combining two independent DNA microarray datasets of NRF3-

630 knockdown (NRF3KD) HCT116, and NRF3-overexpression (NRF3OE) H1299 cells. GFP-

631 overexpression H1299 cells were used as a control. (B) Gene ontology analysis of 146 common

632 genes using the PANTHER Classification System. The term “Molecular function” was selected.

633 (C) Knockdown efficiency of CPEB3 siRNAs. At 48 hours after siRNA transfection, cells were

634 analyzed using RT-qPCR. mRNA levels of CPEB3 were normalized according to levels of β-actin

635 mRNA. Control siRNA (siCont) was used as a control. *p < 0.05 (n = 3, mean ± SD, ANOVA

636 followed by Tukey’s test). (D) Distribution of ribosomal RNAs in HCT116 cells transfected with

637 CPEB3 siRNA. At 48 hours after siRNA transfection, cells were analyzed using sucrose gradient

638 centrifugation followed by agarose gel electrophoresis. Control siRNA (siCont) was used as a

639 control. (E) Impact of CPEB3 knockdown on a global protein synthesis. At 48 hours after siRNA

640 transfection, the cells were stained using an OPP-based protein synthesis assay and analyzed by

641 flow cytometry. Control siRNA (siCont) and CHX were used as controls. n.s., not significant (n

642 = 3, mean ± SD, ANOVA followed by Tukey’s test). (F) Impact of NRF1 knockdown on mRNA

643 levels of CPEB3 in HCT116 cells. At 48 hours after siRNA transfection, the cells were analyzed

644 by RT-qPCR. Control siRNA (siCont) was used as a control. mRNA levels of CPEB3 were

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

645 normalized by β-actin mRNA. *p < 0.05 (n = 3, mean ± SD, t-test) (G) DNA sequence alignment

646 of NRF1- and NRF3-3′UTR. CPEB recognition motifs (5′-UUUUA-3′) in NRF1- and NRF3-

647 3′UTR were highlighted by red and blue, respectively.

648

649 Supplementary Figure 4. Clinical association of NRF1 and CPEB3 expression levels with the

650 prognosis of colorectal cancer patients. (A) Kaplan–Meier analysis comparing overall survival

651 between higher and lower CPEB3/NRF1 groups. The hazard ratio (HR) was calculated based on

652 Cox’s proportional hazards model. Patients having colorectal adenocarcinoma (COAD) or rectal

653 adenocarcinoma (READ) from The Cancer Genome Atlas. (B) Schematic model of the negative

654 feedback regulation of NRF1 through CPEB3-mediated translation repression.

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Waku et al., Fig. 1 (A) (B) siCont siNRF3 *** * 70 n.s. 140 60 n.s. 120 50 n.s. 100 40 80 (Q1, %) (Q1, 30 n.s. siNRF1 siNRF1/3 20 60 Viable cell Viable 40 10 DMSO) of (% Low proteasome activity proteasome Low 0 20 0 ZsGreen siCont siNRF1 siNRF3 siNRF1/3 siCont PI siNRF1 siNRF3 siNRF1/3 (C) 2.0 siCont siNRF1 siNRF3 siNRF1/3 1.5   1.0   †    † 0.5 *  *  † *  0

Relative expression Relative NRF1 NRF3 PSMC1 PSMC2 PSMC5 PSMD3 PSMD12 PSMD14 SHMF1 PSME4 NRF 19S regulatory particle PA200

1.6 1.2 †     0.8   0.4 0 Relative expression Relative PSMA2 PSMA3 PSMA5 PSMA7 PSMB3 PSMB7 PSMG2 PSMG3 POMP

20S proteasome 20S proteasome Assembly chaperons

(D) bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Waku et al., Fig. 2

(A) NRF1 NRF3 (B) NRF1 mRNA NRF3 mRNA 6 6 1.4 1.4 n.s. * n.s. 5 5 1.2 1.2 siCont siNRF1 siNRF3 1 1 intensity 4 4 NRF1 0.8 0.8 3 3 band 0.6 0.6 NRF3 2 2 n.s. 0.4 0.4 1 1 0.2 0.2 Relative expression Relative α-tubulin Relative 0 0 0 0

siCont siCont siCont siCont siNRF3 siNRF1 siNRF1 siNRF3 siNRF1 siNRF3 (C) NRF3 siCont siNRF1 siNRF3 NRF1 CHX(h) 0 0.5 1 1.5 0 0.5 1 1.5 0 0.5 1 1.5 1.5 siCont 1.5 siCont siNRF3 siNRF1 NRF1 intensity 1 1 band NRF3 0.5 0.5

0 0 α-tubulin Relative 05 0. 1 1.5 0 0.5 1 1.5 Time (h) Time (h) (D)

NRF1 mRNA NRF3 0.3 0.3 mRNA siCont siCont mRNA) siNRF3 mRNA) 0.2 0.2 siNRF1 Fold Fold NRF1 0.1 NRF3 0.1 ( / input input / ( 0 input / ( 0 2 3 4 5 6 7 8 9 1011 12 1314151617 1819 20 2 3 4 5 6 7 8 9 1011 12 1314151617 1819 20 non -polysomes polysomes non -polysomes polysomes bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Waku et al., Fig. 3

(A) CPEB3 mRNA (B) (C) CPEB3v1-ARE CPEB3v2-ARE 1.2 * 2 * 0.3 ** 0.3 1 1.5 n.s. 0.8 0.2 0.2 0.6 1 % input % 0.4 0.1 0.1 0.5

Relative expression Relative 0.2

0 0 0 0

GFP GFP GFP siCont NRF3 NRF3 NRF3 siNRF3 (E) NRF1 mRNA NRF1 9 (D) 8 2.5 * (F) 7

* - 2 6 Emp Flag CPEB3 -polysomes 5 intensity 1.5 4 NRF1 / non siCont siCPEB3 #2 siCPEB3 siCPEB3 #1 siCPEB3 band 1 3 CPEB3 NRF1 2 (Flag) 0.5 1

α-tubulin Relative α-tubulin 0 Polysomes 0 #1 #2 #1 #2 siCPEB3 siCont siCont siCPEB3 siNRF3

(G) (H) (I) (J) Luc activity / Luc mRNA 0.06 * 4 * 2.5 * NRF1-3’UTR 0 1 23 4 0.05 2 3 #1 #2 #3 #4 #5 mRNA mRNA Full luciferase

0.04 ** 1.5 n.s. Luc Luc D#3-#5 luciferase

2 ** 0.03 WT % input % 1 0.02 D#2-#5 luciferase ** 1 0.5 0.01 Full luciferase Luc activity / / activity Luc / activity Luc 0 0 0 D#3-#5 luciferase

- mCPE#2

Emp Flag siCont siCont CPEB3 siNRF3

siCPEB3#1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.10.902718; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Waku et al., Fig. 4

(A) NAPSMB3 mR PSMB7 mRNA PSMC2 mRNA PSMD3 mRNA

n.s. ** * 0.08 0.3 0.3 0.3 n.s. * ** 0.25 * n.s. 0.06 0.2 0.2 0.2 0.15 0.04 0.1 0.1 0.1 0.02 0.05 0 0 0 0 Relative expression Relative Emp Flag- Emp Flag- Emp Flag- Emp Flag- CPEB3 CPEB3 CPEB3 CPEB3

PSMG2 mRNA PSMG3 mRNA POMP mRNA 0.1 * 0.07 0.3 n.s. * * * siCont 0.08 0.06 0.25 n.s. 0.05 0.2 siNRF3 0.06 0.04 0.15 0.04 0.03 0.02 0.1 0.02 0.01 0.05

Relative expression Relative 0 0 0 Emp Flag- Emp Flag- Emp Flag- CPEB3 CPEB3 CPEB3

(B) siCont siNRF3 (C) siCont siNRF3 (D) COAD 100 Low CPEB3/NRF3 High CPEB3/NRF3 70 *** 120 n.s. ** 80 Logrank p = 0.1 60 100 HR(high) = 1.5 n(high) = 203 50 80 60 n(Low) = 203 40 60 30 40 n.s. 40 20 (%) survival Overall 20 10 20 Viable cell (% of DMSO) (%of cell Viable

Low proteasome activity (Q1, %) (Q1, activity proteasome Low 0 0 0 Emp Flag- Emp Flag- CPEB3 CPEB3 0 50 100 150 (Months) (E) READ 100

80

60

Low CPEB3/NRF3 40 High CPEB3/NRF3 Logrank p = 0.0019 Overall survival (%) survival Overall 20 HR(high) = 4.1 n(high) = 69 n(Low) = 69 0 0 20 40 60 80 100 120 (Months)