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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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]
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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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.
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 genes 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 gene 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 proteins 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 protein 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
4 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.
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 gene ontology
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
6 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.
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
7 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.
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
8 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.
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
9 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.
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)
10 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.
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
11 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.
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
12 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.
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 human genome,
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
16 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.
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 locus. 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.
17 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.
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)