Renal cell carcinoma (RCC) represents above 3 % of all cancers. At diagnosis, above 25 % of patients with RCC present an advanced disease. Gastric metastasis of RCC is associated with poor outcome. We report the case of a patient treated for a gastric metastasis of RCC and we conducted a systematic review of the literature to report all published cases of RCC patients with gastric metastasis.In December 2010, a 61-year-old man was treated by open partial nephrectomy for a localized right clear cell RCC. In September 2018, a metachronous gastric metastasis was found on CT scan. The lesion was located on the lesser curvature of the stomach, measuring 4.5 cm long axis. No other secondary lesions were identified. A laparoscopic wedge resection, converted to laparotomy was performed. Two years later, in September 2020, a CT scan was performed, revealing a 17 mm adenopathy behind the hepatic hilum and a surgical management was performed, including a lymph node dissection of the hepatic hilum and the hepatic artery. Actually, he remains healthy.Our systematic review suggests that solitary gastric metastasis of RCC are scarce. In comparison of patients with multiple metastatic sites, the median survival of patients with solitary gastric metastasis is longer. At diagThe most common metastatic RCC sites are the lung and bone (up to 60 % and 40 % of patients with metastases) [Gastric metastasis of RCC has been described  and it iThus, these metastases are diagnosed on routine CT scans as part of the monitoring of RCC after local treatment. Gastrointestinal bleeding and anemia are the most common presentations of symptomatic gastric metastasis ,33.We herein report the case of a patient, who underwent a wedge resection for a gastric metastasis of RCC, which is healthy at 2-years of follow-up. Furthermore, we conducted a systematic review of the literature to report all published cases of RCC patients with gastric metastasis.2In December 2010, a 61-year-old man was referred to our academic center for the management of a localized right clear cell RCC, without synchronous metastases. It was an upper polar lesion, measuring 2.5 cm long axis, essentially exophytic, posterior, entirely above the polar lines, without vascular contact, classified 5p RENAL score.An open right partial nephrectomy was performed by an experienced surgeon and histological results highlighted a 2.5 cm clear cell RCC, Furhman grade III, classified pT3aN0R0 due to the extension to perinephric tissues, but not beyond Gerota Fascia.According to EAU (European Association of Urology) guidelines , a surveIn Mars 2012, a localized homolateral recurrence has been highlighted at CT scan. It was a lower polar lesion, measuring 4.3 cm long axis, essentially endophytic, anterior, close to the collecting system, with polar line crosses and venous contact, classified 9 ah RENAL score, Supplemental Fig. 1. There was no evidence of metastasis on this CT scan.An open right radical nephrectomy was performed and histological examination reported a 3.5 cm clear cell RCC, Furhman grade III, classified pT3aN0R0 due to the extension to the renal sinus fat.According to EAU guidelines , a surveIn September 2018, a metachronous gastric metastasis was found on CT scan. The lesion was located on the lesser curvature of the stomach, measuring 4.5 cm long axis. No other secondary lesions were identified, Supplemental Fig. 2. The patient had no digestive symptoms, no hematemesis, no melena.A surgical procedure has been validated: a laparoscopic wedge resection, converted to laparotomy has been performed.Histological examination reported a proliferation of atypical cells containing round clear nuclei and irregularly shaped nuclei with prominent nucleoli using hematoxylin-eosin staining. Immunostaining revealed negativity for all epithelial markers  and positivity for proximal tubular epithelial markers . These characteristics led to the conclusion of gastric metastasis of a clear cell RCC with 30 % sarcomatoid contingent, with negative resection margins.The patient had an uneventful postoperative course and was discharged on the 8th day.Two years later, in September 2020, a CT scan was performed, revealing a 17 mm adenopathy behind the hepatic hilum and a surgical management was performed, by an experienced surgeon, including a lymph node dissection of the hepatic hilum and the hepatic artery. Histological examination reported a node metastasis of the clear cell RCC, in hepatic artery lymph node dissection. Actually, he remains healthy.This case has been reported in line with the SCARE 2020 criteria .3Literature about gastric metastasis of RCC was realized using electronic database Medline via PubMed (1950–2018).Key terms used included “renal cell carcinoma,” “stomach metastasis,” and “gastric metastasis” . ReferenRCC with solitary gastric metastasis cases (n = 11) are summarized in The median patients age (IQR) was 68.0 (62.8–73.3) years, of whom 82 % were men. The most frequent locations of metastasis were the middle body of the stomach (45 %), followed by the upper body of the stomach (36 %) and the lower body of the stomach (18 %). The median time (IQR) between the diagnosis of RCC and metachronous gastric metastasis was 8.5 (3.8–19.3) years. 1 patient presented RCC with synchronous gastric metastasis. A surgical treatment of the metastasis was performed in 64 % of the patients and an endoscopic treatment in 27 % of the cases. Endoscopic treatment was a submucosal resection without positive surgical margins. 1 patient received a palliative chemotherapy. Median patient survival (IQR), after treatment, was 9 months (3.4–15.8).RCC with multiple or unspecified metastatic sites (including stomach) cases (n = 31) are summarized in At RCC gastric metastasis diagnosis, 73 % of the patients presented multiple metastatic sites, mainly lungs and bones. The median patients age (IQR) was 66.0 (60.0–69.0) years, of whom 74 % were men. The median time (IQR) between the diagnosis of RCC and metachronous gastric metastasis was 5.0 (2.0–9.5) years. 3 patients presented RCC with synchronous gastric metastasis. 35 % of the patients had a surgical treatment, 16 % an endoscopic treatment, 23 % a systemic therapy and 26 % a palliative treatment. Endoscopic treatment was mucosal and submucosal resection without positive surgical margins. Median patient survival (IQR), after treatment, was 6 months (2.5–24.0).4Renal cell carcinoma is the most common solid lesion within the kidney and accounts approximately 90 % of all kidney malignancies . It compIndeed, clear cell RCC has an abundant blood supply and can metastasize to several organs . MetastaCurrently, EAU guidelines  and FrenOur systematic review suggests that solitary gastric metastasis of RCC are scarce. Herein, we report the 12th case of solitary gastric metastasis, discovered 6 years after local treatment of RCC. For the 11th cases reported in the literature, the median age of the patients was 68.0 years, of whom 82 % were men. The most frequent locations of metastasis were the middle body of the stomach (45 %), followed by the upper body of the stomach (36 %) and the lower body of the stomach (18 %). The median time between diagnosis of RCC and metachronous gastric metastasis was 8.5 years. A surgical treatment of the metastasis was performed in 64 % of the patients and an endoscopic treatment in 27 % of the cases. Thus, in case of solitary gastric metastasis of RCC, endoscopic or surgical treatment of the metastasis, allows to delay the initiation of systemic treatment, with median patient survival of 9 months after treatment. Our case has a similar age. The location of the metastasis was the middle body of the stomach. The median time between diagnosis of RCC and metachronous gastric metastasis was 6 years, lower than median time reported by the 11th cases. Two years after gastric metastasis surgical treatment, a hepatic hilum adenopathy recurrence occurred, treated by surgery and our median patient survival was higher (26 months versus 9 months).However, in case of multiple metastatic sites, the stomach is a frequent localization of metastases. Thus, at gastric metastases diagnosis, 80 % of patients presented multiple metastatic sites, mainly lungs and bones. The median time between RCC diagnosis and metachronous gastric metastasis diagnosis was shorter in case of multiple metastatic sites (5.0 years versus 8.5 years in case of solitary gastric metastasis).Surgical treatment, when it is feasible remains the best therapeutic option for a solitary gastric metastasis, resulting in significant survival prolongation in eligible patients. Indeed, median patient survival was higher after surgical treatment of gastric metastasis (9 months (3.4–15.8) versus 6 months (2.5–24.0) in case of solitary and multiple metastatic sites).However, after surgical treatment of solitary gastric metastases, the risk of metastatic recurrence is significant. Among the 11 cases of solitary gastric metastasis reported in the literature, 3 patients presented a metastasis recurrence. Our case presented a hepatic hilum adenopathy recurrence, treated by surgery, two years after gastric metastasis surgical treatment.5Our systematic review suggests that solitary gastric metastasis of RCC are scarce. In comparison of patients with multiple metastatic sites, the median survival of patients with solitary gastric metastasis is longer. When it is feasible, surgical or endoscopic treatment of the solitary gastric metastasis should be performed, whenever the patient is fit for surgery, in order to delay the initiation of systemic treatment. Nevertheless, the risk of metastatic recurrence is significant and must be taken into consideration in the therapeutic strategy.None.None.According to French legislation, retrospective studies are not subject to an ethics committee.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Dr. Thomas Prudhomme: Data collection, Data analysis, Manuscript writingDr. Charlotte Maulat: Data collection, Data analysisDr. Guillaume Péré: Data collection, Data analysisDr. Fatima-Zohra Mokrane: Data collection, Data analysisPr. Michel Soulié: Data collection, Data analysisPr. Fabrice Muscari: Data collection, Data analysis, Manuscript writing.Not applicable.Thomas Prudhomme.Not commissioned, externally peer-reviewed.


Bacillus subtilis 6S-1 RNA and its release from σA-RNA polymerase (σA-RNAP) in vitro using truncated and mutated 6S-1 RNA variants. Truncated 6S-1 RNAs, only consisting of the central bubble (CB) flanked by two short helical arms, can still traverse the mechanistic 6S RNA cycle in vitro despite ~10-fold reduced σA-RNAP affinity. This indicates that the RNA’s extended helical arms including the ‘−35′-like region are not required for basic 6S-1 RNA functionality. The role of the ‘central bubble collapse helix’ (CBCH) in pRNA-induced refolding and release of 6S-1 RNA from σA-RNAP was studied by stabilizing mutations. This also revealed base identities in the 5’-part of the CB (5’-CB), upstream of the pRNA transcription start site (nt 40), that impact ground state binding of 6S-1 RNA to σA-RNAP. Stabilization of the CBCH by the C44/45 double mutation shifted the pRNA length pattern to shorter pRNAs and, combined with a weakened P2 helix, resulted in more effective release from RNAP. We conclude that formation of the CBCH supports pRNA-induced 6S-1 RNA refolding and release. Our mutational analysis also unveiled that formation of a second short hairpin in the 3′-CB is detrimental to 6S-1 RNA release. Furthermore, an LNA mimic of a pRNA as short as 6 nt, when annealed to 6S-1 RNA, retarded the RNA’s gel mobility and interfered with σA-RNAP binding. This effect incrementally increased with pLNA 7- and 8-mers, suggesting that restricted conformational flexibility introduced into the 5’-CB by base pairing with pRNAs prevents 6S-1 RNA from adopting an elongated shape. Accordingly, atomic force microscopy of free 6S-1 RNA versus 6S-1:pLNA 8- and 14-mer complexes revealed that 6S-1:pRNA hybrid structures, on average, adopt a more compact structure than 6S-1 RNA alone. Overall, our findings also illustrate that the wild-type 6S-1 RNA sequence and structure ensures an optimal balance of the different functional aspects involved in the mechanistic cycle of 6S-1 RNA.Here we investigated the refolding of  Bacillus subtilis 6S-1 RNA and Escherichia coli 6S RNA is 38 to 45%, depending on the type of alignment . A bioinformatic analysis predicted more than 1700 6S RNAs in >1600 bacterial species . The aforementioned 10 µL reaction mixtures were incubated for 30 min at 37 °C, followed by mixing with 10 µL 2 × native loading dye  and loading onto a native 7.5% native polyacrylamide (PAA) gel , typically run for 3 h at 25 mA. In the experiments shown in Figure 2C, native PAGE was conducted in an electrophoresis buffer containing 0.5 × TBE, 160 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, pH 8.6, with correspondingly adjusted electric field strength to prevent substantial warming of the gel during electrophoresis ; the final concentration of 6S-1 RNA was 10 nM and that of σA-RNAP was 2 µM. After 15 s, 1 min, and 2 min at 37 °C, 10 µL aliquots were withdrawn, mixed with 10 μL of 2 × native loading dye (see above), and put immediately on ice. The three collected aliquots were then loaded onto a 7.5% native PAA gel  that was running at low current (~5 to 10 mA) to avoid diffusion of initially loaded samples from gel pockets until all pockets were loaded. After loading the last aliquot set, the current was increased to 25 mA for 3 h. Phosphor imaging and data evaluation were performed as described in For the NTP concentration variation experiment in Figure 5, a set of 10.5 µL mixtures containing 3.5 µL 6S-1 RNA (100 nM stock concentration with trace amounts of 5′-32P-labeled 6S RNA (~2500 Cherenkov c.p.m per µL) was combined with 5.5 µL of 2 × TE buffer  and refolded as described in A-RNAP (stock concentrations varied between 5.75 and 9.05 µM), and RNase-free ddH2O were added to a total volume of 53.6 µL. The reaction mixture was incubated for 1 h at 37 °C to equilibrate 6S RNA binding to σA-RNAP. Subsequently, 1.4 µL of an A/C/G/UTP mix (2 mM each NTP) was added  to start the reaction at 37 °C. The reaction mixtures contained final concentrations of usually 2 µM  His-tagged σA-RNAP, 50 µM each NTP, and trace amounts (2500 Cherenkov c.p.m per gel lane) of 5′-32P-labeled 6S RNA. ATP omission experiments and the experiment shown in Figure 6A followed the same protocol, except that the concentration of each added NTP was 100 µM instead of 50 µM. Deviations from this protocol are specified in the legend to Figure 2E. Typically, 10 µL aliquots were withdrawn after 15 s and after 2, 5, 10, and 15 min, to which 10 µL of 2 × native loading dye (see above) were added, followed by shock freezing in liquid N2. Samples were loaded onto a native 7.5% PAA gel  that was run for 3 h at 25 mA. Control samples lacking NTPs, or enzyme and NTPs, were prepared and handled in the same manner as the test samples before loading onto the gel (incubation time at 37 °C for at least 15 min). Phosphor imaging and data evaluation were performed as described in A total of 5.5 µL of 5′-× TE buffer and subjected to the refolding protocol (see above). Then, 3 µL of 5 × transcription buffer (see above), 2.4 µL of native σA-RNAP (stock concentration: 15.84 µM), and 3.1 µL of RNase-free ddH2O were added to a volume of 12.5 µL. The reaction mixture was incubated for 15 min at 37 °C to equilibrate binding of 6S RNA to σA-RNAP. Subsequently, 2 µL of NTP mix  plus 0.52 µL [α-32P]UTP  were added. The final concentrations in the reaction mixture were 2 μM for the 6S RNA variant, 2.5 μM for native σA-RNAP, and 200 μM for each NTP. The final reaction mixture (~15 µL) was incubated for up to 1 h at 37 °C. After incubation, 5 µL of the reaction mixture was withdrawn and mixed with 15 µL of 2 × denaturing loading dye , heated for 3 min to 98 °C in a dry bath , followed by cooling on ice for 5 min. Control samples  either lacking native σA-RNAP or without 6S RNA were subjected to the same conditions as the test samples. Samples were loaded onto denaturing 25% PAA gel  that was run for 16 h at 1200 V. Phosphor imaging and data evaluation was performed as described in A-RNAP final concentration of 1 μM, and withdrawal of 5 µL aliquots at time points 5-, 20-, and 60-min post-nucleotide addition.For the experiment shown in Figure 3D, 2 µL of in vitro T7-transcribed 6S RNA (stock concentration: 15 μM) were combined with 2 µL of 2 A-RNAP (stock concentration varied from 5.75 µM to 9.05 µM), and RNase-free ddH2O were added to a total volume of 24.2 µL, followed by equilibration for 15 min at 37 °C. Approximately 12 µL aliquots of this reaction mixture were transferred into two new tubes, followed by the addition of 2.9 µL of A/C/G/UTP mix or C/G/UTP mix (1.39 mM each NTP); the resulting ~15 µL contained [α-32P]UTP , 270 µM end concentration of each indicated NTP, 2 μM 6S RNA, and ~0.4 µM His-tagged σA-RNAP; pRNA transcription was allowed to proceed for 1 h at 37 °C. Thereafter, 5 µL of the reaction mixture was withdrawn and mixed with 15 µL of 2 × denaturing loading dye (see above), heated for 3 min to 98 °C in a dry bath, followed by cooling on ice for 5 min. Control samples either lacking His-tagged σA-RNAP or 6S RNA were treated in the same manner. For details on electrophoresis, see the preceding paragraph. 4 µL of T7-transcribed 6S RNA (stock concentration ~15 μM) and 4 µL of 2 × TE buffer  were mixed and subjected to the RNA refolding procedure (see above). Then, 6 µL of 5 × transcription buffer (see above), ~1 to 5 µL of His-tagged σ32P-labeled 6S-1 RNA  and 0.4 µL 10 × TE buffer. The volume was adjusted to 4 µL with RNase-free ddH2O. Then, 2 µL of a 50 µM stock solution of the respective pLNA or pRNA oligonucleotide was added and the mixture was successively incubated for 5 min each at 95, 90, 80, 70, 60, and 50 °C, followed by transfer to 37 °C for at least 5 min. Subsequently, 1 µL of heparin (400 ng/µL), 2 µL of 5× transcription buffer (see above), and either ~1.1 µL native σA-RNAP (stock concentration: 8 mg/mL) or 1.1 µL double-distilled RNase-free ddH2O (for controls without RNAP) were added. Mixtures were incubated for 30 min at 37 °C, then supplemented with 10 µL of 2 × native RNA loading dye (see above) and analyzed by 10% native PAGE.A total of 10 pmol of 6S-1 RNA were mixed with trace amounts of 5’-× TE buffer. The annealing protocol was the same as described in the preceding paragraph. To remove excess pLNA oligonucleotides, samples containing the pLNA 8- or 14-mer were centrifuged 3 times, with 500 µL 1× TE buffer per round, through Amicon Ultra centrifugal filter units (molecular weight cut-off: 10 kDa). Before the actual measurements, the density of molecules was recorded in a test image; depending on the observed density, RNA mixtures were diluted with 1 × TE buffer to adjust the working concentrations to ~1–4 nM. RNA solutions were transferred to a mica surface treated with poly-L-lysine  as described [2 and a circularity (c = 4π × [area]/[perimeter]2) of 0.2 to 0.6 were annotated as 6S-1 RNA molecules. For each of these particles, an ellipse was fitted. The ratio of the longitudinal (long) and latitudinal (short) axis length  were used to describe the molecule shape. Smaller ratios are indicative of more compact and possibly bent molecules while larger ratios indicate more stretched conformations. For AFM analysis, the RNA was prepared as follows: 1 µL of a 10 µM stock solution of in vitro T7-transcribed 6S-1 RNA was mixed with a tenfold molar excess of the respective pLNA oligonucleotide in a final volume of 10 µL adjusted to 1 escribed . Pictureescribed . Particlhttp://rna.tbi.univie.ac.at, accessed on 23 December 2021) using the default parameters. For simulating complexes of 6S-1 RNA and pRNAs, the corresponding pRNA binding site was blocked using constraint folding with the Enforce Constrained pairing pattern option. The 3D structure predictions were performed with RNAcomposer . For RNAcomposer prediction of the 6S-1:pRNA complex, the 6S-1 RNA sequence was extended at the 5’-end by a linker sequence (here 25 cytidines) and the pRNA sequence at the very 5’-end, mimicking an artificial pseudoknot structure; very similar results were obtained with U25, A25, and G25 linkers or longer homopolymeric linkers of 50, 75, or 100 nt that were analyzed in comparison.Secondary structure predictions were computed on the RNAfold web server 2.4.18  in B. subtilis 6S-1 RNA  and a very similar but circularly permuted variant (6S82cp) that was 82-nt in length, both lacking the ‘−35’ region  6S-1 RNA  forms between nucleotides of the 3’-CB and the 3’-strand of helix P2 , when Pi system . As a sh6S-1 RNA  and no ese in Kd A. Similautations B,C, indi6S-1 RNA D. For a 6S-1 RNA B. This dr of two A and up r of two C. These A-RNAP were preformed and pRNA transcription was induced by the addition of NTP substrates, followed by withdrawal of aliquots at different time points. The experiment illustrated in A-RNAP gains substantial momentum at NTP concentrations >40 µM. Evidently, there are two subpopulations of 6S-1 RNA:σA-RNAP complexes, one reacting in the fast phase of the reaction (≤15 s) and the other reacting with slower kinetics . The subpopulation reacting in the fast phase increases with increasing NTP concentration  kinetics of wt and C44/45 mutant 6S-1 RNA yet provided no evidence for differences between the two . To accet 8M RNA B, but pRt 8M RNA C. The 8Mt 8M RNA A. Howevet 8M RNA D. RNAfolA-RNAP affinity of the variants wt 6M and C44/45 6M, again showing an almost three-fold reduction in RNAP affinity owing to the C44/45 mutations , but still formed more release-resistant complexes than the C44/45 5M RNA . Our findings demonstrate that already stable binding of a 6-meric pRNA mimic constrains or compacts the 6S-1 RNA conformation to an extent that impedes σA-RNAP binding.We previously showed that an iso-sequential analog of a pRNA 8-mer that consists of locked nucleic acid (LNA) residues stably binds to 6S-1 RNA and retards the RNA’s mobility in native PAA gels to a very similar extent as a pRNA 14-mer . Here wep = 2.2 × 10−16, one tailed Welch Two Sample t-test) increase in compactness for 6S-1 RNA:pLNA complexes relative to free 6S-1 RNA (We further employed atomic force microscopy (AFM) to study the shape of free 6S-1 RNA in comparison with complexes annealed to a pLNA 8-mer or 14-mer A,B. Part6S-1 RNA D. For deB. subtilis 6S-1 RNA truncation variant 6S78 and 6S82cp with length reductions of almost 60% still showed hallmarks of 6S RNAs, that is, served as template for transcription of pRNAs including 14-mers to form 6S RNA:pRNA hybrid structures that trigger 6S RNA release from σA-RNAP  caused a release defect and another one (U134A) accelerated the release rate [E. coli 6S RNA:pRNA hybrid structures  in the 5’-CB of E. coli 6S RNA, namely A52U and A50U, were tested for pRNA-induced release from σ70-RNAP [B. subtilis 6S-1 RNA, we saw clear binding defects upon mutation of A50 to U, C, or G and U44/45 to C44/45 upstream of the pRNA TSS (C40), consistent with base-specific direct contacts to the enzyme in this part of the 5’-CB. At present, it can also not be excluded that the C44/45 double mutation more indirectly reduced binding affinity through shifting the conformational equilibrium of unbound 6S-1 RNA toward conformers forming the CBCH instead of helix P2. It is noteworthy that two other 6S-1 RNA mutations in the 5’-CB, U47A, and U53A had no effect on ground state binding  remained indistinguishable between the wt and C44/45 mutant RNA upon pRNA synthesis in the presence of all four NTPs  or 8 (5M) nucleotides. These extensions may be due to NTP misincorporation, non-templated addition of NTPs, or utilization of alternative TSSs.A-RNAP binding. In native gels, elongated/extended/rod-shaped RNAs migrate faster than branched molecules [A. aeolicus 6S RNA that essentially the same gel mobility shift is obtained by annealing a pRNA 15-mer to nucleotides in the 5’-CB/5’-P2 strand compared with an artificial ‘pRNA’ 15-mer annealing to the opposite part of the CB, namely the 3’-strand of P2 and nucleotides in the 3’-CB [We demonstrated here that already stable binding of a hexameric pLNA mimic substantially retards gel mobility of 6S-1 RNA and alters its structure to an extent that is incompatible with σolecules . A 6-bp he 3’-CB . This obA. aeolicus 6S RNA with the artificial ‘pRNA’ 15-mer bound to 3’-P2/3’-CB was inefficient in preventing complex formation with σA-RNAP and rather formed aberrantly migrating complexes with the enzyme in native PAA gels [Remarkably, PAA gels . Thus, cA-RNAP ground state binding affinity, coordination of RNAP scrunching, enzyme switch to the elongation mode, pRNA transcript length, conformational constriction/refolding of 6S-1 RNA, and release of 6S-1 RNA:pRNA hybrids from RNAP.In addition to the pleiotropic and complex phenotypes of the C44/45, 8M, 6M, and 5M mutant RNAs, the sensitivity of 6S-1 RNA function to certain mutations  in the


ETS transcription factors are known to act as either positive or negative regulators of the expression of genes involved in various biological processes. It was reported that ETS variant transcription factor 5 (ETV5), a key member of the ETS family, mainly plays a role as an potential oncogene in various malignant tumors. However, the role and mechanism of ETV5 in high-grade serous ovarian cancer (HGSOC) have not been elucidated.Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to detect ETV5 messenger ribonucleic acid (mRNA) expression in 87 HGSOC tissues and 35 normal fallopian tube tissues. Western blotting and qRT-PCR were used to detect the protein and mRNA expression of ETV5 in six ovarian cancer (OC) and human embryonic cell lines. Knockdown or overexpression of ETV5 in HGSOC cell lines, Cell Counting Kit-8, colony formation, and transwell assays were used to detect HGSOC cell proliferation, invasion, and migration capabilities. The chi-square test was used to analyze the clinicopathological characteristics of HGSOC patients. Survival analysis was performed using the Kaplan-Meier method, and the log-rank test was used to analyze the correlation between ETV5 expression and HGSOC patient prognosis. Univariate and multivariate analyses using the Cox regression model were conducted to determine the independent significance of relevant clinical covariates.p < 0.05). qRT-PCR showed that ETV5 was significantly overexpressed in HGSOC tissues than in fallopian tube tissues (p < 0.05). qRT-PCR and western blotting assays demonstrated that ETV5 was relatively highly expressed in OC cell lines. ETV5 overexpression was positively associated with poor survival in HGSOC patients, therefore making it a high-risk factor for HGSOC progression. Furthermore, ETV5 promoted the proliferation, migration, and invasion capabilities of HGSOC cells.Bioinformatic analysis demonstrated that ETV5 expression was significantly upregulated in OC (ETV5 has a carcinogenic effect in HGSOC and can be used as a clinically effective biomarker to determine the prognosis of HGSOC patients.The online version contains supplementary material available at 10.1186/s13048-021-00899-6. Ovarian cancer (OC) is one of the most lethal gynecological cancers . In the ETS variant transcription factor 5 (ETV5), also known as ERM transcription factor, is a member of the ETS transcription factor family. The ETS family contains an evolutionarily conserved ETS domain that has 85 amino acids and binds to a DNA sequence with a 5′-GGA(A/T)-3′ core ; it playRecent studies have found that ETV5 is overexpressed in several malignant tumors, such as OC, inducing transcription of multiple oncogenes involved in cell apoptosis resistance, angiogenesis, migration, invasion, and other malignant biological behaviors –14. ETV5Thus, this study aimed to investigate the influence of ETV5 on the prognosis and malignant biological behaviors of HGSOC. Towards this goal, ETV5 expression was detected in normal fallopian tubes, HGSOC tissues, and HGSOC cell lines.This was a retrospective study of HGSOC patients who were treated at the First Affiliated Hospital of Shihezi University School of Medicine between 2010 and 2019. The clinicopathological data, including age, International Federation of Gynecology and Obstetrics (FIGO) stage, recurrence, chemotherapy response, ascites, disease-free survival, and overall survival, of these patients were collected from the on-paper medical records and electronic medical record system of the hospital. In total, 87 HGSOC tissues and 35 normal fallopian tube tissues were obtained. All tissues were fixed in 10% neutral formalin, processed, and embedded in paraffin. Diagnoses were confirmed through hematoxylin-eosin and immunohistochemical staining by two experienced pathologists, following the World Health Organization’s Pathology and Genetics Tumors of the Breast and Female Genital Organs (8th edition) guidelines.This study was approved by the Ethics Committee of the First Affiliated Hospital of Shihezi University School of Medicine  and was conducted according to the tenets of the Declaration of Helsinki. All patients provided written informed consent.In the experimental group, HGSOC samples from patients who met the following conditions were included in the study:1. newly diagnosed HGSOC patients who had not undergone radiotherapy and chemotherapy, and 2. patients diagnosed with primary HGSOC. In the control group, Fallopian tube samples from patients who met the following conditions were included in the study:1. patients who had undergone hysterosalpingectomy due to benign disease, and 2. patients who had not undergone radiotherapy and chemotherapy before surgery.Patients were excluded from the study if they had undergone radiotherapy and chemotherapy before surgery, as well as cases where consent according to national regulations was not obtainable, when the investigator was unable to obtain the necessary consent from the patient before inclusion in the study.https://www.ncbi.nlm.nih.gov/geo/) (Affymetrix Human Genome U133 Plus 2.0 Array). GEO is a public functional genomics database that stores data on gene expression profile, methylation profile, chips, and microarrays. GSE18520 contains data from 53 OC and 10 normal ovarian surface epithelial samples. GSE36668 contains data on four serous OC and four normal ovarian epithelial samples. GSE14407 contains data on 12 OC and 12 normal ovarian surface epithelial samples. The GEO2R online tool was used to analyze the differentially expressed genes between OC tissues and normal tissues in the three datasets. A cutoff value of ∣log 2-fold change (FC)∣ > 2 (FC) and a p-value < 0.01 were set as the statistical significance threshold. Gene Expression Profiling Interactive Analysis (GEPIA), an online analysis tool based on data from The Cancer Genome Atlas (TCGA) database, was used to analyze ETV5 differential expression between OC and normal tissues (http://gepia.cancer-pku.cn/).Gene expression profile data  were downloaded from the Gene Expression Omnibus (GEO) database  medium. The human embryonic kidney cell line, 293 T, was cultivated in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS) in a humidified tissue culture incubator containing 5% CO2 at 37 °C. SKOV3, HEYA8, and 293 T cells were purchased from the Chinese Academy of Sciences Type Culture Collection . OVCA433 and A2780 cells were a generous gift from Dr. Gang Cheng . OV2008 and C13* cells were obtained from Prof. Benjamin K. Tsang . The IOSE80 cell line was purchased from Shanghai Gaining Biotechnology Co., Ltd. .Total RNA was extracted from OC cell lines using the miRNeasy Mini Kit  and from HGSOC and fallopian tube tissues using the miRNeasy FFPE Kit  in accordance with the manufacturer’s instructions. Complementary deoxyribonucleic acid (cDNA) was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on a 7500 Fast Real-Time PCR System  using a QuantiFast SYBR Green PCR kit (Qiagen) in accordance with the manufacturer’s instructions. The primers used in this study were prepared by the Shanghai Sangon Biotechnology Company. The primer sequences are listed in Table Western blotting was performed according to standard protocols. The primary antibodies used in this study included mouse anti-human monoclonal ETV5 antibody  and mouse anti-human monoclonal beta (β)-actin antibody . Goat anti-rabbit/mouse immunoglobulin G conjugated to horseradish peroxidase (IgG-HRP), used as the secondary antibody, was purchased from Zhongshan Biotechnology . Protein signals were detected using an enhanced chemiluminescence kit . β-actin was used as an internal control.SKOV3, A2780, and OV2008 cells were incubated and transfected with the different treatments for 48 h, and the Cell Counting Kit-8 (CCK-8) assay was performed to construct a cell growth curve. The optical density (OD) value was determined by measuring the absorbance at 450 nm with a microplate reader at the same time every day until the seventh day, from the beginning of cell attachment. The daily OD values of each group of cells were used to generate a cell growth curve.The effect of ETV5 silencing on the proliferation of A2780 and SKOV3 cells and of ETV5 overexpression on the proliferation of OV2008 cells was analyzed using clone formation assays. Approximately 800 A2780, SKOV3, and OV2008 cells were cultured in a 6-well plate containing 10% FBS and in the RPMI-1640 medium for 10 days at 37 °C with 5% CO2. The colonies were washed twice with 1× phosphate-buffered saline (PBS), fixed for 5 min with 4% paraformaldehyde, and stained for 20 min . The number of colonies with more than 50 cells in a low-magnification visual field was counted, and the colony formation rate was calculated using the following formula: clone formation rate = (number of clones/seeded cells) × 100%.The cell migration and invasion assays were performed using a 24-well Transwell chamber . Cells were seeded at a density of 4 × 104 cells into the upper chamber , the lower chamber was filled with 600 μl RPMI-1640 medium containing 10% FBS. For invasion assays, 1 × 105 cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel. Medium containing 10% FBS was added to the lower chamber. Following a 24 h incubation period at 37 °C (invasion assay incubating for 48 h), the cells remaining on the upper membrane were removed with cotton wool. Cells that had migrated or invaded through the membrane were fixed with methanol, stained with 0.1% crystal violet, imaged, and counted using an inverted microscope .p-value < 0.05 was considered statistically significant.Between-group differences were assessed using a paired two-tailed Student’s t-test. The chi-square test was used to analyze the clinicopathological features associated with ETV5 expression in HGSOC. Survival curves were generated using the Kaplan–Meier survival plots and were compared using log-rank tests. Univariate and multivariate Cox regression models were used to determine the effects of variables on survival. All statistical analyses were performed using SPSS software . A p < 0.05, see Fig. p < 0.05, see Fig. p < 0.01, see Fig. The differentially expressed genes between OC and normal tissues were analyzed separately in three datasets from the GEO database. Genes with a > 2-fold change and p-value < 0.01 were considered discriminatively expressed. The results demonstrated that 64 genes were upregulated in OC tissues, and ETV5 was identified as a candidate see Fig. . Moreovep < 0.05), FIGO stage (p < 0.05), and chemotherapy response (p < 0.05). No significant correlations were observed between ETV5 expression and other clinicopathological parameters, including recurrence and ascites , age (p < 0.05), recurrence (p < 0.05), and FIGO stage (p < 0.001) were associated with a shorter OS. Multivariate analysis identified recurrence (p < 0.01) and FIGO stage (p < 0.05) as independent prognostic factors in HGSOC patients  . The CCK-8 assay showed that, compared to negative controls, ETV5 markedly increased cell proliferation capability from 1 to 7 days see Fig. . These rDespite recent advances in treatment, including the introduction of targeted therapy and immunotherapy, HGSOC patients continue to have a poor clinical treatment benefit and prognosis. This is mainly owing to the unclear pathogenesis, high heterogeneity, low early diagnosis rate, and poor treatment effect for drug-resistant relapses in HGSOC. Therefore, it is crucial to explore the mechanism of HGSOC invasion and metastasis and identify the key molecules involved in its malignant transformation to establish potential therapeutic targets. The results of this study showed that ETV5 expression was significantly higher in OC tissues than in normal tissues.Transcription factors are a set of sequence-specific DNA-binding proteins that regulate gene transcription by binding to specific double-stranded DNA sequences . They caSince the late 1990s, the fallopian tube has been recognized as the main site of origin for HGSOC . In thisWe further analyzed the correlation between ETV5 mRNA expression and prognosis as well as the clinicopathological factors in HGSOC patients. The OS and DFS of patients with ETV5 overexpression were significantly lower than in those with low expression. Regarding the clinical significance of ETV5 expression in HGSOC, we found that ETV5 overexpression was correlated with a high FIGO stage and chemotherapy resistance, indicating that it may be related to HGSOC progression. Univariate Cox regression analysis showed that high ETV5 expression, age, recurrence, and high FIGO stage were risk factors for HGSOC progression. As a member of the PEA3 subfamily of the ETS family, ETV5 and other PEA3 subfamily members play a carcinogenic role in tumors and are independent poor prognostic factors. Segalés L et al. reported that ETV1 transcription factor is highly expressed in prostate cancer, and that high mRNA level of ETV1 is associated with the short recurrence time of prostate-specific antigen . In CRC,Interestingly, immunohistochemistry, used to detect the protein expression of ETV5, showed no significant difference in the positive rate of ETV5 between HGSOC tissues (66.7%) and fallopian tube tissues 61.5%)  gene, which has been shown to induce the EMT process . In endoIn summary, our study provides new insights into the carcinogenic mechanism of HGSOC and identifies a candidate molecular marker for HGSOC prognosis and targeted therapy.Additional file 1. ETV5 protein expression of HGSOC and fallopian tube tissues.


RNA regulates a variety of biological functions by interacting with other molecules. The ligand often binds in the RNA pocket to trigger structural changes or functions. Thus, it is essential to explore and visualize the RNA pocket to elucidate the structural and recognition mechanism for the RNA-ligand complex formation.In this work, we developed one user-friendly bioinformatics tool, RPocket. This database provides geometrical size, centroid, shape, secondary structure element for RNA pocket, RNA-ligand interaction information, and functional sites. We extracted 240 RNA pockets from 94 non-redundant RNA-ligand complex structures. We developed RPDescriptor to calculate the pocket geometrical property quantitatively. The geometrical information was then subjected to RNA-ligand binding analysis by incorporating the sequence, secondary structure, and geometrical combinations. This new approach takes advantage of both the atom-level precision of the structure and the nucleotide-level tertiary interactions. The results show that the higher-level topological pattern indeed improves the tertiary structure prediction. We also proposed a potential mechanism for RNA-ligand complex formation. The electrostatic interactions are responsible for long-range recognition, while the Van der Waals and hydrophobic contacts for short-range binding and optimization. These interaction pairs can be considered as distance constraints to guide complex structural modeling and drug design.http://zhaoserver.com.cn/RPocket/RPocket.html.RPocket database would facilitate RNA-ligand engineering to regulate the complex formation for biological or medical applications. RPocket is available at The online version contains supplementary material available at 10.1186/s12859-021-04349-4. RNA regulates a variety of biological functions by interacting with other molecules. It is currently recognized that more than 70% of the human genome is transcribed into non-coding RNAs . In contAt present, some experimental methods can determine the RNA-ligand structure. Unfortunately, the flexible RNA molecules are challenging to be well-crystallized and determined by X-ray crystallography. Besides, electron microscopy is expensive and time-consuming. The available RNA-ligand experimental structures are few  due to these technical limitations. Some computational methods can predict the RNA and RNA-ligand structures by homologous fragment modeling , moleculThere are several existing RNA-related databases and tools to provide sequence, structure, or interaction information . A workflow of constructing the RPocket database is shown in Fig. (A) The PDB structural files and sequence FASTA files of 1448 RNAs were extracted using the REST API advanced search interface in the Protein Data Bank before February 19, 2020 . Here, w(B) To acquire the non-redundant dataset, we performed the sequence alignment for the 269 structures using the CD-Hit server . We have(C) We identified the RNA-ligand binding sites using a distance-based calculation. A nucleotide is considered one binding site if the distance is less than 4 Å between the RNA and ligand. The detail interactions were generated using Ligplot + with the HBPLUS program , 38. The(D) The functional motifs were identified by the RegRNA program . The Reg(E) The pockets were detected by the 3 V server using the rolling probe method , 43–45. RNA Pocket Descriptor) to calculate the pocket geometric characteristics for RNA molecules. RPDescriptor can generate two descriptors based on Normalized Principal Moments of Inertia Ratios (NPRs) [(F) We developed RPDescriptor (s (NPRs) . The sha(G) The ligands functional groups, hydrogen bond and non-bond interactions, the secondary structure patterns, and pocket topology information were calculated and provided in the RPocket server.One user-friendly bioinformatics tool for RNA pocket information has been missing. This limitation motivated us to develop the RPocket, a user-friendly web server, to analyze the RNA pockets using a simple graphical user interface. Some advanced features implemented in RPocket are (1) contains 240 pocket information extracted from 94 non-redundant RNA-ligand structures; (2) displays the sequence, secondary structure, and RNA-ligand interaction characteristic patterns; (3) constructs a database with the pocket geometric topology information such as volume, surface area, and shape similarity scores; (4) provides a visualization tool for users to scale and rotate the structure; (5) provides one executable script for users to perform pocket topology analysis. (6) offers the related tools to predict or simulate RNA structures. RPocket web server is a reliable and user-friendly tool and facilitates the RNA pocket study without installing programs locally.RPocket consists of eight modules: Home, Search, Visualization, Download, Links, Tutorial, Statistics, and Contacts. The Home module provides a brief introduction to the RPocket database and navigation to other modules. Users can identify and extract the pocket information using the Search module Fig. . The SeaAll the pockets were identified using the rolling probe method by the 3 V program , 43–45. e pocket , 48.RNA Pocket Descriptor) to calculate the pocket geometric characteristics for RNA molecules. Figure The geometric characteristics of the RNA pockets were identified by Normalized Principal Moments of Inertia Ratios (NPRs). NPRs display a three-dimensional molecule's shape by projecting two descriptors calculated using the principal moment of inertia (PMI) onto a two-dimensional plane . PreviouThe first step is to generate the RNA pocket's coordinate file for NPR analysis Fig. A. The 24The second step is PMI and RNA pocket topology descriptors calculation Fig. B. The ceThe third step is to calculate the pocket shape space quantitatively and classify the shape of the pocket Fig. C. The shThe fourth step is to calculate the shape similarity score Fig. D. The \dectively . Here, w formula . The valWe performed a systematic analysis of the 240 RNA pockets extracted from 94 non-redundant RNA-ligand complex structures  is more extensive than sphere-like  and disc-like  pockets. We further analyzed the shape similarity scores to reflect pocket shape quantitatively. The continuous similarity scores are from The pocket topology characteristic is helpful to identify the small molecules for target-specific binding. We analyzed the topology properties  using a rolling probe method by 3 V program . The meamentclass2pt{minim3. The volume and surface area of the ligand-binding pockets (982 Å3 median volume and 622 Å3 median area) are bigger than non-ligand-binding pockets (803 Å3 median volume and 543 Å3 median area). The ligand-binding may affect the pocket breathing motions.We performed the comparative analysis of the 50 ligand-binding and 190 non-ligand-binding pockets to obtain the topological principle for ligand binding. We classified the RNA pockets based on their geometric shapes using RPDescriptor. There are 9 sphere-like, 8 disc-like, and 33 rod-like pockets in 50 ligand-binding pockets. The geometric shape distribution of 190 non-ligand-binding pockets is similar, which are 41 sphere-like, 32 disc-like, 117 rod-like pockets. To further reflect the geometrical characteristic on shape distributions of ligand-binding and non-ligand-binding pockets, an NPR space distribution graph with pocket-size information was generated. Figure The ligand-binding sites usually locate in a specific RNA secondary structure. Binding to the wrong secondary structure may destroy the interactions and the structural stability . Thus, wWe analyzed the distributions of the nucleotides extracted from the RNA-ligand binding sites . There are two hydrogen bonds and eight non-bond interactions involved in the RNA-ligand interactions. It is noted that the hydrogen bonds located the adjacent nucleotides  and eight non-bond interactions dispersed in other parts of the RNA pocket. The other two examples show similar characteristics. The short- and long-range interactions are distributed in different parts of small molecules and stabilize the interaction between RNA and small molecules  are typically in the same shape pockets. To test if the higher-level scale of structural elements can identify native-like RNA structures, we ran four popular RNA tertiary structure prediction programs  on the given testing set to build several tertiary structures and evaluated the prediction accuracy (Additional file In this work, we proposed RPDescritor to calculate the topological properties for RNA pockets quantitatively. The topological information was then subject to RNA-ligand binding analysis by incorporating the sequence and secondary structure information. This new approach takes advantage of both the atom-level precision of the structure and the residue-level tertiary interactions. Together, the results indicate that long-range interactions bring the ligand and RNA together. Then, the short non-bond interactions optimize and stabilize the RNA-ligand binding. We also developed one user-friendly bioinformatics tool, RPocket, to facilitate RNA-ligand engineering to regulate the complex formation for biological or medical applications.Additional file 1. Supplementary material. This file includes the introduction of RPocket, Supplementary material Figure 1–11 and Supplementary material Table 1.Additional file 2. Table S1: RNA-ligand complexes involved in this study.Additional file 3. Table S2: Geometrical information of RNA pockets and secondary structural elements which pocket located.Additional file 4. Table S3: Binding sites of RNA-ligand complexes and functional groups of ligands involved in interaction with RNA.Additional file 5. Folder S1: The structure of all RNA pockets.Additional file 6. Folder S2: Interaction info of RNA-ligand complexes.Additional file 7. Folder S3: Nine other experimental structures and their modeling structures and pockets.Additional file 8. Folder S4: RPDescriptor program code for shape classification of RNA pockets.


Despite recent stereo matching algorithms achieving significant results on public benchmarks, the problem of requiring heavy computation remains unsolved. Most works focus on designing an architecture to reduce the computational complexity, while we take aim at optimizing 3D convolution kernels on the Pyramid Stereo Matching Network (PSMNet) for solving the problem. In this paper, we design a series of comparative experiments exploring the performance of well-known convolution kernels on PSMNet. Our model saves the computational complexity from  To get Classic stereo matching algorithms contain four parts: matching cost computation, cost support aggregation, disparity computation and disparity optimization . Early sOne major problem with current CNN-based stereo matching algorithms is the enormous computation for cost volume regularization. The cost volume aggregation stage builds correspondence between left and right feature maps, aggregating disparity as an additional dimension on left feature maps to form 4D cost volumes . For theA throughout discussion about optimizing already established 3D convolution kernels on stereo matching algorithms;A network design guideline when optimizing 3D convolution kernels on stereo matching algorithms for accurate disparity estimation and less computational complexity;By following the guideline above and without changing the architecture, our model performs comparable results to modern stereo matching algorithms with significantly less computational complexity.Our main contributions are listed below:Plenty of recent studies focus on building lightweight convolution kernels, which comprise the CNN-based application suitable for low resource devices, such as mobile phone and navigation robot. The origin SqueezeNet  achievesThe channel-wise attention mechanism has proven the potential for enhancing the performance with a small implementation. Squeeze and excitation networks (SE-Net)  firstly GC-Net  settles According to We first introduce the details about our network structure, including the architecture of the prototype PSMNet , and a sIn our paper, we take the PSMNet as the baseline and explore a series of 3D kernel-based methods to find a lightweight and accuracy model. In The 4D cost volumes (Equation :(1)C describe the accumulated operations when training neural networks.  explain Memory Access Cost (MAC) is the amount of allocating computational resource during the training process;Model Size shows the storage size of all trained parameters.Before we dive into the network design pipeline, we introduce the metrics for evaluating the computational complexity as follows:In this chapter, we use the 3D kernel-based methods introduced above to replace the standard 3D CNNs in PSMNet, and design a series of comparative experiments to illustrate the impact of different 3D convolution kernels on the performance. Since we focus on discussing the role of 3D convolution kernels in the stereo matching algorithm, we completely follow the original PSMNet on the network layer setting in For all models, we use the same implementation for a fair comparison.Scene Flow is a large scale dataset with synthetic stereo images. It contains 35,454 training and 4370 testing image pairs with KITTI 2015 contains real street scenes taken by driving a car. It includes 200 training image pairs with ground truth disparity maps collected by LiDAR and 200 other test image pairs without ground truth disparity. The size of the training and test images is We evaluate our models on Scene Flow  and KITTWe train all models with an Adam optimizer on one NVIDIA RTX 3090 GPU. During the training process, all input images are randomly cropped to lidation  for prevIn The first 3D convolution layer (3D Head) reduces the number of channels of the cost volume from 64 to 32. Among all 3D convolution kernels, MobileNetV1 builds without residual connection, which is beneficial when operating feature channels. We build 3D MobileNetV1 on the first layer.For all 3D convolution layers  transposed convolutions upsample the input cost volumes to twice the size. It first expands the input size by zero padding the cost volume of each channel, and then compiles the standard 3D convolution to introduce learning parameters for more refined textural information. In our implementation, we follow the ShuffleNetV2 as the 3D convolution. However, when inserting 0 values to restore the size, the pointwise convolution will destroy the learned semantic information and textural information. We add upsamping with trilinear interpolation before ShuffleNetV2 for a continuous and rough cost volume, then the ShuffleNetV2 block restores the cost volume with a series of parameterized convolution layers.Following the architecture of PSMNet in For the last 3D CNN layer (As shown in In We evaluate our model on Scene Flow and KITTI 2015. In In In this paper, based on PSMNet as a prototype, we design a series of kernel-based methods aiming for a lightweight and accurate model without modifying the original architecture. By optimizing 3D convolution kernels with corresponding kernel-based methods, our model greatly reduces computational complexity and achieves comparable results to the modern stereo matching algorithms. In future work, as we mentioned in


Salinity normalization of total alkalinity (TA) and dissolved inorganic carbon (DIC) data is commonly used to account for conservative mixing processes when inferring net metabolic modification of seawater by coral reefs. Salinity (S), TA, and DIC can be accurately and precisely measured, but salinity normalization of TA (nTA) and DIC (nDIC) can generate considerable and unrecognized uncertainties in coral reef metabolic rate estimates. While salinity normalization errors apply to nTA, nDIC, and other ions of interest in coral reefs, here, we focus on nTA due to its application as a proxy for net coral reef calcification and the importance for reefs to maintain calcium carbonate production under environmental change. We used global datasets of coral reef TA, S, and modeled groundwater discharge to assess the effect of different volumetric ratios of multiple freshwater TA inputs  on nTA. Coral reef freshwater endmember TA ranged from -2 up to 3032 μmol/kg in hypothetical reef locations with freshwater inputs dominated by riverine, surface runoff, or precipitation mixing with groundwater. The upper bound of freshwater TA in these scenarios can result in an uncertainty in reef TA of up to 90 μmol/kg per unit S normalization if the freshwater endmember is erroneously assumed to have 0 μmol/kg alkalinity. The uncertainty associated with S normalization can, under some circumstances, even shift the interpretation of whether reefs are net calcifying to net dissolving, or vice versa. Moreover, the choice of reference salinity for normalization implicitly makes assumptions about whether biogeochemical processes occur before or after mixing between different water masses, which can add uncertainties of ±1.4% nTA per unit S normalization. Additional considerations in identifying potential freshwater sources of TA and their relative volumetric impact on seawater are required to reduce uncertainties associated with S normalization of coral reef carbonate chemistry data in some environments. However, at a minimum, researchers should minimize the range of salinities over which the normalization is applied, precisely measure salinity, and normalize TA values to a carefully selected reference salinity that takes local factors into account. Ho. Hooffsh0 μmol/L ), tropic0 μmol/L ), and pr0 μmol/L ) such th unknown , the sys unknown . Moreoveng lines . The preS = 0 on ΔnTA, the application of salinity normalization and the choice of a reference salinity implicitly introduce assumptions about whether conservative mixing occurs before biogeochemical modification  or vice versa [ref = Soffshore) implicitly assumes that calcification occurs prior to mixing with the freshwater endmember. Conversely, by salinity normalizing open ocean TA endmember (point O) to the coral reef salinity (point R) before calculating ΔnTA, O is normalized to R’ and ΔnTA is calculated as the difference in TA between R’ and R (ref = Sreef) implicitly assumes that mixing with the freshwater endmember occurs prior to calcification. The ΔnTA inferred from O and O’ is slightly greater than the ΔnTA inferred from R’ and R , mixing occurs before calcification , or some order of mixing between these two extremes  and generates an additional source of uncertainty in coral reef ΔnTA.In addition to the effects of unknown TAce versa . For exaR’ and R . This scR’ and R . TherefoS = 0) and reference salinities (Sref) using global seawater TA and S, fresh submarine groundwater discharge, a range of different TAS = 0 datasets, and different mixing proportions of freshwater endmembers. We provide a series of suggestions to reduce the uncertainties associated with salinity normalization of coral reef TA data with implications for other carbonate chemistry parameters and ions of interest to coral reef metabolism studies .In a previous study, we suggested that uncertainties in seawater depth and residence time can potentially drive large uncertainties in estimates of coral reef NEC rates while assuming that the accurate determination of TA (±2 μmol/kg) was unlikely to be a significant source of error . Here, wS = 0 using literature derived data in a Monte Carlo approach  assuming three distinct scenarios: (i) riverine + groundwater freshwater inputs  ter inputs ; and  and assuming TAS = 0 = 0. We then calculated the difference between TAreef and nTAreef for each of the 27 coral reefs to estimate the magnitude to which salinity normalization changes TAreef data.We used a global assessment of coral reef seawater carbonate chemistry and salinity data  to quant lmodel2  in R [20reef via ref = Smax) and TAS = 0 values of 0, 1000, 2000, and 3000 μmol/kg, which span the full range of potential simulated freshwater TA endmembers in coral reef ecosystems from this study . Linear regression of these ΔnTA uncertainty vs. ΔS relationships for each TAS = 0 represent the potential range  in the actual ΔnTA values for a given change in S assuming the true TAS = 0 for each reef site is between the 0 and 3000 μmol/kg values explored in this analysis. To further explore how the precision of S can affect nTA, the minimum difference in reported salinity data for each reef was used to estimate the precision of the salinity measurement.We quantified nTAis study . In the offshore = TAoffshore–nTAreef  assuming mixing occurs before calcification; O to R’ to R sensu offshore and ΔnTAreef for each site to evaluate the type II ordinary least squares linear regression between % ΔnTA uncertainty and ΔS for each reef site using the function lmodel2 [R [To quantify how the implicit order of mixing and biogeochemical processes impact coral reef ΔnTA data, we quantified ΔnTA as either ΔnTA lmodel2  in R [20S = 0 for coral reef locations where precipitation, riverine, and freshwater runoff occur in different volumetric proportions (S = 0 estimates were positively skewed with median (2.5th to 97.5th percentile) values of 15 μmol/L (-2 to 1997 μmol/L) for groundwater mixing with precipitation, 1306 μmol/L (293 to 2979 μmol/L) for groundwater mixing with river water, and 598 μmol/L (43 to 2507 μmol/L) for groundwater mixing with surface runoff (S = 0 (± uncertainty) scenarios explored in this study may best represent uncertainties associated with unknown TAS = 0 in future studies where TAS = 0 and their respective volumetric proportions are unknown. However, we nonetheless caution that these simulations are hypothetical  for each of the reefs varied from 0.05 to 2.1   . Mean  scaled with the magnitude of the difference between Smax and Sref (S = 0) equal to 1000, 2000, and 3000 μmol/kg compared to TAS = 0 = 0 μmol/kg were 30, 60, and 90 μmol/kg per unit difference in salinity, respectively  may nonetheless provide a more honest assessment of nTA ± uncertainties for cases where TAS = 0 and the relative volumetric freshwater fluxes are unknown.The alkalinity anomalies for each reef location normalized to different TAnted for . Most imand Sref . Maximumectively . Assuminf TAreef . The canS = 0 of these freshwater endmembers. However, the multitude of endmembers and biogeochemical processes increasingly complicate estimates of the mixing derived component of coral reef TAreef:S variability. While the estimated freshwater TA endmembers for precipitation, riverine, and runoff dominated systems from this study . Moreover, utilizing other tracers to normalize seawater TA data such as silicate, Ra, Rn, Li, Mg, Sr, Ca, Ba, δ18O, and other tracers for submarine groundwater discharge [We recommend viewing coral reefs from an ecosystem-scale perspective to consider the volumetric input of freshwater from different sources  when using ΔTA as a proxy for coral reef calcification and, if warranted, locate and directly measure TAis study  may provischarge –25 warraS = 0  or all mixing is assumed to occur before all calcification (Sref = Sreef)  and ΔnTAreef using Sref = Sreef  quantifies the maximum uncertainty associated with the unknown order of mixing and calcification processes as follows:The ΔnTA uncertainties owing to the implicitly assumed order of mixing and biogeochemical processes in the salinity normalization of TA were much smaller than the uncertainties owing to the unknown TAS = 0 . For exato Sreef . Assumin= Sreef) . Unless offshore and ΔnTAreef results in a ΔnTA that assumes net calcification and mixing occur simultaneously and results in a centered, quantifiable ±1.4% uncertainty in ΔnTA per unit salinity change.Importantly, the average of ΔnTAoffshore and Sreef have the potential to generate non-negligible uncertainties in coral reef salinity normalized total alkalinity anomalies . While we have assumed that Smax = Soffshore to fill in missing data from [offshore should ideally be carefully quantified alongside TAoffshore to reduce actual ΔnTA uncertainties in subsequent studies. Critically, the choice of sampling location for TAoffshore represents an important consideration depending on the aims of the study and characteristics of the coral reef being investigated. For example, choosing an offshore reference that is more proximal to the reef environment can minimize the difference between Soffshore and Sreef to reduce uncertainties owing to a large range in salinity. Depending on the specifics of individual reef systems and nearshore oceanographic processes, TAoffshore of these more proximal offshore waters may be more strongly influenced by other nearshore processes such as upwelling and coastal freshwater inputs [In studies aimed at assessing net reef calcification, the simplest way to reduce uncertainties in coral reef nTA is by selecting sampling sites and time periods with minimal S variability Figs  and 4. Sata from , Soffshor inputs . As a reS = 0 = 3000 μmol/kg relative to TAS = 0 = 0 μmol/kg scenario. With current salinity sensing technology, uncertainties in salinity and, consequently, nTA could be much smaller. Therefore, any such studies that salinity normalize their TA data may be underreporting the true uncertainties in their measurements by simply stating the precision of their TA measurements and not including the uncertainties owing to their S measurements and subsequent salinity normalization. Furthermore, if changes in salinity are within the reported uncertainties of the salinity measurement, then there is not sufficient evidence that salinity has detectably changed across the reef environment. In these cases, salinity normalization may introduce unnecessary error into TA changes, and it may be more appropriate to avoid salinity normalization if the Srange is within the reported precision and/or accuracy of the salinity measurement.While TA is often evaluated with a high degree of precision and accuracy , the esreef data to Sref and especially if TAS = 0 is unknown. We have further provided quantitative simulations of coral reef freshwater TA endmembers (TAS = 0 ± uncertainties) to approximate the potential uncertainties associated with the salinity normalization of coral reef TA data. We conclude that salinity normalization can change both the sign and magnitude of ΔnTA across coral reef environments suggesting that future studies should exercise caution when evaluating and interpreting ΔnTA from coral reef TA and S data. This is especially true for studies with larger ranges in salinity because the uncertainties associated with ΔnTA positively scale with changes in S. We offer several considerations for salinity normalizing coral reef carbonate chemistry data in the future to reduce potential ΔnTA uncertainties when using the alkalinity anomaly technique as a proxy for net coral reef calcification in any reef system:Here we have shown that the uncertainty of ΔnTA measurements is likely greater than the reported ± 2 to 10 μmol/kg precision of TA owing to uncertainties associated with the normalization of TADo not automatically assume salinity normalization is necessary, as it could introduce more error into your measurements than expected.Examine data for any detectable correlations between salinity and TA as these relationships indicate the potential influence of salinity dilution or concentration mechanisms on TA. Even if there are no detectable correlations between salinity and TA, evaluate the potential role that simple dilution or concentration mechanisms may have on coral reef TA data.Precisely measure both salinity and TA. Consider the range and precision of your salinity measurements and the potential impact that S normalization has on TA values. If the range in salinity is within the precision of the salinity measurement, consider not salinity normalizing your data.S = 0) that are mixing with the reef water mass to reduce ΔnTA uncertainties and consider avoiding reef metabolism studies in regions with large salinity changes that may confound conclusions regarding ΔnTA.Take a holistic approach to examine possible sources and relative contributions of freshwater and submarine groundwater discharge to the reef system. Consider taking direct freshwater samples to better determine potential endmembers  and ΔnTAreef  can be used to estimate the ΔnTA ± uncertainties owing to the implicitly assumed order of mixing in the salinity normalization of TA.Carefully select SPropagate the potential uncertainties associated with the salinity normalization of coral reef TA data on calculated ΔnTA to evaluate the relative importance of the salinity normalization process to the resulting estimate of ΔnTA and calculated reef metabolism.2+, Mg2+) for coral reef metabolism studies, which could be exacerbated depending on their background concentrations in seawater. Reducing uncertainties in the evaluation of coral reef metabolism  remains critical for observing and quantifying coral reef function and health under unprecedented environmental change.These findings build upon our previous assessment that depth and residence time are the primary sources of NEC uncertainty  to incluS1 TableSource refers to the following reef locations: 01 = Makapu’u, 03 = Palmyra, 04 = Red Sea 1997, 05 = Red Sea 1998, 06 = Red Sea 2013, 07 = Ofu, 08 = Heron Island, 09 = Palau 1, 11 = Lizard Island, 12 = Ishigaki, 13 = One Tree 1, 14 = Kaiona, 15 = Lady Elliot, 16 = One Tree 2, 17 = Davies, 18 = Palau 2, 19 = Cook Islands, 20 = Cheeca Rocks, 21 = St. John, 22 = West Panama, 23 = Florida Keys, 24 = Bermuda, 25 = Majuro, 26 = Maldives, and 27 = Puerto Rico sensu [method refers to OLS = ordinary least squares regression, term represents the Slope of the relationship, estimate represents the value of the slope, conf.low represents the lower 95% confidence interval of the slope, conf.high represents the upper 95% confidence interval of the slope, and p.value represents the p-value of the slope.(CSV)Click here for additional data file.


Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. In unstimulated cells, p50/p65 is retained by the inhibitor IκBα in the cytoplasm that masks the p65-nuclear localization sequence (NLS). Upon activation, p50/p65 is translocated into the nucleus by the adapter importin α3 and the receptor importin β. Here, we describe a bipartite NLS in p50/p65, analogous to nucleoplasmin NLS but exposed in trans. Importin α3 accommodates the p50- and p65-NLSs at the major and minor NLS-binding pockets, respectively. The p50-NLS is the predominant binding determinant, while the p65-NLS induces a conformational change in the Armadillo 7 of importin α3 that stabilizes a helical conformation of the p65-NLS. Neither conformational change was observed for importin α1, which makes fewer bonds with the p50/p65 NLSs, explaining the preference for α3. We propose that importin α3 discriminates between the transcriptionally active p50/p65 heterodimer and p50/p50 and p65/65 homodimers, ensuring fidelity in NF-κB signaling. Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. Here, the authors identify a bipartite Nuclear Localization Signal in the NF-κB p50/p65 heterodimer that is recognized with high affinity by importin α3. Cytoplasmic cargos targeted for active nuclear import expose a nuclear localization sequence (NLS) recognized by soluble transport receptors. Several pathways for nuclear import have been described, which share similar principles and rely on soluble transport factors of the importin β superfamily (or β-karyopherins)4. These macromolecules promote active, signal-mediated nuclear translocation of cargos by coordinating three activities: high-affinity binding to the NLS-cargo; high-avidity association with the phenylalanine-glycine (FG) repeats lining the inner part of the NPC5; and, in the nucleus, binding to the small GTPase RanGTP, which promotes dissociation from FG-nups and NLS-cargo release.The transport of macromolecules into and out of the cell nucleus is essential for cell physiology and is increasingly linked to the degenerative processes that lead to human diseases. The nuclear pore complex (NPC), the only gateway connecting the cytoplasm and nucleoplasm, functions as a semi-permeable barrier. While smaller molecules (<40 kDa) can diffuse through the NPC passively, most macromolecules in a cell, regardless of their size, shuttle through the NPC via an energy-dependent active transport6 or through specialized adaptor proteins such as snurportin and importin α7. In the classical pathway, importin α binds to importin β through its N-terminal Importin-β binding (IBB) domain9 and to NLS-cargos through its C-terminal 10 stacked Armadillo motifs (Arms) that collectively form an Arm-core11. Importin α possesses two NLS binding pockets located along the concave Arm-core surface: an N-terminal ‘major site’ and C-terminal ‘minor site’ between Arms 2–4 and 6–8, respectively11. There are seven genes for importin α in the human genome, classified into three clades, with importin α1 presumably functioning as the generic adapter for NLS cargos7. Importin α1, and to some extent other isoforms7, is auto-inhibited by the IBB domain that folds back on itself, occupying both NLS sites11. In the presence of importin β and NLS-cargo, the IBB domain is displaced, and the NLS binding sites become accessible for NLS cargo recognition11. A plethora of crystal structures has elucidated the recognition of NLS peptides by importin α12. The classical monopartite NLS, exemplified by the SV40 T-large antigen NLS, contains a short stretch of basic residues (KKKRK) that binds primarily to the major NLS binding site and, to a lesser extent, the minor site10. The bipartite nucleoplasmin NLS (KR-X10-KKKK)12, on the other hand, contains two clusters of basic residues separated by 10–12 residues, which span both NLS-binding sites. Also, several monopartite NLSs bind selectively to either the major14 or minor NLS site17, and their interaction can be regulated by phosphorylation18. Recent work has revealed that importin α isoforms recognize certain NLS-cargos with a great deal of specificity. For instance, importin α3 selectively imports NF-κB p50/p6520 and RCC121. Likewise, viruses infecting human cells hijack specific isoforms, as shown for HIV-1 INV23, influenza PB27, and Nipah and Hendra virus W protein24 that are specific for importin α3 while influenza NP25 and STAT127 appear to use importin α5. Overall, selective recognition of NLS-cargos by importin α isoforms is likely to play a role in cellular differentiation29 and disease states, including viral infections, cancer7, and developmental defects30.Importin β can import NLS-cargos directly by either binding to their exposed NLSs33. There are five known NF-κB subunits; p65 (RelA), RelB, cRel, and the precursors p105 and p100, which are partially digested to form the p50 and p52 subunits, respectively31. These NF-κB subunits exist as a variety of homodimers and heterodimers, of which p50/p65 represents the most abundant and signaling active species31. Overall, all NF-κB subunits contain an N-terminal Rel homology region (RHR) responsible for DNA binding, dimerization, and nuclear localization  pathway, and specific NF-κB subunits regulate each pathway. In the canonical NF-κB signaling pathway, a diverse subset of stimuli, including inflammatory cytokines and chemokines, activates the p65, p50, and cRel subunits and triggers rapid and transient NF-κB response34. The non-canonical NF-κB pathway, on the other hand, is triggered by a smaller subset of stimuli, which activates RelB and p52 and leads to long-term, persistent NF-κB signaling35. The p50/p65 heterodimer, the most abundant signaling complex in the canonical NF-κB pathway, is expressed in all cell types, while the p65/p65 and p50/p50 homodimers are less abundant36. The p50/p65 cellular abundance reflects the greater structural stability of the NF-κB heterodimer compared to homodimers36. The p50/p65 dimerization interface is stabilized by a network of hydrogen bonds and salt bridges, including an energetically favorable interaction between Asp254 from p50 and the equivalently positioned Asn200 in p6538, which is missing in the p50/p50 and p65/p65 homodimers. Comparing homodimers stability, Tyr267 and Phe307 in p50 are replaced by Phe213 and Val248 in p65, respectively, which reduce both the H-bonding and hydrophobic interactions stabilizing the p65 homodimer40. Thus, the NF-κB p50/p65 is more stable than p50/p50, and the p65/p65 homodimer is the least stable of the three NF-κB dimers.The nuclear factor-kappa B (NF-κB) family consists of essential transcription factors mediating various cellular processes, including immune response, cellular differentiation, and apoptosision Fig. . In cont20 identified a functional NLS responsible for nuclear import at the C-termini of p50 and p65 RHR  which mimics IκB and retains the precursor p105/p50 complex in the cytoplasm43. In the resting state, the NF-κB complex is sequestered in the cytoplasm either by IκBα-mediated retention for the p50/p65 and p65/p65 complexes44 or ARD-mediated retention for the precursor p105/p50 complex46. Upon stimulation, the activated inhibitor of NF-κB kinase complex (NEMO/IKKβ/IKKα)47 promotes IκB phosphorylation, ubiquitination, and the eventual dissociation and proteasomal degradation49, which removes the IκB repression from p50/p65 and p65/p65 complexes. The newly liberated NF-κB p50/p65 complex is then shuttled into the nucleus by the isoforms importin α3 and α4, which are 95% identical in sequence20. The p65/p65 homodimer can also localize to the nucleus by binding to importin α3 and α4 as well as other isoforms, including importin α1 and importin 851. Similarly, for the p105/p50 complex, the activated IKK-mediated signaling leads to ARD cleavage by the 20S proteasome and liberation of the p50/p50 homodimer that exposes the NLSs for importin α3/α4-mediated nuclear import52. Overall, the exact mechanisms of NF-κB homodimers (p50/p50 and p65/p65) nuclear import have not been elucidated. In vitro transfection studies of p65 and p5020 could not fully distinguish between the heterodimeric p50/p65, the most abundant NF-κB species in human cells, and the less abundant homodimeric complexes formed by p50/p50 and p65/p65.The nuclear translocation of NF-κB species is tightly regulated in human cells. Fagerlund et al.RHR Fig. . In the Here, we have investigated how NF-κB complexes interact and discriminate between importin α isoforms. Using hybrid structural methods, we found that the p50-NLS and the p65–NLS bind different regions of importin α3, generating a trans bipartite NLS. Unexpectedly, the two NLSs reveal surprising structural polymorphisms in the context of different NF-κB dimers.42. We assembled and purified three NF-κB dimers . To elucidate the association of either importin α isoform with p50/p65, we added an increasing molar excess of purified p50/p65 to 10 μg of purified ΔIBB-importin α1  though had a slightly decreased number of binding sites at equilibrium (Bmax).To determine how the p65- and p50-NLSs mediate binding to importin α, we generated constructs of p65 and p50 lacking the C-terminal NLS and assembled all possible permutations of dimeric NF-κB complexes  are not only superimposable in the two complexes (RMSD of 1.095)  Fig.  but also. 2 Fig.  versus n α1 Fig. . Accordilex Fig. . Compari1–P5 in a p50-NLS-like conformation, with Glu300, Lys301, Arg302, Lys303, and Arg304, making a total of 11 hydrogen bonds, 101 Van der Waals contacts, and burying 724.2 Å. Unlike the p50-NLS, which inserts Arg361 into the P1 position and is stabilized by Van der Waals interactions, the Glu300 in the p65-NLS orients away from the P1 position  retained and bound to both IκBα and importin α3. In contrast, the p65-NLS residues 294-303 fold into α-helix (referred to as helix 3) in the IκB-bound complex but bind importin α3 in an extended conformation. Unexpectedly, in all complexes with importin α1, the p65-NLS adopts a fully extended conformation at the minor NLS-site, with the P1′–P4′ positions occupied by 301KRKR304 residues  is more energetically favorable than the p65-NLS:importin α1 , which possibly explains the physiological preference for importin α3. Additionally, comparing the p65-NLS in both importin α isoforms, there appears to be a shift in the NLS register at the P2′ position. In the importin α1 structures  observed in the importin α3/p65–NLS complex and are part of the α-helix 4 (res. 306–321) responsible for binding to IκBα42.The mechanism by which different isoforms accommodate the p65-NLS at the minor NLS-binding pockets determines the number and types of contacts made in the two complexes. PISAres Fig. , the P2′ α3 Fig. , the P2′8. DynDom analysis56 of the importin α3 crystal structures in the unliganded state and bound to p50-NLS and p65-/p50-NLSs revealed two structural changes in the peripheral arches in response to the p65-NLS binding. At the N-terminus, a hinge movement57 was identified within Arm 4 (res. 198-201) in response to p50-NLS and p50-/p65-NLS binding compared to the apo structure   and even more from the apo α324 (RMSD 2.80 Å between residues 72 and 486). Meanwhile, importin α1 residues 75–497 are perfectly superimposable (RMSD 0.26–0.39 Å) in all complexes of importin α1 bound to either p65-, p50-, and both NLSs  of 44.21 ± 0.28 Å  calculated from the SAXS data indicates a maximum diameter Dmax ~ 137 Å  p65 Fig.  bound to8 Å Fig. , and a v7 Å Fig. . We calcmer Fig. . The dirRg of 48.56 + 0.22 Å  calculated from the SAXS data indicates a maximum diameter Dmax ~ 157 Å   calculated from the SAXS data indicates a maximum diameter Dmax ~ 151 Å, which is slightly larger than the p50/p65:∆IBB-importin α3 complex   and helix 4 (res. 306–315) in complex with IκB67. While our data suggests the p65-NLS is not a major binding determinant for importin α3 in the p50/p65 heterodimer, it is a functional NLS for the p65/p65 homodimer  independently regulated to ensure sustained gene expression76. The p65/p65 is the least stable NF-κB dimer, and while its concentration is likely low, this homodimer can also be inhibited and retained in the cytoplasm by IκBα44 2 2 Fig. , that isIn conclusion, this paper describes the mechanisms of NF-κB dimer recognition by the isoforms α3. Our work paves the way to identifying inhibitors that target the p50-NLS:importin α3 association, reducing the accumulation of NF-κB in the nucleus and dampening aberrant NF-κB signaling.41. ∆NLS-p65 (residues 1–290) was generated by introducing a stop codon at position D291 of p65 using the QuikChange site-directed mutagenesis kit (Stratagene). A synthetic gene encoding human p50 (residues 1–366), p50 lacking the DNA-binding domain ΔDBD-p50, residues 243–366), and p65 lacking the DNA-binding domain ΔDBD-p65, residues 191–316) were purchased from Genewiz and cloned between BamHI and XhoI restriction sites of expression vectors pGEX-6P-1 (GE Life Sciences). Plasmids encoding ∆IBB-importin α1 (residues 70–529) and ∆IBB-importin α3 (residues 68–487 and 63–487) were cloned into vectors pET-30a and pGEX-6P or pET-15b, respectively, as previously described21. Untagged IκBα (residues 67–287) was cloned in the pET-11a67. Peptides encompassing the p65-NLS (294-DRHRIEEKRKRTYETFKSIMKK-315) and p50-NLS (355-DKEEVQRKRQKLMP-368) were synthesized from GenScript .The plasmids encoding human p65 (residues 1–306) and ∆NLS-p50 (residues 41–351) cloned in the pLM1 vector were a generous gift of Dr. Stephen HarrisonE. coli strain in the presence of ampicillin or kanamycin. Plasmids encoding GST-ΔDBD-NF-κB and ΔIBB-Importin α3 were co-transformed in BL21 DE3 E. coli strain and expressed in the presence of ampicillin and kanamycin. Bacterial cultures were grown in LB medium at 37 °C until A600 = ~0.6 AU and all p65 induced at 16 °C with 0.25 mM IPTG for 12–16 h, all p50 induced at 18 °C at 0.25 mM IPTG for 12–16 h, and all importin α1 and α3 at 28 °C with 0.5 mM IPTG for 4 h. Cell pellets expressing all untagged p65 and p50 constructs  were sonicated in lysis buffer 1  glycerol) followed by 0.2% polyethyleneimine precipitation for 1 h at 4 °C and 55% ammonium sulfate precipitation for 2 h at 4 °C. The p65, ∆NLS-p65, and ∆NLS-p50 ammonium sulfate pellets were re-suspended in Re-Suspension Buffer  glycerol), loaded onto 5 mL HiTrapTM SP HP Column , washed with 5 CV of Buffer A , and the proteins were eluted using a linear gradient from 0% to 100% Buffer B . Cell pellets expressing GST-p50, GST-ΔDBD-p50, GST-ΔDBD-p65 and co-expressed GST-ΔDBD-NF-κB and ΔIBB-Importin α3 complexes were sonicated in Lysis Buffer 2  TWEEN 20, 1 mM EDTA), followed by 0.2% polyethyleneimine precipitation for 1 hour at 4 °C. GST-p50, GST-ΔDBD-p50, GST-ΔDBD-p65, and GST-ΔDBD-NF-κB:ΔIBB-Importin α3 complexes were purified by affinity chromatography on Glutathione Resin (GenScript) and incubated overnight with 100 U of PreScission Protease in PPase Cleavage Buffer  TWEEN 20). Untagged p50, ΔDBD-p50, ΔDBD-p65, and ΔDBD-NF-κB:ΔIBB-Importin α3 complexes were recovered off the beads. All GST-tagged7 and His-tagged ∆IBB-Importin α1 and α321 were purified as previously described. IκBα was expressed and purified as previously described67. All purified samples were concentrated with a 10- or 30 kDa Millipore PES concentrator and injected on a Superdex 200 16/60 column  pre-equilibrated with Gel Filtration Buffer 1  glycerol). Sample purity was confirmed by SDS–PAGE (13.5%) analysis. Complexes eluted from beads were further purified on a Superdex 200 16/60 column  pre-equilibrated with Gel Filtration Buffer 1. Co-purified ΔDBD-NF-κB:ΔIBB-Importin α3 complexes were further purified on a Superdex75 10/300 column .Expression plasmids for all human p65, p50 (and relative mutants), ∆IBB-importin α1, ∆IBB-importin α3 were transformed and expressed in BL21 DE3 g at 4 °C for 30 min to remove precipitation41. Homodimeric NF-κB  complexes were concentrated with a 30 kDa Millipore PES concentrator and injected on a Superdex 200 16/60 column  pre-equilibrated with GF Buffer 1. All NF-κB trimeric complexes were assembled by incubating pre-formed NF-κB hetero/homodimers in a 1.5-molar excess of ∆IBB-importin α3 or IκBα, followed by incubation at 4 °C for 1 h. Complexes were concentrated with a 30 kDa Millipore PES concentrator and injected on a Superose 12 10/30 GL column  pre-equilibrated with GF Buffer 1 followed by SDS–PAGE analysis. Trimeric complexes of NF-κB p50 or p65 dimerization domains bound to importin α3 ΔDBD-p50/ΔDBD-p50:α3 and ΔDBD-p65/ΔDBD-p65:α3, Supplementary Fig. Heterodimeric p50/p65 complexes  were assembled by incubating in a 1:1.5 p50:p65 molar ratio, respectively, together at 25 °C for 30 min, then at 37 °C for 1 h, followed by centrifugation at 12,000 × 1, NF-κB hetero/homodimers  were coated on ELISA plates  and incubated at 4 °C overnight. The plates were blocked with PBS containing 5% skim milk for 2 h at room temperature and were washed three times with PBS containing 0.05% Tween-20 (PBST). The His-importin α samples were diluted in PBST (from 1000 to 0 nM) and 100 μL added into the wells, and the plates were incubated at room temperature for 1 h. The plates were washed three times with PBST and incubated with anti-6xHis-4HRP-conjugated rabbit polyclonal antibody (1:3500) (Abcam ab1187) for 1 h at 37 °C. After washing with PBST three times, the reaction was initiated by the addition of 100 μL of tetramethylbenzidine (TMB) substrate (1-Step Ultra TMB-ELISA (Ref 34028), Thermo Scientific). The colorimetric reaction was stopped by adding 100 μL of 2 M H2SO4, and absorbance was read at 450 nm wavelength using a Tecan microplate reader. All experiments were carried out in duplicates. One-site specific binding analysis using least-squares fit was performed using GraphPad Prism 9 for Windows, GraphPad Software, LLC (version 9.2.0). Native gel electrophoresis was performed on a 1.5% agarose gel, as previously described82. In the p50/p65:α titration assay in Fig. For microtiter binding assay−1 and crystallized using the vapor diffusion hanging drop method by mixing 2 µl of purified protein with 0.5 µl containing a 2.5-fold molar excess of NLS peptide. The best crystals for ∆IBB-importin α3 bound to p50-NLS were obtained using 0.2 M potassium thiocyanate, 20% PEG3350, 0.1 M BIS–TRIS pH 6.5. ∆IBB-importin α3 bound to both the p50- and p65-NLS peptides was crystallized in the presence of 0.2 M ammonium sulfate, 0.1 M BIS–TRIS pH 6.5, 25% PEG 3350. For ∆IBB-importin α1, all complexes with the p50-NLS and p65-NLS alone or p50-NLS + p65-NLS peptides were crystallized in the presence of 0.5-0.65 M sodium citrate, 0.1 M HEPES pH 6.0, 10 mM β-Mercaptoethanol. All crystals were harvested in nylon cryo-loops, cryo-protected with 27% ethylene glycol, and flash-frozen in liquid nitrogen. Complete diffraction data were collected at beamlines 9-2 and 12-1 at the Stanford Synchrotron Radiation Lightsource (SSRL) on a Dectris Pilatus 6 M detector (Table 83 (version 2.8). The p50-NLS was built in an unbiased Fo–Fc electron density map using Coot84 (version 0.9.4), and the model was subjected to several rounds of real-space refinement using phenix.real_space_refine85 (version 1.18.2-3874) in combination with reciprocal-space and TLS refinement using phenix.refine86 (version 1.18.2-3874). This model was then used as a search template to solve the structure of ∆IBB-importin α3 bound to both the p65- and p50-NLSs, also using PHASER83. The p65-NLS and p50-NLS were modeled in unbiased Fo-Fc densities readily visible at the minor and major NLS-binding pockets, respectively, using Coot84. The complete model of ∆IBB-importin α3/p50-NLS/p65-NLS was refined as described above. The final models of ∆IBB-importin α3 bound to just the p50-NLS or both the p65- and p50-NLSs have a Rwork/Rfree of 19.9/22.0 and 19.6/23.7, at 2.10 and 2.85 Å resolution, respectively  equilibrated in SEC–SAXS Buffer . SAXS data were recorded on an EIGER 4 M detector  in vacuo at 2 s per frame with a fixed camera length of 1.709 m and 10.03 keV (1.237 Å) energy allowing the collection of the angular range q between 0.008 and 0.54 Å−1. Primary reduction of the SAXS data was performed using RAW88 (versions 1.6.4 and 2.0.3), and ATSAS software89 (version 2.6.0). To minimize the effects of damaged material accumulating on the X-ray sample window and to help compensate for any baseline drift, the buffer profile was constructed by averaging the frames before the sample peak frames. The Guinier plots of the subtracted profiles were linear to the lowest measured q value. GNOM90 was used to calculate P(r) plots from the scattering data. Ab initio model calculations to generate an average electron density from solution scattering data were done using DENSS91, as implemented in RAW. The DENSS densities have a Fourier shell correlation (FSC) of 52.2 Å (p50/p65/∆IBB-importin α3), 64.7 Å (p50/p50/∆IBB-importin α3) and 50.7 Å (p65/p65/∆IBB-importin α3), respectively. Docking of PDB models inside the SAXS density was done manually and improved by rigid-body refinement using Chimera92. Theoretical solution scattering curves were calculated using the FoXS web server93. SEC–SAXS data collection and analysis statistics are in Supplementary Table SEC–SAXS analysis was performed at ID7A1 station at MacCHESS, which is equipped with an AKTA Pure FPLC system . The trimeric p50/p65:∆IBB-importin α3, p65/p65:∆IBB-importin α3 and p50/p50:∆IBB-importin α3 complexes were pre-assembled and concentrated at 4.5, 6.6, and 6.29 mg ml92. Binding interfaces were analyzed using PISA55 and PDBsum94. Secondary structure superimpositions were done in Coot82 and analyzed using SuperPose 1.0 server59. DynDom56 was used to identify importin α3 domain movements upon cargo-binding.All structural illustrations were generated using PyMoL  and ChimeraFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Dear Editor,1. To achieve RNAi-directed knockdown, synthetic small interfering RNAs (siRNAs) or long double-strand RNAs  can be designed to harness the Argonaute-mediated mechanism as well summarized by Kawamata and Tomari2. RNAi typically modulates gene expression post-transcriptionally in the cytoplasm; however, it is poorly understood whether exogenous dsRNAs/siRNAs could affect RNA polymerase II (Pol II) transcription of targeted genes in the nucleus. If so, it will be much more challenging to conclude a cis-acting function for the protein produced or the transcript itself that regulates expression of the encoded gene. Therefore, the raised question is extremely important for the appropriate application of RNAi, and more evidence is needed to fill the gap.As one of the most popular technological breakthroughs, RNA interference (RNAi)-directed transcript knockdown is a rapid, efficient, specific, and potentially high-throughput approach that is not only broadly used in the fields of protein and RNA biology for various experimental purposes, but also provides a therapeutic strategy for widespread clinical/biomedical applicationsMtnA, dati, and Hsp70Aa) and a well-characterized lncRNA roX1 were selected to evaluate the effect of RNAi on targeted gene transcription based on their variable lengths, cytoplasmic or nuclear subcellular localization, and the presence or absence of introns  cells were transfected with the indicated dsRNAs for 2 days. Real-time RT-PCR was performed to examine expression of the steady-state transcript. As expected, these dsRNAs are able to efficiently reduce expression of the targeted genes post-transcriptionally  that only harbor the coding sequence of Ago2 and an Ago2 dsRNA targeting the 3’UTR were used for the rescue assay. Ago2 3’UTR dsRNA only depletes endogenous Ago2 but not transgene-derived Ago2 (Fig. Ago2 dsRNA for 1 day, and dsRNA targeting MtnA, dati, Hsp70Aa, or roX1 was introduced for another 2 days. NRO experiments were performed followed by real-time RT-PCR. In Ago2-knockdown cells, RNAi no longer resulted in a robust reduction in the nascent transcript abundance of the targeted gene (Fig. -knockout cells (Supplementary Fig. PGK1 siRNA transfection led to a 30%–50% decrease in the level of PGK1 pre-mRNA, and depletion of Ago2 or Importin 8, an Ago2 nuclear import factor5, abolished the effect (Fig. Given that RNA cleavage in pre-mRNAs has been shown to exert an important role in Pol II premature terminationgo2 Fig. . S2 cellene Fig. . Reexpreene Fig. . The rolect Fig. . Togethe3. Considering that Ago2-mediated RNA cleavage generates a free 5’ end of the downstream fragment and a free 3’ end of the upstream fragment, we hypothesized that these fragments may provide an entry point for 5’ degradation by Rat1 or 3’ degradation by Dis3. To test our hypothesis, S2 cells were transfected with the control, Dis3, or Rat1 dsRNA for 1 day, and dsRNA targeting MtnA, dati, Hsp70Aa or roX1 was introduced for another 2 days. NRO experiments were then performed followed by real-time RT-PCR. As expected, the level of the upstream fragment was rescued upon Dis3 depletion, and the level of the downstream fragment was restored to a level similar to the control sample in Rat1-depleted cells (Fig. 3, in which the upstream RNA has already been transcribed by Pol II, while Pol II of the downstream RNA is caught up and released by Rat1 (Fig. RNA quality control is a process that is tightly linked to transcription. RNP assembly defect or RNA cleavage triggers RNA degradation within the site of transcription by 5’ and 3’ exonucleasells Fig. . Furtherlls Fig. . This miat1 Fig. .cis effect of a linear lncRNA on its encoded gene transcription is now becoming increasingly an important field of gene regulation; however, informed by Fig. 6 (Fig. circdati, a well-characterized circRNA as described in our previous study8. Transcriptional activity and Pol II occupancy of the dati locus were examined by NRO and Pol II-ChIP assay, respectively. In cells transfected with circdati siRNA, the level of nascent circdati was drastically decreased, whereas the level of nascent dati mRNA was largely unaffected (Fig. circdati siRNA transfection had little effect on Pol II occupancy at the dati gene body (Fig. dati mRNA (Supplementary Fig. dati transcription (Fig. The . 6 Fig. . Emergin. 6 Fig. , it is sted Fig. , suggestody Fig. . These dion Fig. . Therefo9. On the other hand, RNAi-directed knockdown is a vitally important technique to suppress gene expression in eukaryotic system. Instead of investigating the genome-wide picture of RNAi-triggered disturbances, our study focuses on the unexpected effect of exogenous dsRNA/siRNA reagents on their targeted genes in cis. Mirroring the natural mechanism, RNAi attenuates generation of nascent transcripts by inducing premature termination which is combinatorially controlled by Ago2, Dis3, and Rat1 (Fig. 11. Guang et al. first demonstrated that endogenous siRNAs can be transported to the nucleus to pause RNA Pol II, inhibit transcription elongation, and cause premature termination in Caenorhabditis elegans11. Notably, they found that lin-15b pre-mRNA silencing accounted for ~80% of total lin-15b transcript silencing elicited by RNAi10. This suggests that siRNA may function predominantly in the nucleus to nascent transcripts in certain cases, which is consistent with our results (Fig. 13. Therefore, the unexpected effects of these strategies on gene expression should be taken into consideration before any experimental or clinical applications.Although RNAi machinery was first thought to function solely in the cytoplasm, it also has crucial roles in some nuclear events, such as promoter methylation and heterochromatin formationat1 Fig. . In factlts Fig. . Unlike Supplementary Information