Transcription Needs Translation Initiation of the Downstream Gene to Continue Downstream at Intercistronic Junctions in E. Coli.

We have reported a gal mutant called galE stop0, wherein the galE stop codon was changed to a sense codon. The experiment results demonstrated that preventing galE translation termination inhibited the production of galE 3' ends. This implies that when the galE translation termination was prevented, the galE 3' ends generation was reduced or impaired. We anticipated that the translation of galE would continue to galT, producing a chimeric protein GalE-GalT. This study verified that the chimeric protein was produced, but unexpectedly, we found that the GalT protein was also synthesized in the mutant, and its amount equaled that in the wild-type. In the wild-type, we also found that the GalE-GalT chimeric protein was produced in an amount equal to that of the GalE protein. These results suggest that translation termination of galE and translation initiation of galT occur independently, thus, corroborating our transcription-translation model: At the cistron junction, transcription, decoupled from translation due to the translation termination of galE, needs translation initiation of galT to continue downstream; otherwise, transcription would be terminated by Rho. RNase E-mediated transcript cleavage also produces the 3' ends of pre-galE mRNA. These findings indicated that RNase E produces the 3' end of mRNA and establishes gene expression polarity.

sRNA expedites polycistronic mRNA decay in Escherichia coli.

In bacteria, most small RNA (sRNA) elicits RNase E-mediated target mRNA degradation by binding near the translation initiation site at the 5' end of the target mRNA. Spot 42 is an sRNA that binds in the middle of the gal operon near the translation initiation site of galK, the third gene of four, but it is not clear whether this binding causes degradation of gal mRNA. In this study, we measured the decay rate of gal mRNA using Northern blot and found that Spot 42 binding caused degradation of only a specific group of gal mRNA that shares their 3' end with full-length mRNA. The results showed that in the MG1655Δspf strain in which the Spot 42 gene was removed, the half-life of each gal mRNA in the group increased by about 200% compared to the wild type. Since these mRNA species are intermediate mRNA molecules created by the decay process of the full-length gal mRNA, these results suggest that sRNA accelerates the mRNA decaying processes that normally operate, thus revealing an unprecedented role of sRNA in mRNA biology.

Hederacolchiside A1 Suppresses Autophagy by Inhibiting Cathepsin C and Reduces the Growth of Colon Cancer.

While autophagy degrades non-functional or unnecessary cellular components, producing materials for synthesizing cellular components, it can also provide energy for tumor development. Hederacolchiside A1 (HA1) derived from anemone raddeana has anticancer effects on several carcinomas by inducing apoptosis or exhibiting cytotoxicity, but the relationship with autophagy has not been studied. We investigated the association between HA1 and autophagy and evaluated its anticancer effect on colon cancer. HA1 induced accumulation of the autophagy-related markers LC3B and SQSTM1, with distinct vacuolar formation, unlike other autophagy inhibitors; the effects were similar to those of chloroquine. In addition, HA1 decreased the expression and proteolytic activity of lysosomal protein cathepsin C, reduced the growth of colon cancer cells in vitro, and inhibited tumor growth in vivo. It also reduced the expression of Ki-67 and cathepsin C in mouse tissues and reduced the growth of spheroids and organoids composed of cancer cells. Taken together, these results imply that HA1 regulates cell growth and autophagy and has potential as a promising therapeutic agent in colon cancer.

Identification of a Rho-Dependent Termination Site In Vivo Using Synthetic Small RNA.

Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region without secondary structure at the 3' end of mRNA. Determining the exact RDT site in vivo is challenging, because the 3' end of mRNA is rapidly removed after RDT by 3'-to-5' exonuclease processing. Here, we applied synthetic small RNA (sysRNA) to identify the RDT region in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined analyses of rapid amplification of cDNA 3' ends, primer extension, and capillary electrophoresis, we could precisely map and quantify mRNA 3' ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA appeared to protect the cleaved RNA 3' ends from rapid degradation by 3'-to-5' exonuclease, thereby stabilizing the mRNA 3' end. We further verified that the signal intensity at the 3' end was positively correlated with the amount of mRNA. By constructing a series of sysRNAs with close target sites and comparing the difference in signal intensity at the 3' end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within the 21-bp range, which was determined as the RDT region. Our results demonstrated the ability to use sysRNA as a novel tool to identify RDT regions in vivo and expand the range of applications of sysRNA. IMPORTANCE sysRNA, which was formerly widely employed, has steadily lost popularity as more novel techniques for suppressing gene expression come into existence because of issues such as unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new method to identify RDT regions in vivo using sysRNA. This new feature is important because since the discovery of the Rho protein in 1969, specific identification of RDT sites in vivo has been difficult due to the rapid processing of RNA 3' ends by exonucleases, and sysRNA might provide a new approach to address this challenge.

An Evaluation of a New Quantitative Point-of Care Diagnostic to Measure Glucose-6-phosphate Dehydrogenase Activity.

Malaria continues to be one of the most crucial infectious burdens in endemic areas worldwide, as well as for travelers visiting malaria transmission regions. It has been reported that 8-aminoquinolines are effective against the Plasmodium species, particularly primaquine, for anti-hypnozoite therapy in P. vivax malaria. However, primaquine causes acute hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Therefore, G6PD deficiency testing should precede hypnozoite elimination with 8-aminoquinoline. Several point-of-care devices have been developed to detect G6PD deficiency. The aim of the present study was to evaluate the performance of a novel, quantitative G6PD diagnostics based on a metagenomic blue fluorescent protein (mBFP). We comparatively evaluated the sensitivity and specificity of the G6PD diagnostic modality with standard methods using 120 human whole blood samples. The G6PD deficiency was spectrophotometrically confirmed. The performance of the G6PD quantitative test kit was compared with that of a licensed control medical device, the G6PD strip. The G6PD quantitative test kit had a sensitivity of 95% (95% confidence interval (CI): 89.3-100%) and a specificity of 100% (95% CI: 94.3-100%). This study shows that the novel diagnostic G6PD quantitative test kit could be a cost-effective and time-efficient, and universally mandated screening tool for G6PD deficiency.

Failure of Translation Initiation of the Next Gene Decouples Transcription at Intercistronic Sites and the Resultant mRNA Generation.

In Escherichia coli, transcription is coupled with translation. The polar gal operon is transcribed galE-galT-galK-galM; however, about 10% of transcription terminates at the end of galE because of Rho-dependent termination (RDT). When galE translation is complete, galT translation should begin immediately. It is unclear whether RDT at the end of galE is due to decoupling of translation termination and transcription at the cistron junction. In this study, we show that RDT at the galE/galT cistron junction is linked to the failure of translation initiation at the start of galT, rather than translation termination at the end of galE. We also show that transcription pauses 130 nucleotides downstream from the site of galE translation termination, and this pause is required for RDT. IMPORTANCE Transcription of operons is initiated at the promoter of the first gene in the operon, continues through cistron junctions, and terminates at the end of the operon, generating a full-length mRNA. Here, we show that Rho-dependent termination of transcription occurs stochastically at a cistron junction, generating a stable mRNA that is shorter than the full-length mRNA. We further show that stochastic failure in translation initiation of the next gene, rather than the failure of translation termination of the preceding gene, causes the Rho-dependent termination. Thus, stochastic failure in translation initiation at the cistron junction causes the promoter-proximal gene to be transcribed more than promoter-distal genes within the operon.

Decursin inhibits tumor progression in head and neck squamous cell carcinoma by downregulating CXCR7 expression in vitro.

CXC chemokine receptor 7 (CXCR7) is frequently overexpressed in cancer and plays a significant role in tumor growth and metastasis. Consequently, inhibition of CXCR7 is important for treatment strategies. However, little is known concerning the biological role of CXCR7 and its underlying mechanisms in head and neck squamous cell carcinoma (HNSCC). The present study investigated the role of CXCR7 in HNSCC, as well as the effects of decursin, a pyranocoumarin compound isolated from Angelica gigas Nakai, on CXCR7 and its downstream signaling. Expression levels of CXCR7 in HNSCC cells were examined using flow cytometry, reverse transcriptase PCR, western blot analysis, and immunofluorescence. The effects of CXCR7 on cell proliferation, migration, and invasion were studied using CCK­8, gap closure, and transwell assays. The results revealed that decursin significantly reduced CXCR7 expression and inhibited cell proliferation, migration, and invasion of human HNSCC cell lines. In addition, decursin induced G0/G1 cell cycle arrest in CXCR7­overexpressing cells and decreased the levels of cyclin A, cyclin E, and CDK2. Furthermore, CXCR7 promoted cancer progression via the STAT3/c­Myc pathway in HNSCC; suppression of CXCR7 with decursin prevented this effect. These results suggest that CXCR7 promotes cancer progression through the STAT3/c­Myc pathway and that the natural compound decursin targets CXCR7 and may be valuable in the treatment of HNSCC.

sRNA-mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho-dependent transcription termination.

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3'-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation.

MITF Promotes Cell Growth, Migration and Invasion in Clear Cell Renal Cell Carcinoma by Activating the RhoA/YAP Signal Pathway.

Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper transcription factor involved in the lineage-specific regulation of melanocytes, osteoclasts and mast cells. MITF is also involved in the progression of melanomas and other carcinomas, including the liver, pancreas and lung. However, the role of MITF in clear cell renal cell carcinoma (ccRCC) is largely unknown. This study investigates the functional role of MITF in cancer and the molecular mechanism underlying disease progression in ccRCC. MITF knockdown inhibited cell proliferation and shifted the cell cycle in ccRCC cells. In addition, MITF knockdown reduced wound healing, cell migration and invasion compared with the controls. Conversely, MITF overexpression in SN12C and SNU482 cells increased cell migration and invasion. Overexpression of MITF activated the RhoA/YAP signaling pathway, which regulates cell proliferation and invasion, and increased YAP signaling promoted cell cycle-related protein expression. Additionally, tumor formation was impaired by MITF knockdown and enhanced by MITF overexpression in vivo. In summary, MITF expression was associated with aggressive tumor behavior, and increased the migratory and invasive capabilities of ccRCC cells. These effects were reversed by MITF suppression. These results suggest that MITF is a potential therapeutic target for the treatment of ccRCC.

Decursin inhibits cell growth and autophagic flux in gastric cancer via suppression of cathepsin C.

Autophagy plays an important role in the survival of cancer cells under stressful conditions, such as nutrient or oxygen deficiency. Therefore, autophagy inhibition is being considered as a novel therapeutic strategy for cancer. Decursin is a natural compound derived from Angelica gigas; it has been used in the treatment of various diseases, including cancer. However, the mechanism by which decursin regulates autophagy in gastric cancer and other carcinomas remains unclear. Here, we demonstrated that decursin reduced the growth and induced cell cycle arrest in gastric cancer cells in vitro. Decursin blocked autophagic flux by reducing the expression of lysosomal protein cathepsin C (CTSC) and attenuating its activity, thereby causing autophagic dysregulation (i.e., accumulation of LC3 and SQSTM1). Decursin also inhibited cell proliferation and cell cycle progression by inhibiting CTSC and E2F3, both of which were linked to gastric cancer aggressiveness. The antitumor effects of decursin were confirmed in vivo. We established spheroid and patient-derived organoid models and found that decursin decreased the growth of spheroids and patient-derived gastric organoids, as well as modulated the expression of CTSC and autophagy-related proteins. Hence, our findings uncovered a previously unknown mechanism by which decursin regulates cell growth and autophagy and suggests that decursin may act as a potential therapeutic agent that simultaneously inhibits cell growth and autophagy.

Loss of desmoglein-2 promotes gallbladder carcinoma progression and resistance to EGFR-targeted therapy through Src kinase activation.

Gallbladder carcinoma (GBC) exhibits poor prognosis due to local recurrence, metastasis, and resistance to targeted therapies. Using clinicopathological analyses of GBC patients along with molecular in vitro and tumor in vivo analysis of GBC cells, we showed that reduction of Dsg2 expression was highly associated with higher T stage, more perineural, and lymphatic invasion. Dsg2-depleted GBC cells exhibited significantly enhanced proliferation, migration, and invasiveness in vitro and tumor growth and metastasis in vivo through Src-mediated signaling activation. Interestingly, Dsg2 binding inhibited Src activation, whereas its loss activated cSrc-mediated EGFR plasma membrane clearance and cytoplasmic localization, which was associated with acquired EGFR-targeted therapy resistance and decreased overall survival. Inhibition of Src activity by dasatinib enhanced therapeutic response to anti-EGFR therapy. Dsg2 status can help stratify predicted patient response to anti-EGFR therapy and Src inhibition could be a promising strategy to improve the clinical efficacy of EGFR-targeted therapy.

Translation Initiation Control of RNase E-Mediated Decay of Polycistronic gal mRNA.

In bacteria, mRNA decay is a major mechanism for regulating gene expression. In Escherichia coli, mRNA decay initiates with endonucleolytic cleavage by RNase E. Translating ribosomes impede RNase E cleavage, thus providing stability to mRNA. In transcripts containing multiple cistrons, the translation of each cistron initiates separately. The effect of internal translation initiations on the decay of polycistronic transcripts remains unknown, which we have investigated here using the four-cistron galETKM transcript. We find that RNase E cleaves a few nucleotides (14-36) upstream of the translation initiation site of each cistron, generating decay intermediates galTKM, galKM, and galM mRNA with fewer but full cistrons. Blocking translation initiation reduced stability, particularly of the mutated cistrons and when they were the 5'-most cistrons. This indicates that, together with translation failure, the location of the cistron is important for its elimination. The instability of the 5'-most cistron did not propagate to the downstream cistrons, possibly due to translation initiation there. Cistron elimination from the 5' end was not always sequential, indicating that RNase E can also directly access a ribosome-free internal cistron. The finding in gal operon of mRNA decay by cistron elimination appears common in E. coli and Salmonella.

CXCR7 promotes migration and invasion in head and neck squamous cell carcinoma by upregulating TGF-ß1/Smad2/3 signaling.

The chemokine receptor CXCR7 has been suggested to play important roles in the progression of several types of cancers. However, few studies have investigated the biological roles of CXCR7 in head and neck squamous cell carcinoma (HNSCC). CXCR7 expression and its clinical implications were examined in 103 HNSCC tissues using immunohistochemistry (IHC). The biological roles and mechanisms of CXCR7-mediated signaling pathways were investigated in HNSCC cells through CXCR7 overexpression in vitro and in vivo. High expression of CXCR7 was significantly associated with tumor size (P = 0.007), lymph node metastasis (P = 0.004), and stage (P = 0.020) in HNSCC. Overexpression of CXCR7 in HNSCC cells enhanced cell migration and invasion in vitro and promoted lymph node metastasis in vivo. CXCR7 also induced epithelial-mesenchymal transition through PI3K/AKT. CXCR7 increased secretion of transforming growth factor-ß1 (TGF-ß1) and promoted EMT through phosphorylated Smad2/3. Taken together, our results provide functional and mechanistic roles of CXCR7 as a master regulator of oncogenic TGF-ß1/Smad2/3 signaling in HNSCC, suggesting that CXCR7 might be a therapeutic target for the treatment of HNSCC.

Decursin inhibits tumor growth, migration, and invasion in gastric cancer by down-regulating CXCR7 expression.

CXC chemokine receptor 7 (CXCR7) is highly expressed in various type of cancers and promotes cancer progression and metastasis. However, the biological role and regulation of CXCR7 in gastric cancer remains unclear, and little is known about compounds that modulate CXCR7. Here, we investigated the role of CXCR7 in gastric tumorigenesis, and the effects of decursin, which is derived from Angelica gigas Nakai, on CXCR7. Our results showed that CXCR7 significantly promoted growth of gastric cancer cells and increased migration and invasion, which was mediated by the STAT3/c-Myc pathway. We also confirmed that decursin had an antitumor effect through down-regulating the expression of CXCR7 in gastric cancer. Furthermore, apoptotic cell death was induced through the reduction of anti-apoptotic factors such as Bcl-2 in vitro and in vivo. Our findings show that CXCR7 in gastric cancer promotes cancer progression through the STAT3/c-Myc pathway and that decursin is a natural compound that may target CXCR7 in gastric cancer treatment.

Processing generates 3' ends of RNA masking transcription termination events in prokaryotes.

Two kinds of signal-dependent transcription termination and RNA release mechanisms have been established in prokaryotes in vitro by: (i) binding of Rho to cytidine-rich nascent RNA [Rho-dependent termination (RDT)], and (ii) the formation of a hairpin structure in the nascent RNA, ending predominantly with uridine residues [Rho-independent termination (RIT)]. As shown here, the two signals act independently of each other and can be regulated (suppressed) by translation-transcription coupling in vivo. When not suppressed, both RIT- and RDT-mediated transcription termination do occur, but ribonucleolytic processing generates defined new 3' ends in the terminated RNA molecules. The actual termination events at the end of transcription units are masked by generation of new processed 3' RNA ends; thus the in vivo 3' ends do not define termination sites. We predict generation of 3' ends of mRNA by processing is a common phenomenon in prokaryotes as is the case in eukaryotes.

Visualization of RNA 3' ends in Escherichia coli Using 3' RACE Combined with Primer Extension.

In this assay, 3' RACE (Rapid Amplification of cDNA 3' Ends) followed by PE (primer extension), abbreviated as 3' RACE-PE is used to identify the mRNA 3' ends. The following protocol describes the amplification of the mRNA 3' ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, the definite primer is 5' end-labeled using [γ-(32) P] ATP and T4 polynucleotide kinase, which anneals to the specific DNA molecules within the PCR product of the 3' RACE. The conventional PE can only be used to locate the 5' end of an mRNA transcript since reverse transcriptase (RTase) polymerizes only in the 5' → 3' direction. Thus, Taq polymerase is used instead of RTase, PCR is performed. Therefore, we are able to locate the 3' end of the mRNA using this assay. The relative amount of the 3' end can be directly visualized and quantified by way of separating DNA products in a denaturing 8% urea-PAGE (Polyacrylamide Gel Electrophoresis) gel. The exact position of the 3' ends can be sequenced by comparison of these final DNA products with the corresponding DNA sequencing ladder.

Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene.

The Escherichia coli gal operon has the structure Pgal-galE-galT-galK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the down-regulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of galT.

Expression of each cistron in the gal operon can be regulated by transcription termination and generation of a galk-specific mRNA, mK2.

The gal operon of Escherichia coli has 4 cistrons, galE, galT, galK, and galM. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. 378: 318-327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3' ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5' ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galE-galT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5' ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3' ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5' portion of pre-mK2 is likely to become mE2. These results also suggested that galK expression could be regulated through mK2 production independent from natural polarity.

The CnuK9E H-NS complex antagonizes DNA binding of DicA and leads to temperature-dependent filamentous growth in E. coli.

Cnu (an OriC-binding nucleoid protein) associates with H-NS. A variant of Cnu was identified as a key factor for filamentous growth of a wild-type Escherichia coli strain at 37°C. This variant (CnuK9E) bears a substitution of a lysine to glutamic acid, causing a charge reversal in the first helix. The temperature-dependent filamentous growth of E. coli bearing CnuK9E could be reversed by either lowering the temperature to 25°C or lowering the CnuK9E concentration in the cell. Gene expression analysis suggested that downregulation of dicA by CnuK9E causes a burst of dicB transcription, which, in turn, elicits filamentous growth. In vivo assays indicated that DicA transcriptionally activates its own gene, by binding to its operator in a temperature-dependent manner. The antagonizing effect of CnuK9E with H-NS on DNA-binding activity of DicA was stronger at 37°C, presumably due to the lower operator binding of DicA at 37°C. These data suggest that the temperature-dependent negative effect of CnuK9E on DicA binding plays a major role in filamentous growth. The C-terminus of DicA shows significant amino acid sequence similarity to the DNA-binding domains of RovA and SlyA, regulators of pathogenic genes in Yersinia and Salmonella, respectively, which also show better DNA-binding activity at 25°C.

A mutational study of Cnu reveals attractive forces between Cnu and H-NS.

Cnu is a small 71-amino acid protein that complexes with H-NS and binds to a specific sequence in the replication origin of the E. coli chromosome. To understand the mechanism of interaction between Cnu and H-NS, we used bacterial genetics to select and analyze Cnu variants that cannot complex with H-NS. Out of 2,000 colonies, 40 Cnu variants were identified. Most variants (82.5%) had a single mutation, but a few variants (17.5%) had double amino acid changes. An in vitro assay was used to identify Cnu variants that were truly defective in H-NS binding. The changes in these defective variants occurred exclusively at charged amino acids (Asp, Glu, or Lys) on the surface of the protein. We propose that the attractive force that governs the Cnu-H-NS interaction is an ionic bond, unlike the hydrophobic interaction that is the major attractive force in most proteins.