Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation.

Cleavage and polyadenylation (CPA) is responsible for 3' end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets CPSF-73, is the first known CPA inhibitor (CPAi) in mammalian cells. Here we show that JTE-607 perturbs gene expression through both transcriptional readthrough and alternative polyadenylation (APA). Sensitive genes are associated with features similar to those previously identified for PCF11 knockdown, underscoring a unified transcriptomic signature of CPAi. The degree of inhibition of an APA site by JTE-607 correlates with its usage level and, consistently, cells with elevated CPA activities, such as those with induced overexpression of FIP1, display greater transcriptomic disturbances when treated with JTE-607. Moreover, JTE-607 causes S phase crisis and is hence synergistic with inhibitors of DNA damage repair pathways. Together, our data reveal CPA activity and proliferation rate as determinants of CPAi-mediated cell death, raising the possibility of using CPAi as an adjunct therapy to suppress certain cancers.

Clinical Characteristics and Survival Analysis of Patients With Second Primary Malignancies After Hepatocellular Carcinoma Liver Transplantation: A SEER-based Analysis.

BACKGROUND: Second primary malignancies (SPMs) after liver transplantation (LT) are becoming the leading causes of death in LT recipients. The purpose of this study was to explore prognostic factors for SPMs and to establish an overall survival nomogram. METHODS: A retrospective analysis was conducted of data from the Surveillance, Epidemiology, and End Results (SEER) database on adult patients with primary hepatocellular carcinoma who had undergone LT between 2004 and 2015. Cox regression analysis was used to explore the independent prognostic factors for SPMs. Nomogram was constructed using R software to predict the overall survival at 2, 3, and 5 years. The concordance index, calibration curves, and decision curve analysis were used to evaluate the clinical prediction model. RESULTS: Data from a total of 2078 patients were eligible, of whom 221 (10.64%) developed SPMs. A total of 221 patients were split into a training cohort (n=154) or a validation cohort (n=67) with a 7:3 ratio. The 3 most common SPMs were lung cancer, prostate cancer, and non-Hodgkin lymphoma. Age at initial diagnosis, marital status, year of diagnosis, T stage, and latency were the prognostic factors for SPMs. The C-index of the nomogram for overall survival in the training and validation cohorts were 0.713 and 0.729, respectively. CONCLUSIONS: We analyzed the clinical characteristics of SPMs and developed a precise prediction nomogram, with a good predictive performance. The nomogram we developed may help clinicians provide personalized decisions and clinical treatment for LT recipients.

CRISPRpas: programmable regulation of alternative polyadenylation by dCas9.

Most human protein-coding genes produce alternative polyadenylation (APA) isoforms that differ in 3' UTR size or, when coupled with splicing, have variable coding sequences. APA is an important layer of gene expression program critical for defining cell identity. Here, by using a catalytically dead Cas9 and coupling its target site with polyadenylation site (PAS), we develop a method, named CRISPRpas, to alter APA isoform abundance. CRISPRpas functions by enhancing proximal PAS usage, whose efficiency is influenced by several factors, including targeting strand of DNA, distance between PAS and target sequence and strength of the PAS. For intronic polyadenylation (IPA), splicing features, such as strengths of 5' splice site and 3' splice site, also affect CRISPRpas efficiency. We show modulation of APA of multiple endogenous genes, including IPA of PCF11, a master regulator of APA and gene expression. In sum, CRISPRpas offers a programmable tool for APA regulation that impacts gene expression.

Enzymatic Synthesis of Nucleoside Triphosphates and Deoxynucleoside Triphosphates by Surface-Displayed Kinases.

Nucleoside triphosphates and deoxynucleoside triphosphates are important biochemical molecules. In this study, recombinant Escherichia coli that could display nucleotide kinases (INP-N-NMKases) and acetate kinase (INP-N-ACKase) on the cell surface were constructed by fusing an enzyme (NMKase/ACKase) to the N-terminus of ice nucleation protein (INP-N). By using intact recombinant bacteria cells as a catalyst coupled with an ACKase-catalyzed adenosine-5'-triphosphate (ATP) regeneration system, nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) could be synthesized efficiently. In a reaction system with 5 mmol/l substrate, the conversion rates of cytidine-5'-triphosphate (CTP) and deoxycytidine-5'-triphosphate (dCTP) were 96% and 93%, respectively, the conversion rate of ATP and deoxyadenosine-5'-triphosphate (dATP) was 96%, the conversion rate of deoxythymidine-5'-triphosphate (dTTP) was 91%, and the conversion rate of uridine-5'-triphosphate (UTP) was 80%. There was no obvious degradation. At 37 °C, the stability of the surface-displayed fusion protein, especially in the presence of the substrate, was significantly improved. Each whole cell could be reused more than 8 times.

Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes.

Regulation of cleavage and polyadenylation (CPA) affects gene expression and polyadenylation site (PAS) choice. Here, we report that the CPA and termination factor PCF11 modulates gene expression on the basis of gene size. Although downregulation of PCF11 leads to inhibition of short gene expression, long genes are upregulated because of suppressed intronic polyadenylation (IPA) enriched in large introns. We show that this regulatory scheme, named PCF11-mediated expression regulation through IPA (PEIPA), takes place in cell differentiation, during which downregulation of PCF11 is coupled with upregulation of long genes with functions in cell morphology, adhesion, and migration. PEIPA targets distinct gene sets in different cell contexts with similar rules. Furthermore, PCF11 is autoregulated through a conserved IPA site, the removal of which leads to global activation of PASs close to gene promotors. Therefore, PCF11 uses distinct mechanisms to regulate genes of different sizes, and its autoregulation maintains homeostasis of PAS usage in the cell.

Biosynthesis of (deoxy)guanosine-5'-triphosphate by GMP kinase and acetate kinase fixed on the surface of E. coli.

(Deoxy)guanosine-5'-triphosphate (5'-(d)GTP), the precursor for synthesizing DNA or RNA in vivo, is an important raw material for various modern biotechnologies based on PCR. In this study, we investigated the application of whole-cell catalysts constructed by bacterial cell surface display in biosynthetic reactions of 5'-(d)GTP from (deoxy)guanosine-5'-monophosphate (5'-(d)GMP). By N-terminal or N- and C-terminal fusion of the ice nucleation protein, we successfully displayed the GMP kinase of Lactobacillus bulgaricus and the acetate kinase of E. coli on the surface of E. coli cells. A large amount of soluble target protein was obtained upon induction with 0.2 mM IPTG at 25 °C for 30 h. The conversion of dGMP was up to 91% when catalysed by the surface-displayed enzymes at 37 °C for 4 h. Up to 95% of the GMP was converted after 3 h of reaction. The stability of the whole-cell catalyst at 37 °C was very good. The enzyme activity was maintained above 50% after 9 rounds of recovery. Our research showed that only one-twentieth of the initial substrate concentration of added ATP was sufficient to meet the reaction requirements.

Cellular stress alters 3'UTR landscape through alternative polyadenylation and isoform-specific degradation.

Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different 3'UTR lengths, production of which is influenced by cellular conditions. Here, we show that arsenic stress elicits global shortening of 3'UTRs through preferential usage of proximal polyadenylation sites during stress and enhanced degradation of long 3'UTR isoforms during recovery. We demonstrate that RNA-binding protein TIA1 preferentially interacts with alternative 3'UTR sequences through U-rich motifs, correlating with stress granule association and mRNA decay of long 3'UTR isoforms. By contrast, genes with shortened 3'UTRs due to stress-induced APA can evade mRNA clearance and maintain transcript abundance post stress. Furthermore, we show that stress causes distinct 3'UTR size changes in proliferating and differentiated cells, highlighting its context-specific impacts on the 3'UTR landscape. Together, our data reveal a global, 3'UTR-based mRNA stability control in stressed cells and indicate that APA can function as an adaptive mechanism to preserve mRNAs in response to stress.

NTP regeneration and its application in the biosynthesis of nucleotides and their derivatives.

BACKGROUND: Nucleoside 5'-triphosphates (NTPs) play an important role in cells in the transfer of phosphate groups or bioenergy. In vivo, they are ready to be produced, regenerated and consumed in different kinds of metabolic pathways, and their concentrations are strictly controlled. NTPs are useful reagents that take part in many biosynthetic processes. However, NTPs are expensive and unstable, which greatly increases the cost of the final product if a large amount of NTPs is used directly in biosynthesis. Furthermore, during reactions, NTPs degrade into NDPs and need to be separated from the reaction mixture, making the operation complicated. Therefore, NTPs are normally regenerated from NDPs, and only very few NTPs are used in the reaction. METHOD: Mechanisms of NTP regeneration were analysed, and their applications in the biosynthesis of nucleotides and their derivates were described. Basically, NTP regeneration involves isolated enzyme systems and whole-cell systems. RESULT: As one type of cofactor regeneration, NTPs can be effectively regenerated by acetate kinase, pyruvate kinase, and polyphosphate kinase from acetyl phosphate, phosphoenol pyruvate, and polyphosphate, respectively, or by whole cells of yeast and Corynebacterium ammoniagenes from simple carbohydrates and phosphate. The NTP-regeneration method is selected primarily due to the main reaction that it is being coupled with. The cost of phosphate donors and the convenience of integration with the main process should be considered. CONCLUSION: Significant advances have been made when NTP regeneration is coupled with other biosynthetic processes, especially in the preparation of nucleotides, 2'-deoxynucleotides, sugar-nucleotides and their derivatives.

Enzymatic Manufacture of Deoxythymidine-5'-Triphosphate with Permeable Intact Cells of E. coli Coexpressing Thymidylate Kinase and Acetate Kinase.

A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25°C for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'- triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.

Phosphorylation of uridine and cytidine by uridine-cytidine kinase.

Uridine 5'-monophosphate (5'-UMP) and cytidine 5'-monophosphate (5'-CMP) were biosynthesized by recombinant uridine-cytidine kinase (UCK) and acetate kinase (ACK). The ack and uck genes from Escherichia coli K12 and the uck1, uck2 and ack genes from Lactobacillus bulgaricus ATCC 11842 were cloned and inserted into the plasmid pET-28a. All of the recombinant E. coli strains were capable of overexpressing UCK and ACK, which catalyzed the reaction using guanosine 5'-triphosphate (GTP) as a phosphate intermediate that was regenerated by ACK from acetyl phosphate. The effect of several parameters, including the substrate concentration, the GTP concentration, the temperature and the reaction pH, were optimized. High efficiency was achieved if uridine or cytidine was phosphorylated by UCK encoded by uck from E. coli and ACK encoded by ack from L. bulgaricus. The maximum conversion yield of 5'-UMP and 5'-CMP was 97% at 37 °C and pH 7.5 when 30 mM uridine/cytidine and 0.5mM GTP in a total of 1 mL were used. In addition, the 5'-UMP and 5'-CMP products were very stable in the reaction system and did not undergo significant degradation.

Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system.

A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na2, 0.5 mM ATP, 5 mM Mg²âº, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.

Efficient production of deoxynucleoside-5'-monophosphates using deoxynucleoside kinase coupled with a GTP-regeneration system.

Deoxynucleoside-5'-monophosphates (5'-dNMPs) are the basic components of DNA and are widely used in medicine and as chemical and biochemical reagents. A large amount of effort has been expended to obtain 5'-dNMPs of high quality and at a low cost. However, these procedures are inefficient and inconvenient. In this study, deoxyadenosine-5'-monophosphate (5'-dAMP), 2,6-diaminopurine deoxynucleoside-5'-monophosphate (5'-dDAMP), and deoxycytidine-5'-monophosphate (5'-dCMP) were biosynthesized using recombinant N-deoxyribosyltransferase II (NDT-II), deoxycytidine kinase, and acetate kinase in a one-pot reaction system. The ndt-II gene from Lactobacillus delbrueckii, dck from Bacillus subtilus, and ack from Escherichia coli K12 were overexpressed in E. coli BL21 (DE3). Thymidine was used as the deoxyribose donor; GTP was used as the phosphate donor, and acetyl phosphate was used to regenerate GTP. Under optimized conditions, each 10 mM adenine, 10 mM 2,6-diaminopurine, or 10 mM cytosine were converted into 9.01 mM 5'-dAMP, 8.68 mM 5'-dDAMP, or 6.23 mM 5'-dCMP, respectively. The high yield indicated that this process of biosynthesis of 5'-dAMP, 5'-dDAMP, or 5'-dCMP was efficient and economical, and this one-pot system may also potentially be used for the preparation of other types of 5'-dNMPs.

Optimum induction of recombinant thymidine phosphorylase and its application.

Recombinant E. coli pDEOA was constructed and lactose can be used instead of IPTG to induce the expression of thymidine phosphorylase by pDEOA. The use of lactose at concentrations higher than 0.5 mmol/L had an induction effect similar to that of IPTG but resulted in a longer initial induction time and better cell growth. The thymidine phosphorylase induced by lactose was very stable at 50°C. Intact pDEOA cells induced by lactose can be used as a source of thymidine phosphorylase. Under standard reaction conditions, several deoxynucleosides were effectively produced from thymidine.

Enzymatic synthesis of nucleosides by nucleoside phosphorylase co-expressed in Escherichia coli.

Nucleoside phosphorylase is an important enzyme involved in the biosynthesis of nucleosides. In this study, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase were co-expressed in Escherichia coli and the intact cells were used as a catalyst for the biosynthesis of nucleosides. For protein induction, lactose was used in place of isopropyl ß-D-1-thiogalactopyranoside (IPTG). When the concentration of lactose was above 0.5 mmol/L, the ability to induce protein expression was similar to that of IPTG. We determined that the reaction conditions of four bacterial strains co-expressing these genes (TUD, TAD, DUD, and DAD) were similar for the biosyntheses of 2,6-diaminopurine nucleoside and 2,6-diaminopurine deoxynucleoside. When the substrate concentration was 30 mmol/L and 0.5% of the recombinant bacterial cell volume was used as the catalyst (pH 7.5), a greater than 90% conversion yield was reached after a 2-h incubation at 50 °C. In addition, several other nucleosides and nucleoside derivatives were efficiently synthesized using bacterial strains co-expressing these recombinant enzymes.

Co-expression of recombinant nucleoside phosphorylase from Escherichia coli and its application.

The genes encoding purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase), and thymidine phosphorylase (TPase) from Escherichia coli K12 were cloned respectively into expression vector pET-11a or pET-28a. The recombinant plasmids were transformed into the host strain E. coli BL21(DE3) to construct four co-expression recombinant strains. Two of them had double recombinant plasmids (DUD and DAD) and the other two had tandem recombinant plasmid (TDU and TDA) in them. Under the repression of antibiotic, recombinant plasmids stably existed in host strains. Enzymes were abundantly expressed after induction with IPTG and large amount of target proteins were expressed in soluble form analyzed with SDS-PAGE. Compared with the host strain, enzyme activity of the recombinant strains had been notably improved. In the transglycosylation reaction, yield of 2,6-diaminopurine-2'-deoxyriboside (DAPdR) from 2,6-diaminopurine (DAP) and thymidine reached 40.2% and 51.8% catalyzed by DAD and TDA respectively; yield of 2,6-diaminopurine riboside (DAPR) from DAP and uridine reached 88.2% and 58.0% catalyzed by TDU and DUD respectively.

Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside.

Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5'-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.