En-Duo Wang

A novel miR‐155/miR‐143 cascade controls glycolysis by regulating hexokinase 2 in breast cancer cells

Cancer cells preferentially metabolize glucose through aerobic glycolysis. This phenomenon, known as the Warburg effect, is an anomalous characteristic of glucose metabolism in cancer cells. Chronic inflammation is a key promoting factor of tumourigenesis. It remains, however, largely unexplored whether and how pro‐tumourigenic inflammation regulates glucose metabolism in cancer cells. Here, we show that pro‐inflammatory cytokines promote glycolysis in breast cancer cells, and that the inflammation‐induced miR‐155 functions as an important mediator in this process. We further show that miR‐155 acts to upregulate hexokinase 2 (hk2), through two distinct mechanisms. First, miR‐155 promotes hk2 transcription by activation of signal transducer and activator of transcription 3 (STAT3), a transcriptional activator for hk2. Second, via targeting C/EBPβ (a transcriptional activator for mir‐143), miR‐155 represses mir‐143, a negative regulator of hk2, thus resulting in upregulation of hk2 expression at the post‐transcriptional level. The miR‐155‐mediated hk2 upregulation also appears to operate in other types of cancer cells examined. We suggest that the miR‐155/miR‐143/HK2 axis may represent a common mechanism linking inflammation to the altered metabolism in cancer cells.

Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis.

Spermatogenesis in mammals is characterized by two waves of piRNA expression: one corresponds to classic piRNAs responsible for silencing retrotransponsons and the second wave is predominantly derived from nontransposon intergenic regions in pachytene spermatocytes, but the function of these pachytene piRNAs is largely unknown. Here, we report the involvement of pachytene piRNAs in instructing massive mRNA elimination in mouse elongating spermatids (ES). We demonstrate that a piRNA-induced silencing complex (pi-RISC) containing murine PIWI (MIWI) and deadenylase CAF1 is selectively assembled in ES, which is responsible for inducing mRNA deadenylation and decay via a mechanism that resembles the action of miRNAs in somatic cells. Such a highly orchestrated program appears to take full advantage of the enormous repertoire of diversified targeting capacity of pachytene piRNAs derived from nontransposon intergenic regions. These findings suggest that pachytene piRNAs are responsible for inactivating vast cellular programs in preparation for sperm production from ES.

tRNA-dependent pre-transfer editing by prokaryotic leucyl-tRNA synthetase

To prevent genetic code ambiguity due to misincorporation of amino acids into proteins, aminoacyl-tRNA synthetases have evolved editing activities to eliminate intermediate or final non-cognate products. In this work we studied the different editing pathways of class Ia leucyl-tRNA synthetase (LeuRS). Different mutations and experimental conditions were used to decipher the editing mechanism, including the recently developed compound AN2690 that targets the post-transfer editing site of LeuRS. The study emphasizes the crucial importance of tRNA for the pre- and post-transfer editing catalysis. Both reactions have comparable efficiencies in prokaryotic Aquifex aeolicus and Escherichia coli LeuRSs, although the E. coli enzyme favors post-transfer editing, whereas the A. aeolicus enzyme favors pre-transfer editing. Our results also indicate that the entry of the CCA-acceptor end of tRNA in the editing domain is strictly required for tRNA-dependent pre-transfer editing. Surprisingly, this editing reaction was resistant to AN2690, which inactivates the enzyme by forming a covalent adduct with tRNALeu in the post-transfer editing site. Taken together, these data suggest that the binding of tRNA in the post-transfer editing conformation confers to the enzyme the capacity for pre-transfer editing catalysis, regardless of its capacity to catalyze post-transfer editing.

piRNA-Triggered MIWI Ubiquitination and Removal by APC/C in Late Spermatogenesis

The PIWI/PIWI-interacting RNA (piRNA) machinery has been well documented to maintain genome integrity by silencing transposons in animal germ cells. Recent studies have advanced our understanding of the biogenesis and function of this machinery; however, its metabolism has remained largely unexplored. Here, we show that murine PIWI (MIWI) is degraded through the APC/C-26S proteasome pathway and that piRNAs play an indispensable role in this process by enhancing MIWI interaction with an APC/C substrate-binding subunit. Interestingly, piRNA-triggered MIWI destruction occurs in late spermatids, which in turn leads to piRNA elimination, suggesting a feedforward mechanism for coordinated removal of the MIWI/piRNA machinery at a specific developmental stage. Importantly, the proper removal of MIWI/piRNA is essential for sperm maturation. Together, our results reveal a role for piRNAs in regulating the clearance of the MIWI/piRNA machinery via the ubiquitin-proteosome pathway and demonstrate the critical importance of proper temporal regulation of MIWI/piRNA in male germ cell development.

tRNA-independent Pretransfer Editing by Class I Leucyl-tRNA Synthetase

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNA in a two-step reaction starting with amino acid activation followed by aminoacyl group transfer to tRNA. To clear mistakes that occasionally occur, some of these enzymes carry out editing activities, acting on the misactivated amino acid (pretransfer editing) or after the transfer on the tRNA (post-transfer editing). The post-transfer editing pathway of leucyl-tRNA synthetase has been extensively studied by structural and biochemical approaches. Here, we report the finding of a tRNA-independent pretransfer editing pathway in leucyl-tRNA synthetases from Aquifex aeolicus. Using a CP1-mutant defective in its post-transfer editing function, we showed that this new editing pathway is distinct from the post-transfer editing site and may occur at the synthetic catalytic site, as recently proposed for other aminoacyl-tRNA synthetases.

Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNALeu. It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNALeu (hctRNALeu) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit α-amino butyrate, although both amino acids can be charged to tRNALeu. Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution.

Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

The editing reactions catalyzed by aminoacyl‐tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl‐tRNA synthetase from the deep‐rooted bacterium Aquifex aeolicus (αβ‐LeuRS) catalyzes the hydrolytic editing of both mischarged tRNALeu and minihelixLeu. Within the domain, we have identified a crucial 20‐amino‐acid peptide that confers editing capacity when transplanted into the inactive Escherichia coli LeuRS editing domain. Likewise, fusion of the β‐subunit of αβ‐LeuRS to the E. coli editing domain activates its editing function. These results suggest that αβ‐LeuRS still carries the basic features from a primitive synthetase molecule. It has a remarkable capacity to transfer autonomous active modules, which is consistent with the idea that modern synthetases arose after exchange of small idiosyncratic domains. It also has a unique αβ‐heterodimeric structure with separated catalytic and tRNA‐binding sites. Such an organization supports the tRNA/synthetase coevolution theory that predicts sequential addition of tRNA and synthetase domains.

Role of tRNA amino acid-accepting end in aminoacylation and its quality control

Aminoacyl–tRNA synthetases (aaRSs) are remarkable enzymes that are in charge of the accurate recognition and ligation of amino acids and tRNA molecules. The greatest difficulty in accurate aminoacylation appears to be in discriminating between highly similar amino acids. To reduce mischarging of tRNAs by non-cognate amino acids, aaRSs have evolved an editing activity in a second active site to cleave the incorrect aminoacyl–tRNAs. Editing occurs after translocation of the aminoacyl–CCA76 end to the editing site, switching between a hairpin and a helical conformation for aminoacylation and editing. Here, we studied the consequence of nucleotide changes in the CCA76 accepting end of tRNALeu during the aminoacylation and editing reactions. The analysis showed that the terminal A76 is essential for both reactions, suggesting that critical interactions occur in the two catalytic sites. Substitutions of C74 and C75 selectively decreased aminoacylation keeping nearly unaffected editing. These mutations might favor the regular helical conformation required to reach the editing site. Mutating the editing domain residues that contribute to CCA76 binding reduced the aminoacylation fidelity leading to cell-toxicity in the presence of non-cognate amino acids. Collectively, the data show how protein synthesis quality is controlled by the CCA76 homogeneity of tRNAs.

The tRNA recognition mechanism of the minimalist SPOUT methyltransferase, TrmL

Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition.

Overproduction and purification ofEscherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA (1) (Leu) and tRNA (2) (Leu) were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A(260), respectively: the content of tRNA (1) (Leu) was 50% of total tRNA from MT-Leu1, while that of tRNA (2) (Leu) was 30% of total tRNA from MT-Leu2. Both tRNA(Leu)s from their rotal tRNs were fractionated to 1 600 pmol/A(260) after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNA(Leu) catalyzed by leucyl-tRNA synthetase were determined.Chemically synthesized genes encodingEscherichia coli tRNA1Leu and tRNA2Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A260, respectively: the content of tRNA1Leu was 50% of total tRNA from MT-Leu1, while that of tRNA2Leu was 30% of total tRNA from MT-Leu2. Both tRNALeus from their rotal tRNs were fractionated to 1 600 pmol/A260 after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNALeu catalyzed by leucyl-tRNA synthetase were determined.