A new transcriptome-wide method produces reliable m ⁶ A maps at single-base resolution and reveals site conservation in mammals.

Charcot–Marie–Tooth (CMT) diseases are the most common heritable peripheral neuropathy. At least 10 different mutant alleles of GARS (the gene for glycyl-tRNA synthetase) have been reported to cause a dominant axonal form of CMT (type 2D). A unifying connection between these mutations and CMT has been unclear. Here, mapping mutations onto the recently determined crystal structure of human GlyRS showed them within a band encompassing both sides of the dimer interface, with two CMT-causing mutations being at sites that are complementary partners of a “kissing” contact across the dimer interface. The CMT phenotype is shown here to not correlate with aminoacylation activity. However, most mutations affect dimer formation (to enhance or weaken). Seven CMT-causing variants and the wild-type protein were expressed in transfected neuroblastoma cells that sprout primitive neurites. Wild-type GlyRS distributed into the nascent neurites and was associated with normal neurite sprouting. In contrast, all mutant proteins were distribution-defective. Thus, CMT-causing mutations of GlyRS share a common defect in localization. This defect may be connected in some way to a change in the surfaces at the dimer interface.

Cleavage of transfer (t)RNA and ribosomal (r)RNA are critical and conserved steps of translational control for cells to overcome varied environmental stresses. However, enzymes that are responsible for this event have not been fully identified in high eukaryotes. Here, we report a mammalian tRNA/rRNA-targeting endoribonuclease: SLFN13, a member of the Schlafen family. Structural study reveals a unique pseudo-dimeric U-pillow-shaped architecture of the SLFN13 N′-domain that may clamp base-paired RNAs. SLFN13 is able to digest tRNAs and rRNAs in vitro, and the endonucleolytic cleavage dissevers 11 nucleotides from the 3′-terminus of tRNA at the acceptor stem. The cytoplasmically localised SLFN13 inhibits protein synthesis in 293T cells. Moreover, SLFN13 restricts HIV replication in a nucleolytic activity-dependent manner. According to these observations, we term SLFN13 RNase S13. Our study provides insights into the modulation of translational machinery in high eukaryotes, and sheds light on the functional mechanisms of the Schlafen family.

Most β-glucosidases reported are sensitive to the end product (glucose), making it the rate limiting component of cellulase for efficient degradation of cellulose through enzymatic route. Thus, there are ongoing interests in searching for glucose-tolerant β-glucosidases, which are still active at high glucose concentration. Although many β-glucosidases with different glucose-tolerance levels have been isolated and characterized in the past decades, the effects of glucose-tolerance on the hydrolysis of cellulose are not thoroughly studied. In the present study, a novel β-glucosidase (Bgl6) with the half maximal inhibitory concentration (IC 50) of 3.5 M glucose was isolated from a metagenomic library and characterized. However, its poor thermostability at 50 °C hindered the employment in cellulose hydrolysis. To improve its thermostability, random mutagenesis was performed. A thermostable mutant, M3, with three amino acid substitutions was obtained. The half-life of M3 at 50 °C is 48 h, while that of Bgl6 is 1 h. The K cat/K m value of M3 is 3-fold higher than that of Bgl6. The mutations maintained its high glucose-tolerance with IC 50 of 3.0 M for M3. In a 10-h hydrolysis of cellobiose, M3 completely converted cellobiose to glucose, while Bgl6 reached a conversion of 80 %. Then their synergistic effects with the commercial cellulase (Celluclast 1.5 L) on hydrolyzing pretreated sugarcane bagasse (SCB) were investigated. The supplementation of Bgl6 or mutant M3 to Celluclast 1.5 L significantly improved the SCB conversion from 64 % (Celluclast 1.5 L alone) to 79 % (Bgl6) and 94 % (M3), respectively. To further evaluate the application potential of M3 in high-solids cellulose hydrolysis, such reactions were performed at initial glucose concentration of 20–500 mM. Results showed that the supplementation of mutant M3 enhanced the glucose production from SCB under all the conditions tested, improving the SCB conversion by 14–35 %. These results not only clearly revealed the significant role of glucose-tolerance in cellulose hydrolysis, but also showed that mutant M3 may be a potent candidate for high-solids cellulose refining.