Full-Range Profiling of tRNA Modifications Using LC-MS/MS at Single-Base Resolution through a Site-Specific Cleavage Strategy.

Transfer RNAs (tRNAs) are the most heavily modified RNA species. Liquid chromatography coupled with mass spectrometry (LC-MS/MS) is a powerful tool for characterizing tRNA modifications, which involves pretreating tRNAs with base-specific ribonucleases to produce smaller oligonucleotides amenable to MS. However, the quality and quantity of products from base-specific digestions are severely impacted by the base composition of tRNAs. This often leads to a loss of sequence information. Here, we report a method for the full-range profiling of tRNA modifications at single-base resolution by combining site-specific RNase H digestion with the LC-MS/MS and RNA-seq techniques. The key steps were designed to generate high-quality products of optimal lengths and ionization properties. A linear correlation between collision energies and the m/z of oligonucleotides significantly improved the information content of collision-induced dissociation (CID) spectra. False positives were eliminated by up to 95% using novel inclusion criteria for collecting a census of modifications. This method is illustrated by the mapping of mouse mitochondrial tRNAHis(GUG) and tRNAVal(UAC), which were hitherto not investigated. The identities and locations of the five species of modifications on these tRNAs were fully characterized. This approach is universally applicable to any tRNA species and provides an experimentally realizable pathway to the de novo sequencing of post-transcriptionally modified tRNAs with high sequence coverage.

cDNA cloning and heterologous expression of an endo-ß-1,4-glucanase from the fungus-growing termite Macrotermes barneyi.

Major ß-glucosidase (BG) and endo-ß-1,4-glucanase (EG) activities were localized to the midgut of the fungus-growing termite Macrotermes barneyi. Previously, we obtained the endogenous BG gene (MbmgBG1) from the midgut of M. barneyi. Here, we report the cDNA cloning of another endogenous cellulase, the EG protein MbEG1. This cellulase was partially purified from crude extract of the midgut of worker termites using zymogram analysis. Based on the N-terminal amino acid sequence and using rapid amplification of cDNA ends (RACE), a full-length cDNA of 1,843 base pairs was obtained. This encoded 448 amino acids and the sequence was similar to that of the members of glycoside hydrolase family 9. The MbEG1 transcript was detected primarily in the midgut using quantitative real-time polymerase chain reaction (PCR). To confirm functional activity of MbEG1, heterologous expression was conducted in both Escherichia coli and Pichia pastoris expression systems. Results indicated that MbEG1 could be functionally expressed in P. pastoris. This study provides the information that may facilitate understanding of cellulolytic systems in fungus-growing termites.

The role of post-transcriptional modification on a new tRNAIle(GAU) identified from Ganoderma lucidum in its fragments' cytotoxicity on cancer cells.

Ganoderma lucidum (Ganoderma) is a famous Chinese herbal medicine which has been used clinically for thousands of years in China. Despite numerous studies on triterpenes and polysaccharides, the bioactivity of RNAs abundant in Ganoderma remains unknown. Here, based on LC-MS techniques, dihydrouracil, 5-methyluridine (m5U) and pseudouridine were identified at position 19, 52 and 53 of a new tRNAIle(GAU) which was isolated as the most abundant tRNA species in Ganoderma, and is the first purified tRNA from fungus. Cytotoxic screening of tRNA-half (t-half) and tRNA fragment (tRF) derived from this tRNA, as well as their mimics (t-half or tRF as antisense strand), demonstrated that the double-stranded form, i.e., tRF and t-halve mimics, exhibited stronger cytotoxicity than their single-stranded form, and the cytotoxicity of t-half mimic is significantly stronger than that of tRF mimic. Notably, the cytotoxicity of 3'-t-half mimic is not only much more potent than that of taxol, but also is much more potent than that of ganoderic acids, the major bioactive components in Ganoderma. Furthermore, 3'-t-half mimic_M2 (m5U modified) exhibited significantly stronger cytotoxicity than unmodified 3'-t-half mimic, which is consistent with the computational simulation showing that m5U modification enhances the stability of the tertiary structure of 3'-t-half mimic. Overall, the present study not only indicates t-halves are bioactive components in Ganoderma which should not be neglected, but also reveals an important role of post-transcriptional modification on tRNA in its fragments' cytotoxicity against cancer cells, which benefits the design and development of RNAi drugs from natural resource.

The complete mitochondrial genome of Macrotermes barneyi Light (Isoptera: Termitidae).

The complete nucleotide sequence of the mitochondrial genome of Macrotermes barneyi Light (Isoptera: Termitidae) was determined. This mitochondrial genome is 15,940 bp with 37 typical animal mitochondrial genes and an A+T-rich region. Gene positions and directions are identical to that of the pupative ancestral arrangement of insects. All protein-coding genes start with ATN codon. Seven protein-coding genes stop with termination codon TAA. Four protein-coding genes use incomplete stop codons TA and two use T. The base composition of M. barneyi mitochondrial genome is normal to most insects. All of the 22 tRNA genes, ranging from 63 to 76 bp, have a typical cloverleaf structure except for trnS2. In trnS2, D-stem pairings in the DHU (dihydrouridine) arm is absent as in many sequenced insect species. Five elements characterized by A+T-rich region of the insect mitochondrial genome are found on their conserved arrangement. However, no repeat sequence is present on the downstream of the A+T-rich region.