Human Thg1 displays tRNA-inducible GTPase activity.

tRNAHis guanylyltransferase (Thg1) catalyzes the 3'-5' incorporation of guanosine into position -1 (G-1) of tRNAHis. G-1 is unique to tRNAHis and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNAHis opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNAHis opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAsHis with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74-76), how HsThg1 prevents successive 3'-5' incorporation of G-1/G-2/G-3 into mitochondrial tRNAHis (tRNAmHis) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNAmHis indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNAmHis by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNAmHisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1. This study suggests that HsThg1 may downregulate the level of GTP through its GTPase activity to prevent multiple-GTP incorporation into tRNAmHis.

Large-scale data analysis for robotic yeast one-hybrid platforms and multi-disciplinary studies using GateMultiplex.

BACKGROUND: Yeast one-hybrid (Y1H) is a common technique for identifying DNA-protein interactions, and robotic platforms have been developed for high-throughput analyses to unravel the gene regulatory networks in many organisms. Use of these high-throughput techniques has led to the generation of increasingly large datasets, and several software packages have been developed to analyze such data. We previously established the currently most efficient Y1H system, meiosis-directed Y1H; however, the available software tools were not designed for processing the additional parameters suggested by meiosis-directed Y1H to avoid false positives and required programming skills for operation. RESULTS: We developed a new tool named GateMultiplex with high computing performance using C++. GateMultiplex incorporated a graphical user interface (GUI), which allows the operation without any programming skills. Flexible parameter options were designed for multiple experimental purposes to enable the application of GateMultiplex even beyond Y1H platforms. We further demonstrated the data analysis from other three fields using GateMultiplex, the identification of lead compounds in preclinical cancer drug discovery, the crop line selection in precision agriculture, and the ocean pollution detection from deep-sea fishery. CONCLUSIONS: The user-friendly GUI, fast C++ computing speed, flexible parameter setting, and applicability of GateMultiplex facilitate the feasibility of large-scale data analysis in life science fields.

Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling.

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5' splice-site (5'SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28's ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.

Naturally occurring dual recognition of tRNAHis substrates with and without a universal identity element.

The extra 5' guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis (CetRNAnHis) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNAHis (CetRNAmHis) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNAmHis strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNAnHis without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNAHis identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNAHis effectively converted a non-cognate tRNA to a tRNAHis-like substrate. Our study suggests that a new balance between identity elements of tRNAHis relieves HisRS from the absolute requirement for G-1.

A novel synthetic-genetic-array-based yeast one-hybrid system for high discovery rate and short processing time.

Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor-DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF-DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.

Integrative transcriptome sequencing reveals extensive alternative trans-splicing and cis-backsplicing in human cells.

Transcriptionally non-co-linear (NCL) transcripts can originate from trans-splicing (trans-spliced RNA; 'tsRNA') or cis-backsplicing (circular RNA; 'circRNA'). While numerous circRNAs have been detected in various species, tsRNAs remain largely uninvestigated. Here, we utilize integrative transcriptome sequencing of poly(A)- and non-poly(A)-selected RNA-seq data from diverse human cell lines to distinguish between tsRNAs and circRNAs. We identified 24,498 NCL events and found that a considerable proportion (20-35%) of them arise from both tsRNAs and circRNAs, representing extensive alternative trans-splicing and cis-backsplicing in human cells. We show that sequence generalities of exon circularization are also observed in tsRNAs. Recapitulation of NCL RNAs further shows that inverted Alu repeats can simultaneously promote the formation of tsRNAs and circRNAs. However, tsRNAs and circRNAs exhibit quite different, or even opposite, expression patterns, in terms of correlation with the expression of their co-linear counterparts, expression breadth/abundance, transcript stability, and subcellular localization preference. These results indicate that tsRNAs and circRNAs may play different regulatory roles and analysis of NCL events should take the joint effects of different NCL-splicing types and joint effects of multiple NCL events into consideration. This study describes the first transcriptome-wide analysis of trans-splicing and cis-backsplicing, expanding our understanding of the complexity of the human transcriptome.

The conserved AU dinucleotide at the 5' end of nascent U1 snRNA is optimized for the interaction with nuclear cap-binding-complex.

Splicing is initiated by a productive interaction between the pre-mRNA and the U1 snRNP, in which a short RNA duplex is established between the 5' splice site of a pre-mRNA and the 5' end of the U1 snRNA. A long-standing puzzle has been why the AU dincucleotide at the 5'-end of the U1 snRNA is highly conserved, despite the absence of an apparent role in the formation of the duplex. To explore this conundrum, we varied this AU dinucleotide into all possible permutations and analyzed the resulting molecular consequences. This led to the unexpected findings that the AU dinucleotide dictates the optimal binding of cap-binding complex (CBC) to the 5' end of the nascent U1 snRNA, which ultimately influences the utilization of U1 snRNP in splicing. Our data also provide a structural interpretation as to why the AU dinucleotide is conserved during evolution.

Feedback Control of Snf1 Protein and Its Phosphorylation Is Necessary for Adaptation to Environmental Stress.

Snf1, a member of the AMP-activated protein kinase family, plays a critical role in metabolic energy control in yeast cells. Snf1 activity is activated by phosphorylation of Thr-210 on the activation loop of its catalytic subunit; following activation, Snf1 regulates stress-responsive transcription factors. Here, we report that the level of Snf1 protein is dramatically decreased in a UBP8- and UBP10-deleted yeast mutant (ubp8Δ ubp10Δ), and this is independent of transcriptional regulation and proteasome-mediated degradation. Surprisingly, most Snf1-mediated functions, including glucose limitation regulation, utilization of alternative carbon sources, stress responses, and aging, are unaffected in this strain. Snf1 phosphorylation in ubp8Δ ubp10Δ cells is hyperactivated upon stress, which may compensate for the loss of the Snf1 protein and protect cells against stress and aging. Furthermore, artificial elevation of Snf1 phosphorylation (accomplished through deletion of REG1, which encodes a protein that regulates Snf1 dephosphorylation) restored Snf1 protein levels and the regulation of Snf1 activity in ubp8Δ ubp10Δ cells. Our results reveal the existence of a feedback loop that controls Snf1 protein level and its phosphorylation, which is masked by Ubp8 and Ubp10 through an unknown mechanism. We propose that this dynamic modulation of Snf1 phosphorylation and its protein level may be important for adaptation to environmental stress.