Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus.

Cell type annotation and lineage construction are two of the most critical tasks conducted in the analyses of single-cell RNA sequencing (scRNA-seq). Four recent scRNA-seq studies of differentiating xylem propose four models on differentiating xylem development in Populus. The differences are mostly caused by the use of different strategies for cell type annotation and subsequent lineage interpretation. Here, we emphasize the necessity of using in situ transcriptomes and anatomical information to construct the most plausible xylem development model.

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.

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.

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.

Adaptive transcription-splicing resynchronization upon losing an essential splicing factor.

Essential genes form the core of a genome and are therefore thought to be indispensable for cellular viability. However, recent findings have challenged this notion in that cells may survive in the absence of some essential genes provided that relevant genetic modifiers are in existence. We therefore hypothesized that the loss of an essential gene may not always be fatefully detrimental; instead, it may pave the way towards genome evolution. We experimentally tested this hypothesis in the context of pre-messenger RNA splicing by evolving yeast cells harbouring a permanent loss of the essential splicing factor Prp28 in the presence of a genetic modifier. Here, we show that cellular fitness can be restored by compensatory mutations that alter either the splicing machinery per se or the Spt-Ada-Gcn5 acetyltransferase transcription co-activator complex in the cells with no Prp28. Biochemical and genetic analysis revealed that slowing down transcription compensates for splicing deficiency, which in turn boosts cellular fitness. In addition, we found that inefficient splicing also conversely decreases nascent RNA production. Taken together, our data suggest that transcription-splicing synchronization contributes to robustness in the gene-expression pathway and argue that the intrinsic interconnectivity within a biological system can be exploited for compensatory evolution and system re-optimization.

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.

Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion.

BACKGROUND/PURPOSE: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. METHODS: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. RESULTS: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. CONCLUSION: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs.

Alternative Splicing of Toll-Like Receptor 9 Transcript in Teleost Fish Grouper Is Regulated by NF-κB Signaling via Phosphorylation of the C-Terminal Domain of the RPB1 Subunit of RNA Polymerase II.

Similar to its mammalian counterparts, teleost Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA presented in the genome of bacteria or DNA viruses and initiates signaling pathway(s) for immune responses. We have previously shown that the TLR9 pathway in grouper, an economically important teleost, can be debilitated by an inhibitory gTLR9B isoform, whose production is mediated by RNA alternative splicing. However, how does grouper TLR9 (gTLR9) signaling impinge on the RNA splicing machinery to produce gTlr9B is unknown. Here we show that the gTlr9 alternative splicing is regulated through ligand-induced phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). We first observed that ligand-activated NF- κB pathway biased the production of the gTlr9B isoform. Because NF- κB is known to recruit p-TEFb kinase, which phosphorylates the Pol II CTD at Ser2 residues, we examined p-TEFb's role in alternative splicing. We found that promoting p-TEFb kinase activity significantly favored the production of the gTlr9B isoform, whereas inhibiting p-TEFb yielded an opposite result. We further showed that p-TEFb-mediated production of the gTlr9B isoform down-regulates its own immune responses, suggesting a self-limiting mechanism. Taken together, our data indicate a feedback mechanism of the gTLR9 signaling pathway to regulate the alternative splicing machinery, which in turn produces an inhibitor to the pathway.

Detection of Protein-Protein Interaction Within an RNA-Protein Complex Via Unnatural-Amino-Acid-Mediated Photochemical Crosslinking.

Although DExD/H-box proteins are known to unwind RNA duplexes and modulate RNA structures in vitro, it is highly plausible that, in vivo, some may function to remodel RNA-protein complexes. Precisely how the latter is achieved remains a mystery. We investigated this critical issue by using yeast Prp28p, an evolutionarily conserved DExD/H-box splicing factor, as a model system. To probe how Prp28p interacts with spliceosome, we strategically placed p-benzoyl-phenylalanine (BPA), a photoactivatable unnatural amino acid, along the body of Prp28p in vivo. Extracts prepared from these engineered strains were then used to assemble in vitro splicing reactions for BPA-mediated protein-protein crosslinkings. This enabled us, for the first time, to "capture" Prp28p in action. This approach may be applicable to studying the roles of other DExD/H-box proteins functioning in diverse RNA-related pathways, as well as to investigating protein-protein contacts within an RNA-protein complex.

A population study of killer viruses reveals different evolutionary histories of two closely related Saccharomyces sensu stricto yeasts.

Microbes have evolved ways of interference competition to gain advantage over their ecological competitors. The use of secreted killer toxins by yeast cells through acquiring double-stranded RNA viruses is one such prominent example. Although the killer behaviour has been well studied in laboratory yeast strains, our knowledge regarding how killer viruses are spread and maintained in nature and how yeast cells co-evolve with viruses remains limited. We investigated these issues using a panel of 81 yeast populations belonging to three Saccharomyces sensu stricto species isolated from diverse ecological niches and geographic locations. We found that killer strains are rare among all three species. In contrast, killer toxin resistance is widespread in Saccharomyces paradoxus populations, but not in Saccharomyces cerevisiae or Saccharomyces eubayanus populations. Genetic analyses revealed that toxin resistance in S. paradoxus is often caused by dominant alleles that have independently evolved in different populations. Molecular typing identified one M28 and two types of M1 killer viruses in those killer strains. We further showed that killer viruses of the same type could lead to distinct killer phenotypes under different host backgrounds, suggesting co-evolution between the viruses and hosts in different populations. Taken together, our data suggest that killer viruses vary in their evolutionary histories even within closely related yeast species.

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.

Functions of the DExD/H-box proteins in nuclear pre-mRNA splicing.

In eukaryotes, many genes are transcribed as precursor messenger RNAs (pre-mRNAs) that contain exons and introns, the latter of which must be removed and exons ligated to form the mature mRNAs. This process is called pre-mRNA splicing, which occurs in the nucleus. Although the chemistry of pre-mRNA splicing is identical to that of the self-splicing Group II introns, hundreds of proteins and five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, are essential for executing pre-mRNA splicing. Spliceosome, arguably the most complex cellular machine made up of all those proteins and snRNAs, is responsible for carrying out pre-mRNA splicing. In contrast to the transcription and the translation machineries, spliceosome is formed anew onto each pre-mRNA and undergoes a series of highly coordinated reconfigurations to form the catalytic center. This amazing process is orchestrated by a number of DExD/H-proteins that are the focus of this article, which aims to review the field in general and to project the exciting challenges and opportunities ahead. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

Mammalian α arrestins link activated seven transmembrane receptors to Nedd4 family e3 ubiquitin ligases and interact with ß arrestins.

The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination - which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1-5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.

Attenuation of 40S ribosomal subunit abundance differentially affects host and HCV translation and suppresses HCV replication.

For Hepatitis C virus (HCV), initiation of translation is cap-independently mediated by its internal ribosome entry site (IRES). Unlike other IRES-containing viruses that shut off host cap-dependent translation, translation of HCV coexists with that of the host. How HCV IRES-mediated translation is regulated in the infected cells remains unclear. Here, we show that the intracellular level of 40S ribosomal subunit plays a key role in facilitating HCV translation over host translation. In a loss-of-function screen, we identified small subunit ribosomal protein 6 (RPS6) as an indispensable host factor for HCV propagation. Knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation. Such preferential suppression of HCV translation correlated well with the reduction of the abundance of 40S ribosomal subunit following knockdown of RPS6 or other RPS genes. In contrast, reduction of the amount of ribosomal proteins of the 60S subunit did not produce similar effects. Among the components of general translation machineries, only knockdowns of RPS genes caused inhibitory effects on HCV translation, pointing out the unique role of 40S subunit abundance in HCV translation. This work demonstrates an unconventional notion that the translation initiation of HCV and host possess different susceptibility toward reduction of 40S ribosomal subunit, and provides a model of selective modulation of IRES-mediated translation through manipulating the level of 40S subunit.

Analysis of stress granule assembly in Schizosaccharomyces pombe.

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.

H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast.

BACKGROUND: The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. RESULTS: Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. CONCLUSION: These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

Genome-wide analysis of the cis-regulatory modules of divergent gene pairs in yeast.

In budding yeast, approximately a quarter of adjacent genes are divergently transcribed (divergent gene pairs). Whether genes in a divergent pair share the same regulatory system is still unknown. By examining transcription factor (TF) knockout experiments, we found that most TF knockout only altered the expression of one gene in a divergent pair. This prompted us to conduct a comprehensive analysis in silico to estimate how many divergent pairs are regulated by common sets of TFs (cis-regulatory modules, CRMs) using TF binding sites and expression data. Analyses of ten expression datasets show that only a limited number of divergent gene pairs share CRMs in any single dataset. However, around half of divergent pairs do share a regulatory system in at least one dataset. Our analysis suggests that genes in a divergent pair tend to be co-regulated in at least one condition; however, in most conditions, they may not be co-regulated.