D-Seq: Genome-wide detection of dihydrouridine modifications in RNA.

In addition to A, C, G and U, RNA contains over 100 additional chemically distinct residues. An abundant modified base frequently found in tRNAs, dihydrouridine (D) has recently been mapped to over 100 positions in mRNAs in yeast and human cells. Multiple highly conserved dihydrouridine synthases associate with and modify mRNA, suggesting there are many D sites yet to be found. Because D alters RNA structure, installation of D in mRNA is likely to effect multiple steps in mRNA metabolism including processing, trafficking, translation, and degradation. Here, we introduce D-seq, a method to chart the D landscape at single nucleotide resolution. The included protocols start with RNA isolation and carry through D-seq library preparation and data analysis. While the protocols below are tailored to map Ds in mRNA, the D-seq method is generalizable to any RNA type of interest, including non-coding RNAs, which have also recently been identified as dihydrouridine synthase targets.

Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA.

Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease.

Integrative Genome-Scale Metabolic Modeling Reveals Versatile Metabolic Strategies for Methane Utilization in Methylomicrobium album BG8.

Methylomicrobium album BG8 is an aerobic methanotrophic bacterium with promising features as a microbial cell factory for the conversion of methane to value-added chemicals. However, the lack of a genome-scale metabolic model (GEM) of M. album BG8 has hindered the development of systems biology and metabolic engineering of this methanotroph. To fill this gap, a high-quality GEM was constructed to facilitate a system-level understanding of the biochemistry of M. album BG8. Flux balance analysis, constrained with time-series data derived from experiments with various levels of methane, oxygen, and biomass, was used to investigate the metabolic states that promote the production of biomass and the excretion of carbon dioxide, formate, and acetate. The experimental and modeling results indicated that M. album BG8 requires a ratio of ∼1.5:1 between the oxygen- and methane-specific uptake rates for optimal growth. Integrative modeling revealed that at ratios of >2:1 oxygen-to-methane uptake flux, carbon dioxide and formate were the preferred excreted compounds, while at ratios of <1.5:1 acetate accounted for a larger fraction of the total excreted flux. Our results showed a coupling between biomass production and the excretion of carbon dioxide that was linked to the ratio between the oxygen- and methane-specific uptake rates. In contrast, acetate excretion was experimentally detected during exponential growth only when the initial biomass concentration was increased. A relatively lower growth rate was also observed when acetate was produced in the exponential phase, suggesting a trade-off between biomass and acetate production. IMPORTANCE A genome-scale metabolic model (GEM) is an integrative platform that enables the incorporation of a wide range of experimental data. It is used to reveal system-level metabolism and, thus, clarify the link between the genotype and phenotype. The lack of a GEM for Methylomicrobium album BG8, an aerobic methane-oxidizing bacterium, has hindered its use in environmental and industrial biotechnology applications. The diverse metabolic states indicated by the GEM developed in this study demonstrate the versatility in the methane metabolic processes used by this strain. The integrative GEM presented here will aid the implementation of the design-build-test-learn paradigm in the metabolic engineering of M. album BG8. This advance will facilitate the development of a robust methane bioconversion platform and help to mitigate methane emissions from environmental systems.

Direct analysis of ribosome targeting illuminates thousand-fold regulation of translation initiation.

Translational control shapes the proteome in normal and pathophysiological conditions. Current high-throughput approaches reveal large differences in mRNA-specific translation activity but cannot identify the causative mRNA features. We developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5' untranslated regions that confer 1,000-fold differences in ribosome recruitment in biochemically accessible cell lysates. Using DART, we determined a functional role for most alternative 5' UTR isoforms expressed in yeast, revealed a general mode of increased translation via direct binding to a core translation factor, and identified numerous translational control elements including C-rich silencers that are sufficient to repress translation both in vitro and in vivo. DART enables systematic assessment of the translational regulatory potential of 5' UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems.

Interplay between Position-Dependent Codon Usage Bias and Hydrogen Bonding at the 5' End of ORFeomes.

Codon usage bias exerts control over a wide variety of molecular processes. The positioning of synonymous codons within coding sequences (CDSs) dictates protein expression by mechanisms such as local translation efficiency, mRNA Gibbs free energy, and protein cotranslational folding. In this work, we explore how codon usage affects the position-dependent content of hydrogen bonding, which in turn influences energy requirements for unwinding double-stranded DNA (dsDNA). We categorized codons according to their hydrogen bond content and found differential effects on hydrogen bonding encoded by codon variants. The specific positional disposition of codon variants within CDSs creates a ramp of hydrogen bonding at the 5' end of the ORFeome in Escherichia coli CDSs occupying the first position of operons are subjected to selective pressure that reduces their hydrogen bonding compared to internal CDSs, and highly transcribed CDSs demand a lower maximum capacity of hydrogen bonds per codon, suggesting that the energetic requirement for unwinding the dsDNA in highly transcribed CDSs has evolved to be minimized in E. coli Subsequent analysis of over 14,000 ORFeomes showed a pervasive ramp of hydrogen bonding at the 5' end in Bacteria and Archaea that positively correlates with the probability of mRNA secondary structure formation. Both the ramp and the correlation were not found in Fungi The position-dependent hydrogen bonding might be part of the mechanism that contributes to the coordination between transcription and translation in Bacteria and Archaea A Web-based application to analyze the position-dependent hydrogen bonding of ORFeomes has been developed and is publicly available (https://juanvillada.shinyapps.io/hbonds/).IMPORTANCE Redundancy of the genetic code creates a vast space of alternatives to encode a protein. Synonymous codons exert control over a variety of molecular and physiological processes of cells mainly through influencing protein biosynthesis. Recent findings have shown that synonymous codon choice affects transcription by controlling mRNA abundance, mRNA stability, transcription termination, and transcript biosynthesis cost. In this work, by analyzing thousands of Bacteria, Archaea, and Fungi genomes, we extend recent findings by showing that synonymous codon choice, corresponding to the number of hydrogen bonds in a codon, can also have an effect on the energetic requirements for unwinding double-stranded DNA in a position-dependent fashion. This report offers new perspectives on the mechanism behind the transcription-translation coordination and complements previous hypotheses on the resource allocation strategies used by Bacteria and Archaea to manage energy efficiency in gene expression.

Human SNORA31 variations impair cortical neuron-intrinsic immunity to HSV-1 and underlie herpes simplex encephalitis.

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.

Genomic Evidence for Simultaneous Optimization of Transcription and Translation through Codon Variants in the pmoCAB Operon of Type Ia Methanotrophs.

Understanding the interplay between genotype and phenotype is a fundamental goal of functional genomics. Methane oxidation is a microbial phenotype with global-scale significance as part of the carbon biogeochemical cycle and a sink for greenhouse gas. Microorganisms that oxidize methane (methanotrophs) are taxonomically diverse and widespread around the globe. In methanotrophic bacteria, enzymes in the methane oxidation metabolic module (KEGG module M00174, conversion of methane to formaldehyde) are encoded in four operons (pmoCAB, mmoXYZBCD, mxaFI, and xoxF). Recent reports have suggested that methanotrophs in Proteobacteria acquired methane monooxygenases through horizontal gene transfer. Here, we used a genomic meta-analysis to infer the transcriptional and translational advantages of coding sequences from the methane oxidation metabolic modules of different types of methanotrophs. By analyzing isolate and metagenome-assembled genomes from phylogenetically and geographically diverse sources, we detected an anomalous nucleotide composition bias in the coding sequences of particulate methane monooxygenase genes (pmoCAB) from type Ia methanotrophs. We found that this nucleotide bias increases the level of codon bias by decreasing the GC content in the third base of codons, a strategy that contrasts with that of other coding sequences in the module. Further codon usage analyses uncovered that codon variants of the type Ia pmoCAB coding sequences deviate from the genomic signature to match ribosomal protein-coding sequences. Subsequently, computation of transcription and translation metrics revealed that the pmoCAB coding sequences of type Ia methanotrophs optimize the usage of codon variants to maximize translation efficiency and accuracy, while minimizing the synthesis cost of transcripts and proteins.IMPORTANCE Microbial methane oxidation plays a fundamental role in the biogeochemical cycle of Earth's system. Recent reports have provided evidence for the acquisition of methane monooxygenases by horizontal gene transfer in methane-oxidizing bacteria from different environments, but how evolution has shaped the coding sequences to execute methanotrophy efficiently remains unexplored. In this work, we provide genomic evidence that among the different types of methanotrophs, type Ia methanotrophs possess a unique coding sequence of the pmoCAB operon that is under positive selection for optimal resource allocation and efficient synthesis of transcripts and proteins. This adaptive trait possibly enables type Ia methanotrophs to respond robustly to fluctuating methane availability and explains their global prevalence.

Designing and Engineering Methylorubrum extorquens AM1 for Itaconic Acid Production.

Methylorubrum extorquens (formerly Methylobacterium extorquens) AM1 is a methylotrophic bacterium with a versatile lifestyle. Various carbon sources including acetate, succinate and methanol are utilized by M. extorquens AM1 with the latter being a promising inexpensive substrate for use in the biotechnology industry. Itaconic acid (ITA) is a high-value building block widely used in various industries. Given that no wildtype methylotrophic bacteria are able to utilize methanol to produce ITA, we tested the potential of M. extorquens AM1 as an engineered host for this purpose. In this study, we successfully engineered M. extorquens AM1 to express a heterologous codon-optimized gene encoding cis-aconitic acid decarboxylase. The engineered strain produced ITA using acetate, succinate and methanol as the carbon feedstock. The highest ITA titer in batch culture with methanol as the carbon source was 31.6 ± 5.5 mg/L, while the titer and productivity were 5.4 ± 0.2 mg/L and 0.056 ± 0.002 mg/L/h, respectively, in a scaled-up fed-batch bioreactor under 60% dissolved oxygen saturation. We attempted to enhance the carbon flux toward ITA production by impeding poly-ß-hydroxybutyrate accumulation, which is used as carbon and energy storage, via mutation of the regulator gene phaR. Unexpectedly, ITA production by the phaR mutant strain was not higher even though poly-ß-hydroxybutyrate concentration was lower. Genome-wide transcriptomic analysis revealed that phaR mutation in the ITA-producing strain led to complex rewiring of gene transcription, which might result in a reduced carbon flux toward ITA production. Besides poly-ß-hydroxybutyrate metabolism, we found evidence that PhaR might regulate the transcription of many other genes including those encoding other regulatory proteins, methanol dehydrogenases, formate dehydrogenases, malate:quinone oxidoreductase, and those synthesizing pyrroloquinoline quinone and thiamine co-factors. Overall, M. extorquens AM1 was successfully engineered to produce ITA using acetate, succinate and methanol as feedstock, further supporting this bacterium as a feasible host for use in the biotechnology industry. This study showed that PhaR could have a broader regulatory role than previously anticipated, and increased our knowledge of this regulator and its influence on the physiology of M. extorquens AM1.

Lso2 is a conserved ribosome-bound protein required for translational recovery in yeast.

Ribosome-binding proteins function broadly in protein synthesis, gene regulation, and cellular homeostasis, but the complete complement of functional ribosome-bound proteins remains unknown. Using quantitative mass spectrometry, we identified late-annotated short open reading frame 2 (Lso2) as a ribosome-associated protein that is broadly conserved in eukaryotes. Genome-wide crosslinking and immunoprecipitation of Lso2 and its human ortholog coiled-coil domain containing 124 (CCDC124) recovered 25S ribosomal RNA in a region near the A site that overlaps the GTPase activation center. Consistent with this location, Lso2 also crosslinked to most tRNAs. Ribosome profiling of yeast lacking LSO2 (lso2Δ) revealed global translation defects during recovery from stationary phase with translation of most genes reduced more than 4-fold. Ribosomes accumulated at start codons, were depleted from stop codons, and showed codon-specific changes in occupancy in lso2Δ. These defects, and the conservation of the specific ribosome-binding activity of Lso2/CCDC124, indicate broadly important functions in translation and physiology.

Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs.

Translational control of gene expression plays essential roles in cellular stress responses and organismal development by enabling rapid, selective, and localized control of protein production. Translational regulation depends on context-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. Here, we comprehensively determined the RNA-binding preferences of the eukaryotic initiation factor 4G (eIF4G) to assess whether this core translation initiation factor has intrinsic sequence preferences that may contribute to preferential translation of specific mRNAs. We identified a simple RNA sequence motif-oligo-uridine-that mediates high-affinity binding to eIF4G in vitro. Oligo(U) motifs occur naturally in the transcript leader (TL) of hundreds of yeast genes, and mRNAs with unstructured oligo(U) motifs were enriched in immunoprecipitations against eIF4G. Ribosome profiling following depletion of eIF4G in vivo showed preferentially reduced translation of mRNAs with long TLs, including those that contain oligo(U). Finally, TL oligo(U) elements are enriched in genes with regulatory roles and are conserved between yeast species, consistent with an important cellular function. Taken together, our results demonstrate RNA sequence preferences for a general initiation factor, which cells potentially exploit for translational control of specific mRNAs.

Loss-of-function mutations in the RNA biogenesis factor NAF1 predispose to pulmonary fibrosis-emphysema.

Chronic obstructive pulmonary disease and pulmonary fibrosis have been hypothesized to represent premature aging phenotypes. At times, they cluster in families, but the genetic basis is not understood. We identified rare, frameshift mutations in the gene for nuclear assembly factor 1, NAF1, a box H/ACA RNA biogenesis factor, in pulmonary fibrosis-emphysema patients. The mutations segregated with short telomere length, low telomerase RNA levels, and extrapulmonary manifestations including myelodysplastic syndrome and liver disease. A truncated NAF1 was detected in cells derived from patients, and, in cells in which the frameshift mutation was introduced by genome editing, telomerase RNA levels were reduced. The mutant NAF1 lacked a conserved carboxyl-terminal motif, which we show is required for nuclear localization. To understand the disease mechanism, we used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) to generate Naf1(+/-) mice and found that they had half the levels of telomerase RNA. Other box H/ACA RNA levels were also decreased, but rRNA pseudouridylation, which is guided by snoRNAs, was intact. Moreover, first-generation Naf1(+/-) mice showed no evidence of ribosomal pathology. Our data indicate that disease in NAF1 mutation carriers is telomere-mediated; they show that NAF1 haploinsufficiency selectively disturbs telomere length homeostasis by decreasing the levels of telomerase RNA while sparing rRNA pseudouridylation.

The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs.

Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational 'closed loop' complex comprised of eIF4E, eIF4G, and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions.

Transcriptome-Wide Identification of Pseudouridine Modifications Using Pseudo-seq.

A diverse array of post-transcriptional modifications is found in RNA molecules from all domains of life. While the locations of RNA modifications are well characterized in abundant noncoding RNAs, modified sites in less abundant mRNAs are just beginning to be discovered. Recent work has revealed hundreds of previously unknown and dynamically regulated pseudouridines (Ψ) in mRNAs from diverse organisms. This unit describes Pseudo-seq, an efficient, high-resolution method for identification of Ψs genome-wide. This unit includes methods for isolation of RNA from S. cerevisiae, preparation of Pseudo-seq libraries from RNA samples, and identification of sites of pseudouridylation from the sequencing data. Pseudo-seq is applicable to any organism or cell type, facilitating rapid identification of novel pseudouridylation events.

Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA.

RNA molecules contain a variety of chemically diverse, posttranscriptionally modified bases. The most abundant modified base found in cellular RNAs, pseudouridine (Ψ), has recently been mapped to hundreds of sites in mRNAs, many of which are dynamically regulated. Though the pseudouridine landscape has been determined in only a few cell types and growth conditions, the enzymes responsible for mRNA pseudouridylation are universally conserved, suggesting many novel pseudouridylated sites remain to be discovered. Here, we present Pseudo-seq, a technique that allows the identification of sites of pseudouridylation genome-wide with single-nucleotide resolution. In this chapter, we provide a detailed description of Pseudo-seq. We include protocols for RNA isolation from Saccharomyces cerevisiae, Pseudo-seq library preparation, and data analysis, including descriptions of processing and mapping of sequencing reads, computational identification of sites of pseudouridylation, and assignment of sites to specific pseudouridine synthases. The approach presented here is readily adaptable to any cell or tissue type from which high-quality mRNA can be isolated. Identification of novel pseudouridylation sites is an important first step in elucidating the regulation and functions of these modifications.

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

Alternative transcription start site selection leads to large differences in translation activity in yeast.

mRNA levels do not accurately predict protein levels in eukaryotic cells. To investigate contributions of 5' untranslated regions (5' UTRs) to mRNA-specific differences in translation, we determined the 5' UTR boundaries of 96 yeast genes for which in vivo translational efficiency varied by 80-fold. A total of 25% of genes showed substantial 5' UTR heterogeneity. We compared the capacity of these genes' alternative 5' UTR isoforms for cap-dependent and cap-independent translation using quantitative in vitro and in vivo translation assays. Six out of nine genes showed mRNA isoform-specific translation activity differences of greater than threefold in at least one condition. For three genes, in vivo translation activities of alternative 5' UTR isoforms differed by more than 100-fold. These results show that changing genes' 5' UTR boundaries can produce large changes in protein output without changing the overall amount of mRNA. Because transcription start site (TSS) heterogeneity is common, we suggest that TSS choice is greatly under-appreciated as a quantitatively significant mechanism for regulating protein production.