Functions and mechanisms of A-to-I RNA editing in filamentous ascomycetes.

Although lack of ADAR (adenosine deaminase acting on RNA) orthologs, genome-wide A-to-I editing occurs specifically during sexual reproduction in a number of filamentous ascomycetes, including Fusarium graminearum and Neurospora crassa. Unlike ADAR-mediated editing in animals, fungal A-to-I editing has a strong preference for hairpin loops and U at -1 position, which leads to frequent editing of UAG and UAA stop codons. Majority of RNA editing events in fungi are in the coding region and cause amino acid changes. Some of these editing events have been experimentally characterized for providing heterozygote and adaptive advantages in F. graminearum. Recent studies showed that FgTad2 and FgTad3, 2 ADAT (adenosine deaminase acting on tRNA) enzymes that normally catalyze the editing of A34 in the anticodon of tRNA during vegetative growth mediate A-to-I mRNA editing during sexual reproduction. Stage specificity of RNA editing is conferred by stage-specific expression of short transcript isoforms of FgTAD2 and FgTAD3 as well as cofactors such as AME1 and FIP5 that facilitate the editing of mRNA in perithecia. Taken together, fungal A-to-I RNA editing during sexual reproduction is catalyzed by ADATs and it has the same sequence and structural preferences with editing of A34 in tRNA.

Single-molecule reconstruction of eukaryotic factor-dependent transcription termination.

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.

Loss of Conserved rRNA Modifications in the Peptidyl Transferase Center Leads to Diminished Protein Synthesis and Cell Growth in Budding Yeast.

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (snR34, snR52, and snR65) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m5C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.

Potential global cis and trans regulation of lncRNAs in Saccharomyces cerevisiae subjected to ethanol stress.

Long noncoding RNAs (lncRNAs) are regulatory RNAs. Saccharomyces cerevisiae strains transcribe hundreds of lncRNAs. LncRNAs can regulate the expression of adjacent genes (cis-regulation) or distant genes from lncRNAs (trans-regulation). Here, we analyzed the potential global cis and trans-regulation of lncRNAs of yeast subjected to ethanol stress. For potential cis regulation, for BMA641-A and S288C strains, we observed that most lncRNA-neighbor gene pairs increased the expression at a certain point followed by a decrease, and vice versa. Based on the transcriptome profile and triple helix prediction between lncRNAs and promoters of coding genes, we observed nine different ways of potential trans regulation that work in a strain-specific manner. Our data provide an initial landscape of potential cis and trans regulation in yeast, which seems to be strain-specific.

Role of Rmd9p in 3'-end processing of mitochondrial 15S rRNA in Saccharomyces cerevisiae.

Ribosome biogenesis, involving processing/assembly of rRNAs and r-proteins is a vital process. In Saccharomyces cerevisiae mitochondria, ribosomal small subunit comprises 15S rRNA (15S). While the 15S 5'-end processing uses Ccm1p and Pet127p, the mechanisms of the 3'-end processing remain unclear. We reveal involvement of Rmd9p in safeguarding/processing 15S 3'-end. Rmd9p deficiency results in a cleavage at a position 183 nucleotides upstream of 15S 3'-end, and in the loss of the 3'-minor domain. Rmd9p binds to the sequences in the 3'-end region of 15S, and a genetic interaction between rmd9 and dss1 indicates that Rmd9p regulates/limits mtEXO activity during the 3'-end spacer processing.

eIF4F is a thermo-sensing regulatory node in the translational heat shock response.

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.

Highly Reactive Group I Introns Ubiquitous in Pathogenic Fungi.

Systemic fungal infections are a growing public health threat, and yet viable antifungal drug targets are limited as fungi share a similar proteome with humans. However, features of RNA metabolism and the noncoding transcriptomes in fungi are distinctive. For example, fungi harbor highly structured RNA elements that humans lack, such as self-splicing introns within key housekeeping genes in the mitochondria. However, the location and function of these mitochondrial riboregulatory elements has largely eluded characterization. Here we used an RNA-structure-based bioinformatics pipeline to identify the group I introns interrupting key mitochondrial genes in medically relevant fungi, revealing their fixation within a handful of genetic hotspots and their ubiquitous presence across divergent phylogenies of fungi, including all highest priority pathogens such as Candida albicans, Candida auris, Aspergillus fumigatus and Cryptococcus neoformans. We then biochemically characterized two representative introns from C. albicans and C. auris, demonstrating their exceptionally efficient splicing catalysis relative to previously-characterized group I introns. Indeed, the C. albicans mitochondrial intron displays extremely rapid catalytic turnover, even at ambient temperatures and physiological magnesium ion concentrations. Our results unmask a significant new set of players in the RNA metabolism of pathogenic fungi, suggesting a promising new type of antifungal drug target.

MicroRNA-like RNA Functions Are Required for the Biosynthesis of Active Compounds in the Medicinal Fungus Sanghuangporus vaninii.

miRNA-like RNAs (milRNAs) have been recognized as sequence-specific regulators of posttranscriptional regulation of gene expression in eukaryotes. However, the functions of hundreds of fungal milRNAs in the biosynthesis of metabolic components are obscure. Sanghuangporus produces diverse bioactive compounds and is widely used in Asian countries. Here, genes encoding two Dicers, four Argonautes, and four RdRPs were identified and characterized in Sanghuangporus vanini. Due to the lack of an efficient gene manipulation system, the efficacy of spray-induced gene silencing (SIGS) was determined in S. vanini, which showed efficient double-stranded RNA (dsRNA) uptake and gene silencing efficiency. SIGS-mediated gene knockdown showed that SVRDRP-3, SVRDRP-4, SVDICER-1, and SVDICER-2 were critical for mycelial biomass, flavonoid, triterpenoid, and polysaccharide production. Illumina deep sequencing was performed to characterize the milRNAs from S. vanini mycelium and fruiting body. A total of 31 milRNAs were identified, out of which, SvmilR10, SvmilR17, and SvmilR33 were Svrdrp-4- and Svdicer-1-dependent milRNAs. Importantly, SIGS-mediated overexpression of SvmilR10 and SvmilR33 resulted in significant changes in the yields of flavonoids, triterpenoids, and polysaccharides. Further analysis showed that these milRNA target genes encoding the retrotransposon-derived protein PEG1 and histone-lysine N-methyltransferase were potentially downregulated in the milRNA overexpressing strain. Our results revealed that S. vanini has high external dsRNA and small RNA uptake efficiency and that milRNAs may play crucial regulatory roles in the biosynthesis of bioactive compounds. IMPORTANCE Fungi can take up environmental RNA that can silence fungal genes with RNA interference, which prompts the development of SIGS. Efficient dsRNA and milRNA uptake in S. vanini, successful dsRNA-targeted gene block, and the increase in intracellular miRNA abundance showed that SIGS technology is an effective and powerful tool for the functional dissection of fungal genes and millRNAs. We found that the RdRP, Dicer, and Argonaute genes are critical for mycelial biomass and bioactive compound production. Our study also demonstrated that overexpressed SVRDRP-4- and SVDICER-1-dependent milRNAs (SvmilR10 and SvmilR33) led to significant changes in the yields of the three active compounds. This study not only provides the first report on SIGS-based gene and milRNA function exploration, but also provides a theoretical platform for exploration of the functions of milRNAs involved in biosynthesis of metabolic compounds in fungi.

Chemical Modifications of Ribosomal RNA.

Cellular RNAs in all three kingdoms of life are modified with diverse chemical modifications. These chemical modifications expand the topological repertoire of RNAs, and fine-tune their functions. Ribosomal RNA in yeast contains more than 100 chemically modified residues in the functionally crucial and evolutionary conserved regions. The chemical modifications in the rRNA are of three types-methylation of the ribose sugars at the C2-positionAbstract (Nm), isomerization of uridines to pseudouridines (Ψ), and base modifications such as (methylation (mN), acetylation (acN), and aminocarboxypropylation (acpN)). The modifications profile of the yeast rRNA has been recently completed, providing an excellent platform to analyze the function of these modifications in RNA metabolism and in cellular physiology. Remarkably, majority of the rRNA modifications and the enzymatic machineries discovered in yeast are highly conserved in eukaryotes including humans. Mutations in factors involved in rRNA modification are linked to several rare severe human diseases (e.g., X-linked Dyskeratosis congenita, the Bowen-Conradi syndrome and the William-Beuren disease). In this chapter, we summarize all rRNA modifications and the corresponding enzymatic machineries of the budding yeast.

Transcriptional neighborhoods regulate transcript isoform lengths and expression levels.

Sequence features of genes and their flanking regulatory regions are determinants of RNA transcript isoform expression and have been used as context-independent plug-and-play modules in synthetic biology. However, genetic context-including the adjacent transcriptional environment-also influences transcript isoform expression levels and boundaries. We used synthetic yeast strains with stochastically repositioned genes to systematically disentangle the effects of sequence and context. Profiling 120 million full-length transcript molecules across 612 genomic perturbations, we observed sequence-independent alterations to gene expression levels and transcript isoform boundaries that were influenced by neighboring transcription. We identified features of transcriptional context that could predict these alterations and used these features to engineer a synthetic circuit where transcript length was controlled by neighboring transcription. This demonstrates how positional context can be leveraged in synthetic genome engineering.

RNA-dependent synthesis of ergosteryl-3ß-O-glycine in Ascomycota expands the diversity of steryl-amino acids.

A wide range of bacteria possess virulence factors such as aminoacyl-tRNA transferases (ATTs) that are capable of rerouting aminoacyl-transfer RNAs away from protein synthesis to conjugate amino acids onto glycerolipids. We recently showed that, although these pathways were thought to be restricted to bacteria, higher fungi also possess ergosteryl-3ß-O-L-aspartate synthases (ErdSs), which transfer the L-Asp moiety of aspartyl-tRNAAsp onto the 3ß-OH group of ergosterol (Erg), yielding ergosteryl-3ß-O-L-aspartate (Erg-Asp). Here, we report the discovery, in fungi, of a second type of fungal sterol-specific ATTs, namely, ergosteryl-3ß-O-glycine (Erg-Gly) synthase (ErgS). ErgS consists of a freestanding DUF2156 domain encoded by a gene distinct from and paralogous to that of ErdS. We show that the enzyme only uses Gly-tRNAGly produced by an independent glycyl-tRNA synthetase (GlyRS) to transfer glycine onto the 3ß-OH of Erg, producing Erg-Gly. Phylogenomics analysis also show that the Erg-Gly synthesis pathway exists only in Ascomycota, including species of biotechnological interest, and more importantly, in human pathogens, such as Aspergillus fumigatus. The discovery of a second type of Erg-aa not only expands the repertoire of this particular class of fungal lipids but suggests that Erg-aa synthases might constitute a genuine subfamily of lipid-modifying ATTs.

Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation in Saccharomyces cerevisiae.

Rap1 is the main protein that binds double-stranded telomeric DNA in Saccharomyces cerevisiae. Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. tlc1-tm cells grow similar to wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. Rif2 is a protein normally recruited to telomeres by Rap1, but we show that Rif2 can still associate with Rap1-depleted tlc1-tm telomeres, and that this association is required to inhibit telomere degradation by the MRX complex. Rif2 and the Ku complex work in parallel to prevent tlc1-tm telomere degradation; tlc1-tm cells lacking Rif2 and the Ku complex are inviable. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.

Determinants of the temperature adaptation of mRNA degradation.

The rate of chemical reactions increases proportionally with temperature, but the interplay of biochemical reactions permits deviations from this relation and adaptation. The degradation of individual mRNAs in yeast increased to varying degrees with temperature. We examined how these variations are influenced by the translation and codon composition of mRNAs. We developed a method that revealed the existence of a neutral half-life above which mRNAs are stabilized by translation but below which they are destabilized. The proportion of these two mRNA subpopulations remained relatively constant under different conditions, even with slow cell growth due to nutrient limitation, but heat shock reduced the proportion of translationally stabilized mRNAs. At the same time, the degradation of these mRNAs was partially temperature-compensated through Upf1, the mediator of nonsense-mediated decay. Compensation was also promoted by some asparagine and serine codons, whereas tyrosine codons promote temperature sensitization. These codons play an important role in the degradation of mRNAs encoding key cell membrane and cell wall proteins, which promote cell integrity.

Functional profiling of long intergenic non-coding RNAs in fission yeast.

Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analysed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse coding-gene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug, and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analysed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analysed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions.

Single-Base Resolution Mapping Reveals Distinct 5-Formylcytidine in Saccharomyces cerevisiae mRNAs.

5-Formylcytidine (f5C) is one type of post-transcriptional RNA modification, which is known at the wobble position of tRNA in mitochondria and essential for mitochondrial protein synthesis. Here, we show a method to detect f5C modifications in RNA and a transcriptome-wide f5C mapping technique, named f5C-seq. It is developed based on the treatment of pyridine borane, which can reduce f5C to 5,6-dihydrouracil, thus inducing C-to-T transition in f5C sites during PCR to achieve single-base resolution detection. More than 1000 f5C sites were identified after mapping in Saccharomyces cerevisiae by f5C-seq. Moreover, codon composition demonstrated a preference for f5C within wobble sites in mRNA, suggesting the potential role in regulation of translation. These findings expand the scope of the understanding of cytosine modifications in mRNA.

Transcription-wide mapping of dihydrouridine reveals that mRNA dihydrouridylation is required for meiotic chromosome segregation.

The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.

Decoupling of degradation from deadenylation reshapes poly(A) tail length in yeast meiosis.

Nascent messenger RNA is endowed with a poly(A) tail that is subject to gradual deadenylation and subsequent degradation in the cytoplasm. Deadenylation and degradation rates are typically correlated, rendering it difficult to dissect the determinants governing each of these processes and the mechanistic basis of their coupling. Here we developed an approach that allows systematic, robust and multiplexed quantification of poly(A) tails in Saccharomyces cerevisiae. Our results suggest that mRNA deadenylation and degradation rates are decoupled during meiosis, and that transcript length is a major determinant of deadenylation rates and a key contributor to reshaping of poly(A) tail lengths. Meiosis-specific decoupling also leads to unique positive associations between poly(A) tail length and gene expression. The decoupling is associated with a focal localization pattern of the RNA degradation factor Xrn1, and can be phenocopied by Xrn1 deletion under nonmeiotic conditions. Importantly, the association of transcript length with deadenylation rates is conserved across eukaryotes. Our study uncovers a factor that shapes deadenylation rate and reveals a unique context in which degradation is decoupled from deadenylation.

RNA-DNA hybrids regulate meiotic recombination.

RNA-DNA hybrids are often associated with genome instability and also function as a cellular regulator in many biological processes. In this study, we show that accumulated RNA-DNA hybrids cause multiple defects in budding yeast meiosis, including decreased sporulation efficiency and spore viability. Further analysis shows that these RNA-DNA hybrid foci colocalize with RPA/Rad51 foci on chromosomes. The efficient formation of RNA-DNA hybrid foci depends on Rad52 and ssDNA ends of meiotic DNA double-strand breaks (DSBs), and their number is correlated with DSB frequency. Interestingly, RNA-DNA hybrid foci and recombination foci show similar dynamics. The excessive accumulation of RNA-DNA hybrids around DSBs competes with Rad51/Dmc1, impairs homolog bias, and decreases crossover and noncrossover recombination. Furthermore, precocious removal of RNA-DNA hybrids by RNase H1 overexpression also impairs meiotic recombination similarly. Taken together, our results demonstrate that RNA-DNA hybrids form at ssDNA ends of DSBs to actively regulate meiotic recombination.

Biochemical approach for isolation of polyadenylated RNAs with bound proteins from yeast.

In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress. For complete details on the use and execution of this protocol, please refer to Matia-González et al. (2021, 2015).

A nuclear pore sub-complex restricts the propagation of Ty retrotransposons by limiting their transcription.

Beyond their canonical function in nucleocytoplasmic exchanges, nuclear pore complexes (NPCs) regulate the expression of protein-coding genes. Here, we have implemented transcriptomic and molecular methods to specifically address the impact of the NPC on retroelements, which are present in multiple copies in genomes. We report a novel function for the Nup84 complex, a core NPC building block, in specifically restricting the transcription of LTR-retrotransposons in yeast. Nup84 complex-dependent repression impacts both Copia and Gypsy Ty LTR-retrotransposons, all over the S. cerevisiae genome. Mechanistically, the Nup84 complex restricts the transcription of Ty1, the most active yeast retrotransposon, through the tethering of the SUMO-deconjugating enzyme Ulp1 to NPCs. Strikingly, the modest accumulation of Ty1 RNAs caused by Nup84 complex loss-of-function is sufficient to trigger an important increase of Ty1 cDNA levels, resulting in massive Ty1 retrotransposition. Altogether, our study expands our understanding of the complex interactions between retrotransposons and the NPC, and highlights the importance for the cells to keep retrotransposons under tight transcriptional control.