Deciphering the cis- regulatory landscape of natural yeast Transcript Leaders.

Protein synthesis is a vital process that is highly regulated at the initiation step of translation. Eukaryotic 5' transcript leaders (TLs) contain a variety of cis -regulatory features that influence translation and mRNA stability. However, the relative influences of these features in natural TLs are poorly characterized. To address this, we used massively parallel reporter assays (MPRAs) to quantify RNA levels, ribosome loading, and protein levels from 11,027 natural yeast TLs in vivo and systematically compared the relative impacts of their sequence features on gene expression. We found that yeast TLs influence gene expression over two orders of magnitude. While a leaky scanning model using Kozak contexts and uAUGs explained half of the variance in expression across transcript leaders, the addition of other features explained ∼70% of gene expression variation. Our analyses detected key cis -acting sequence features, quantified their effects in vivo, and compared their roles to motifs reported from an in vitro study of ribosome recruitment. In addition, our work quantitated the effects of alternative transcription start site usage on gene expression in yeast. Thus, our study provides new quantitative insights into the roles of TL cis-acting sequences in regulating gene expression.

Unraveling the influences of sequence and position on yeast uORF activity using massively parallel reporter systems and machine learning.

Upstream open-reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that both uORF sequences and locations within transcript leaders predict their effect on gene expression. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as predictive as uORF sequences.

Reinforcement amid genetic diversity in the Candida albicans biofilm regulatory network.

Biofilms of the fungal pathogen Candida albicans include abundant long filaments called hyphae. These cells express hypha-associated genes, which specify diverse virulence functions including surface adhesins that ensure biofilm integrity. Biofilm formation, virulence, and hypha-associated gene expression all depend upon the transcription factor Efg1. This transcription factor has been characterized extensively in the C. albicans type strain SC5314 and derivatives, but only recently has its function been explored in other clinical isolates. Here we define a principal set of Efg1-responsive genes whose expression is significantly altered by an efg1Δ/Δ mutation across 17 clinical isolates. This principal gene set includes 68 direct Efg1 targets, whose 5' regions are bound by Efg1 in five clinical isolates, and 42 indirect Efg1 targets, whose 5' regions are not detectably bound by Efg1. Three direct Efg1 target genes encode transcription factors-BRG1, UME6, and WOR3 -whose increased expression in an efg1Δ/Δ mutant restores expression of multiple indirect and direct principal targets, as well as biofilm formation ability. Although BRG1 and UME6 are well known positive regulators of hypha-associated genes and biofilm formation, WOR3 is best known as an antagonist of Efg1 in the sexual mating pathway. We confirm the positive role of WOR3 in biofilm formation with the finding that a wor3Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo biofilm model. Positive control of Efg1 direct target genes by other Efg1 direct target genes-BRG1, UME6, and WOR3 -may buffer principal Efg1-responsive gene expression against the impact of genetic variation in the C. albicans species.

False-positive IRESes from Hoxa9 and other genes resulting from errors in mammalian 5' UTR annotations.

Hyperconserved genomic sequences have great promise for understanding core biological processes. It has been recently proposed that scores of hyperconserved 5' untranslated regions (UTRs), also known as transcript leaders (hTLs), encode internal ribosome entry sites (IRESes) that drive cap-independent translation, in part, via interactions with ribosome expansion segments. However, the direct functional significance of such interactions has not yet been definitively demonstrated. We provide evidence that the putative IRESes previously reported in Hox gene hTLs are rarely included in transcript leaders. Instead, these regions function independently as transcriptional promoters. In addition, we find the proposed RNA structure of the putative Hoxa9 IRES is not conserved. Instead, sequences previously shown to be essential for putative IRES activity encode a hyperconserved transcription factor binding site (E-box) that contributes to its promoter activity and is bound by several transcription factors, including USF1 and USF2. Similar E-box sequences enhance the promoter activities of other putative Hoxa gene IRESes. Moreover, we provide evidence that the vast majority of hTLs with putative IRES activity overlap transcriptional promoters, enhancers, and 3' splice sites that are most likely responsible for their reported IRES activities. These results argue strongly against recently reported widespread IRES-like activities from hTLs and contradict proposed interactions between ribosomal expansion segment ES9S and putative IRESes. Furthermore, our work underscores the importance of accurate transcript annotations, controls in bicistronic reporter assays, and the power of synthesizing publicly available data from multiple sources.

Collaboration between Antagonistic Cell Type Regulators Governs Natural Variation in the Candida albicans Biofilm and Hyphal Gene Expression Network.

Candida albicans is among the most significant human fungal pathogens. However, the vast majority of C. albicans studies have focused on a single clinical isolate and its marked derivatives. We investigated natural variation among clinical C. albicans isolates in gene regulatory control of biofilm formation, a process crucial to virulence. The transcription factor Efg1 is required for biofilm-associated gene expression and biofilm formation. Previously, we found extensive variation in Efg1-responsive gene expression among 5 diverse clinical isolates. However, chromatin immunoprecipitation sequencing analysis showed that Efg1 binding to genomic loci was uniform among the isolates. Functional dissection of strain differences identified three transcription factors, Brg1, Tec1, and Wor1, for which small changes in expression levels reshaped the Efg1 regulatory network. Brg1 and Tec1 are known biofilm activators, and their role in Efg1 network variation may be expected. However, Wor1 is a known repressor of EFG1 expression and an inhibitor of biofilm formation. In contrast, we found that a modest increase in WOR1 RNA levels, reflecting the expression differences between C. albicans strains, could augment biofilm formation and expression of biofilm-related genes. The analysis of natural variation here reveals a novel function for a well-characterized gene and illustrates that strain diversity offers a unique resource for elucidation of network interactions. IMPORTANCE Clinical isolates of all pathogens vary in the strength of traits linked to disease. In this study, we focused on variation in a pathogenicity trait of the fungal pathogen Candida albicans, biofilm formation. This trait is under the control of the cell type regulator Efg1. Expression of Efg1 is known from previous studies to be repressed by a second cell type regulator, Wor1. However, we found that natural variation in biofilm formation and biofilm-related gene expression was driven by collaboration between Efg1 and Wor1. Our findings show that analysis of natural isolates can reveal unexpected features of gene function, even for well-studied genes.

Multiplexed Analysis of Human uORF Regulatory Functions During the ISR Using PoLib-Seq.

Protein synthesis is a highly regulated essential process. As such, it is subjected to substantial regulation in response to stress. One hallmark of the Integrated Stress Response (ISR) is the immediate shutdown of most translation through phosphorylation of the alpha subunit of translation initiation factor eIF2 and activation of eIF4E binding proteins. While these posttranslational modifications largely inhibit cap-dependent translation, many mRNA resist this inhibition by alternative translation mechanisms involving cis-regulatory sequences and structures in 5' transcript leaders, including upstream Open Reading Frames (uORFs), Internal Ribosome Entry Sites (IRESes), and Cap-Independent Translation Elements (CITEs). Studies of uORF and IRES activity are often performed on a gene-by-gene basis; however, high-throughput methods have recently emerged. Here, we describe a protocol for Polysome Library Sequencing (PoLib-Seq; Fig. 1), a multiplexed assay of reporter gene translation that can be used during the ISR. A designer library of reporter RNAs are transfected into tissue-culture cells, and their translation is assayed via sucrose gradient fractionation followed by high-throughput sequencing. As an example, we include PoLib-seq results simultaneously assaying translation of wildtype and uORF mutant human ATF4 reporter RNAs, recapitulating the known function of uORF1 in resisting translational inhibition during the ISR.

High-Throughput Quantitation of Yeast uORF Regulatory Impacts Using FACS-uORF.

Eukaryotic upstream Open Reading Frames (uORFs) are short translated regions found in many transcript leaders (Barbosa et al. PLoS Genet 9:e1003529, 2013; Zhang et al. Trends Biochem Sci 44:782-794, 2019). Modern transcript annotations and ribosome profiling studies have found thousands of AUG-initiated uORFs, and many more uORFs initiated by near-cognate codons (CUG, GUG, UUG, etc.). Their translation generally decreases the expression of the main encoded protein by preventing ribosomes from reaching the main ORF of each gene, and by inducing nonsense mediated decay (NMD) through premature termination. Under many cellular stresses, uORF containing transcripts are de-repressed due to decreased translation initiation (Young et al. J Biol Chem 291:16927-16935, 2016). Traditional experimental evaluation of uORFs involves comparing expression from matched uORF-containing and start-codon mutated transcript leader reporter plasmids. This tedious process has precluded analysis of large numbers of uORFs. We recently used FACS-uORF to simultaneously assay thousands of yeast uORFs in order to evaluate the impact of codon usage on their functions (Lin et al. Nucleic Acids Res 2:1-10, 2019). Here, we provide a step-by-step protocol for this assay.

Roles of Candida albicans Mig1 and Mig2 in glucose repression, pathogenicity traits, and SNF1 essentiality.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.

Impacts of uORF codon identity and position on translation regulation.

Translation regulation plays an important role in eukaryotic gene expression. Upstream open reading frames (uORFs) are potent regulatory elements located in 5' mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frames, but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the importance of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of an uORF coding region on gene regulation by assaying the functions of thousands of variants in the yeast YAP1 uORF. Varying uORF codons resulted in a wide range of functions, including repressing and enhancing expression of the downstream ORF. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.

Circuit diversification in a biofilm regulatory network.

Genotype-phenotype relationships can vary extensively among members of a species. One cause of this variation is circuit diversification, the alteration of gene regulatory relationships among members of a species. Circuit diversification is thought to be a starting point for the circuit divergence or rewiring that occurs during speciation. How widespread is circuit diversification? Here we address this question with the fungal pathogen Candida albicans, which forms biofilms rich in distinctive hyphal cells as a prelude to infection. Our understanding of the biofilm/hyphal regulatory network comes primarily from studies of one clinical isolate, strain SC5314, and its marked derivatives. We used CRISPR-based methods to create mutations of four key biofilm transcription factor genes-BCR1, UME6, BRG1, and EFG1 -in SC5314 and four additional clinical isolates. Phenotypic analysis revealed that mutations in BCR1 or UME6 have variable impact across strains, while mutations in BRG1 or EFG1 had uniformly severe impact. Gene expression, sampled with Nanostring probes and examined comprehensively for EFG1 via RNA-Seq, indicates that regulatory relationships are highly variable among isolates. Our results suggest that genotype-phenotype relationships vary in this strain panel in part because of differences in control of BRG1 by BCR1, a hypothesis that is supported through engineered constitutive expression of BRG1. Overall, the data show that circuit diversification is the rule, not the exception, in this biofilm/hyphal regulatory network.

Correction to: DNA copy number evolution in Drosophila cell lines.

Following publication of the original article [1], the authors reported the following errors.

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data.

Upstream open reading frames (uORFs), located in transcript leaders (5' UTRs), are potent cis-acting regulators of translation and mRNA turnover. Recent genome-wide ribosome profiling studies suggest that thousands of uORFs initiate with non-AUG start codons. Although intriguing, these non-AUG uORF predictions have been made without statistical control or validation; thus, the importance of these elements remains to be demonstrated. To address this, we took a comparative genomics approach to study AUG and non-AUG uORFs. We mapped transcription leaders in multiple Saccharomyces yeast species and applied a novel machine learning algorithm (uORF-seqr) to ribosome profiling data to identify statistically significant uORFs. We found that AUG and non-AUG uORFs are both frequently found in Saccharomyces yeasts. Although most non-AUG uORFs are found in only one species, hundreds have either conserved sequence or position within Saccharomyces uORFs initiating with UUG are particularly common and are shared between species at rates similar to that of AUG uORFs. However, non-AUG uORFs are translated less efficiently than AUG-uORFs and are less subject to removal via alternative transcription initiation under normal growth conditions. These results suggest that a subset of non-AUG uORFs may play important roles in regulating gene expression.

Exploring Ribosome Positioning on Translating Transcripts with Ribosome Profiling.

Recent technological advances (e.g., microarrays and massively parallel sequencing) have facilitated genome-wide measurement of many aspects of gene regulation. Ribosome profiling is a high-throughput sequencing method used to measure gene expression at the level of translation. This is accomplished by quantifying both the number of translating ribosomes and their locations on mRNA transcripts. The inventors of this approach have published several methods papers detailing its implementation and addressing the basics of ribosome profiling data analysis. Here we describe our lab's procedure, which differs in some respects from those published previously. In addition, we describe a data analysis pipeline, Ribomap, for ribosome profiling data. Ribomap allocates sequence reads to alternative mRNA isoforms, normalizes sequencing bias along transcripts using RNA-seq data, and outputs count vectors of per-codon ribosome occupancy for each transcript.

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila.

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified "high occupancy target" (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteins and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. From the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.

Regulation of alternative splicing in Drosophila by 56 RNA binding proteins.

Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

Mod-seq: A High-Throughput Method for Probing RNA Secondary Structure.

It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecules are currently unknown (Hennelly & Sanbonmatsu, 2012). While chemical probing can reveal single-stranded regions of RNA, traditional approaches to identify sites of chemical modification are time consuming. Mod-seq is a high-throughput method used to map chemical modification sites on RNAs of any size, including complex mixtures of RNA. In this protocol, we describe preparation of Mod-seq high-throughput sequencing libraries from chemically modified RNA. We also describe a software package "Mod-seeker," which is a compilation of scripts written in Python, for the analysis of Mod-seq data. Mod-seeker returns statistically significant modification sites, which can then be used to aid in secondary structure prediction.

DNA copy number evolution in Drosophila cell lines.

BACKGROUND: Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles. RESULTS: Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). CONCLUSION: Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

Mod-seq: high-throughput sequencing for chemical probing of RNA structure.

The functions of RNA molecules are intimately linked to their ability to fold into complex secondary and tertiary structures. Thus, understanding how these molecules fold is essential to determining how they function. Current methods for investigating RNA structure often use small molecules, enzymes, or ions that cleave or modify the RNA in a solvent-accessible manner. While these methods have been invaluable to understanding RNA structure, they can be fairly labor intensive and often focus on short regions of single RNAs. Here we present a new method (Mod-seq) and data analysis pipeline (Mod-seeker) for assaying the structure of RNAs by high-throughput sequencing. This technique can be utilized both in vivo and in vitro, with any small molecule that modifies RNA and consequently impedes reverse transcriptase. As proof-of-principle, we used dimethyl sulfate (DMS) to probe the in vivo structure of total cellular RNAs in Saccharomyces cerevisiae. Mod-seq analysis simultaneously revealed secondary structural information for all four ribosomal RNAs and 32 additional noncoding RNAs. We further show that Mod-seq can be used to detect structural changes in 5.8S and 25S rRNAs in the absence of ribosomal protein L26, correctly identifying its binding site on the ribosome. While this method is applicable to RNAs of any length, its high-throughput nature makes Mod-seq ideal for studying long RNAs and complex RNA mixtures.

Diversity and dynamics of the Drosophila transcriptome.

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.

Ribosome profiling reveals post-transcriptional buffering of divergent gene expression in yeast.

Understanding the patterns and causes of phenotypic divergence is a central goal in evolutionary biology. Much work has shown that mRNA abundance is highly variable between closely related species. However, the extent and mechanisms of post-transcriptional gene regulatory evolution are largely unknown. Here we used ribosome profiling to compare transcript abundance and translation efficiency in two closely related yeast species (S. cerevisiae and S. paradoxus). By comparing translation regulatory divergence to interspecies differences in mRNA sequence features, we show that differences in transcript leaders and codon bias substantially contribute to divergent translation. Globally, we find that translation regulatory divergence often buffers species differences in mRNA abundance, such that ribosome occupancy is more conserved than transcript abundance. We used allele-specific ribosome profiling in interspecies hybrids to compare the relative contributions of cis- and trans-regulatory divergence to species differences in mRNA abundance and translation efficiency. The mode of gene regulatory divergence differs for these processes, as trans-regulatory changes play a greater role in divergent mRNA abundance than in divergent translation efficiency. Strikingly, most genes with aberrant transcript abundance in F1 hybrids (either over- or underexpressed compared to both parent species) did not exhibit aberrant ribosome occupancy. Our results show that interspecies differences in translation contribute substantially to the evolution of gene expression. Compensatory differences in transcript abundance and translation efficiency may increase the robustness of gene regulation.