1. Dynamics of ribosome scanning and recycling revealed by translation complex profiling.

    Archer SK, Shirokikh NE, Beilharz TH, Preiss T.

    Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5' cap; the SSU then scans in the 3' direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5' UTRs. Scanning SSU have 5'-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5' start site but differ at the 3' end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.

    Nature. 2016
  2. Understanding the regulation of coding and noncoding transcription in cell populations.

    Beilharz TH.

    Whole transcriptome analyses have unveiled the uncomfortable truth that we know less about how transcription is regulated then we thought. In addition to its role in classic promoter-driven transcription of coding RNA, it is now clear that RNA Pol II also drives abundant expression of noncoding RNA. For the majority of this the functional significance remains unclear. Moreover, its regulation and impact are hard to predict because it often proceeds in unexpected ways from cryptic promoters, including by driving convergent antisense transcription from within 3' UTRs. This review suggests that its time to rethink how we envisage gene expression by inclusion of the regulatory architecture of the full genetic locus, and expanding our thinking to encompass the fact that we generally study cells within heterogeneous populations.

    Curr Genet. 2016
  3. Epitope-tagged yeast strains reveal promoter driven changes to 3'-end formation and convergent antisense-transcription from common 3' UTRs.

    Swaminathan A, Beilharz TH.

    Epitope-tagging by homologous recombination is ubiquitously used to study gene expression, protein localization and function in yeast. This is generally thought to insulate the regulation of gene expression to that mediated by the promoter and coding regions because native 3' UTR are replaced. Here we show that the 3' UTRs, CYC1 and ADH1, contain cryptic promoters that generate abundant convergent antisense-transcription in Saccharomyces cerevisiae. Moreover we show that aberrant, truncating 3' -end formation is often associated with regulated transcription in TAP-tagged strains. Importantly, the steady-state level of both 3' -truncated and antisense transcription products is locus dependent. Using TAP and GFP-tagged strains we show that the transcriptional state of the gene-of-interest induces changes to 3' -end formation by alternative polyadenylation and antisense transcription from a universal 3' UTR. This means that these 3' UTRs contains plastic features that can be molded to reflect the regulatory architecture of the locus rather than bringing their own regulatory paradigm to the gene-fusions as would be expected. Our work holds a cautionary note for studies utilizing tagged strains for quantitative biology, but also provides a new model for the study of promoter driven rewiring of 3' -end formation and regulatory non-coding transcription.

    Nucleic Acids Res. 2016
  4. Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans.

    Verma-Gaur J, Qu Y, Harrison PF, Lo TL, Quenault T, Dagley MJ, Bellousoff M, Powell DR, Beilharz TH, Traven A.

    The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease.

    PLoS Genet. 2015
    3'UTR Tracks for upload to the Candida Genome Database
  5. POS-1 Promotes Endo-mesoderm Development by Inhibiting the Cytoplasmic Polyadenylation of neg-1 mRNA.

    Elewa A, Shirayama M, Kaymak E, Harrison PF, Powell DR, Du Z, Chute CD, Woolf H, Yi D, Ishidate T, Srinivasan J, Bao Z, Beilharz TH, Ryder SP, Mello CC.

    The regulation of mRNA translation is of fundamental importance in biological mechanisms ranging from embryonic axis specification to the formation of long-term memory. POS-1 is one of several CCCH zinc-finger RNA-binding proteins that regulate cell fate specification during C. elegans embryogenesis. Paradoxically, pos-1 mutants exhibit striking defects in endo-mesoderm development but have wild-type distributions of SKN-1, a key determinant of endo-mesoderm fates. RNAi screens for pos-1 suppressors identified genes encoding the cytoplasmic poly(A)-polymerase homolog GLD-2, the Bicaudal-C homolog GLD-3, and the protein NEG-1. We show that NEG-1 localizes in anterior nuclei, where it negatively regulates endo-mesoderm fates. In posterior cells, POS-1 binds the neg-1 3' UTR to oppose GLD-2 and GLD-3 activities that promote NEG-1 expression and cytoplasmic lengthening of the neg-1 mRNA poly(A) tail. Our findings uncover an intricate series of post-transcriptional regulatory interactions that, together, achieve precise spatial expression of endo-mesoderm fates in C. elegans embryos.

    (2015) Developmental Cell
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  6. PAT-seq: a method to study the integration of 3'-UTR dynamics with gene expression in the eukaryotic transcriptome.

    Harrison PF, Powell DR, Clancy JL, Preiss T, Boag PR, Traven A, Seemann T, Beilharz TH.

    A major objective of systems biology is to quantitatively integrate multiple parameters from genome-wide measurements. To integrate gene expression with dynamics in poly(A) tail length and adenylation site, we developed a targeted next-generation sequencing approach, Poly(A)-Test RNA-sequencing. PAT-seq returns (i) digital gene expression, (ii) polyadenylation site/s, and (iii) the polyadenylation-state within and between eukaryotic transcriptomes. PAT-seq differs from previous 3' focused RNA- seq methods in that it depends strictly on 3' adenylation within total RNA samples and that the full-native poly(A) tail is included in the sequencing libraries. Here, total RNA samples from budding yeast cells were analyzed to identify the intersect between adenylation state and gene expression in response to loss of the major cytoplasmic deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in the classic Crabtree-Warburg metabolic shift. Because all polyadenylated RNA is interrogated by the approach, alternative adenylation sites, noncoding RNA and RNA-decay intermediates were also identified. Most important, the PAT-seq approach uses standard sequencing procedures, supports significant multiplexing, and thus replication and rigorous statistical analyses can for the first time be brought to the measure of 3' -UTR dynamics genome wide.

    RNA (2015)
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  7. Probing the closed-loop model of mRNA translation in living cells.

    Archer SK, Shirokikh NE, Hallwirth CV, Beilharz TH, Preiss T.

    The mRNA closed-loop, formed through interactions between the cap structure, poly(A) tail, eIF4E, eIF4G and PAB, features centrally in models of eukaryotic translation initiation, although direct support for its existence in vivo is not well established. Here, we investigated the closed-loop using a combination of mRNP isolation from rapidly cross-linked cells and high-throughput qPCR. Using the interaction between these factors and the opposing ends of mRNAs as a proxy for the closed-loop, we provide evidence that it is prevalent for eIF4E/4G-bound but unexpectedly sparse for PAB1-bound mRNAs, suggesting it primarily occurs during a distinct phase of polysome assembly. We observed mRNA-specific variation in the extent of closed-loop formation, consistent with a role for polysome topology in the control of gene expression.

    RNA Biology Volume 12, Issue 3, 2015
  8. Using Klenow-mediated extension to measure poly(A)-tail length and position in the transcriptome.

    Lee MC1, Jänicke A, Beilharz TH.

    The poly(A)-tail that terminates most mRNA and many noncoding RNA is a convenient "hook" to isolate mRNA. However the length of this tail and its position within the primary RNA transcript can also hold diagnostic value for RNA metabolism. In general, mRNA with a long poly(A)-tail is well translated, whereas a short poly(A)-tail can indicate translational silencing. A short poly(A)-tail is also appended to RNA-decay intermediates via the TRAMP complex. A number of approaches have been developed to measure the length and position of the poly(A)-tail. Here, we describe a simple method to "tag" adenylated RNA using the native function of DNA polymerase I to extend an RNA primer on a DNA template in second-strand DNA synthesis. This function can be harnessed as a means to purify, visualize, and quantitate poly(A)-dynamics of individual RNA and the transcriptome en masse.

    Methods Mol Biol. 2014;1125:25-42
  9. Introns regulate gene expression in Cryptococcus neoformans in a Pab2p dependent pathway.

    Goebels C, Thonn A, Gonzalez-Hilarion S, Rolland O, Moyrand F, Beilharz TH, Janbon G.

    Most Cryptococccus neoformans genes are interrupted by introns, and alternative splicing occurs very often. In this study, we examined the influence of introns on C. neoformans gene expression. For most tested genes, elimination of introns greatly reduces mRNA accumulation. Strikingly, the number and the position of introns modulate the gene expression level in a cumulative manner. A screen for mutant strains able to express functionally an intronless allele revealed that the nuclear poly(A) binding protein Pab2 modulates intron-dependent regulation of gene expression in C. neoformans. PAB2 deletion partially restored accumulation of intronless mRNA. In addition, our results demonstrated that the essential nucleases Rrp44p and Xrn2p are implicated in the degradation of mRNA transcribed from an intronless allele in C. neoformans. Double mutant constructions and over-expression experiments suggested that Pab2p and Xrn2p could act in the same pathway whereas Rrp44p appears to act independently. Finally, deletion of the RRP6 or the CID14 gene, encoding the nuclear exosome nuclease and the TRAMP complex associated poly(A) polymerase, respectively, has no effect on intronless allele expression.

    PLoS Genet. 2013;9(8):e1003686
  10. Yeast hEST1A/B (SMG5/6)-like proteins contribute to environment-sensing adaptive gene expression responses.

    Lai X, Beilharz T, Au WC, Hammet A, Preiss T, Basrai MA, Heierhorst J.

    During its natural life cycle, budding yeast (Saccharomyces cerevisiae) has to adapt to drastically changing environments, but how environmental-sensing pathways are linked to adaptive gene expression changes remains incompletely understood. Here, we describe two closely related yeast hEST1A-B (SMG5-6)-like proteins termed Esl1 and Esl2 that contain a 14-3-3-like domain and a putative PilT N-terminus ribonuclease domain. We found that, unlike their metazoan orthologs, Esl1 and Esl2 were not involved in nonsense-mediated mRNA decay or telomere maintenance pathways. However, in genome-wide expression array analyses, absence of Esl1 and Esl2 led to more than two-fold deregulation of ~50 transcripts, most of which were expressed inversely to the appropriate metabolic response to environmental nutrient supply; for instance, normally glucose-repressed genes were derepressed in esl1Δ esl2Δ double mutants during growth in a high-glucose environment. Likewise, in a genome-wide synthetic gene array screen, esl1Δ esl2Δ double mutants were synthetic sick with null mutations for Rim8 and Dfg16, which form the environmental-sensing complex of the Rim101 pH response gene expression pathway. Overall, these results suggest that Esl1 and Esl2 contribute to the regulation of adaptive gene expression responses of environmental sensing pathways.

    G3 (Bethesda). 2013 Oct 3;3(10):1649-59
  11. In vivo mutation of pre-mRNA processing factor 8 (Prpf8) affects transcript splicing, cell survival and myeloid differentiation.

    Keightley MC, Crowhurst MO, Layton JE, Beilharz T, Markmiller S, Varma S, Hogan BM, de Jong-Curtain TA, Heath JK, Lieschke GJ.

    Mutated spliceosome components are recurrently being associated with perturbed tissue development and disease pathogenesis. Cephalopho╠ćnus (cph), is a zebrafish mutant carrying an early premature STOP codon in the spliceosome component Prpf8 (pre-mRNA processing factor 8). Cph initially develops normally, but then develops widespread cell death, especially in neurons, and is embryonic lethal. Cph mutants accumulate aberrantly spliced transcripts retaining both U2- and U12-type introns. Within early haematopoiesis, myeloid differentiation is impaired, suggesting Prpf8 is required for haematopoietic development. Cph provides an animal model for zygotic PRPF8 dysfunction diseases and for evaluating therapeutic interventions.

    FEBS Lett. 2013 Jul 11;587(14):2150-7
  12. ifet-1 is a broad-scale translational repressor required for normal P granule formation in C. elegans.

    Sengupta MS, Low WY, Patterson JR, Kim HM, Traven A, Beilharz TH, Colaiácovo MP, Schisa JA, Boag PR.

    Large cytoplasmic ribonucleoprotein germ granule complexes are a common feature in germ cells. In C. elegans these are called P granules and for much of the life-cycle they associate with nuclear pore complexes in germ cells. P granules are rich in proteins that function in diverse RNA pathways. Here we report that the C. elegans homolog of the eIF4E-transporter IFET-1 is required for oogenesis but not spermatogenesis. We show that IFET-1 is required for translational repression of several maternal mRNAs in the distal gonad and functions in conjunction with the broad-scale translational regulators CGH-1, CAR-1 and PATR-1 to regulate germ cell sex determination. Furthermore we have found that IFET-1 localizes to P granules throughout the gonad and in the germ cell lineage in the embryo. Interestingly, IFET-1 is required for the normal ultrastructure of P granules and for the localization of CGH-1 and CAR-1 to P granules. Our findings suggest that IFET-1 is a key translational regulator and is required for normal P granule formation.

    J Cell Sci. 2013 Feb 1;126(Pt 3):850-9
  13. A model system for mitochondrial biogenesis reveals evolutionary rewiring of protein import and membrane assembly pathways.

    Hewitt VL, Heinz E, Shingu-Vazquez M, Qu Y, Jelicic B, Lo TL, Beilharz TH, Dumsday G, Gabriel K, Traven A, Lithgow T.

    The controlled biogenesis of mitochondria is a key cellular system coordinated with the cell division cycle, and major efforts in systems biology currently are directed toward understanding of the control points at which this coordination is achieved. Here we present insights into the function, evolution, and regulation of mitochondrial biogenesis through the study of the protein import machinery in the human fungal pathogen, Candida albicans. Features that distinguish C. albicans from baker's yeast (Saccharomyces cerevisiae) include the stringency of metabolic control at the level of oxygen consumption, the potential for ATP exchange through the porin in the outer membrane, and components and domains in the sorting and assembling machinery complex, a molecular machine that drives the assembly of proteins in the outer mitochondrial membrane. Analysis of targeting sequences and assays of mitochondrial protein import show that components of the electron transport chain are imported by distinct pathways in C. albicans and S. cerevisiae, representing an evolutionary rewiring of mitochondrial import pathways. We suggest that studies using this pathogen as a model system for mitochondrial biogenesis will greatly enhance our knowledge of how mitochondria are made and controlled through the course of the cell-division cycle.

    Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):E3358-66
  14. Transcriptional profiling of a yeast colony provides new insight into the heterogeneity of multicellular fungal communities.

    Traven A, Jänicke A, Harrison P, Swaminathan A, Seemann T, Beilharz TH.

    Understanding multicellular fungal structures is important for designing better strategies against human fungal pathogens. For example, the ability to form multicellular biofilms is a key virulence property of the yeast Candida albicans. C. albicans biofilms form on indwelling medical devices and are drug resistant, causing serious infections in hospital settings. Multicellular fungal communities are heterogeneous, consisting of cells experiencing different environments. Heterogeneity is likely important for the phenotypic characteristics of communities, yet it is poorly understood. Here we used colonies of the yeast Saccharomyces cerevisiae as a model fungal multicellular structure. We fractionated the outside colony layers from the cells in the center by FACS, using a Cit1-GFP marker expressed exclusively on the outside. Transcriptomics analysis of the two subpopulations revealed that the outside colony layers are actively growing by fermentative metabolism, while the cells residing on the inside are in a resting state and experience changes to mitochondrial activity. Our data shows several parallels with C. albicans biofilms providing insight into the contributions of heterogeneity to biofilm phenotypes. Hallmarks of C. albicans biofilms - the expression of ribosome and translation functions and activation of glycolysis and ergosterol biosynthesis occur on the outside of colonies, while expression of genes associates with sulfur assimilation is observed in the colony center. Cell wall restructuring occurs in biofilms, and cell wall functions are enriched in both fractions: the outside cells display enrichment of cell wall biosynthesis enzymes and cell wall proteins, while the inside cells express cell wall degrading enzymes. Our study also suggests that noncoding transcription and posttranscriptional mRNA regulation play important roles during growth of yeast in colonies, setting the scene for investigating these pathways in the development of multicellular fungal communities.

    PLoS One. 2012;7(9):e46243
  15. The mRNA decay pathway regulates the expression of the Flo11 adhesin and biofilm formation in Saccharomyces cerevisiae.

    Lo TL, Qu Y, Uwamahoro N, Quenault T, Beilharz TH, Traven A.

    Regulation of the FLO11 adhesin is a model for gene expression control by extracellular signals and developmental switches. We establish that the major mRNA decay pathway regulates FLO11 expression. mRNA deadenylation of transcriptional repressors of FLO11 by the exonuclease Ccr4 keeps their levels low, thereby allowing FLO11 transcription.

    Genetics. 2012 Aug;191(4):1387-91
  16. ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications.

    Jänicke A, Vancuylenberg J, Boag PR, Traven A, Beilharz TH.

    The addition of a poly(A)-tail to the 3' termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3'-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3' UTR usage in 3' RACE applications.

    RNA. 2012 Jun;18(6):1289-95
  17. The functions of Mediator in Candida albicans support a role in shaping species-specific gene expression.

    Uwamahoro N, Qu Y, Jelicic B, Lo TL, Beaurepaire C, Bantun F, Quenault T, Boag PR, Ramm G, Callaghan J, Beilharz TH, Nantel A, Peleg AY, Traven A.

    The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species.

    PLoS Genet. 2012;8(4):e1002613.
  18. Mitochondrial sorting and assembly machinery subunit Sam37 in Candida albicans: insight into the roles of mitochondria in fitness, cell wall integrity, and virulence.

    Qu Y, Jelicic B, Pettolino F, Perry A, Lo TL, Hewitt VL, Bantun F, Beilharz TH, Peleg AY, Lithgow T, Djordjevic JT, Traven A.

    Recent studies indicate that mitochondrial functions impinge on cell wall integrity, drug tolerance, and virulence of human fungal pathogens. However, the mechanistic aspects of these processes are poorly understood. We focused on the mitochondrial outer membrane SAM (Sorting and Assembly Machinery) complex subunit Sam37 in Candida albicans. Inactivation of SAM37 in C. albicans leads to a large reduction in fitness, a phenotype not conserved with the model yeast Saccharomyces cerevisiae. Our data indicate that slow growth of the sam37ΔΔ mutant results from mitochondrial DNA loss, a new function for Sam37 in C. albicans, and from reduced activity of the essential SAM complex subunit Sam35. The sam37ΔΔ mutant was hypersensitive to drugs that target the cell wall and displayed altered cell wall structure, supporting a role for Sam37 in cell wall integrity in C. albicans. The sensitivity of the mutant to membrane-targeting antifungals was not significantly altered. The sam37ΔΔmutant was avirulent in the mouse model, and bioinformatics showed that the fungal Sam37 proteins are distant from their animal counterparts and could thus represent potential drug targets. Our study provides the first direct evidence for a link between mitochondrial function and cell wall integrity in C. albicans and is further relevant for understanding mitochondrial function in fitness, antifungal drug tolerance, and virulence of this major pathogen. Beyond the relevance to fungal pathogenesis, this work also provides new insight into the mitochondrial and cellular roles of the SAM complex in fungi.

    Eukaryot Cell. 2012 Apr;11(4):532-44
  19. Polyadenylation state microarray (PASTA) analysis.

    Beilharz TH, Preiss T.

    Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

    Methods Mol Biol. 2011;759:133-48
  20. mRNA isoform diversity can obscure detection of miRNA-mediated control of translation.

    Clancy JL, Wei GH, Echner N, Humphreys DT, Beilharz TH, Preiss T.

    Reporter-based studies support inhibition of translation at the level of initiation as a substantial component of the miRNA mechanism, yet recent global analyses have suggested that they predominantly act through decreasing target mRNA stability. Cells commonly coexpress several processing isoforms of an mRNA, which may also differ in their regulatory untranslated regions (UTR). In particular, cancer cells are known to express high levels of short 3' UTR isoforms that evade miRNA-mediated regulation, whereas longer 3' UTRs predominate in nontransformed cells. To test whether mRNA isoform diversity can obscure detection of miRNA-mediated control at the level of translation, we assayed the responses of 11 endogenous let-7 targets to inactivation of this miRNA in HeLa cells, an intensively studied model system. We show that translational regulation in many cases appears to be modest when measuring the composite polysome profile of all extant isoforms of a given mRNA by density ultracentrifugation. In contrast, we saw clear effects at the level of translation initiation for multiple examples when selectively profiling mRNA isoforms carrying the 5' or 3' untranslated regions that were actually permissive to let-7 action, or when let-7 and a second targeting miRNA were jointly manipulated. Altogether, these results highlight a caveat to the mechanistic interpretation of data from global miRNA target analyses in transformed cells. Importantly, they reaffirm the importance of translational control as part of the miRNA mechanism in animal cells.

    RNA. 2011 Jun;17(6):1025-31.
  21. Cell wall integrity is linked to mitochondria and phospholipid homeostasis in Candida albicans through the activity of the post-transcriptional regulator Ccr4-Pop2.

    Dagley MJ, Gentle IE, Beilharz TH, Pettolino FA, Djordjevic JT, Lo TL, Uwamahoro N, Rupasinghe T, Tull DL, McConville M, Beaurepaire C, Nantel A, Lithgow T, Mitchell AP, Traven A.

    The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post-transcriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall ╬▓-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.

    Mol Microbiol. 2011 Feb;79(4):968-89
  22. miRNA Effects on mRNA closed-loop formation during translation initiation.

    Beilharz TH, Humphreys DT, Preiss T.

    A flurry of recent studies, carried out primarily in transfected cells or in vitro translation systems, have attempted to reveal the molecular means by which animal microRNAs (miRNAs) attenuate mRNA translation. Despite these intense efforts it has not yet been possible to derive a consensus model for such a mechanism. Here we summarise our own experimental contributions to this topic, which led us to propose that miRNAs control early translation initiation by affecting eukaryotic initiation factor 4E/cap structure and poly(A) tail function, and place them in a current context of this rapidly moving and challenging field.

    Prog Mol Subcell Biol. 2010;50:99-112.
  23. microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells

    Beilharz TH, Humphreys DT, Clancy JL, Thermann R, Martin DI, Hentze MW, Preiss T.

    Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3' untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3' poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3' UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs.

    PLoS One. 2009 Aug 27;4(8):e6783
  24. The Ccr4-Pop2-NOT mRNA deadenylase contributes to septin organization in Saccharomyces cerevisiae.

    Traven A, Beilharz TH, Lo TL, Lueder F, Preiss T, Heierhorst J.

    In yeast, assembly of the septins at the cell cortex is required for a series of key cell cycle events: bud-site selection, the morphogenesis and mitotic exit checkpoints, and cytokinesis. Here we establish that the Ccr4-Pop2-NOT mRNA deadenylase contributes to septin organization. mRNAs encoding regulators of septin assembly (Ccd42, Cdc24, Rga1, Rga2, Bem3, Gin4, Cla4, and Elm1) presented with short poly(A) tails at steady state in wild-type (wt) cells, suggesting their translation could be restricted by deadenylation. Deadenylation of septin regulators was dependent on the major cellular mRNA deadenylase Ccr4-Pop2-NOT, whereas the alternative deadenylase Pan2 played a minor role. Consistent with deadenylation of septin regulators being important for function, deletion of deadenylase subunits CCR4 or POP2, but not PAN2, resulted in septin morphology defects (e.g., ectopic bud-localized septin rings), particularly upon activation of the Cdc28-inhibitory kinase Swe1. Aberrant septin staining was also observed in the deadenylase-dead ccr4-1 mutant, demonstrating the deadenylase activity of Ccr4-Pop2 is required. Moreover, ccr4Delta, pop2Delta, and ccr4-1 mutants showed aberrant cell morphology previously observed in septin assembly mutants and exhibited genetic interactions with mutations that compromise septin assembly (shs1Delta, cla4Delta, elm1Delta, and gin4Delta). Mutations in the Not subunits of Ccr4-Pop2-NOT, which are thought to predominantly function in transcriptional control, also resulted in septin organization defects. Therefore, both mRNA deadenylase and transcriptional functions of Ccr4-Pop2-NOT contribute to septin organization in yeast.

    Genetics. 2009 Aug;182(4):955-66
  25. Transcriptome-wide measurement of mRNA polyadenylation state.

    Beilharz TH, Preiss T

    The 3' poly(A) tail has important roles throughout the eukaryotic mRNA life cycle. A characteristic aspect of poly(A) tail function is furthermore that it can be modulated by changes in its length. This is in turn a well-recognised cellular means to regulate both, mRNA translation and stability, and a positive correlation has often been found between the efficiency of mRNA translation and the length of its poly(A) tail. Here we describe methodology to measure mRNA polyadenylation state in a transcriptome-wide manner, using separation of cellular mRNA populations on poly(U) sepharose in combination with microarray analysis of the resulting fractions. We further detail methods for bulk and mRNA-specific poly(A) tail length measurements to monitor the efficiency of initial mRNA separation and to verify candidates selected from the microarray data. Although detailed here for the study of yeast mRNAs, these methods are adaptable to the investigation of any cellular context in which poly(A) tail length control is known or suspected to operate.

    Methods. 2009 Jul;48(3):294-300.
  26. Methods to analyze microRNA-mediated control of mRNA translation.

    Clancy JL, Nousch M, Humphreys DT, Westman BJ, Beilharz TH, Preiss T.

    MicroRNAs (miRs) are an important class of gene regulators that affect a wide range of biological processes. Despite the early recognition of miRs as translational regulators and intense interest in studying this phenomenon, it has so far not been possible to derive a consensus model for the underlying molecular mechanism(s). The potential of miRs to act in a combinatorial manner and to also promote mRNA decay creates conceptual and technical challenges in their study. Here, we discuss critical parameters in design and analysis of experiments used to study miR function including creation of synthetic miR and mRNA partners for assay of translational inhibition using luciferase reporters; measurement of mRNA stability after miR action; defining poly(A) tail length in miR target mRNA; determining the distribution of miRs and their target mRNAs in polysome profiles; and visualization of P-body components. We describe protocols for each of these procedures.

    Methods Enzymol. 2007;431:83-111.
  27. Widespread use of poly(A) tail length control to accentuate expression of the yeast transcriptome.

    Beilharz TH, Preiss T.

    Control of poly(A) tail length can affect translation and stability of eukaryotic mRNAs. Although well established for individual cases, it was not known to what extent this type of adjustable gene control is used to shape expression of eukaryotic transcriptomes. Here we report on microarray-based measurements of mRNA poly(A) tail lengths and association with the poly(A)-binding protein Pab1 in S. cerevisiae, revealing extensive correlation between tail length and other physical and functional mRNA characteristics. Gene ontology analyses and further directed experiments indicate coregulation of tail length on functionally and cytotopically related mRNAs to coordinate cell-cycle progression, ribosome biogenesis, and retrotransposon expression. We show that the 3'-untranslated region drives transcript-specific adenylation control and translational efficiency of multiple mRNAs. Our findings suggest a wide-spread interdependence between 3'-untranslated region-mediated poly(A) tail length control, Pab1 binding, and mRNA translation in budding yeast. They further provide a molecular explanation for deadenylase function in the cell cycle and suggest additional cellular processes that depend on control of mRNA polyadenylation.

    RNA. 2007 Jul;13(7):982-97.
  28. A network of multiple regulatory layers shapes gene expression in fission yeast.

    Lackner DH, Beilharz TH, Marguerat S, Mata J, Watt S, Schubert F, Preiss T, Bähler J.

    Gene expression is controlled at multiple layers, and cells may integrate different regulatory steps for coherent production of proper protein levels. We applied various microarray-based approaches to determine key gene-expression intermediates in exponentially growing fission yeast, providing genome-wide data for translational profiles, mRNA steady-state levels, polyadenylation profiles, start-codon sequence context, mRNA half-lives, and RNA polymerase II occupancy. We uncovered widespread and unexpected relationships between distinct aspects of gene expression. Translation and polyadenylation are aligned on a global scale with both the lengths and levels of mRNAs: efficiently translated mRNAs have longer poly(A) tails and are shorter, more stable, and more efficiently transcribed on average. Transcription and translation may be independently but congruently optimized to streamline protein production. These rich data sets, all acquired under a standardized condition, reveal a substantial coordination between regulatory layers and provide a basis for a systems-level understanding of multilayered gene-expression programs.

    Mol Cell. 2007 Apr 13;26(1)
  29. Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length.

    Woolstencroft RN, Beilharz TH, Cook MA, Preiss T, Durocher D, Tyers M.

    In Saccharomyces cerevisiae, DNA replication stress activates the replication checkpoint, which slows S-phase progression, stabilizes slowed or stalled replication forks, and relieves inhibition of the ribonucleotide reductase (RNR) complex. To identify novel genes that promote cellular viability after replication stress, the S. cerevisiae non-essential haploid gene deletion set (4812 strains) was screened for sensitivity to the RNR inhibitor hydroxyurea (HU). Strains bearing deletions in either CCR4 or CAF1/POP2, which encode components of the cytoplasmic mRNA deadenylase complex, were particularly sensitive to HU. We found that Ccr4 cooperated with the Dun1 branch of the replication checkpoint, such that ccr4Delta dun1Delta strains exhibited irreversible hypersensitivity to HU and persistent activation of Rad53. Moreover, because ccr4Delta and chk1Delta exhibited epistasis in several genetic contexts, we infer that Ccr4 and Chk1 act in the same pathway to overcome replication stress. A counterscreen for suppressors of ccr4Delta HU sensitivity uncovered mutations in CRT1, which encodes the transcriptional repressor of the DNA-damage-induced gene regulon. Whereas Dun1 is known to inhibit Crt1 repressor activity, we found that Ccr4 regulates CRT1 mRNA poly(A) tail length and may subtly influence Crt1 protein abundance. Simultaneous overexpression of RNR2, RNR3 and RNR4 partially rescued the HU hypersensitivity of a ccr4Delta dun1Delta strain, consistent with the notion that the RNR genes are key targets of Crt1. These results implicate the coordinated regulation of Crt1 via Ccr4 and Dun1 as a crucial nodal point in the response to DNA replication stress.

    J Cell Sci. 2006 Dec 15;119(Pt 24)
  30. Integral membrane proteins in the mitochondrial outer membrane of Saccharomyces cerevisiae.

    Burri L, Vascotto K, Gentle IE, Chan NC, Beilharz T, Stapleton DI, Ramage L, Lithgow T.

    Mitochondria evolved from a bacterial endosymbiont ancestor in which the integral outer membrane proteins would have been beta-barrel structured within the plane of the membrane. Initial proteomics on the outer membrane from yeast mitochondria suggest that while most of the protein components are integral in the membrane, most of these mitochondrial proteins behave as if they have alpha-helical transmembrane domains, rather than beta-barrels. These proteins are usually predicted to have a single alpha-helical transmembrane segment at either the N- or C-terminus, however, more complex topologies are also seen. We purified the novel outer membrane protein Om14 and show it is encoded in the gene YBR230c. Protein sequencing revealed an intron is spliced from the transcript, and both transcription from the YBR230c gene and steady-state level of the Om14 protein is dramatically less in cells grown on glucose than in cells grown on nonfermentable carbon sources. Hydropathy predictions together with data from limited protease digestion show three alpha-helical transmembrane segments in Om14. The alpha-helical outer membrane proteins provide functions derived after the endosymbiotic event, and require the translocase in the outer mitochondrial membrane complex for insertion into the outer membrane.

    FEBS J. 2006 Apr;273(7)
  31. Translational profiling: the genome-wide measure of the nascent proteome.

    Beilharz TH, Preiss T.

    Translation in eukaryotic cells is both physically and temporally separated from transcription. This provides cells with extended options to alter their proteome: (1) directly, by synchronizing translation with an altering transcriptional profile; (2) by imposing a changed translational control over transcripts already present in the transcriptome; or (3) by a combination of (1) and (2). In this paper, recent findings in the controlled translation of the transcriptome using microarray analyses are reviewed. A guide to the current technologies and data analysis is also provided, and future directions in the study of translational control as the interface between the transcriptome and the proteome are outlined. This survey is focused on the yeast Saccharomyces cerevisiae, but the topics covered have universal relevance to the control of translation in eukaryotic cells.

    Brief Funct Genomic Proteomic. 2004 Aug;3(2)
  32. Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast.

    Youker RT, Walsh P, Beilharz T, Lithgow T, Brodsky JL.

    Aberrant secreted proteins can be destroyed by ER-associated protein degradation (ERAD), and a prominent, medically relevant ERAD substrate is the cystic fibrosis transmembrane conductance regulator (CFTR). To better define the chaperone requirements during CFTR maturation, the protein was expressed in yeast. Because Hsp70 function impacts CFTR biogenesis in yeast and mammals, we first sought ER-associated Hsp40 cochaperones involved in CFTR maturation. Ydj1p and Hlj1p enhanced Hsp70 ATP hydrolysis but CFTR degradation was slowed only in yeast mutated for both YDJ1 and HLJ1, suggesting functional redundancy. In contrast, CFTR degradation was accelerated in an Hsp90 mutant strain, suggesting that Hsp90 preserves CFTR in a folded state, and consistent with this hypothesis, Hsp90 maintained the solubility of an aggregation-prone domain (NBD1) in CFTR. Soluble ERAD substrate degradation was unaffected in the Hsp90 or the Ydj1p/Hlj1p mutants, and surprisingly CFTR degradation was unaffected in yeast mutated for Hsp90 cochaperones. These results indicate that Hsp90, but not the Hsp90 complex, maintains CFTR structural integrity, whereas Ydj1p/Hlj1p catalyze CFTR degradation.

    Mol Biol Cell. 2004 Nov;15(11)
  33. Multiple cargo binding sites on the COPII subunit Sec24p ensure capture of diverse membrane proteins into transport vesicles.

    Miller EA, Beilharz TH, Malkus PN, Lee MC, Hamamoto S, Orci L, Schekman R.

    We have characterized the mechanisms of cargo selection into ER-derived vesicles by the COPII subunit Sec24p. We identified a site on Sec24p that recognizes the v-SNARE Bet1p and show that packaging of a number of cargo molecules is disrupted when mutations are introduced at this site. Surprisingly, cargo proteins affected by these mutations did not share a single common sorting signal, nor were proteins sharing a putative class of signal affected to the same degree. We show that the same site is conserved as a cargo-interaction domain on the Sec24p homolog Lst1p, which only packages a subset of the cargoes recognized by Sec24p. Finally, we identified an additional mutation that defines another cargo binding domain on Sec24p, which specifically interacts with the SNARE Sec22p. Together, our data support a model whereby Sec24p proteins contain multiple independent cargo binding domains that allow for recognition of a diverse set of sorting signals.

    Cell. 2003 Aug 22;114(4)
  34. A SNARE required for retrograde transport to the endoplasmic reticulum.

    Burri L, Varlamov O, Doege CA, Hofmann K, Beilharz T, Rothman JE, Söllner TH, Lithgow T.

    SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells. Sequence analysis of the yeast genome revealed a previously uncharacterized SNARE, SNARE-like tail-anchored protein 1 (Slt1). Slt1 is an essential protein localized in the endoplasmic reticulum (ER). It forms a SNARE complex with Sec22 and the ER syntaxin Ufe1. Down-regulation of Slt1 levels leads to improper secretion of proteins normally resident in the ER. We suggest that Slt1 is a component of the SNAREpin required for retrograde traffic to the ER. Based on the previously reported association with Ufe1 and Sec22, Sec20 likely contributes the fourth SNARE to the SNAREpin.

    Proc Natl Acad Sci U S A. 2003 Aug 19;100(17)
  35. Bipartite signals mediate subcellular targeting of tail-anchored membrane proteins in Saccharomyces cerevisiae.

    Beilharz T, Egan B, Silver PA, Hofmann K, Lithgow T.

    Tail-anchored proteins have an NH(2)-terminal cytosolic domain anchored to intracellular membranes by a single, COOH-terminal, transmembrane segment. Sequence analysis identified 55 tail-anchored proteins in Saccharomyces cerevisiae, with several novel proteins, including Prm3, which we find is required for karyogamy and is tail-anchored in the nuclear envelope. A total of six tail-anchored proteins are present in the mitochondrial outer membrane and have relatively hydrophilic transmembrane segments that serve as targeting signals. The rest, by far the majority, localize via a bipartite system of signals: uniformly hydrophobic tail anchors are first inserted into the endoplasmic reticulum, and additional segments within the cytosolic domain of each protein can dictate subsequent sorting to a precise destination within the cell.

    J Biol Chem. 2003 Mar 7;278(10)
  36. A conserved proline residue is present in the transmembrane-spanning domain of Tom7 and other tail-anchored protein subunits of the TOM translocase.

    Allen R, Egan B, Gabriel K, Beilharz T, Lithgow T.

    The TOM translocase consists of several integral membrane proteins organised around the channel forming protein Tom40. Here we show that one of these protein subunits, Tom7, is a tail-anchored protein. The carboxy-terminal 33 amino acids of Tom7 contain the information for targeting the protein to the mitochondrial outer membrane, and a conserved proline residue within the transmembrane segment is required for efficient targeting of Tom7 to the outer membrane. An equivalent proline residue is important in targeting each of the other three tail-anchored proteins that associate with Tom40 to form the core of the TOM translocase.

    FEBS Lett. 2002 Mar 13;514(2-3)
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