Specific features of telomerase RNA from Hansenula polymorpha.
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ABSTRACT:
SUBMITTER: Smekalova EM
PROVIDER: S-EPMC3851723 | biostudies-literature | 2013 Nov
REPOSITORIES: biostudies-literature
Publications
Specific features of telomerase RNA from Hansenula polymorpha.
Smekalova Elena M EM Malyavko Alexander N AN Zvereva Maria I MI Mardanov Andrey V AV Ravin Nikolai V NV Skryabin Konstantin G KG Westhof Eric E Dontsova Olga A OA
RNA (New York, N.Y.) 20130917 11
Telomerase, a ribonucleoprotein, is responsible for the maintenance of eukaryotic genome integrity by replicating the ends of chromosomes. The core enzyme comprises the conserved protein TERT and an RNA subunit (TER) that, in contrast, displays large variations in size and structure. Here, we report the identification of the telomerase RNA from thermotolerant yeast Hansenula polymorpha (HpTER) and describe its structural features. We show further that the H. polymorpha telomerase reverse transcr ...[more]
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Project description:Telomerase is a ribonucleoprotein enzyme, which maintains genome integrity in eukaryotes and ensures continuous cellular proliferation. Telomerase holoenzyme from the thermotolerant yeast Hansenula polymorpha, in addition to the catalytic subunit (TERT) and telomerase RNA (TER), contains accessory proteins Est1 and Est3, which are essential for in vivo telomerase function. Here we report the high-resolution structure of Est3 from Hansenula polymorpha (HpEst3) in solution, as well as the characterization of its functional relationships with other components of telomerase. The overall structure of HpEst3 is similar to that of Est3 from Saccharomyces cerevisiae and human TPP1. We have shown that telomerase activity in H. polymorpha relies on both Est3 and Est1 proteins in a functionally symmetrical manner. The absence of either Est3 or Est1 prevents formation of a stable ribonucleoprotein complex, weakens binding of a second protein to TER, and decreases the amount of cellular TERT, presumably due to the destabilization of telomerase RNP. NMR probing has shown no direct in vitro interactions of free Est3 either with the N-terminal domain of TERT or with DNA or RNA fragments mimicking the probable telomerase environment. Our findings corroborate the idea that telomerase possesses the evolutionarily variable functionality within the conservative structural context.
| S-EPMC7338525 | biostudies-literature
Project description:BackgroundIn the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity.ResultsMutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones.ConclusionsComparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.
| S-EPMC5490237 | biostudies-literature
Project description:Telomeres are special structures at the ends of chromosomes that play an important role in the protection of the genetic material. Telomere composition is very diverse; noticeable differences can often be observed even among closely related species. Here, we identify the homolog of telomeric protein Cdc13 in the thermotolerant yeast Hansenula polymorpha. We show that it can specifically bind single-stranded telomeric DNA, as well as interact with the Stn1 protein. In addition, we have uncovered an interaction between Cdc13 and TERT (one of the core components of the telomerase complex), which suggests that Cdc13 is potentially involved in telomerase recruitment to telomeres in H. polymorpha.
| S-EPMC7245964 | biostudies-literature
Project description:Telomerase RNA is an integral part of telomerase, the ribonucleoprotein enzyme that catalyzes the synthesis of telomeric DNA. The RNA moiety contains a templating domain that directs the synthesis of a species-specific telomeric repeat and may also be important for enzyme structure and/or catalysis. Phylogenetic comparisons of telomerase RNA sequences from various Tetrahymena spp. and hypotrich ciliates have revealed two conserved secondary structure models that share many features. We have cloned and sequenced the telomerase RNA genes from an additional six Tetrahymena spp. (T. vorax, T. borealis, T. australis, T. silvana, T. capricornis and T. paravorax). Inclusion of these sequences, most notably that from T. paravorax, in a phylogenetic comparative analysis allowed us to more narrowly define structural elements that may be necessary for a minimal telomerase RNA. A primary sequence element, positioned 5' of the template and conserved between all previously known ciliate telomerase RNAs, has been reduced from 5'-(C)UGUCA-3' to the 4 nt sequence 5'-GUCA-3'. Conserved secondary structural features and the impact they have on the general organization of ciliate telomerase RNAs is discussed.
| S-EPMC306816 | biostudies-other
Project description:BACKGROUND: Methylotrophic yeast species (e.g. Hansenula polymorpha, Pichia pastoris) can grow on methanol as sole source of carbon and energy. These organisms are important cell factories for the production of recombinant proteins, but are also used in fundamental research as model organisms to study peroxisome biology. During exponential growth on glucose, cells of H. polymorpha typically contain a single, small peroxisome that is redundant for growth while on methanol multiple, enlarged peroxisomes are present. These organelles are crucial to support growth on methanol, as they contain key enzymes of methanol metabolism.In this study, changes in the transcriptional profiles during adaptation of H. polymorpha cells from glucose- to methanol-containing media were investigated using DNA-microarray analyses. RESULTS: Two hours after the shift of cells from glucose to methanol nearly 20% (1184 genes) of the approximately 6000 annotated H. polymorpha genes were significantly upregulated with at least a two-fold differential expression. Highest upregulation (> 300-fold) was observed for the genes encoding the transcription factor Mpp1 and formate dehydrogenase, an enzyme of the methanol dissimilation pathway. Upregulated genes also included genes encoding other enzymes of methanol metabolism as well as of peroxisomal beta-oxidation.A moderate increase in transcriptional levels (up to 4-fold) was observed for several PEX genes, which are involved in peroxisome biogenesis. Only PEX11 and PEX32 were higher upregulated. In addition, an increase was observed in expression of the several ATG genes, which encode proteins involved in autophagy and autophagy processes. The strongest upregulation was observed for ATG8 and ATG11.Approximately 20% (1246 genes) of the genes were downregulated. These included glycolytic genes as well as genes involved in transcription and translation. CONCLUSION: Transcriptional profiling of H. polymorpha cells shifted from glucose to methanol showed the expected downregulation of glycolytic genes together with upregulation of the methanol utilisation pathway. This serves as a confirmation and validation of the array data obtained. Consistent with this, also various PEX genes were upregulated. The strong upregulation of ATG genes is possibly due to induction of autophagy processes related to remodeling of the cell architecture required to support growth on methanol. These processes may also be responsible for the enhanced peroxisomal beta-oxidation, as autophagy leads to recycling of membrane lipids. The prominent downregulation of transcription and translation may be explained by the reduced growth rate on methanol (td glucose 1 h vs td methanol 4.5 h).
| S-EPMC2827406 | biostudies-literature
Project description:Rif1 is a large multifaceted protein involved in various processes of DNA metabolism - from telomere length regulation and replication to double-strand break repair. The mechanistic details of its action, however, are often poorly understood. Here, we report functional characterization of the Rif1 homologue from methylotrophic thermotolerant budding yeast Hansenula polymorpha DL-1. We show that, similar to other yeast species, H. polymorpha Rif1 suppresses telomerase-dependent telomere elongation. We uncover two novel modes of Rif1 recruitment at H. polymorpha telomeres: via direct DNA binding and through the association with the Ku heterodimer. Both of these modes (at least partially) require the intrinsically disordered N-terminal extension - a region of the protein present exclusively in yeast species. We also demonstrate that Rif1 binds Stn1 and promotes its accumulation at telomeres in H. polymorpha.
| S-EPMC8820739 | biostudies-literature
Project description:Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by β-1,4 and β-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors' ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.
| S-EPMC7913781 | biostudies-literature
Project description:BackgroundHansenula polymorpha DL1 is a methylotrophic yeast, widely used in fundamental studies of methanol metabolism, peroxisome biogenesis and function, and also as a microbial cell factory for production of recombinant proteins and metabolic engineering towards the goal of high temperature ethanol production.ResultsWe have sequenced the 9 Mbp H. polymorpha DL1 genome and performed whole-genome analysis for the H. polymorpha transcriptome obtained from both methanol- and glucose-grown cells. RNA-seq analysis revealed the complex and dynamic character of the H. polymorpha transcriptome under the two studied conditions, identified abundant and highly unregulated expression of 40% of the genome in methanol grown cells, and revealed alternative splicing events. We have identified subtelomerically biased protein families in H. polymorpha, clusters of LTR elements at G + C-poor chromosomal loci in the middle of each of the seven H. polymorpha chromosomes, and established the evolutionary position of H. polymorpha DL1 within a separate yeast clade together with the methylotrophic yeast Pichia pastoris and the non-methylotrophic yeast Dekkera bruxellensis. Intergenome comparisons uncovered extensive gene order reshuffling between the three yeast genomes. Phylogenetic analyses enabled us to reveal patterns of evolution of methylotrophy in yeasts and filamentous fungi.ConclusionsOur results open new opportunities for in-depth understanding of many aspects of H. polymorpha life cycle, physiology and metabolism as well as genome evolution in methylotrophic yeasts and may lead to novel improvements toward the application of H. polymorpha DL-1 as a microbial cell factory.
| S-EPMC3866509 | biostudies-literature
Project description:Background and objectivesDuring past decades Hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer.Materials and methodsThe corresponding nucleotide sequences for the expression of heterologous genes in Hansenula poylmorpha were extracted and assembled in an E. coli vector. The constructed expression cassette included formate dehydrogenase promoter (pFMD), a secretory signal sequence, a multiple cloning site (MCS) and methanol oxidase (MOX) terminator. Zeocin resistance gene fragment and complete cDNA encoding granulocyte colony stimulating factor (GCSF) were cloned downstream of the expression cassette in-frame with signal sequence. Restriction mapping and sequence analysis confirmed the correct cloning procedures. Final vector was transformed into Hansenula and recombinant host was induced for the expression of GCSF protein by adding methanol. SDS-PAGE and immuno-blotting were performed to confirm the identity of r-GCSF.ResultsThe expression cassette containing gcsf gene (615bp) and zeocin resistance marker (sh-ble, 1200bp) was prepared and successfully transformed into competent Hansenula polymorpha cells via electroporation. Zeocin resistant colonies were selected and GCSF expression was induced in recombinant Hansenula transformants using 0.5% methanol and an approximately 19kDa protein was observed on SDS-PAGE. Western blot analysis using serum isolated from GCSF-treated rabbit confirmed the identity of the protein.ConclusionsMolecular studies confirmed the designed expression cassette containing gcsf gene along with pFMD and signal sequence. The expressed 19kDa protein also confirmed the ability of designed vector in expressing heterologous genes in Hansenula cells.
| S-EPMC4833737 | biostudies-literature
Project description:The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed. Copper amine oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches. A crystal structure of H. polymorpha amine oxidase in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O(2) free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O(2) migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall k(cat)/K(m) (O(2)), supporting multiple dynamic pathways for molecular oxygen to reach the active site.
| S-EPMC3081669 | biostudies-literature