Tay1 Protein, a Novel Telomere Binding Factor from Yarrowia lipolytica*

Inspection of the complete genome of the yeast Yarrowia lipolytica for the presence of genes encoding homologues of known telomere-binding proteins surprisingly revealed no counterparts of typical yeast Myb domain-containing telomeric factors including Rap1 or Taz1. Instead, we identified a gene, YALIOD10923g, encoding a protein containing two Myb domains, exhibiting a high degree of similarity to the Myb domain of human telomeric proteins TRF1 and TRF2 and homologous to an essential fission yeast protein Mug152 whose expression is elevated during meiosis. The protein, which we named Tay1p (telomere-associated in Yarrowia lipolytica 1), was purified for biochemical studies. Using a model Y. lipolytica telomere, we demonstrate that the protein preferentially binds to Y. lipolytica telomeric tracts. Tay1p binds along the telomeric tract as dimers and larger oligomers, and it is able to remodel the telomeric DNA into both looped structures and synaptic complexes of two model telomere DNAs. The ability of Tay1p to induce dimerization of telomeres in vitro goes in line with its oligomeric nature, where each oligomer can employ several Myb domains to form intermolecular telomere clusters. We also provide experimental evidence that Tay1p may be associated with Y. lipolytica telomeres in vivo. Together with its homologues from Schizosaccharomyces pombe and several basidiomycetous fungi (Sánchez-Alonso, P., and Guzman, P. (2008) Fungal Genet. Biol. 45, S54–S62), Tay1p constitutes a novel family of putative telomeric factors whose analysis may be instrumental in understanding the function and evolution of double-stranded DNA telomeric proteins

Structural integrity of the PCI domain of eIF3a TIF32 is required for mRNA recruitment to the 43S pre initiation complexes

Transfer of genetic information from genes into proteins is mediated by messenger RNA (mRNA) that must be first recruited to ribosomal pre-initiation complexes (PICs) by a mechanism that is still poorly understood. Recent studies showed that besides eIF4F and poly(A)-binding protein, eIF3 also plays a critical role in this process, yet the molecular mechanism of its action is unknown. We showed previously that the PCI domain of the eIF3c/NIP1 subunit of yeast eIF3 is involved in RNA binding. To assess the role of the second PCI domain of eIF3 present in eIF3a/TIF32, we performed its mutational analysis and identified a 10-Ala-substitution (Box37) that severely reduces amounts of model mRNA in the 43–48S PICs in vivo as the major, if not the only, detectable defect. Crystal structure analysis of the a/TIF32-PCI domain at 2.65-Å resolution showed that it is required for integrity of the eIF3 core and, similarly to the c/NIP1-PCI, is capable of RNA binding. The putative RNA-binding surface defined by positively charged areas contains two Box37 residues, R363 and K364. Their substitutions with alanines severely impair the mRNA recruitment step in vivo suggesting that a/TIF32-PCI represents one of the key domains ensuring stable and efficient mRNA delivery to the PICs

Identification and comparative analysis of telomerase RNAs from Candida species reveal conservation of functional elements

The RNA component of telomerase (telomerase RNA; TER) varies substantially both in sequence composition and size (from ∼150 nucleotides [nt] to >1500 nt) across species. This dramatic divergence has hampered the identification of TER genes and a large-scale comparative analysis of TER sequences and structures among distantly related species. To identify by phylogenetic analysis conserved sequences and structural features of TER that are of general importance, it is essential to obtain TER sequences from evolutionarily distant groups of species, providing enough conservation within each group and enough variation among the groups. To this end, we identified TER genes in several yeast species with relatively large (>20 base pairs) and nonvariant telomeric repeats, mostly from the genus Candida. Interestingly, several of the TERs reported here are longer than all other yeast TERs known to date. Within these TERs, we predicted a pseudoknot containing U-A·U base triples (conserved in vertebrates, budding yeasts, and ciliates) and a three-way junction element (conserved in vertebrates and budding yeasts). In addition, we identified a novel conserved sequence (CS2a) predicted to reside within an internal-loop structure, in all the budding yeast TERs examined. CS2a is located near the Est1p-binding bulge-stem previously identified in Saccharomyces cerevisiae. Mutational analyses in both budding yeasts S. cerevisiae and Kluyveromyces lactis demonstrate that CS2a is essential for in vivo telomerase function. The comparative and mutational analyses of conserved TER elements reported here provide novel insights into the structure and function of the telomerase ribonucleoprotein complex

The conserved histone deacetylase Rpd3 and the DNA binding regulator Ume6 repress BOI1's meiotic transcript isoform during vegetative growth in Saccharomyces cerevisiae.

International audienceBOI1 and BOI2 are paralogs important for the actin cytoskeleton andpolar growth. BOI1 encodes a meiotic transcript isoform with an extended5-untranslated region predicted to impair protein translation. It is,however, unknown how the isoform is repressed during mitosis, and ifBoi1 is present during sporulation. By interpreting microarray data fromMATa cells, MATa/ cells, a starving MAT/ control, and a meiosis-impairedrrp6 mutant, we classified BOI1's extended isoform as earlymeiosis-specific. These results were confirmed by RNA-Sequencing, andextended by a 5-RACE assay and Northern blotting, showing that meioticcells induce the long isoform while the mitotic isoform remainsdetectable during meiosis. We provide evidence via motif predictions, anin vivo binding assay and genetic experiments that the Rpd3/Sin3/Ume6histone deacetylase complex, which represses meiotic genes duringmitosis, also prevents the induction of BOI1's 5-extended isoform inmitosis by direct binding of Ume6 to its URS1 target. Finally, we findthat Boi1 protein levels decline when cells switch from fermentation torespiration and sporulation. The histone deacetylase Rpd3 is conserved,and eukaryotic genes frequently encode transcripts with variable 5-UTRs.Our findings are therefore relevant for regulatory mechanisms involvedin the control of transcript isoforms in multi-cellular organisms

Nontelomeric splice variant of telomere repeat-binding factor 2 maintains neuronal traits by sequestering repressor element 1-silencing transcription factor

Telomere repeat-binding factor 2 (TRF2) is critical for telomere integrity in dividing stem and somatic cells, but its role in postmitotic neurons is unknown. Apart from protecting telomeres, nuclear TRF2 interacts with the master neuronal gene-silencer repressor element 1-silencing transcription factor (REST), and disruption of this interaction induces neuronal differentiation. Here we report a developmental switch from the expression of TRF2 in proliferating neural progenitor cells to expression of a unique short nontelomeric isoform of TRF2 (TRF2-S) as neurons establish a fully differentiated state. Unlike nuclear TRF2, which enhances REST-mediated gene repression, TRF2-S is located in the cytoplasm where it sequesters REST, thereby maintaining the expression of neuronal genes, including those encoding glutamate receptors, cell adhesion, and neurofilament proteins. In neurons, TRF2-S–mediated antagonism of REST nuclear activity is greatly attenuated by either overexpression of TRF2 or administration of the excitatory amino acid kainic acid. Overexpression of TRF2-S rescues kainic acid-induced REST nuclear accumulation and its gene-silencing effects. Thus, TRF2-S acts as part of a unique developmentally regulated molecular switch that plays critical roles in the maintenance and plasticity of neurons