Investigating the role of the Est3 protein in yeast telomere replication

The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Δ strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of the S. castellii telomerase RNA. Furthermore, using nuclear magnetic resonance chemical shift perturbation, no chemical shift change was observed at any site in the protein upon addition of single-stranded DNA, arguing against a role for Est3 in recognition of telomeric substrates by telomerase. Addition of exogenous Est3 protein, including mutant Est3 proteins that are severely impaired for telomere replication in vivo, fully restored activity in est3-Δ telomerase reactions. Thus, Est3 performs an in vivo regulatory function in telomere replication, which is distinct from any potential contribution that Est3 might make to telomerase activity

Structure of the Essential Yeast Telomerase Protein Est3 from S. cerevisiae and Structure-Guided Investigation of Function

Telomeres are the nucleoprotein complexes at the ends of linear chromosomes that protect the genome from degradation and chromosomal fusions. Telomeres are replicated by the specialized enzyme telomerase. The telomerase holoenzyme in S. cerevisiae contains an RNA template and three known protein subunits, Est1, Est2 and Est3 (est = ever shorter telomeres for the phenotype observed upon their deletion). The reverse transcriptase Est2 and the RNA template TLC1 constitute the catalytic core of the telomerase holoenzyme. While Est1 and Est3 are not required for catalysis in vitro, they are strictly required for activity in vivo. The function of Est3 has remained elusive, although genetic data suggests that one mode of Est3\u27s function might be carried out via its interaction with Est2. To provide insights into Est3 function, we have solved its high resolution structure. Because Est3 is a difficult protein target for structure elucidation, the structure was solved using a relatively novel strategy of combining minimal NMR experimental data (chemical shifts, RDCs and NOEs) with Rosetta de novo structure prediction. The structure is an OB-fold, with five-stranded β-barrel, capped with an α-helix and has some specialized features that distinguish it from other OB folds. The canonical loop L45 is quite unusual in the case of Est3, in that it is long and highly structured and plugs on top of the OB-fold canonical ligand binding face. Even in the absence of appreciable sequence relationship, the Est3 structure shows remarkable similarity to HsTPP1-OB structure, not only in the core β-barrel, but also in the positioning of the α-helix at the base and placement of C-terminal tail partially covering the canonical OB-fold binding face. Mapping residues involved in telomerase-association onto the structure reveals a novel protein interaction surface at the base of the β-barrel for Est3 and TPP1-OB. In vivo analysis, using structure-guided mutagenesis of Est3 surface also identified several new, functionally relevant, residues of Est3. The structure also served as a validation tool for an in vivo guided in vitro study that showed a direct correlation of in vivo dominant-negative mutants with their structural retention in vitro

Partners in crime: the TGFβ and MAPK pathways in cancer progression

Abstract The TGFβ and Ras-MAPK pathways play critical roles in cell development and cell cycle regulation, as well as in tumor formation and metastasis. In the absence of cellular transformation, these pathways operate in opposition to one another, where TGFβ maintains an undifferentiated cell state and suppresses proliferation, while Ras-MAPK pathways promote proliferation, survival and differentiation. However, in colorectal and pancreatic cancers, the opposing pathways' mechanisms are simultaneously activated in order to promote cancer progression and metastasis. Here, we highlight the roles of the TGFβ and Ras-MAPK pathways in normal and malignant states, and provide an explanation for how the concomitant activation of these pathways drives tumor biology. Finally, we survey potential therapeutic targets in these pathways.</p

Drosophila polypyrimidine tract-binding protein is necessary for spermatid individualization

Although mammalian polypyrimidine tract-binding (PTB) protein functions in most or all cell types to regulate a wide spectrum of transcripts, Drosophila PTB encodes an abundant male germline-specific mRNA isoform (dmPTB) whose expression correlates with male fertility. The biological function of this isoform is unknown. Using selection–amplification, we show that mammalian and Drosophila PTB have similar RNA sequence preference, suggesting that cell-specific expression rather than unique RNA-binding properties account for the sex-specific function of dmPTB. We also show that the dmPTB protein isoform expressed in the male germline is by far the most abundant isoform, and reduction of its levels correlates with male sterility. Finally, we show that dmPTB expression is necessary for proper spermatid individualization, the terminal step necessary for production of motile sperm. Loss of dmPTB results in severe disruption of the actin cones of the spermatid individualization complex. This represents a cytological defect resulting from PTB loss. We discuss the basis for functional differences between mammalian and Drosophila PTB orthologs