Maria Teresa Teixeira

Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.

Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs. We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy‐terminal domain (C‐Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG‐containing amino‐terminal domain (N‐Nup145p) which is not part of this complex. Whereas the essential C‐Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N‐Nup145p, which is homologous to the GLFG‐containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth. However, the N‐Nup145p becomes essential in a nup188 mutant background. Strikingly, generation of a free N‐domain is a prerequisite for complementation of this peculiar synthetic lethal mutant. These data suggest that N‐ and C‐domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance.

Telomere maintenance, function and evolution: the yeast paradigm

Telomeres are multifunctional genetic elements that cap chromosome ends, playing essential roles in genome stability, chromosome higher-order organization and proliferation control. The telomere field has largely benefited from the study of unicellular eukaryotic organisms such as yeasts. Easy cultivation in laboratory conditions and powerful genetics have placed mainly Saccharomyces cerevisiae, Kluveromyces lactis and Schizosaccharomyces pombe as crucial model organisms for telomere biology research. Studies in these species have made it possible to elucidate the basic mechanisms of telomere maintenance, function and evolution. Moreover, comparative genomic analyses show that telomeres have evolved rapidly among yeast species and functional plasticity emerges as one of the driving forces of this evolution. This provides a precious opportunity to further our understanding of telomere biology.

Genome-wide nuclear morphology screen identifies novel genes involved in nuclear architecture and gene-silencing in Saccharomyces cerevisiae

Organisation of the cell nucleus is crucial for the regulation of gene expression but little is known about how nuclei are structured. To address this issue, we designed a genomic screen to identify factors involved in nuclear architecture in Saccharomyces cerevisiae. This screen is based on microscopic monitoring of nuclear pore complexes and nucleolar proteins fused with the green fluorescent protein in a collection of approximately 400 individual deletion mutants. Among the 12 genes identified by this screen, most affect both the nuclear envelope and the nucleolar morphology. Corresponding gene products are localised preferentially to the nucleus or close to the nuclear periphery. Interestingly, these nuclear morphology alterations were associated with chromatin-silencing defects. These genes provide a molecular context to explore the functional link between nuclear architecture and gene silencing.

The Length of the Shortest Telomere as the Major Determinant of the Onset of Replicative Senescence

The absence of telomerase in many eukaryotes leads to the gradual shortening of telomeres, causing replicative senescence. In humans, this proliferation barrier constitutes a tumor suppressor mechanism and may be involved in cellular aging. Yet the heterogeneity of the senescence phenotype has hindered the understanding of its onset. Here we investigated the regulation of telomere length and its control of senescence heterogeneity. Because the length of the shortest telomeres can potentially regulate cell fate, we focus on their dynamics in Saccharomyces cerevisiae. We developed a stochastic model of telomere dynamics built on the protein-counting model, where an increasing number of protein-bound telomeric repeats shift telomeres into a nonextendable state by telomerase. Using numerical simulations, we found that the length of the shortest telomere is well separated from the length of the others, suggesting a prominent role in triggering senescence. We evaluated this possibility using classical genetic analyses of tetrads, combined with a quantitative and sensitive assay for senescence. In contrast to mitosis of telomerase-negative cells, which produces two cells with identical senescence onset, meiosis is able to segregate a determinant of senescence onset among the telomerase-negative spores. The frequency of such segregation is in accordance with this determinant being the length of the shortest telomere. Taken together, our results substantiate the length of the shortest telomere as being the key genetic marker determining senescence onset in S. cerevisiae.

Self-catalyzed Cleavage of the Yeast Nucleoporin Nup145p Precursor

Nup145p is a component of the nuclear pore complex of Saccharomyces cerevisiae and is essential for mRNA export. Nup145p and its apparent vertebrate homologue are the only known nucleoporins to be composed of two functionally independent peptide moieties resulting from the post-translational cleavage of a large precursor molecule. In this study, the proteolytic cleavage site of Nup145p has been mapped upstream of an evolutionary conserved serine residue. Cleavage occurs at the same site when a precursor is artificially expressed in Escherichia coli. A hydroxyl-containing residue is critical for the reaction, although a thiol-containing residue offers an acceptable replacement. In vitro kinetics experiments using a purified precursor molecule demonstrate that the cleavage is self-catalyzed and that the catalytic domain lies within the N-terminal moiety. Taken altogether, our data are consistent with a proteolytic mechanism involving an N>O acyl rearrangement and a subsequent ester intermediate uncovered in other self-processing proteins.

Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle

Maintenance of a minimal telomere length is essential to prevent cellular senescence. When critically short telomeres arise in the absence of telomerase, they can be repaired by homology-directed repair (HDR) to prevent premature senescence onset. It is unclear why specifically the shortest telomeres are targeted for HDR. We demonstrate that the non-coding RNA TERRA accumulates as HDR-promoting RNA-DNA hybrids (R-loops) preferentially at very short telomeres. The increased level of TERRA and R-loops, exclusively at short telomeres, is due to a local defect in RNA degradation by the Rat1 and RNase H2 nucleases, respectively. Consequently, the coordination of TERRA degradation with telomere replication is altered at shortened telomeres. R-loop persistence at short telomeres contributes to activation of the DNA damage response (DDR) and promotes recruitment of the Rad51 recombinase. Thus, the telomere length-dependent regulation of TERRA and TERRA R-loops is a critical determinant of the rate of replicative senescence.

Large telomerase RNA, telomere length heterogeneity and escape from senescence in Candida glabrata

Telomerase, the key enzyme essential for the maintenance of eukaryotic chromosome ends, contains a reverse transcriptase and an RNA that provides the template for the synthesis of telomeric repeats. Here, we characterize the telomerase subunits in the hemiascomycete yeast Candida glabrata. We propose a secondary structure model for the telomerase RNA that is the largest described to date. Telomerase deletion mutants show a progressive shortening of telomeres and a modest loss of viability. Frequent post‐senescence survivors emerge that possess long telomeric repeat tracts. We suggest that the high telomere length heterogeneity accounts for this distinct senescence phenotype.

Length-dependent processing of telomeres in the absence of telomerase

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5′-3′ resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5′-3′ resection at the shortest telomeres.

Trans-generational Immune Priming Protects the Eggs Only against Gram-Positive Bacteria in the Mealworm Beetle.

In many vertebrates and invertebrates, offspring whose mothers have been exposed to pathogens can exhibit increased levels of immune activity and/or increased survival to infection. Such phenomena, called “Trans-generational immune priming” (TGIP) are expected to provide immune protection to the offspring. As the offspring and their mother may share the same environment, and consequently similar microbial threats, we expect the immune molecules present in the progeny to be specific to the microbes that immune challenged the mother. We provide evidence in the mealworm beetle Tenebrio molitor that the antimicrobial activity found in the eggs is only active against Gram-positive bacteria, even when females were exposed to Gram-negative bacteria or fungi. Fungi were weak inducers of TGIP while we obtained similar levels of anti-Gram-positive activity using different bacteria for the maternal challenge. Furthermore, we have identified an antibacterial peptide from the defensin family, the tenecin 1, which spectrum of activity is exclusively directed toward Gram-positive bacteria as potential contributor to this antimicrobial activity. We conclude that maternal transfer of antimicrobial activity in the eggs of T. molitor might have evolved from persistent Gram-positive bacterial pathogens between insect generations.

Saccharomyces cerevisiae as a Model to Study Replicative Senescence Triggered by Telomere Shortening.

In many somatic human tissues, telomeres shorten progressively because of the DNA-end replication problem. Consequently, cells cease to proliferate and are maintained in a metabolically viable state called replicative senescence. These cells are characterized by an activation of DNA damage checkpoints stemming from eroded telomeres, which are bypassed in many cancer cells. Hence, replicative senescence has been considered one of the most potent tumor suppressor pathways. However, the mechanism through which short telomeres trigger this cellular response is far from being understood. When telomerase is removed experimentally in Saccharomyces cerevisiae, telomere shortening also results in a gradual arrest of population growth, suggesting that replicative senescence also occurs in this unicellular eukaryote. In this review, we present the key steps that have contributed to the understanding of the mechanisms underlying the establishment of replicative senescence in budding yeast. As in mammals, signals stemming from short telomeres activate the DNA damage checkpoints, suggesting that the early cellular response to the shortest telomere(s) is conserved in evolution. Yet closer analysis reveals a complex picture in which the apparent single checkpoint response may result from a variety of telomeric alterations expressed in the absence of telomerase. Accordingly, the DNA replication of eroding telomeres appears as a critical challenge for senescing budding yeast cells and the easy manipulation of S. cerevisiae is providing insights into the way short telomeres are integrated into their chromatin and nuclear environments. Finally, the loss of telomerase in budding yeast triggers a more general metabolic alteration that remains largely unexplored. Thus, telomerase-deficient S. cerevisiae cells may have more common points than anticipated with somatic cells, in which telomerase depletion is naturally programed, thus potentially inspiring investigations in mammalian cells.