Regulation of telomerase reverse transcriptase expression in Schizosaccharomyces pombe

Eukaryotic cells undergo chromosome shortening during each cell division due to the inability of DNA polymerase to replicate chromosome ends. This end replication problem is counteracted by the ribonucleoprotein telomerase, which functions as a reverse transcriptase to add DNA repeats to chromosome ends. These DNA repeats, called telomeres, delay cellular senescence and recruit DNA binding proteins which protect chromosome ends and distinguish them from double-strand breaks. Telomere attrition is thought to function as a means of tumor suppression as cells can only undergo a limited number of divisions in the absence of telomerase or an alternative mechanism for replenishing DNA at chromosome ends. In multicellular organisms, telomerase is present and active during the early stages of development, but is later downregulated, resulting in little or no activity in most somatic cell types. In contrast, approximately 90% of cancer cells do have detectable telomerase activity. This discovery has identified telomerase components, and other molecules regulating telomerase function, as potential targets for cancer treatment. Unlike multicellular organisms, single-celled organisms, such as the fission yeast Schizosaccharomyces pombe, are widely believed to constitutively express telomerase, despite insufficient studies on expression or activity in this species outside of typical laboratory conditions. Surprisingly, we have observed that the level of telomerase reverse transcriptase (Trt1) protein in this organism decreases in cells which have been arrested by entry into stationary phase, which corresponds with a decrease in in vitro telomerase activity from these cells. This decrease in Trt1 level is similarly observed in cells which have been starved of nitrogen or glucose, and the rate of turnover appears to be quite rapid. The downregulation of Trt1 protein is independent of trt1 mRNA transcript level, and regions within the protein coding sequence appear to be sufficient for causing the observed decrease in expression. These results indicate that telomerase reverse transcriptase expression in the fission yeast S. pombe is regulated in nutrient-starved cells, likely through degradation of the protein, as triggered by multiple protein sequence or structural elements. Further studies in this model organism will likely reveal great insights into mechanisms and functions of telomerase regulation

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Evolutionary Emergence, Optimization, and Co-Option of Aminoacylation Ribozymes

Understanding molecular evolution can reveal a great deal about the past, present, and future of biological systems. The evolution of catalytic RNA is of particular interest because of its potential role in an ‘RNA World’ at the origin of life. Two crucial aspects in the evolution of biomolecules are optimization on the fitness landscape and co-option for new functions. The fitness landscape describes a function of fitness in the space of all possible sequences. Molecules evolve through a random walk on the fitness landscape, with a bias toward climbing peaks. In addition, the ability of enzymes, including ribozymes, to catalyze side reactions is believed to be essential to the evolution of novel biochemical activities. It has been speculated that the earliest ribozymes were low in activity but high in promiscuity, which then gave rise to specialized descendants with higher activity and specificity. One particularly essential activity for the origin of life would be the reaction of ribozymes with activated amino acids to form aminoacyl-RNAs, with co-option of these aminoacyl-RNAs leading to genetic code expansion. In this work, self-aminoacylating ribozymes were identified through in vitro selection from full coverage of sequence space and characterized using a massively parallel kinetic assay. Three major sequence motifs were identified on the landscape and analysis of evolutionary pathways revealed that, while local optimization within a ribozyme family would be possible, optimization of activity over the entire landscape would be frustrated by large valleys of low activity. The sequence motifs associated with each peak represent different solutions for catalysis, so the inability to traverse the landscape globally corresponds to an inability to restructure the ribozyme without losing activity. In addition, five families representing the three sequence motifs were further investigated to measure their activity with alternative substrates. Ribozymes in each family displayed high levels of co-optability, with activity on multiple substrates, demonstrating that co-option for a new function can occur more readily than optimization of an existing one. Related ribozymes exhibited preferences for biophysically similar substrates, indicating that co-option of existing ribozymes to adopt additional amino acids into the genetic code would itself lead to error minimization. Furthermore, ribozyme activity was positively correlated with specificity, indicating that selection for increased activity would also lead to increased specificity. These results demonstrate how the genetic code may have evolved through the emergence and co-option of aminoacylation ribozymes

Evolutionary Emergence, Optimization, and Co-Option of Aminoacylation Ribozymes

Understanding molecular evolution can reveal a great deal about the past, present, and future of biological systems. The evolution of catalytic RNA is of particular interest because of its potential role in an ‘RNA World’ at the origin of life. Two crucial aspects in the evolution of biomolecules are optimization on the fitness landscape and co-option for new functions. The fitness landscape describes a function of fitness in the space of all possible sequences. Molecules evolve through a random walk on the fitness landscape, with a bias toward climbing peaks. In addition, the ability of enzymes, including ribozymes, to catalyze side reactions is believed to be essential to the evolution of novel biochemical activities. It has been speculated that the earliest ribozymes were low in activity but high in promiscuity, which then gave rise to specialized descendants with higher activity and specificity. One particularly essential activity for the origin of life would be the reaction of ribozymes with activated amino acids to form aminoacyl-RNAs, with co-option of these aminoacyl-RNAs leading to genetic code expansion. In this work, self-aminoacylating ribozymes were identified through in vitro selection from full coverage of sequence space and characterized using a massively parallel kinetic assay. Three major sequence motifs were identified on the landscape and analysis of evolutionary pathways revealed that, while local optimization within a ribozyme family would be possible, optimization of activity over the entire landscape would be frustrated by large valleys of low activity. The sequence motifs associated with each peak represent different solutions for catalysis, so the inability to traverse the landscape globally corresponds to an inability to restructure the ribozyme without losing activity. In addition, five families representing the three sequence motifs were further investigated to measure their activity with alternative substrates. Ribozymes in each family displayed high levels of co-optability, with activity on multiple substrates, demonstrating that co-option for a new function can occur more readily than optimization of an existing one. Related ribozymes exhibited preferences for biophysically similar substrates, indicating that co-option of existing ribozymes to adopt additional amino acids into the genetic code would itself lead to error minimization. Furthermore, ribozyme activity was positively correlated with specificity, indicating that selection for increased activity would also lead to increased specificity. These results demonstrate how the genetic code may have evolved through the emergence and co-option of aminoacylation ribozymes

Kinetic sequencing (k-Seq) as a massively parallel assay for ribozyme kinetics: utility and critical parameters.

Characterizing genotype-phenotype relationships of biomolecules (e.g. ribozymes) requires accurate ways to measure activity for a large set of molecules. Kinetic measurement using high-throughput sequencing (e.g. k-Seq) is an emerging assay applicable in various domains that potentially scales up measurement throughput to over 106 unique nucleic acid sequences. However, maximizing the return of such assays requires understanding the technical challenges introduced by sequence heterogeneity and DNA sequencing. We characterized the k-Seq method in terms of model identifiability, effects of sequencing error, accuracy and precision using simulated datasets and experimental data from a variant pool constructed from previously identified ribozymes. Relative abundance, kinetic coefficients, and measurement noise were found to affect the measurement of each sequence. We introduced bootstrapping to robustly quantify the uncertainty in estimating model parameters and proposed interpretable metrics to quantify model identifiability. These efforts enabled the rigorous reporting of data quality for individual sequences in k-Seq experiments. Here we present detailed protocols, define critical experimental factors, and identify general guidelines to maximize the number of sequences and their measurement accuracy from k-Seq data. Analogous practices could be applied to improve the rigor of other sequencing-based assays

Molecular Fitness Landscapes from High-Coverage Sequence Profiling.

The function of fitness (or molecular activity) in the space of all possible sequences is known as the fitness landscape. Evolution is a random walk on the fitness landscape, with a bias toward climbing hills. Mapping the topography of real fitness landscapes is fundamental to understanding evolution, but previous efforts were hampered by the difficulty of obtaining large, quantitative data sets. The accessibility of high-throughput sequencing (HTS) has transformed this study, enabling large-scale enumeration of fitness for many mutants and even complete sequence spaces in some cases. We review the progress of high-throughput studies in mapping molecular fitness landscapes, both in vitro and in vivo, as well as opportunities for future research. Such studies are rapidly growing in number. HTS is expected to have a profound effect on the understanding of real molecular fitness landscapes

Promiscuous Ribozymes and Their Proposed Role in Prebiotic Evolution.

The ability of enzymes, including ribozymes, to catalyze side reactions is believed to be essential to the evolution of novel biochemical activities. It has been speculated that the earliest ribozymes, whose emergence marked the origin of life, were low in activity but high in promiscuity, and that these early ribozymes gave rise to specialized descendants with higher activity and specificity. Here, we review the concepts related to promiscuity and examine several cases of highly promiscuous ribozymes. We consider the evidence bearing on the question of whether de novo ribozymes would be quantitatively more promiscuous than later evolved ribozymes or protein enzymes. We suggest that while de novo ribozymes appear to be promiscuous in general, they are not obviously more promiscuous than more highly evolved or active sequences. Promiscuity is a trait whose value would depend on selective pressures, even during prebiotic evolution

Promiscuous Ribozymes and Their Proposed Role in Prebiotic Evolution.

The ability of enzymes, including ribozymes, to catalyze side reactions is believed to be essential to the evolution of novel biochemical activities. It has been speculated that the earliest ribozymes, whose emergence marked the origin of life, were low in activity but high in promiscuity, and that these early ribozymes gave rise to specialized descendants with higher activity and specificity. Here, we review the concepts related to promiscuity and examine several cases of highly promiscuous ribozymes. We consider the evidence bearing on the question of whether de novo ribozymes would be quantitatively more promiscuous than later evolved ribozymes or protein enzymes. We suggest that while de novo ribozymes appear to be promiscuous in general, they are not obviously more promiscuous than more highly evolved or active sequences. Promiscuity is a trait whose value would depend on selective pressures, even during prebiotic evolution