Mutational Patterns in RNA Secondary Structure Evolution Examined in Three RNA Families

The goal of this work was to study mutational patterns in the evolution of RNA secondary structure. We analyzed bacterial tmRNA, RNaseP and eukaryotic telomerase RNA secondary structures, mapping structural variability onto phylogenetic trees constructed primarily from rRNA sequences. We found that secondary structures evolve both by whole stem insertion/deletion, and by mutations that create or disrupt stem base pairing. We analyzed the evolution of stem lengths and constructed substitution matrices describing the changes responsible for the variation in the RNA stem length. In addition, we used principal component analysis of the stem length data to determine the most variable stems in different families of RNA. This data provides new insights into the evolution of RNA secondary structures and patterns of variation in the lengths of double helical regions of RNA molecules. Our findings will facilitate design of improved mutational models for RNA structure evolution

Ancestral Mutation in Telomerase Causes Defects in Repeat Addition Processivity and Manifests As Familial Pulmonary Fibrosis

The telomerase reverse transcriptase synthesizes new telomeres onto chromosome ends by copying from a short template within its integral RNA component. During telomere synthesis, telomerase adds multiple short DNA repeats successively, a property known as repeat addition processivity. However, the consequences of defects in processivity on telomere length maintenance are not fully known. Germline mutations in telomerase cause haploinsufficiency in syndromes of telomere shortening, which most commonly manifest in the age-related disease idiopathic pulmonary fibrosis. We identified two pulmonary fibrosis families that share two non-synonymous substitutions in the catalytic domain of the telomerase reverse transcriptase gene hTERT: V791I and V867M. The two variants fell on the same hTERT allele and were associated with telomere shortening. Genealogy suggested that the pedigrees shared a single ancestor from the nineteenth century, and genetic studies confirmed the two families had a common founder. Functional studies indicated that, although the double mutant did not dramatically affect first repeat addition, hTERT V791I-V867M showed severe defects in telomere repeat addition processivity in vitro. Our data identify an ancestral mutation in telomerase with a novel loss-of-function mechanism. They indicate that telomere repeat addition processivity is a critical determinant of telomere length and telomere-mediated disease

Telomerase Mechanism of Telomere Synthesis

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines

Structure of human telomerase holoenzyme with bound telomeric DNA

Telomerase adds telomeric repeats at chromosome ends to compensate for telomere loss caused by incomplete genome end replication1. In humans, telomerase is upregulated during embryogenesis and in cancers, while mutations that compromise its function result in diseases2. Our previous 8 Å human telomerase structure revealed vertebrate-specific composition and architecture3, consisting of a catalytic core flexibly tethered to an H/ACA ribonucleoprotein (RNP) lobe by telomerase RNA. To effectively modulate telomerase activity as a therapeutic approach against cancers and diseases, high-resolution structural information is necessary. Here we present the structure of human telomerase holoenzyme bound to a telomeric DNA, determined by cryo-electron microscopy (cryo-EM) at 3.4 Å resolution for the H/ACA RNP and 3.8 Å resolution for the catalytic core. The structure reveals crucial DNA/RNA binding interfaces in telomerase active site and locations of mutations that alter telomerase activity. We identified a histone H2A-H2B dimer within the holoenzyme bound to an essential telomerase RNA motif, suggesting an unexpected role for histones in telomerase RNA folding and function. Furthermore, the first high-resolution structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs and new interactions crucial for understanding the molecular pathology of many disease mutations. Our findings illuminate unanticipated structural details of human telomerase assembly and active site, paving the way for the development of telomerase-targeting therapeutics