Beginning to Understand the End of the Chromosome

In their 1985 Cell paper, Greider and Blackburn announced the discovery of an enzyme that extended the DNA at chromosome telomeres in the ciliate, Tetrahymena. Since then, there has been an explosion of knowledge about both the RNA and protein subunits of this unusual ribonucleoprotein enzyme in organisms ranging from the ciliates to yeast to humans. The regulation of telomerase is now understood to take place both at the level of synthesis of the enzyme and via the state of its substrate, the telomere itself. The roles of telomerase in both cellular immortality and cancer are vibrant areas of current research

Pot1, the Putative Telomere End-binding Protein in Fission Yeast and Humans

Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes. We have now identified homologous proteins in fission yeast and in humans. These Pot1 (protection oftelomeres) proteins each bind the G-rich strand of their own telomeric repeat sequence, consistent with a direct role in protecting chromosome ends. Deletion of the fission yeastpot1 +gene has an immediate effect on chromosome stability, causing rapid loss of telomeric DNA and chromosome circularization. It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom

Engineering Disulfide Cross‐Links in RNA Using Thiol‐Disulfide Interchange Chemistry

Protocols for postsynthetic modification of 2‐amino‐containing oligoribonucleotides with either an alkyl‐phenyl disulfide or an alkyl thiol group are described. These groups react under mild conditions to form disulfide cross‐links by thiol‐disulfide interchange. These reactants do not form a disulfide bond when incorporated on opposite faces of a short continuous RNA helix, but do form disulfide bonds rapidly when they are placed in proximity. In addition, by incorporating these groups at various positions on large RNAs by semisynthesis, the dynamics of thermal motions can be detected. Such motions are believed to be linked to biological function, and the protocols presented in this unit are among the few simple ways to assess such dynamics.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143724/1/cpnc0501.pd

Crawling Out of the RNA World

Comparison of phylogenetically diverse ribonucleoprotein (RNP) enzymes and information about their biochemistry have stimulated hypotheses about their evolution. Instead of the canonical view, in which catalysis proceeds from ribozyme to RNP enzyme to protein enzyme, RNP enzymes and proteins are seen to share contemporary catalysis. Furthermore, the RNA components of RNP enzymes show no evidence of fading out but instead, in some cases, have elaborated new functions

RNase P Branches Out from RNP to Protein: Organelle-Triggered Diversification?

RNase P is the enzyme that removes 5′ leader sequences from precursor tRNAs. Remarkably, in most organisms, RNase P is a ribonucleoprotein particle where the RNA component is responsible for catalysis. In this issue of Genes \u26 Development, Gutmann and colleagues (pp. 1022–1027) report the first organism,Arabidopsis thaliana, to employ protein-only RNase P in both its nucleus and organelles. An intriguing possibility is that replacement of RNase P ribonucleoprotein particles (RNPs) by proteins may have been triggered by the acquisition of organelles

Local RNA Structural Changes Induced by Crystallization are Revealed by SHAPE

We present a simple approach to locate sites that undergo conformational changes upon crystallization by comparative structural mapping of the same RNA in three different environments. As a proof of principle, we probed the readily crystallized P4–P6ΔC209 domain from the Tetrahymena thermophila group I intron in a native solution, in a solution mimicking the crystallization drop, and in crystals. We chose the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, which monitors the flexibility and the conformation of each nucleotide. First, SHAPE successfully revealed the structural changes that occur during the crystallization process. Specifically, 64% of the nucleotides implicated in packing contacts and present in the portion of the molecule analyzed were identified. Second, reactivity differences for some of these nucleotides were already observed in the crystallization solution, suggesting that the crystallization buffer locked down a particular structure that was favorable to crystal formation. Third, the probing of a known structure extends our understanding of the structural basis for the SHAPE reaction by suggesting that reactivity is enhanced by a C2′-endo sugar pucker. Furthermore, by identifying local conformational changes of the RNA that take place during crystallization, SHAPE could be combined with the in vitro selection of stable mutants to rationalize the design of RNA candidates for crystallization

In vivo Selection of Better Self-Splicing Introns in E. coli: the Role of the P1 Extension Helix of the Tetrahymena Intron

In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.</p

Structure and Function of Steroid Receptor RNA Activator Protein, the Proposed partner of SRA noncoding RNA

In a widely accepted model, the steroid receptor RNA activator protein (SRA protein; SRAP) modulates the transcriptional regulatory activity of SRA RNA by binding a specific stem–loop of SRA. We first confirmed that SRAP is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, where it is expressed at the level of about 105 molecules per cell. However, our SRAP–RNA binding experiments, both in vitro with recombinant protein and in cultured cells with plasmid-expressed protein and RNA, did not reveal a specific interaction between SRAP and SRA. We determined the crystal structure of the carboxy-terminal domain of human SRAP and found that it does not have the postulated RRM (RNA recognition motif). The structure is a five-helix bundle that is distinct from known RNA-binding motifs and instead is similar to the carboxy-terminal domain of the yeast spliceosome protein PRP18, which stabilizes specific protein–protein interactions within a multisubunit mRNA splicing complex. SRA binding experiments with this domain gave negative results. Transcriptional regulation by SRA/SRAP was examined with siRNA knockdown. Effects on both specific estrogen-responsive genes and genes identified by RNA-seq as candidates for regulation were examined in MCF-7 cells. Only a small effect (~ 20% change) on one gene resulting from depletion of SRA/SRAP could be confirmed. We conclude that the current model for SRAP function must be reevaluated; we suggest that SRAP may function in a different context to stabilize specific intermolecular interactions in the nucleus

Global expression changes resulting from loss of telomeric DNA in fission yeast

BACKGROUND: Schizosaccharomyces pombe cells lacking the catalytic subunit of telomerase (encoded by trt1(+)) lose telomeric DNA and enter crisis, but rare survivors arise with either circular or linear chromosomes. Survivors with linear chromosomes have normal growth rates and morphology, but those with circular chromosomes have growth defects and are enlarged. We report the global gene-expression response of S. pombe to loss of trt1(+). RESULTS: Survivors with linear chromosomes had expression profiles similar to cells with native telomeres, whereas survivors with circular chromosomes showed continued upregulation of core environmental stress response (CESR) genes. In addition, survivors with circular chromosomes had altered expression of 51 genes compared to survivors with linear chromosomes, providing an expression signature. S. pombe progressing through crisis displayed two waves of altered gene expression. One coincided with crisis and consisted of around 110 genes, 44% of which overlapped with the CESR. The second was synchronized with the emergence of survivors and consisted of a single class of open reading frames (ORFs) with homology both to RecQ helicases and to dh repeats at centromeres targeted for heterochromatin formation via an RNA interference (RNAi) mechanism. Accumulation of transcript from the ORF was found not only in trt1(- )cells, but also in dcr1(- )and ago1(- )RNAi mutants, suggesting that RNAi may control its expression. CONCLUSIONS: These results demonstrate a correlation between a state of cellular stress, short telomeres and growth defects in cells with circular chromosomes. A putative new RecQ helicase was expressed as survivors emerged and appears to be transcriptionally regulated by RNAi, suggesting that this mechanism operates at telomeres