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

Guidelines for Negotiating Scientific Collaboration

Whether it's sharing reagents with a laboratory on the other side of the world or working with the postdoc at the neighboring bench, some simple rules of collaboration might help

LGBTQ Inequality in Engineering Education

BackgroundResearchers over the past three decades have documented processes of gender and racial/ethnic inequality in engineering education but little is known about other axes of difference, including the experiences of lesbian, gay, bisexual, transgender, and queer (LGBTQ) persons in engineering. Despite growing interest in LGBTQ inequality generally, prior research has yet to systematically document day‐to‐day experiences of inequality in engineering education along LGBTQ status.Purpose/HypothesisIn this article, we use survey data from students enrolled in eight universities to examine LGBTQ inequality in engineering education. Specifically, we explore whether LGBTQ students experience greater marginalization than their classmates, whether their engineering work is more likely to be devalued, and whether they experience more negative health and wellness outcomes. We hypothesize that LGBTQ students experience greater marginalization and devaluation and more negative health and wellness outcomes compared to their non‐LGBTQ peers.Data/MethodWe analyzed novel survey data from 1,729 undergraduate students (141 of whom identify as LGBTQ) enrolled in eight U.S. engineering programs.ResultsWe found that LGBTQ students face greater marginalization, devaluation, and health and wellness issues relative to their peers, and that these health and wellness inequalities are explained in part by LGBTQ students’ experiences of marginalization and devaluation in their engineering programs. Furthermore, there is little variation in the climate for LGBTQ students across the eight schools, suggesting that anti‐LGBTQ bias may be widespread in engineering education.ConclusionsWe call for reflexive research on LGBTQ inequality in engineering education and the institutional and cultural shifts needed to mitigate these processes and better support LGBTQ students.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146822/1/jee20239.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146822/2/jee20239_am.pd

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

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

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

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

RNA Recognition by the DNA End-Binding Ku Heterodimer

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem–loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem–loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer