CGG repeats trigger translational frameshifts that generate aggregation-prone chimeric proteins.

CGG repeat expansions in the FMR1 5'UTR cause the neurodegenerative disease Fragile X-associated tremor/ataxia syndrome (FXTAS). These repeats form stable RNA secondary structures that support aberrant translation in the absence of an AUG start codon (RAN translation), producing aggregate-prone peptides that accumulate within intranuclear neuronal inclusions and contribute to neurotoxicity. Here, we show that the most abundant RAN translation product, FMRpolyG, is markedly less toxic when generated from a construct with a non-repetitive alternating codon sequence in place of the CGG repeat. While exploring the mechanism of this differential toxicity, we observed a +1 translational frameshift within the CGG repeat from the arginine to glycine reading frame. Frameshifts occurred within the first few translated repeats and were triggered predominantly by RNA sequence and structural features. Short chimeric R/G peptides form aggregates distinct from those formed by either pure arginine or glycine, and these chimeras induce toxicity in cultured rodent neurons. Together, this work suggests that CGG repeats support translational frameshifting and that chimeric RAN translated peptides may contribute to CGG repeat-associated toxicity in FXTAS and related disorders.

The cAMP signaling pathway regulates Epe1 protein levels and heterochromatin assembly.

The epigenetic landscape of a cell frequently changes in response to fluctuations in nutrient levels, but the mechanistic link is not well understood. In fission yeast, the JmjC domain protein Epe1 is critical for maintaining the heterochromatin landscape. While loss of Epe1 results in heterochromatin expansion, overexpression of Epe1 leads to defective heterochromatin. Through a genetic screen, we found that mutations in genes of the cAMP signaling pathway suppress the heterochromatin defects associated with Epe1 overexpression. We further demonstrated that the activation of Pka1, the downstream effector of cAMP signaling, is required for the efficient translation of epe1+ mRNA to maintain Epe1 overexpression. Moreover, inactivation of the cAMP-signaling pathway, either through genetic mutations or glucose deprivation, leads to the reduction of endogenous Epe1 and corresponding heterochromatin changes. These results reveal the mechanism by which the cAMP signaling pathway regulates heterochromatin landscape in fission yeast.

Pseudouridine synthase 7 is an opportunistic enzyme that binds and modifies substrates with diverse sequences and structures.

Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.

Investigating the consequences of mRNA modifications on protein synthesis using in vitro translation assays.

The ribosome translates the information stored in the genetic code into functional proteins. In this process messenger RNAs (mRNAs) serve as templates for the ribosome, ensuring that amino acids are linked together in the correct order. Chemical modifications to mRNA nucleosides have the potential to influence the rate and accuracy of protein synthesis. Here, we present an in vitro Escherichia coli translation system utilizing highly purified components to directly investigate the impact of mRNA modifications on the speed and accuracy of the ribosome. This system can be used to gain insights into how individual chemical modifications influence translation on the molecular level. While the fully reconstituted system described in this chapter requires a lengthy time investment to prepare experimental materials, it is highly verstaile and enables the systematic assessment of how single variables influence protein synthesis by the ribosome.

A molecular-level perspective on the frequency, distribution, and consequences of messenger RNA modifications.

Cells use chemical modifications to alter the sterics, charge, and conformations of large biomolecules, modulating their biogenesis, function, and stability. Until recently post-transcriptional RNA modifications were thought to be largely limited to nonprotein coding RNA species. However, this dogma has rapidly transformed with the discovery of a host of modifications in protein coding messenger RNAs (mRNAs). Recent advancements in genome-wide sequencing technologies have enabled the identification of mRNA modifications as a potential new frontier in gene regulation-leading to the development of the epitranscriptome field. As a result, there has been a flurry of multiple groundbreaking discoveries, including new modifications, nucleoside modifying enzymes ("writers" and "erasers"), and RNA binding proteins that recognize chemical modifications ("readers"). These discoveries opened the door to understanding how post-transcriptional mRNA modifications can modulate the mRNA lifecycle, and established a link between the epitranscriptome and human health and disease. Despite a rapidly growing recognition of their importance, fundamental questions regarding the identity, prevalence, and functional consequences of mRNA modifications remain to be answered. Here, we highlight quantitative studies that characterize mRNA modification abundance, frequency, and interactions with cellular machinery. As the field progresses, we see a need for the further integration of quantitative and reductionist approaches to complement transcriptome wide studies in order to establish a molecular-level framework for understanding the consequences of mRNA chemical modifications on biological processes. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Processing > RNA Editing and Modification.

Size-Dependent Relationships between Protein Stability and Thermal Unfolding Temperature Have Important Implications for Analysis of Protein Energetics and High-Throughput Assays of Protein-Ligand Interactions.

Changes in protein stability are commonly reported as changes in the melting temperature, Δ TM, or as changes in unfolding free energy at a particular temperature, ΔΔ G°. Using data for 866 mutants from 16 proteins, we examine the relationship between ΔΔ G° and Δ TM. A linear relationship is observed for each protein. The slopes of the plots of Δ TM vs ΔΔ G° for different proteins scale as N-1, where N is the number of residues in the protein. Thus, a given change in Δ G° causes a much larger change in TM for a small protein relative to the effect observed for a large protein. The analysis suggests that reasonable estimates of ΔΔ G° for a mutant can be obtained by interpolating measured values of TM. The relationship between ΔΔ G° and Δ TM has implications for the design and interpretation of high-throughput assays of protein-ligand binding. So-called thermal shift assays rely upon the increase in stability which results from ligand binding to the folded state. Quantitative relationships are derived which show that the observed thermal shift, Δ TM, scales as N-1. Hence, thermal shift assays are considerably less sensitive for ligand binding to larger proteins.

Redundancy among three herbivorous insects across an experimental current velocity gradient.

We conducted streamside experiments to determine if the ability of herbivorous insects to remove algal periphyton varies with local current velocity. We used two mayfly species (Baetis bicaudatusand Drunella grandis) and one caddisfly species (Glossosoma verdona), which differ from one another in body morphology and mobility. Periphyton was grown for 30 days on ceramic tiles in constant velocity to create similar initial forage conditions for grazers. Tiles were transferred to three velocity regimes characteristic of the natural streambed: slow (3-5 cm s(-1)), medium (15-20 cm s(-1)) and fast (32-41 cm s(-1)). Four grazer treatments (Baetis, Drunella, and Glossosoma alone, and all species combined) were repeated for each velocity treatment to isolate the effect of local current on grazer ability to crop periphyton. Grazers differed in their abilities to remove periphyton across current treatments. Glossosoma removed significantly (P<0.05) more periphyton at fast versus either slow or medium velocities; Baetis showed a similar (but non-significant) trend; and, Drunella always removed about 75% of periphyton, irrespective of current. At fast current, periphyton removal was equivalent among the species. At medium current, Drunella removed significantly more than both Baetis and Glossosoma, whereas at slow current, Drunella removed more than Baetis, which removed more than Glossosoma. Periphyton removal under the combined three-grazer treatment was similar qualitatively to the combined effects of individual grazers. More periphyton tended to be removed as current increased, with the fast versus slow contrast showing marginal significance (P=0.10). Under all current regimes, the quantity of periphyton removed did not differ from the null model expectation of simple additive effects among individual grazers (i.e., no facilitation or inhibition). These experiments show that for some species, herbivory varies with current, which suggests that the herbivore "function" of cropping periphyton may vary with the environmental context of local current. Under some local velocities, however, different herbivore species "function" similarly and are potentially redundant with respect to periphytic removal. In naturally heterogeneous streams characterized by sharp gradients in local current velocity, we expect current-dependent species interactions to be common and at least partially contribute to intra-guild co-existence of species.