Inhibition of Ribosome Assembly and Ribosome Translation Has Distinctly Different Effects on Abundance and Paralogue Composition of Ribosomal Protein mRNAs in Saccharomyces cerevisiae.

Many mutations in genes for ribosomal proteins (r-proteins) and assembly factors cause cell stress and altered cell fate, resulting in congenital diseases collectively called ribosomopathies. Even though all such mutations depress the cell's protein synthesis capacity, they generate many different phenotypes, suggesting that the diseases are not due simply to insufficient protein synthesis capacity. To learn more, we investigated how the global transcriptome in Saccharomyces cerevisiae responds to reduced protein synthesis generated in two different ways: abolishing the assembly of new ribosomes and inhibiting ribosomal function. Our results showed that the mechanism by which protein synthesis is obstructed affects the ribosomal protein transcriptome differentially: ribosomal protein mRNA abundance increases during the abolition of ribosome formation but decreases during the inhibition of ribosome function. Interestingly, the ratio between mRNAs from some, but not all, pairs of paralogous ribosomal protein genes encoding slightly different versions of a given r-protein changed differently during the two types of stress, suggesting that expression of specific ribosomal protein paralogous mRNAs may contribute to the stress response. Unexpectedly, the abundance of transcripts for ribosome assembly factors and translation factors remained relatively unaffected by the stresses. On the other hand, the state of the translation apparatus did affect cell physiology: mRNA levels for some other proteins not directly related to the translation apparatus also changed differentially, though not coordinately with the r-protein genes, in response to the stresses. IMPORTANCE Mutations in genes for ribosomal proteins or assembly factors cause a variety of diseases called ribosomopathies. These diseases are typically ascribed to a reduction in the cell's capacity for protein synthesis. Paradoxically, ribosomal mutations result in a wide variety of disease phenotypes, even though they all reduce protein synthesis. Here, we show that the transcriptome changes differently depending on how the protein synthesis capacity is reduced. Most strikingly, inhibiting ribosome formation and ribosome function had opposite effects on the abundance of mRNA for ribosomal proteins, while genes for ribosome translation and assembly factors showed no systematic responses. Thus, the process by which the protein synthesis capacity is reduced contributes decisively to global mRNA composition. This emphasis on process is a new concept in understanding ribosomopathies and other stress responses.

Upregulated Blood miR-150-5p in Alzheimer's Disease Dementia Is Associated with Cognition, Cerebrospinal Fluid Amyloid-ß, and Cerebral Atrophy.

BACKGROUND: There is an urgent need for noninvasive, cost-effective biomarkers for Alzheimer's disease (AD), such as blood-based biomarkers. They will not only support the clinical diagnosis of dementia but also allow for timely pharmacological and nonpharmacological interventions and evaluations. OBJECTIVE: To identify and validate a novel blood-based microRNA biomarker for dementia of the Alzheimer's type (DAT). METHODS: We conducted microRNA sequencing using peripheral blood mononuclear cells isolated from a discovery cohort and validated the identified miRNAs in an independent cohort and AD postmortem tissues. miRNA correlations with AD pathology and AD clinical-radiological imaging were conducted. We also performed bioinformatics and cell-based assay to identify miRNA target genes. RESULTS: We found that miR-150-5p expression was significantly upregulated in DAT compared to mild cognitive impairment and healthy subjects. Upregulation of miR-150-5p was observed in AD hippocampus. We further found that higher miR-150-5p levels were correlated with the clinical measures of DAT, including lower global cognitive scores, lower CSF Aß42, and higher CSF total tau. Interestingly, we observed that higher miR-150-5p levels were associated with MRI brain volumes within the default mode and executive control networks, two key networks implicated in AD. Furthermore, pathway analysis identified the targets of miR-150-5p to be enriched in the Wnt signaling pathway, including programmed cell death 4 (PDCD4). We found that PDCD4 was downregulated in DAT blood and was downregulated by miR-150-5p at both the transcriptional and protein levelsConclusion:Our findings demonstrated that miR-150-5p is a promising clinical blood-based biomarker for DAT.

The small and large ribosomal subunits depend on each other for stability and accumulation.

The 1:1 balance between the numbers of large and small ribosomal subunits can be disturbed by mutations that inhibit the assembly of only one of the subunits. Here, we have investigated if the cell can counteract an imbalance of the number of the two subunits. We show that abrogating 60S assembly blocks 40S subunit accumulation. In contrast, cessation of the 40S pathways does not prevent 60S accumulation, but does, however, lead to fragmentation of the 25S rRNA in 60S subunits and formation of a 55S ribosomal particle derived from the 60S. We also present evidence suggesting that these events occur post assembly and discuss the possibility that the turnover of subunits is due to vulnerability of free subunits not paired with the other subunit to form 80S ribosomes.

Analysis of cell cycle parameters during the transition from unhindered growth to ribosomal and translational stress conditions.

Abrogation of ribosome synthesis (ribosomal stress) leads to cell cycle arrest. However, the immediate cell response to cessation of ribosome formation and the transition from normal cell proliferation to cell cycle arrest have not been characterized. Furthermore, there are conflicting conclusions about whether cells are arrested in G2/M or G1, and whether the cause is dismantling ribosomal assembly per se, or the ensuing decreased number of translating ribosomes. To address these questions, we have compared the time kinetics of key cell cycle parameters after inhibiting ribosome formation or function in Saccharomyces cerevisiae. Within one-to-two hours of repressing genes for individual ribosomal proteins or Translation Elongation factor 3, configurations of spindles, spindle pole bodies began changing. Actin began depolarizing within 4 hours. Thus the loss of ribosome formation and function is sensed immediately. After several hours no spindles or mitotic actin rings were visible, but membrane ingression was completed in most cells and Ace2 was localized to daughter cell nuclei demonstrating that the G1 stage was reached. Thus cell division was completed without the help of a contractile actin ring. Moreover, cell wall material held mother and daughter cells together resulting in delayed cell separation, suggesting that expression or function of daughter gluconases and chitinases is inhibited. Moreover, cell development changes in very similar ways in response to inhibition of ribosome formation and function, compatible with the notion that decreased translation capacity contributes to arresting the cell cycle after abrogation of ribosome biogenesis. Potential implications for the mechanisms of diseases caused by mutations in ribosomal genes (ribosomopathies) are discussed.

High-throughput RNA profiling via up-front sample parallelization.

We describe a method called modular, early-tagged amplification (META) RNA profiling that can quantify a broad panel of microRNAs or mRNAs simultaneously across many samples and requires far less sequence depth than existing digital profiling technologies. The method assigns quantitative tags during reverse transcription to permit up-front sample pooling before competitive amplification and deep sequencing. This simple, scalable and inexpensive approach improves the practicality of large-scale gene expression studies.

Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes.

The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

Shaping the nucleus: factors and forces.

Take a look at a textbook illustration of a cell and you will immediately be able to locate the nucleus, which is often drawn as a spherical or ovoid shaped structure. But not all cells have such nuclei. In fact, some disease states are diagnosed by the presence of nuclei that have an abnormal shape or size. What defines nuclear shape and nuclear size, and how does nuclear geometry affect nuclear function? While the answer to the latter question remains largely unknown, significant progress has been made towards understanding the former. In this review, we provide an overview of the factors and forces that affect nuclear shape and size, discuss the relationship between ER structure and nuclear morphology, and speculate on the possible connection between nuclear size and its shape. We also note the many interesting questions that remain to be explored.

Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae.

The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both gram-positive and gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes.