High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq

Measurements of cellular tRNA abundance are hampered by pervasive blocks to cDNA synthesis at modified nucleosides and the extensive similarity among tRNA genes. We overcome these limitations with modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which combines a workflow for full-length cDNA library construction from endogenously modified tRNA with a comprehensive and user-friendly computational analysis toolkit. Our method accurately captures tRNA abundance and modification status in yeast, fly, and human cells and is applicable to any organism with a known genome. We applied mim-tRNAseq to discover a dramatic heterogeneity of tRNA isodecoder pools among diverse human cell lines and a surprising interdependence of modifications at distinct sites within the same tRNA transcript

Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity

SummaryProteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress, accumulate aggregates of endogenous proteins, and are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA modifications in maintaining proteome integrity

Exploring regulatory functions and enzymatic activities in the nidovirus replicase

Members of the order Nidovirales (Coronaviridae, Arteriviridae, and Roniviridae) employ genomes with mRNA polarity (plus-strand) and encode one of the most complex RNA virus replicases currently known. This replicase is expressed from the viral genome by translation of two large 5__-proximal ORFs into two polyproteins, which are processed by virus-encoded proteases in 13-16 individual nonstructural proteins (nsps). The nsps direct the formation of an RNA-synthesizing complex that mediates viral genome replication, as well as the synthesis of a 3__-coterminal nested set of subgenomic (sg) mRNAs, from which the viral genes located downstream of the replicase gene are expressed. Arteriviruses and coronaviruses presumably employ a unique mechanism of discontinuous minus-strand extension to generate subgenome-length templates for sg mRNA synthesis. This thesis focused on the functional characterization of two replicase subunits and their roles in coupling different processes in the replicative cycle of equine arteritis virus (EAV), the arterivirus prototype. The biological importance of a conserved domain unique to nidoviruses (NendoU), mapping to arterivirus nsp11 and proposed to function as an endoribonuclease, was addressed. We demonstrated the recombinant nsp11 exhibits pyrimidine-specific endoribonuclease activity, and showed the critical importance of the NendoU domain for EAV RNA synthesis. In addition, we identified a multidomain replicase subunit, EAV nsp1, as a key coordinator of EAV genome replication, sg mRNA synthesis, and virus production. Our results reveal that the relative abundance of EAV mRNAs is tightly controlled by nsp1 and is critical for efficient production of new virus particles. The protein was implicated in modulating the accumulation of full-length and subgenome-length minus-strand templates for EAV mRNA synthesis. A protocol for purification of soluble recombinant nsp1, which can be used in future research on the molecular mechanisms of nsp1 function, is described.UBL - phd migration 201

Screening the Antimicrobial activity of Actinomycetes strains isolated from Antarctica

A total of 40 actinomycete strains, isolated from Antarctica, were tested for antagonistic activity against 7 Gram-positive and Gram negative bacteria and yeasts, 16 phytopathogenic fungi and bacteria. During the initial screening 60 % of the strains showed inhibition potential against test-microorganisms. Ten of them had a broader spectrum of antibacterial activity and could be used in the development of new substances for pharmaceutical or agricultural purposes

Deciphering Flavio Pereira’s Reading of Dostoevsky after the The House of the Dead

This article provides a “dechiffrage” of the form, style, compositional techniques, and referential musical meanings employed by the Brazilian composer and pedagogue Flavio Santos Pereira in the composition of a seven-part suite entitled Reading of Dostoevsky, written in 2016 and based on the book The House of the Dead by Fyodor Dostoevsky. This chamber work is a premonitory work about the present pandemic time, which tests not only the human existential instincts and fears but also the spiritual, philosophical, and moral values upon which a mature and complete personality is built. The author manages to turn the economic structure of a dodecaphonic material into a source of polyphonic, polyrhythmic, stylistic, and timbre diversity. Oscillating and incremental textures, often reaching four-voice overlays, find their counterbalance in the asymmetric movements that synthesize complex subharmonic timbre combinations. The work can be classified as program music, as it employs characteristics of expressionist and impressionist styles mixed with free improvisatory polyphonic techniques. This paper also aims at inducing young performers to consider the paradigmatic model of “dechiffrage” for interpretation supported by stylistic and formal analysis based on classical and modern models. The article includes the full score of Reading of Dostoevsky by Flavio Santos Pereira

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response

The exonuclease Xrn1 activates transcription and translation of mRNAs encoding membrane proteins

The highly conserved 5'-3' exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay. By integrating transcriptome-wide analyses of translation with biochemical and functional studies, we demonstrate an unanticipated regulatory role of Xrn1 in protein synthesis. Xrn1 promotes translation of a specific group of transcripts encoding membrane proteins. Xrnl-dependence for translation is linked to poor structural RNA contexts for translation initiation, is mediated by interactions with components of the translation initiation machinery and correlates with an Xrnl-dependence for mRNA localization at the endoplasmic reticulum, the translation compartment of membrane proteins. Importantly, for this group of mRNAs, Xrn1 stimulates transcription, mRNA translation and decay. Our results uncover a crosstalk between the three major stages of gene expression coordinated by Xrn1 to maintain appropriate levels of membrane proteins