Roles for the canonical polarity machinery in the establishment of polarity in budding yeast spores.

The yeast buds at sites pre-determined by cortical landmarks deposited during prior budding. During mating between haploid cells in the lab, external pheromone cues override the cortical landmarks to drive polarization and cell fusion. By contrast, in haploid gametes (called spores) produced by meiosis, a pre-determined polarity site drives initial polarized morphogenesis independent of mating partner location.

Mechanism-based approach in designing patient-specific combination therapies for nonsense mutation diseases.

Premature termination codon (PTC) diseases, arising as a consequence of nonsense mutations in a patient's DNA, account for approximately 12% of all human disease mutations. Currently there are no FDA approved treatments for increasing PTC readthrough in nonsense mutation diseases, although one translational readthrough inducing drug, ataluren, has had conditional approval for treatment of Duchenne muscular dystrophy in Europe and elsewhere for 10 years. Ataluren displays consistent low toxicity in clinical trials for treatment of several different PTC diseases, but its therapeutic effects on such diseases are inconsistent.

Roles for the canonical polarity machinery in the establishment of polarity in budding yeast spores.

The yeast buds at sites pre-determined by cortical landmarks deposited during prior budding. During mating between haploid cells in the lab, external pheromone cues override the cortical landmarks to drive polarization and cell fusion. By contrast, in haploid gametes (called spores) produced by meiosis, a pre-determined polarity site drives initial polarized morphogenesis independent of mating partner location.

Single-Molecule Studies of Cognate and Near-Cognate Elongation in an Eukaryotic Translation System.

The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation.

Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation.

Assembly of the Mitochondrial Translation Initiation Complex.

Mitochondria maintain their own translational machinery that is responsible for the synthesis of essential components of the oxidative phosphorylation system. The mammalian mitochondrial translation system differs significantly from its cytosolic and bacterial counterparts. Here, we describe detailed protocols for efficient in vitro reconstitution of the mammalian mitochondrial translation initiation complex, which can be further used for mechanistic analyses of different aspects of mitochondrial translation.

A High-Throughput Assay for In Vitro Determination of Release Factor-Dependent Peptide Release from a Pretermination Complex by Fluorescence Anisotropy-Application to Nonsense Suppressor Screening and Mechanistic Studies.

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases.

Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation.

Translation initiation of leaderless and polycistronic transcripts in mammalian mitochondria.

The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5' leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes.

Human mitochondria require mtRF1 for translation termination at non-canonical stop codons.

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons.

Ataluren binds to multiple protein synthesis apparatus sites and competitively inhibits release factor-dependent termination.

Genetic diseases are often caused by nonsense mutations, but only one TRID (translation readthrough inducing drug), ataluren, has been approved for clinical use. Ataluren inhibits release factor complex (RFC) termination activity, while not affecting productive binding of near-cognate ternary complex (TC, aa-tRNA.eEF1A.

Post-Transcriptional Control of Mating-Type Gene Expression during Gametogenesis in .

Gametogenesis in diploid cells of the budding yeast produces four haploid meiotic products called spores. Spores are dormant until nutrients trigger germination, when they bud asexually or mate to return to the diploid state. Each sporulating diploid produces a mix of spores of two haploid mating types, and α.

Site-Specific Fluorescent Labeling of RNA Interior Positions.

The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Here I briefly review methods, some well-established and others newly developed, which have been successfully exploited to site-specifically fluorescently label interior positions of RNAs, as a guide to investigators seeking to apply this approach to their studies. Most of these methods can be applied directly to intact RNAs, including (1) the exploitation of natural posttranslational modifications, (2) the repurposing of enzymatic transferase reactions, and (3) the nucleic acid-assisted labeling of intact RNAs.

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms.

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through.

Saccharomyces spores are born prepolarized to outgrow away from spore-spore connections and penetrate the ascus wall.

How nonspore haploid Saccharomyces cells choose sites of budding and polarize towards pheromone signals in order to mate has been a subject of intense study. Unlike nonspore haploids, sibling spores produced via meiosis and sporulation by a diploid cell are physically interconnected and encased in a sac derived from the old cell wall of the diploid, called the ascus. Nonspore haploids bud adjacent to previous sites of budding, relying on stable cortical landmarks laid down during prior divisions, but because spore membranes are made de novo, it was assumed that, as is known for fission yeast, Saccharomyces spores break symmetry and polarize at random locations.

Small RNA Deep Sequencing Identifies a Unique miRNA Signature Released in Serum Exosomes in a Mouse Model of Sjögren's Syndrome.

Sjögren's Syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and loss of function of moisture-producing exocrine glands as well as systemic inflammation. SS diagnosis is cumbersome, subjective and complicated by manifestation of symptoms that overlap with those of other rheumatic and ocular diseases. Definitive diagnosis averages 4-5 years and this delay may lead to irreversible tissue damage.

Dynamics of intracellular stress-induced tRNA trafficking.

Stress is known to induce retrograde tRNA translocation from the cytoplasm to the nucleus but translocation kinetics and tRNA-spatial distribution have not been characterized previously. We microinject fluorescently-labeled tRNA into living cells and use confocal microscopy to image tRNA spatial distribution in single cells at various levels of starvation and to determine translocation rate constants. Retrograde tRNA translocation occurs reversibly, within minutes after nutrition depletion of the extracellular medium.

E. coli elongation factor Tu bound to a GTP analogue displays an open conformation equivalent to the GDP-bound form.

According to the traditional view, GTPases act as molecular switches, which cycle between distinct 'on' and 'off' conformations bound to GTP and GDP, respectively. Translation elongation factor EF-Tu is a GTPase essential for prokaryotic protein synthesis. In its GTP-bound form, EF-Tu delivers aminoacylated tRNAs to the ribosome as a ternary complex.

Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome.

The GTPase elongation factor EF-Tu delivers aminoacyl-tRNAs to the mRNA-programmed ribosome during translation. Cognate codon-anticodon interaction stimulates GTP hydrolysis within EF-Tu. It has been proposed that EF-Tu undergoes a large conformational change subsequent to GTP hydrolysis, which results in the accommodation of aminoacyl-tRNA into the ribosomal A-site.

Translocation kinetics and structural dynamics of ribosomes are modulated by the conformational plasticity of downstream pseudoknots.

Downstream stable mRNA secondary structures can stall elongating ribosomes by impeding the concerted movements of tRNAs and mRNA on the ribosome during translocation. The addition of a downstream mRNA structure, such as a stem-loop or a pseudoknot, is essential to induce -1 programmed ribosomal frameshifting (-1 PRF). Interestingly, previous studies revealed that -1 PRF efficiencies correlate with conformational plasticity of pseudoknots, defined as their propensity to form incompletely folded structures, rather than with the mechanical properties of pseudoknots.

tRNA Fluctuations Observed on Stalled Ribosomes Are Suppressed during Ongoing Protein Synthesis.

The pretranslocation complex of the ribosome can undergo spontaneous fluctuations of messenger RNA and transfer RNAs (tRNAs) between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classical and hybrid state tRNA positions have been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation. However, during multiple ongoing elongation cycles when both EF-G and ternary complexes are present, EF-G can bind to the pretranslocation complex much faster than the timescale of the classic-hybrid transitions.

Probing the interaction between NatA and the ribosome for co-translational protein acetylation.

N-terminal acetylation is among the most abundant protein modifications in eukaryotic cells. Over the last decade, significant progress has been made in elucidating the function of N-terminal acetylation for a number of diverse systems, involved in a wide variety of biological processes. The enzymes responsible for the modification are the N-terminal acetyltransferases (NATs).

The kinetic mechanism of bacterial ribosome recycling.

Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon bound to the 70S ribosome. The translation factors, elongation factor G and ribosome recycling factor, are known to be required for recycling, but there is controversy concerning whether these factors act primarily to effect the release of mRNA and tRNA from the ribosome, with the splitting of the ribosome into subunits being somewhat dispensable, or whether their main function is to catalyze the splitting reaction, which necessarily precedes mRNA and tRNA release. Here, we utilize three assays directly measuring the rates of mRNA and tRNA release and of ribosome splitting in several model PoTCs.

Stringent Nucleotide Recognition by the Ribosome at the Middle Codon Position.

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon University of California base formation during protein synthesis. Our results suggest that codon:anticodon base pairing is subject to tighter constraints at the middle position than at the 5'- and 3'-positions, and further suggest a sequential mechanism of formation of the three base pairs in the codon:anticodon helix.