[Show abstract][Hide abstract] ABSTRACT: Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter
at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5′ ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.
[Show abstract][Hide abstract] ABSTRACT: The filamentous fungus Neurospora crassa responds to light in complex ways. To thoroughly study the transcriptional response of this organism to light, RNA-seq was used to analyze capped and polyadenylated mRNA prepared from mycelium grown for 24 hr in the dark and then exposed to light for 0 (control) 15, 60, 120 and 240 min. More than three quarters of all defined protein coding genes (79%) were expressed in these cells. The increased sensitivity of RNA-seq compared to previous microarray studies revealed that the RNA levels for 31% of expressed genes were affected 2-fold or more by exposure to light. Additionally, a large class of mRNAs, enriched for transcripts specifying products involved in rRNA metabolism, showed decreased expression in response to light, indicating a heretofore undocumented effect of light on this pathway. Based on measured changes in mRNA levels, light generally increases cellular metabolism, and at the same time causes significant oxidative stress to the organism. To deal with this stress, protective photopigments are made, antioxidants are produced and genes involved in ribosome biogenesis are transiently repressed.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cell-free in vitro translation systems have been in use since the 1970s. These systems can faithfully synthesize polypeptides when programmed with mRNA, enabling the production of polypeptides for analysis as well as permitting analyses of the cis- and trans-acting factors that regulate translation. Here we describe the preparation and use of cell-free translation systems from the yeast Saccharomyces cerevisiae.
Methods in enzymology 03/2014; 539:17-28. DOI:10.1016/B978-0-12-420120-0.00002-5 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome's ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome.
Nucleic Acids Research 10/2013; 42(2). DOI:10.1093/nar/gkt923 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The filamentous fungus Neurospora crassa played a central role in the development of twentieth-century genetics, biochemistry and molecular biology, and continues to serve as a model organism for eukaryotic biology. Here, we have reconstructed a genome-scale model of its metabolism. This model consists of 836 metabolic genes, 257 pathways, 6 cellular compartments, and is supported by extensive manual curation of 491 literature citations. To aid our reconstruction, we developed three optimization-based algorithms, which together comprise Fast Automated Reconstruction of Metabolism (FARM). These algorithms are: LInear MEtabolite Dilution Flux Balance Analysis (limed-FBA), which predicts flux while linearly accounting for metabolite dilution; One-step functional Pruning (OnePrune), which removes blocked reactions with a single compact linear program; and Consistent Reproduction Of growth/no-growth Phenotype (CROP), which reconciles differences between in silico and experimental gene essentiality faster than previous approaches. Against an independent test set of more than 300 essential/non-essential genes that were not used to train the model, the model displays 93% sensitivity and specificity. We also used the model to simulate the biochemical genetics experiments originally performed on Neurospora by comprehensively predicting nutrient rescue of essential genes and synthetic lethal interactions, and we provide detailed pathway-based mechanistic explanations of our predictions. Our model provides a reliable computational framework for the integration and interpretation of ongoing experimental efforts in Neurospora, and we anticipate that our methods will substantially reduce the manual effort required to develop high-quality genome-scale metabolic models for other organisms.
[Show abstract][Hide abstract] ABSTRACT: Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes. Codon usage is also implicated in the control of transcription, splicing and RNA structure. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotic cells, initiation may occur from near-cognate codons that differ from AUG by a single nucleotide. The stringency of start codon selection impacts the efficiency of initiation at near-cognate codons and the efficiency of initiation at AUG codons in different contexts. We used a codon-optimized firefly luciferase reporter initiated with AUG or each of the nine near-cognate codons in preferred context to examine the stringency of start codon selection in the model filamentous fungus Neurospora crassa. In vivo results indicated that the hierarchy of initiation at start codons in N. crassa (AUG > CUG > GUG > ACG > AUA ≈ UUG > AUU > AUC) is similar to that in human cells. Similar results were obtained by translating mRNAs in a homologous N. crassa in vitro translation system or in rabbit reticulocyte lysate. We next examined the efficiency of initiation at AUG, CUG and UUG codons in different contexts in vitro. The preferred context was more important for efficient initiation from near-cognate codons than from AUG. These studies demonstrated that near-cognate codons are used for initiation in N. crassa. Such events could provide additional coding capacity or have regulatory functions. Analyses of the 5'-leader regions in the N. crassa transcriptome revealed examples of highly conserved near-cognate codons in preferred contexts that could extend the N-termini of the predicted polypeptides.
[Show abstract][Hide abstract] ABSTRACT: Therapeutic treatment for systemic mycoses is severely hampered by the extremely limited number of antifungals. The difficulty
of treatment of fungal infections in the central nervous system is further compounded by the poor central nervous system (CNS)
penetration of most antifungals due to the blood-brain barrier. Only a few fungistatic azole drugs, such as fluconazole, show
reasonable CNS penetration. Here we demonstrate that sertraline (Zoloft), the most frequently prescribed antidepressant, displays
potent antifungal activity against Cryptococcus neoformans, the major causative agent of fungal meningitis. In in vitro assays, this neurotropic drug is fungicidal to all natural Cryptococcus isolates tested at clinically relevant concentrations. Furthermore, sertraline interacts synergistically or additively with
fluconazole against Cryptococcus. Importantly, consistent with our in vitro observations, sertraline used alone reduces the brain fungal burden at an efficacy comparable to that of fluconazole in a
murine model of systemic cryptococcosis. It works synergistically with fluconazole in reducing the fungal burden in brain,
kidney, and spleen. In contrast to its potency against Cryptococcus, sertraline is less effective against strains of Candida species and its interactions with fluconazole against Candida strains are often antagonistic. Therefore, our data suggest the unique application of sertraline against cryptococcosis.
To understand the antifungal mechanisms of sertraline, we screened a whole-genome deletion collection of Saccharomyces cerevisiae for altered sertraline susceptibility. Gene ontology analyses of selected mutations suggest that sertraline perturbs translation.
In vitro translation assays using fungal cell extracts show that sertraline inhibits protein synthesis. Taken together, our findings
indicate the potential of adopting this antidepressant in treating cryptococcal meningitis.
[Show abstract][Hide abstract] ABSTRACT: The fungal arginine attenuator peptide (AAP) is encoded by a regulatory upstream open reading frame (uORF). The AAP acts as
a nascent peptide within the ribosome tunnel to stall translation in response to arginine (Arg). The effect of AAP and Arg
on ribosome peptidyl transferase center (PTC) function was analyzed in Neurospora crassa and wheat germ translation extracts using the transfer of nascent AAP to puromycin as an assay. In the presence of a high
concentration of Arg, the wild-type AAP inhibited PTC function, but a mutated AAP that lacked stalling activity did not. While
AAP of wild-type length was most efficient at stalling ribosomes, based on primer extension inhibition (toeprint) assays and
reporter synthesis assays, a window of inhibitory function spanning four residues was observed at the AAP's C terminus. The
data indicate that inhibition of PTC function by the AAP in response to Arg is the basis for the AAP's function of stalling
ribosomes at the uORF termination codon. Arg could interfere with PTC function by inhibiting peptidyltransferase activity
and/or by restricting PTC A-site accessibility. The mode of PTC inhibition appears unusual because neither specific amino
acids nor a specific nascent peptide chain length was required for AAP to inhibit PTC function.
[Show abstract][Hide abstract] ABSTRACT: The fungal arginine attenuator peptide (AAP) is a regulatory peptide that controls ribosome function. As a nascent peptide within the ribosome exit tunnel, it acts to stall ribosomes in response to arginine (Arg). We used three approaches to probe the molecular basis for stalling. First, PEGylation assays revealed that the AAP did not undergo overall compaction in the tunnel in response to Arg. Second, site-specific photocross-linking showed that Arg altered the conformation of the wild-type AAP, but not of nonfunctional mutants, with respect to the tunnel. Third, using time-resolved spectral measurements with a fluorescent probe placed in the nascent AAP, we detected sequence-specific changes in the disposition of the AAP near the peptidyltransferase center in response to Arg. These data provide evidence that an Arg-induced change in AAP conformation and/or environment in the ribosome tunnel is important for stalling.
[Show abstract][Hide abstract] ABSTRACT: An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection.
Nucleic Acids Research 12/2011; 40(7):2898-906. DOI:10.1093/nar/gkr1192 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNA(Pro) peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation. Such protection was not observed in mutant ribosomes containing changes in 23S rRNA nucleotides of the A748-A752 region. Nucleotides A752 and U2609 establish a base-pair interaction. Most replacements of either A752 or U2609 affected Trp induction of a TnaC-regulated LacZ reporter. However, the single change A752G, or the dual replacements A752G and U2609C, maintained Trp induction. Replacements at the conserved TnaC residues W12 and D16 also abolished the protection of U2609 by TnaC-tRNA(Pro) against chemical methylation. These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive. This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.
Nucleic Acids Research 11/2011; 40(5):2247-57. DOI:10.1093/nar/gkr1052 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ribosomes catalyze protein synthesis using transfer RNAs and auxiliary proteins. Historically, ribosomes have been considered nonspecific translational machines, having no regulatory functions. However, a new class of regulatory mechanisms has been discovered that is based on interactions occurring within the ribosomal peptide exit tunnel that result in ribosome stalling during translation of an appropriate mRNA segment. These discoveries reveal an unexpectedly dynamic role ribosomes play in regulating their own activity. By using nascent leader peptides in combination with bound specific amino acids or antibiotics, ribosome functions can be altered significantly resulting in regulated expression of downstream coding regions. This review summarizes relevant findings in recent articles and outlines our current understanding of nascent peptide-induced ribosome stalling in regulating gene expression.
Current opinion in microbiology 02/2011; 14(2):160-6. DOI:10.1016/j.mib.2011.01.011 · 5.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The central feature of standard eukaryotic translation initiation is small ribosome subunit loading at the 5' cap followed by its 5' to 3' scanning for a start codon. The preferred start is an AUG codon in an optimal context. Elaborate cellular machinery exists to ensure the fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) plays a central role in this process. Here we show that the translation of eIF1 homologs in eukaryotes from diverse taxa involves initiation from an AUG codon in a poor context. Using human eIF1 as a model, we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own translation, establishing that variability in the stringency of start codon selection is used for gene regulation in eukaryotes. We show that the stringency of start codon selection (preferential utilization of optimal start sites) is increased to a surprising degree by overexpressing eIF1. The capacity for the cellular level of eIF1 to impact initiation through the variable stringency of initiation codon selection likely has significant consequences for the proteome in eukaryotes.
Proceedings of the National Academy of Sciences 10/2010; 107(42):18056-60. DOI:10.1073/pnas.1009269107 · 9.67 Impact Factor