Human and rat gut microbiome composition is maintained following sleep restriction.

Insufficient sleep increasingly characterizes modern society, contributing to a host of serious medical problems. Loss of sleep is associated with metabolic diseases such as obesity and diabetes, cardiovascular disorders, and neurological and cognitive impairments. Shifts in gut microbiome composition have also been associated with the same pathologies; therefore, we hypothesized that sleep restriction may perturb the gut microbiome to contribute to a disease state. In this study, we examined the fecal microbiome by using a cross-species approach in both rat and human studies of sleep restriction. We used DNA from hypervariable regions (V1-V2) of 16S bacteria rRNA to define operational taxonomic units (OTUs) of the microbiome. Although the OTU richness of the microbiome is decreased by sleep restriction in rats, major microbial populations are not altered. Only a single OTU, TM7-3a, was found to increase with sleep restriction of rats. In the human microbiome, we find no overt changes in the richness or composition induced by sleep restriction. Together, these results suggest that the microbiome is largely resistant to changes during sleep restriction.

Molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis virus infection.

The dicistrovirus Cricket Paralysis virus contains a unique dicistronic RNA genome arrangement, encoding two main open reading frames that are driven by distinct internal ribosome entry sites (IRES). The intergenic region (IGR) IRES adopts an unusual structure that directly recruits the ribosome and drives translation of viral structural proteins in a factor-independent manner. While structural, biochemical, and biophysical approaches have provided mechanistic details into IGR IRES translation, these studies have been limited to in vitro systems and little is known about the behavior of these IRESs during infection. Here, we examined the role of previously characterized IGR IRES mutations on viral yield and translation in CrPV-infected Drosophila S2 cells. Using a recently generated infectious CrPV clone, introduction of a subset of mutations that are known to disrupt IRES activity failed to produce virus, demonstrating the physiological relevance of specific structural elements within the IRES for virus infection. However, a subset of mutations still led to virus production, thus revealing the key IRES-ribosome interactions for IGR IRES translation in infected cells, which highlights the importance of examining IRES activity in its physiological context. This is the first study to examine IGR IRES translation in its native context during virus infection.

Ribosome profiling reveals an important role for translational control in circadian gene expression.

Physiological and behavioral circadian rhythms are driven by a conserved transcriptional/translational negative feedback loop in mammals. Although most core clock factors are transcription factors, post-transcriptional control introduces delays that are critical for circadian oscillations. Little work has been done on circadian regulation of translation, so to address this deficit we conducted ribosome profiling experiments in a human cell model for an autonomous clock. We found that most rhythmic gene expression occurs with little delay between transcription and translation, suggesting that the lag in the accumulation of some clock proteins relative to their mRNAs does not arise from regulated translation. Nevertheless, we found that translation occurs in a circadian fashion for many genes, sometimes imposing an additional level of control on rhythmically expressed mRNAs and, in other cases, conferring rhythms on noncycling mRNAs. Most cyclically transcribed RNAs are translated at one of two major times in a 24-h day, while rhythmic translation of most noncyclic RNAs is phased to a single time of day. Unexpectedly, we found that the clock also regulates the formation of cytoplasmic processing (P) bodies, which control the fate of mRNAs, suggesting circadian coordination of mRNA metabolism and translation.

Temperature oscillations drive cycles in the activity of MMP-2,9 secreted by a human trabecular meshwork cell line.

PURPOSE: Aqueous humor inflow falls 50% during sleeping hours without proportional fall in IOP, partly reflecting reduced outflow facility. The mechanisms underlying outflow facility cycling are unknown. One outflow facility regulator is matrix metalloproteinase (MMP) release from trabecular meshwork (TM) cells. Because anterior segment temperature must oscillate due to core temperature cycling and eyelid closure during sleep, we tested whether physiologically relevant temperature oscillations drive cycles in the activity of secreted MMP. METHODS: Temperature of transformed normal human TM cells (hTM5 line) was fixed or alternated 12 hours/12 hours between 33°C and 37°C. Activity of secreted MMP-2 and MMP-9 was measured by zymography, and gene expression by RT-PCR and quantitative PCR. RESULTS: Raising temperature to 37°C increased, and lowering to 33°C reduced, activity of secreted MMP. Switching between 37°C and 33°C altered MMP-9 by 40% ± 3% and MMP-2 by 22% ± 2%. Peripheral circadian clocks did not mediate temperature-driven cycling of MMP secretion because MMP-release oscillations did not persist at constant temperature after 3 to 6 days of alternating temperatures, and temperature cycles did not entrain clock-gene expression in these cells. Furthermore, inhibiting heat shock transcription factor 1, which links temperature and peripheral clock-gene oscillations, inhibited MMP-9 but not MMP-2 temperature-driven MMP cycling. Inhibition of heat-sensitive TRPV1 channels altered total MMP secretion but not temperature-induced modulations. Inhibiting cold-sensitive TRPM-8 channels had no effect. CONCLUSIONS: Physiologically relevant temperature oscillations drive fluctuations of secreted MMP-2 and MMP-9 activity in hTM5 cells independent of peripheral clock genes and temperature-sensitive TRP channels.

An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila.

Little is known about molecular links between circadian clocks and steroid hormone signalling, although both are important for normal physiology. Here we report a circadian function for a nuclear receptor, ecdysone-induced protein 75 (Eip75/E75), which we identified through a gain-of-function screen for circadian genes in Drosophila melanogaster. Overexpression or knockdown of E75 in clock neurons disrupts rest:activity rhythms and dampens molecular oscillations. E75 represses expression of the gene encoding the transcriptional activator, CLOCK (CLK), and may also affect circadian output. PER inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of PER on Clk transcription. The ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms. We find that E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila.

Severity of X-linked dyskeratosis congenita (DKCX) cellular defects is not directly related to dyskerin (DKC1) activity in ribosomal RNA biogenesis or mRNA translation.

Dyskerin (encoded by the DKC1 locus) is the pseudouridine synthase responsible for the modification of noncoding RNA. Dyskerin is also an obligate member of the telomerase enzyme, and participates in the biogenesis of telomerase. Genetic lesions at the DKC1 locus are associated with X-linked dyskeratosis congenita (X-DC) and the Hoyeraal-Hreidarsson Syndrome (HHS). Both syndromes have been linked to deficient telomere maintenance, but little is known about the RNA modification activities of dyskerin in X-DC and HHS cells. To evaluate whether X-DC-associated dyskerin mutations affect the modification or function of ribosomal RNA, we studied five telomerase-rescued X-DC cells (X-DC(T) ). Our data revealed a small reproducible loss of pseudouridines in mature rRNA in two X-DC variants. However, we found no difference in protein synthesis between telomerized wild-type (WT(T) ) and X-DC(T) cells, with an internal ribosomal entry site translation assay, or by measuring total protein synthesis in live cells. X-DC(T) cells and WT(T) cells also exhibited similar tolerances to ionizing radiation and endoplasmic reticulum stress. Despite the loss in rRNA pseudouridine modification, functional perturbations from these changes are secondary to the telomere maintenance defects of X-DC. Our data show that telomere dysfunction is the primary and unifying etiology of X-DC.

Translation-competent 48S complex formation on HCV IRES requires the RNA-binding protein NSAP1.

Translation of many cellular and viral mRNAs is directed by internal ribosomal entry sites (IRESs). Several proteins that enhance IRES activity through interactions with IRES elements have been discovered. However, the molecular basis for the IRES-activating function of the IRES-binding proteins remains unknown. Here, we report that NS1-associated protein 1 (NSAP1), which augments several cellular and viral IRES activities, enhances hepatitis C viral (HCV) IRES function by facilitating the formation of translation-competent 48S ribosome-mRNA complex. NSAP1, which is associated with the solvent side of the 40S ribosomal subunit, enhances 80S complex formation through correct positioning of HCV mRNA on the 40S ribosomal subunit. NSAP1 seems to accomplish this positioning function by directly binding to both a specific site in the mRNA downstream of the initiation codon and a 40S ribosomal protein (or proteins).

Modular domains of the Dicistroviridae intergenic internal ribosome entry site.

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae viral family can directly assemble 80S ribosomes and initiate translation at a non-AUG codon from the ribosomal A-site. These functions are directed by two independently folded domains of the IGR IRES. One domain, composed of overlapping pseudoknots II and III (PKII/III), mediates ribosome recruitment. The second domain, composed of PKI, mimics a tRNA anticodon-codon interaction to position the ribosome at the ribosomal A-site. Although adopting a common secondary structure, the dicistrovirus IGR IRESs can be grouped into two classes based on distinct features within each domain. In this study, we report on the modularity of the IGR IRESs and show that the ribosome-binding domain and the tRNA anticodon mimicry domain are functionally interchangeable between the Type I and the Type II IGR IRESs. Using structural probing, ribosome-binding assays, and ribosome positioning analysis by toeprinting assays, we show that the chimeric IRESs fold properly, assemble 80S ribosomes, and can mediate IRES translation in rabbit reticulocyte lysates. We also demonstrate that the chimeric IRESs can stimulate the ribosome-dependent GTPase activity of eEF2, which suggests that the ribosome is primed for a step downstream from IRES binding. Overall, the results demonstrate that the dicistrovirus IGR IRESs are composed of two modular domains that work in concert to manipulate the ribosome and direct translation initiation.

Conserved element of the dicistrovirus IGR IRES that mimics an E-site tRNA/ribosome interaction mediates multiple functions.

The internal ribosome entry site within the intergenic region (IGR IRES) of the Dicistroviridae family mimics a tRNA to directly assemble 80 S ribosomes and initiate translation at a non-AUG codon from the ribosomal A-site. A comparison of IGR IRESs within this viral family reveals structural similarity but little sequence similarity. However, a few specific conserved elements exist, which likely have important roles in IRES function. In this study, we have generated a battery of mutations to characterize the role of a conserved loop (L1.1) region of the IGR IRES. Mutating specific nucleotides within the L1.1 region inhibited IGR IRES-mediated translation in rabbit reticulocyte lysates. By assaying different steps in IRES function, we found that the mutant L1.1 IRESs had reduced affinity for 80 S ribosomes but not 40 S subunits, indicating that the L1.1 region mediated either binding to preformed 80 S or 60 S joining. Furthermore, mutations in L1.1 altered the position of the ribosome on the mutant IRES, indicating that the tRNA-like anticodon/codon mimic within the ribosomal P-site is disrupted. Structural studies have revealed that the L1.1 region interacts with the L1 stalk of the 60 S subunit, which is similar to the interactions between the T-loop of the E-site tRNA and ribosomal protein rpL1. Our results demonstrate that the conserved L1.1 region directs multiple steps in IGR IRES-mediated translation including ribosome binding and positioning, which are functions that the E-site tRNA may normally mediate during translation.

Expression, purification and structural characterization of the scaffoldin hydrophilic X-module from the cellulosome of Clostridium thermocellum.

The cellulosome is a membrane-bound, extracellular multi-subunit complex responsible for the degradation of crystalline cellulose by a number of organisms including anaerobic bacteria and fungi. The hydrophilic X-module (CipA-X) from the modular scaffoldin subunit of Clostridium thermocellum cellulosome has been proposed to play various roles in cellulosomal function, including thermal and structural stability. Towards elucidating the function of CipA-X using structural and biophysical studies, the region comprising residues 1692-1785 from the C. thermocellum CipA cDNA encoding CipA-X was cloned into a pET21b expression vector. When expressed in Escherichia coli, the C-terminal His-tagged protein accumulated in the insoluble fraction. Cell fractionation experiments showed that the recombinant protein was localized to inclusion bodies. Refolding and purification involved denaturation of the whole cell lysate by addition of urea, followed by a nickel-Sepharose chromatography step and dialysis into native conditions (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 10 mM EDTA). A final gel filtration step purified the protein to homogeneity, yielding 40 mg/L. The two-dimensional 1H-15N correlation spectrum of uniformly 15N-labelled CipA-X showed the characteristics of a well-folded protein comprising significant beta-structure, which is in agreement with the circular dichroism data.