Arts and Humanities: Senate Report (1976): Report 02

<p>(A) Translation profile of wild-type M5bG7 (top diagram, original profile) and upon introduction of translational attenuation sites (bottom diagram, adapted profile) in <i>E</i>. <i>coli</i>. Secondary structure alignment (β-sheets—blue bars, α-helices—dark red bars, uncolored empty space—linking structural elements) of M5bG7 to EH-Ar to identify regions for introduction of slow-translating stretches (indicated with arrows). The domains in M5bG7 were delineated based on the domain architecture of EH-Ar represented in the color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127039#pone.0127039.g001" target="_blank">Fig 1A</a>. Note the longer N-terminal domain of M5bG7 than that of EH-Ar. (B) Representative immunoblot of M5bG7 (abbreviated M5) variants. L1 (Leu 189, CUC/A, numbering is according to the M5bG7 sequence) and L2 (Leu 257, CUG/A) denote the synonymous exchange of a fast-translating Leu codons to Leu CUA in the first and second attenuation, respectively, and LL (Leu189, CUC/A, Leu257, CUG/A) in both simultaneously. T, total protein, S, soluble and I, insoluble fraction. GAPDH served as loading control. (C) Quantification of immunoblots of three biological replicates ± SEM. **, p<0.01, Tukey’s test. For details refer to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127039#pone.0127039.g001" target="_blank">Fig 1</a>. (D) Quantification of mRNA levels by qRT-PCR of the M5bG7 variants. Values were normalized to GAPDH mRNA expression, represented as a fold-change to the wild-type mRNA and are means ± SEM (n = 3).</p

An expanded CAG repeat in Huntingtin Causes +1 frameshifting

Maintenance of triplet decoding is crucial for the expression of functional protein because deviations either into the −1 or +1 reading frames are often non-functional. We report here that expression of huntingtin (Htt) exon 1 with expanded CAG repeats, implicated in Huntington pathology, undergoes a sporadic +1 frameshift to generate from the CAG repeat a trans-frame AGC repeat-encoded product. This +1 recoding is exclusively detected in pathological Htt variants, i.e. those with expanded repeats with more than 35 consecutive CAG codons. An atypical +1 shift site, UUC C at the 5′ end of CAG repeats, which has some resemblance to the influenza A virus shift site, triggers the +1 frameshifting and is enhanced by the increased propensity of the expanded CAG repeats to form a stem-loop structure. The +1 trans-frame-encoded product can directly influence the aggregation of the parental Htt exon 1

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation.

In addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process1,2. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage

EBV Tegument Protein BNRF1 Disrupts DAXX-ATRX to Activate Viral Early Gene Transcription

Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus

Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs

Enhanced Coagulation of Surface Water Sources In The State of South Dakota for TOC Removal

Data was collected by the SDDENR and by a survey sent out and collected by Paul Saffert and Dr. Delvin DeBoer from the water treatment utilities utilizing surface water sources. Enhanced coagulation jar tests, as defined by the D/DBP Rule, were performed on a representative sample of the surface waters of South Dakota. TOC removals were established from the 3x3 matrix of the D/DBP Rule and from the Stage 2 Enhanced Coagulation Jar Test Protocol. Simulated Distribution System tests were set up to establish a link between TOC removal and THM formation. Generally speaking, the source waters in the State of South Dakota are amenable to enhanced coagulation. Ferric chloride generally removed higher levels of TOC at lower doses than alum during coagulation. THM concentration was dependent on several factors including TOC concentration, pH, chlorination time and temperature

Structure and Function of p97 and Pex1/6 Type II AAA+ Complexes

Protein complexes of the Type II AAA+ (ATPases associated with diverse cellular activities) family are typically hexamers of 80–150 kDa protomers that harbor two AAA+ ATPase domains. They form double ring assemblies flanked by associated domains, which can be N-terminal, intercalated or C-terminal to the ATPase domains. Most prominent members of this family include NSF (N-ethyl-maleimide sensitive factor), p97/VCP (valosin-containing protein), the Pex1/Pex6 complex and Hsp104 in eukaryotes and ClpB in bacteria. Tremendous efforts have been undertaken to understand the conformational dynamics of protein remodeling type II AAA+ complexes. A uniform mode of action has not been derived from these works. This review focuses on p97/VCP and the Pex1/6 complex, which both structurally remodel ubiquitinated substrate proteins. P97/VCP plays a role in many processes, including ER- associated protein degradation, and the Pex1/Pex6 complex dislocates and recycles the transport receptor Pex5 from the peroxisomal membrane during peroxisomal protein import. We give an introduction into existing knowledge about the biochemical and cellular activities of the complexes before discussing structural information. We particularly emphasize recent electron microscopy structures of the two AAA+ complexes and summarize their structural differences

Optimization of translation profiles enhances protein expression and solubility.

mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein

Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin

SummaryHuntington disease (HD), a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ) repeat in huntingtin (Htt), displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNA)Gln-CUG that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNAGln-CUG. The concentration of tRNAGln-CUG also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels

Translation attenuation sites delineate domain boundaries and impact protein solubility.

<p>(A) Translation profile of EH-Ar predicted with RiboTempo. Vertical gray bars represent the rate of translation of each single codon which is averaged (red line) along the whole ORF with a window of 19 codons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127039#pone.0127039.ref042" target="_blank">42</a>]. Translation minima below the genome-wide threshold (blue horizontal line) denote the putative slow-translating attenuation sites. AA denotes amino acid number, kDa marks the corresponding molecular weight and SS denotes the predicted secondary structure (β-sheets—blue bars, α-helices—dark red bars, uncolored empty space—linking structural elements). The rainbow-colored bar visualizes the putative structural domains, colored in the same way in the 3D- structure (PDB 1EHY). (B) Summary of the exchanged codons in EH-Ar. The position of the exchanged amino acids for each variant is indicated. (C) Translation profiles of EH-Ar variants with exchanged slow translating patches (B) predicted with RiboTempo. (D, E). Removal of the translational attenuation sites reduces the solubility of EH-Ar. (D) Representative immunoblot of EH-Ar variants (summarized in C). The total (T) protein content was fractionated into soluble (S) and insoluble (I) fractions and 0.05 OD₆₀₀ of cells were applied per lane. GAPDH served as a loading control; note that it is a completely soluble protein and its absence in the insoluble fraction confirms the good quality of the fractionation procedure. (E) Quantification of the immunoblots of three biological replicates ± SEM. Each total fraction was normalized to GAPDH intensity to allow for comparison between the samples; the soluble and insoluble fractions were determined as a percentage of this normalized value. *, p<0.05, Tukey’s test. (F) Quantification of mRNA levels by qRT-PCR of the EH-Ar variants. Values were normalized to GAPDH mRNA expression, represented as a fold-change to the wild-type mRNA and are means ± SEM (n = 3).</p