In Depth Proteome Analysis of Ripening Muscadine Grape Berry cv. Carlos Reveals Proteins Associated with Flavor and Aroma Compounds.

Ripening in nonclimacteric fruits such as grape involves complex chemical changes that have a profound influence on the accumulation of flavor and aroma compounds distinct to a particular grape genotype. In this study, proteome characterization of wine type bronze muscadine grape (Vitis rotundifolia cv. Carlos), primarily grown in the Southeastern United States was performed during berry ripening. Stage-specific protein expression was obtained among different stages of berries. Differential analysis showed the expression of 522 proteins that regulate diverse biological processes and metabolic pathways. Of these, 30 proteins are associated with the production of key phenolic compounds, whereas 25 are associated with the production of muscadine aroma compounds. These proteins are involved in the phenylpropanoid pathway, terpene synthesis, fatty acid derived volatiles and esters that affect muscadine berry flavor and aroma characteristics. Further, gene expression analysis during ripening validated the expression pattern of 12 proteins. Catechin, epicatechin, and four stilbenes were quantified to correlate observed proteome changes. This study not only revealed biochemical changes during muscadine berry ripening but also offers indicators for marker-assisted breeding to enhance organoleptic properties of muscadine grape to improve its flavor and aroma properties.

Post-transcriptional Mechanisms Contribute Little to Phenotypic Variation in Snake Venoms.

Protein expression is a major link in the genotype-phenotype relationship, and processes affecting protein abundances, such as rates of transcription and translation, could contribute to phenotypic evolution if they generate heritable variation. Recent work has suggested that mRNA abundances do not accurately predict final protein abundances, which would imply that post-transcriptional regulatory processes contribute significantly to phenotypes. Post-transcriptional processes also appear to buffer changes in transcriptional patterns as species diverge, suggesting that the transcriptional changes have little or no effect on the phenotypes undergoing study. We tested for concordance between mRNA and protein expression levels in snake venoms by means of mRNA-seq and quantitative mass spectrometry for 11 snakes representing 10 species, six genera, and three families. In contrast to most previous work, we found high correlations between venom gland transcriptomes and venom proteomes for 10 of our 11 comparisons. We tested for protein-level buffering of transcriptional changes during species divergence by comparing the difference between transcript abundance and protein abundance for three pairs of species and one intraspecific pair. We found no evidence for buffering during divergence of our three species pairs but did find evidence for protein-level buffering for our single intraspecific comparison, suggesting that buffering, if present, was a transient phenomenon in venom divergence. Our results demonstrated that post-transcriptional mechanisms did not contribute significantly to phenotypic evolution in venoms and suggest a more prominent and direct role for cis-regulatory evolution in phenotypic variation, particularly for snake venoms.

Linking the transcriptome and proteome to characterize the venom of the eastern diamondback rattlesnake (Crotalus adamanteus).

Understanding the molecular basis of the phenotype is key to understanding adaptation, and the relationship between genes and specific traits is represented by the genotype-phenotype map. The specialization of the venom-gland towards toxin production enables the use of transcriptomics to identify a large number of loci that contribute to a complex phenotype (i.e., venom), while proteomic techniques allow verification of the secretion of the proteins produced by these loci, creating a genotype-phenotype map. We used the extensive database of mRNA transcripts generated by the venom-gland transcriptome of Crotalus adamanteus along with proteomic techniques to complete the genotype-phenotype map for the C. adamanteus venom system. Nanospray LC/MS(E) analysis of a whole venom sample identified evidence for 52 of the 78 unique putative toxin transcript clusters, including 44 of the 50 most highly expressed transcripts. Tandem mass spectrometry and SDS-PAGE of reversed-phase high-performance liquid chromatography fractions identified 40 toxins which clustered into 20 groups and represented 10 toxin families, creating a genotype-phenotype map. By using the transcriptome to understand the proteome we were able to achieve locus-specific resolution and provide a detailed characterization of the C. adamanteus venom system. BIOLOGICAL SIGNIFICANCE: Identifying the mechanisms by which genetic variation presents itself to the sieve of selection at the phenotypic level is key to understanding the molecular basis of adaptation, and the first step in understanding this relationship is to identify the genetic basis of the phenotype through the construction of a genotype-phenotype map. We used the high-throughput venom-gland transcriptomic characterization of the eastern diamondback rattlesnake (C. adamanteus) and proteomic techniques to complete and confirm the genotype-phenotype map, providing a detailed characterization of the C. adamanteus venom system.

Regional and subcellular distribution of HDAC4 in mouse brain.

Histone deacetylases (HDACs) are part of a system that links epigenetic control of gene expression to a variety of environmental stimuli. Some HDACs, including HDAC4, shuttle between the cytoplasm and nucleus in response to physiological cues such as calcium signaling. HDAC4 mRNA is enriched in the brain, but the regional and subcellular protein expression pattern of HDAC4 is not known. Here we show that HDAC4 is more highly expressed in some brain regions than in others. HDAC4 is present in the perikaryial cytoplasm of most neurons but its nuclear localization is variable. In some areas, such as the dentate gyrus, nuclear expression is not detectable, whereas in other areas some neuronal nuclei contain HDAC4 immunoreactivity whereas others do not. In the cytoplasm, HDAC4 immunoreactivity is punctate. Some of these puncta are present in dendritic spines where the strongest immunoreactivity is associated with the postsynaptic density. These data demonstrate that the regional and subcellular distribution of HDAC4 is heterogeneous and raise the possibilities that HDAC4 acts on nonhistone substrates in dendritic spines or that it shuttles between spine and nucleus to coordinate synaptic activity with gene expression.

Probing the catalytic triad of an archaeal RNA splicing endonuclease.

Among the four known mechanisms of intron removal, three are reputedly catalyzed by RNA molecules. In the fourth mechanism, a protein endonuclease removes introns from nuclear tRNA and all archaeal RNAs. Three strictly conserved residues of the splicing endonuclease, a histidine, a lysine, and a tyrosine, were predicted to catalyze the intron cleavage reaction in a manner similar to that of the catalytic triad of ribonuclease A. Single-turnover kinetic parameters were obtained for the wild-type enzyme and two triad mutants. Mutation of histidine to alanine produced an at least approximately 28-fold reduction; mutation of tyrosine to phenylalanine produced an at least approximately 7-fold reduction in activity, while a histidine and tyrosine double mutation abolished cleavage. The single mutation of lysine to glutamic acid abolished RNA cleavage activity in the absence of a divalent metal but maintained a substantial level of activity in the presence of specific divalent metals. These data support important functional roles already proposed for the catalytic triad and suggest an intriguing hypothesis in which the splicing endonuclease is an intermediate in the transition from the RNA to the RNP world.

Long-distance placement of substrate RNA by H/ACA proteins.

The structural basis for accurate placement of substrate RNA by H/ACA proteins is studied using a nonintrusive fluorescence assay. A model substrate RNA containing 2-aminopurine immediately 3' of the uridine targeted for modification produces distinct fluorescence signals that report the substrate's docking status within the enzyme active site. We combined substrate RNA with complete and subcomplexes of H/ACA ribonucleoprotein particles and monitored changes in the substrate conformation. Our results show that each of the three accessory proteins, as well as an active site residue, have distinct effects on substrate conformations, presumably as docking occurs. Interestingly, in some cases these effects are exerted far from the active site. Application of our data to an available structural model of the holoenzyme, enables the functional role of each accessory protein in substrate placement to come into view.

Achieving specific RNA cleavage activity by an inactive splicing endonuclease subunit through engineered oligomerization.

Protein-protein interaction is a common strategy exploited by enzymes to control substrate specificity and catalytic activities. RNA endonucleases, which are involved in many RNA processing and regulation processes, are prime examples of this. How the activities of RNA endonucleases are tightly controlled such that they act on specific RNA is of general interest. We demonstrate here that an inactive RNA splicing endonuclease subunit can be switched "on" solely by oligomerization. Furthermore, we show that the mode of assembly correlates with different RNA specificities. The recently identified splicing endonuclease homolog from Sulfolobus solfataricus, despite possessing all of the putatively catalytic residues, has no detectable RNA cleavage activity on its own but is active upon mixing with its structural subunit. Guided by the previously determined three-dimensional structure of the catalytic subunit, we altered its sequence such that it could potentially self-assemble thereby enabling its catalytic activity. We present the evidence for the specific RNA cleavage activity of the engineered catalytic subunit and for its formation of a functional tetramer. We also identify a higher order oligomer species that possesses distinct RNA cleavage specificity from that of previously characterized RNA splicing endonucleases.

RNA recognition and cleavage by a splicing endonuclease.

The RNA splicing endonuclease cleaves two phosphodiester bonds within folded precursor RNAs during intron removal, producing the functional RNAs required for protein synthesis. Here we describe at a resolution of 2.85 angstroms the structure of a splicing endonuclease from Archaeglobus fulgidus bound with a bulge-helix-bulge RNA containing a noncleaved and a cleaved splice site. The endonuclease dimer cooperatively recognized a flipped-out bulge base and stabilizes sharply bent bulge backbones that are poised for an in-line RNA cleavage reaction. Cooperativity arises because an arginine pair from one catalytic domain sandwiches a nucleobase within the bulge cleaved by the other catalytic domain.

The heteromeric Nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge-helix-bulge motifs of joined tRNA halves.

Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5' and 3' tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge-helix-bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate.

Structural characterization of the catalytic subunit of a novel RNA splicing endonuclease.

The RNA splicing endonuclease is responsible for recognition and excision of nuclear tRNA and all archaeal introns. Despite the conserved RNA cleavage chemistry and a similar enzyme assembly, currently known splicing endonuclease families have limited RNA specificity. Different from previously characterized splicing endonucleases in Archaea, the splicing endonuclease from archaeum Sulfolobus solfataricus was found to contain two different subunits and accept a broader range of substrates. Here, we report a crystal structure of the catalytic subunit of the S.solfataricus endonuclease at 3.1 angstroms resolution. The structure, together with analytical ultracentrifugation analysis, identifies the catalytic subunit as an inactive but stable homodimer, thus suggesting the possibility of two modes of functional assembly for the active enzyme.