Quality criteria for finding genes with high mRNA-protein expression correlation and coexpression correlation

mRNA expression is widely used as a proxy for protein expression. However, their true relation is not known and two genes with the same mRNA levels might have different abundances of respective proteins. A related question is whether the coexpression of mRNA for gene pairs is reflected by the corresponding protein pairs. We examined the mRNA-protein correlation for both expression and coexpression. This analysis yielded insights into the relationship between mRNA and protein abundance, and allowed us to identify subsets of greater mRNA-protein coherence. The correlation between mRNA and protein was low for both expression and coexpression, 0.12 and 0.06 respectively. However, applying the best-performing quality measure, high-quality subsets reached a Spearman correlation of 0.31 for expression, 0.34 for coexpression and 0.49 for coexpression when restricted to functionally coupled genes. Our methodology can thus identify subsets for which the mRNA levels are expected to be the strongest correlated with protein levels.     

Relation between mRNA expression and sequence information in Desulfovibrio vulgaris: combinatorial contributions of upstream regulatory motifs and coding sequence features to variations in mRNA abundance

The context-dependent expression of genes is the core for biological activities, and significant attention has been given to identification of various factors contributing to gene expression at genomic scale. However, so far this type of analysis has been focused either on relation between mRNA expression and non-coding sequence features such as upstream regulatory motifs or on correlation between mRNA abundance and non-random features in coding sequences (e.g., codon usage and amino acid usage). In this study multiple regression analyses of the mRNA abundance and all sequence information in Desulfovibrio vulgaris were performed, with the goal to investigate how much coding and non-coding sequence features contribute to the variations in mRNA expression, and in what manner they act together. Using the AlignACE program, 442 over-represented motifs were identified from the upstream 100bp region of 293 genes located in the known regulons. Regression of mRNA expression data against the measures of coding and non-coding sequence features indicated that 54.1% of the variations in mRNA abundance can be explained by the presence of upstream motifs, while coding sequences alone contribute to 29.7% of the variations in mRNA abundance. Interestingly, most of contribution from coding sequences is overlapping with that from upstream motifs; thereby a total of 60.3% of the variations in mRNA abundance can be explained when coding and non-coding information was included. This result demonstrates that upstream regulatory motifs and coding sequence information contribute to the overall mRNA expression in a combinatorial rather than an additive manner.     

Ultraviolet light-induced crosslinking of mRNA to proteins.

Irradiation of intact or EDTA-dissociated L-cell polyribosomes with 254 nm UV light at doses of 1-2 x 10(5) ergs/mm2 extensively crosslinks mRNA to proteins. The crosslinked mRNA-protein complexes can be isolated on the basis of buoyant density in urea-containing CS2SO4 gradients that dissociate non-covalent complexes. Crosslinking of mRNA can also be assayed by phenolchloroform extraction. mRNA recovered from the crosslinked complexes by digestion with proteinase K has the same electrophoretic mobility in polyacrylamide gels as unirradiated mRNA. Therefore, irradiation does not either crosslink RNA molecules to RNA molecules or break phosphodiester bonds. With these methods it has been found that more than 70% of high molecular weight polydisperse mRNA, but only 25-40% of histone mRNA, can be crosslinked to protein. On the basis of buoyant density the histone mRNA-protein complex had a protein content of 26%, whereas the mean protein content of most non-histone mRNA-protein complexes was 65%. It is concluded that most mRNA in polyribosomes is in close contact with proteins, and that histone mRNA can be crosslinked to many fewer proteins that most other mRNAs.     

How is mRNA expression predictive for protein expression? A correlation study on human circulating monocytes

A key assumption in studying mRNA expression is that it is informative in the prediction of protein expression. However, only limited studies have explored the mRNA-protein expression correlation in yeast or human tissues and the results have been relatively inconsistent. We carried out correlation analyses on mRNA-protein expressions in freshly isolated human circulating monocytes from 30 unrelated women. The expressed proteins for 71 genes were quantified and identified by 2-D electrophoresis coupled with mass spectrometry. The corresponding mRNA expressions were quantified by Affymetrix gene chips. Significant correlation ( r =0.235, P <0.0001) was observed for the whole dataset including all studied genes and all samples. The correlations varied in different biological categories of gene ontology. For example, the highest correlation was achieved for genes of the extracellular region in terms of cellular component ( r =0.643, P <0.0001) and the lowest correlation was obtained for genes of regulation ( r =0.099, P=0.213) in terms of biological process. In the genome, half of the samples showed significant positive correlation for the 71 genes and significant correlation was found between the average mRNA and the average protein expression levels in all samples ( r =0.296, P <0.01). However, at the study group level, only five studied genes had significant positive correlation across all the samples. Our results showed an overall positive correlation between mRNA and protein expression levels. However, the moderate and varied correlations suggest that mRNA expression might be sometimes useful, but certainly far from perfect, in predicting protein expression levels.     

Determinants of variation in human serum paraoxonase activity

Paraoxonase-1 (PON1) is associated with high-density lipoprotein (HDL) particles and is believed to contribute to antiatherogenic properties of HDLs. We assessed the determinants of PON1 activity variation using different substrates of the enzyme. PON1 activity in serum samples from 922 participants in the San Antonio Family Heart Study was assayed using a reliable microplate format with three substrates: paraoxon, phenyl acetate and the lactone dihydrocoumarin. There were major differences among results from the three substrates in degree of effect by various environmental and genetic factors, suggesting that knowledge of one substrate activity alone may not provide a complete sense of PON1 metabolism. Three significant demographic covariates (age, smoking status and contraceptive usage) together explained 1-6% of phenotypic variance, whereas four metabolic covariates representing lipoprotein metabolism (apoAII, apoAI, triglycerides and non-HDL cholesterol) explained 4-19%. Genes explained 65-92% of phenotypic variance and the dominant genetic effect was exerted by a locus mapping at or near the protein structural locus (PON1) on chromosome 7. Additional genes influencing PON1 activity were localized to chromosomes 3 and 14. Our study identified environmental and genetic determinants of PON1 activity that accounted for 88-97% of total phenotypic variance, suggesting that few, if any, major biological determinants are unrepresented in the models.     

Discordant protein and mRNA expression in lung adenocarcinomas

The relationship between gene expression measured at the mRNA level and the corresponding protein level is not well characterized in human cancer. In this study, we compared mRNA and protein expression for a cohort of genes in the same lung adenocarcinomas. The abundance of 165 protein spots representing 98 individual genes was analyzed in 76 lung adenocarcinomas and nine non-neoplastic lung tissues using two-dimensional polyacrylamide gel electrophoresis. Specific polypeptides were identified using matrix-assisted laser desorption/ionization mass spectrometry. For the same 85 samples, mRNA levels were determined using oligonucleotide microarrays, allowing a comparative analysis of mRNA and protein expression among the 165 protein spots. Twenty-eight of the 165 protein spots (17%) or 21 of 98 genes (21.4%) had a statistically significant correlation between protein and mRNA expression (r > 0.2445; p < 0.05); however, among all 165 proteins the correlation coefficient values (r) ranged from -0.467 to 0.442. Correlation coefficient values were not related to protein abundance. Further, no significant correlation between mRNA and protein expression was found (r = -0.025) if the average levels of mRNA or protein among all samples were applied across the 165 protein spots (98 genes). The mRNA/protein correlation coefficient also varied among proteins with multiple isoforms, indicating potentially separate isoform-specific mechanisms for the regulation of protein abundance. Among the 21 genes with a significant correlation between mRNA and protein, five genes differed significantly between stage I and stage III lung adenocarcinomas. Using a quantitative analysis of mRNA and protein expression within the same lung adenocarcinomas, we showed that only a subset of the proteins exhibited a significant correlation with mRNA abundance.     

Increasing precision in randomised field experiments: barnacle microtopography as a predictor of Littorina abundance

We investigated whether measurements of barnacle microtopography could be used as covariates to increase precision in randomised field experiments with intertidal Littorina populations. We used image analysis to quantify the microtopography of the barnacles within 100 cm2 quadrats in July 1994 using as variables: number of barnacles, number of dead empty barnacles, mean distance to nearest neighbouring barnacle, total area covered by barnacles, and number of pockets of different size-classes [extra-small (< 0.15 cm2), small (0.15 and < 0.30 cm2), medium ( 0.30 and < 0.45 cm2), large ( 0.45 and < 0.60 cm2), and extra-large (0.60 cm2)] at least 75% surrounded by barnacles. We then used these variables to predict the abundance of Littorina spp. in the same quadrat. Two variables: the number of extra-small pockets and the number of small pockets accounted for 50% of the variation in total littorinid snail abundance among quadrats for January 1995 and for 47% of the variation for August 1994 but for only 16% of the variation for June 1994. However the single variable, the number of barnacles, accounted for 29–36% of the variation in snail abundance on these dates and can be measured without special equipment. The best combination of covariates was the number of extra-small pockets alone. The number of extra-small pockets between barnacles may be influencing the snail abundance by providing a refuge from wave shock and heat stress/desiccation. We suggest that the availability of these refuges among the barnacles could act as a density-dependent regulator of the total population size of a particular species of Littorina.     

Relation of growth- and sterol-related regulatory pathways for low density lipoprotein receptor gene expression

The relationship between growth- and sterol-related regulation of low density lipoprotein (LDL) receptor gene expression was examined in human skin fibroblasts. Platelet-derived growth factor (PDGF) and high density lipoprotein (HDL3) each produced increased cellular LDL protein and mRNA levels. However, HDL, when added with PDGF or insulin produced no additional effect. Coincubations with 25-hydroxycholesterol markedly attenuated fibroblast LDL receptor protein and mRNA response to growth activation. Acute transfection experiments using sterol-responsive elements of the LDL receptor gene indicated that these elements are also responsive to growth activation with PDGF. These data indicate that alteration of cellular sterol balance or metabolism leading to release of end product repression contributes to growth-related stimulation of LDL receptor gene expression.     

Protective role of 20-hydroxyecdysone against lead stress in Chlorella vulgaris cultures

Treatment of cultured C. vulgaris cells with 10(-6)-10(-4) M lead decreased their growth and chemical composition during the first 48 h of cultivation. However, at concentrations above 10(-4) M, lead is cytotoxic to Chlorella vulgaris cells, resulting in cellular fragmentation and lysis. In contrast, at concentrations below 10(-6) M lead had no influence on the growth and metabolism of C. vulgaris cells. 20-Hydroxyecdysone (20E) (10(-10)-10(-8) M) increased growth and chemical composition of C. vulgaris cells over a concentration range. Levels per cell of chlorophylls, protein, sugars are all increased by 20E treatment, when compared to non-treated control cells. However, the cultures treated with 20E and lead show a lower stimulation than the cultures treated with 20E alone. The effects of 20E mixed with lead on the growth and the level of cellular lead, chlorophyll, sugar and protein in C. vulgaris are also reported. The decreased growth and composition of C. vulgaris cells treated with lead was restored by the 20E. Application of 20E to C. vulgaris cultures reduced the impact of lead stress on growth, prevented chlorophyll, sugar and protein loss and increased phytochelatins synthesis. Furthermore, 20E did not restore toxic effect of lead on C. vulgaris cells. The combined treatment with lead and 20E appeared to have a stimulatory effect on the above parameters during the 48 h of cultivation, as compared to the control. 20E reduced the toxicity of lead and the growth recovered to the level of cells treated with 20E alone. Concentration-dependent stimulation was observed with increasing concentration of 20E and decreasing concentration of lead.     

Influence of Protein Abundance on High-Throughput Protein-Protein Interaction Detection

Experimental protein-protein interaction (PPI) networks are increasingly being exploited in diverse ways for biological discovery. Accordingly, it is vital to discern their underlying natures by identifying and classifying the various types of deterministic (specific) and probabilistic (nonspecific) interactions detected. To this end, we have analyzed PPI networks determined using a range of high-throughput experimental techniques with the aim of systematically quantifying any biases that arise from the varying cellular abundances of the proteins. We confirm that PPI networks determined using affinity purification methods for yeast and Eschericia coli incorporate a correlation between protein degree, or number of interactions, and cellular abundance. The observed correlations are small but statistically significant and occur in both unprocessed (raw) and processed (high-confidence) data sets. In contrast, the yeast two-hybrid system yields networks that contain no such relationship. While previously commented based on mRNA abundance, our more extensive analysis based on protein abundance confirms a systematic difference between PPI networks determined from the two technologies. We additionally demonstrate that the centrality-lethality rule, which implies that higher-degree proteins are more likely to be essential, may be misleading, as protein abundance measurements identify essential proteins to be more prevalent than nonessential proteins. In fact, we generally find that when there is a degree/abundance correlation, the degree distributions of nonessential and essential proteins are also disparate. Conversely, when there is no degree/abundance correlation, the degree distributions of nonessential and essential proteins are not different. However, we show that essentiality manifests itself as a biological property in all of the yeast PPI networks investigated here via enrichments of interactions between essential proteins. These findings provide valuable insights into the underlying natures of the various high-throughput technologies utilized to detect PPIs and should lead to more effective strategies for the inference and analysis of high-quality PPI data sets.     

Isolation and structural characterization of cap-binding proteins from poliovirus-infected HeLa cells.

In poliovirus-infected HeLa cells, poliovirus RNA is translated at times when cellular mRNA translation is strongly inhibited. It is thought that this translational control mechanism is mediated by inactivation of a cap-binding protein complex (comprising polypeptides of 24 [24-kilodalton cap-binding protein], 50, and approximately 220 kilodaltons). This complex can restore the translation of capped mRNAs in extracts from poliovirus-infected cells. We have previously shown that the virally induced defect prevents interaction between cap recognition factors and mRNA. Here, we show that the cap-binding protein complex (and not the 24-kilodalton cap-binding protein) has activity that restores the cap-specific mRNA-protein interaction when added to initiation factors from poliovirus-infected cells. Thus, the activity that restores the cap-specific mRNA-protein interaction and that which restores the translation of capped mRNAs in extracts from poliovirus-infected cells, copurify. The results also indicate, by an alternative assay, that the cap-binding protein complex is the only factor inactivated by poliovirus. We also purified cap-binding proteins from uninfected and poliovirus-infected HeLa cells. By various criteria, the 24-kilodalton cap-binding protein is not structurally modified as a result of infection. However, the 220-kilodalton polypeptide of the cap-binding protein complex is apparently cleaved by a putative viral (or induced) protease. By in vivo labeling and m7GDP affinity chromatography, we isolated a modified cap-binding protein complex from poliovirus-infected cells, containing proteolytic cleavage fragments of the 220-kilodalton polypeptide.