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Transcription, mRNA decay, translation and protein degradation are essential processes during eukaryotic gene expression, but their relative global contributions to steady-state protein concentrations in multi-cellular eukaryotes are largely unknown. Using measurements of absolute protein and mRNA abundances in cellular lysate from the human Daoy medulloblastoma cell line, we quantitatively evaluate the impact of mRNA concentration and sequence features implicated in translation and protein degradation on protein expression. Sequence features related to translation and protein degradation have an impact similar to that of mRNA abundance, and their combined contribution explains two-thirds of protein abundance variation. mRNA sequence lengths, amino-acid properties, upstream open reading frames and secondary structures in the 5' untranslated region (UTR) were the strongest individual correlates of protein concentrations. In a combined model, characteristics of the coding region and the 3'UTR explained a larger proportion of protein abundance variation than characteristics of the 5'UTR. Further, we used data from human and six other organisms (bacteria, yeast, worm, fly, and plant) and established that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data suggest strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge. The absolute protein and mRNA concentration measurements for >1000 human genes and for other organisms represent one of the largest datasets currently available, and reveal both general trends and specific examples of post-transcriptional regulation.

β-adrenergic receptors (β-ARs) are G protein-coupled receptors that activate signal transduction pathways involved in angiogenesis, resulting in enhanced tumor vascularization and more aggressive growth. In this study, we evaluated the expression of β-ARs in a population of 12 children affected by malignant primary brain tumors. We found a significant expression of β1- and β2-ARs in all 12 samples as well as the 3 cell lines tested (U87MG, T98G and DAOY). The mean absolute β1-AR mRNA level standardized to GAPDH was 5.81 (range, -7.91 to 11.29) for brain tumors and 8.59 (range, 6.046 to 12.59) for cell lines (U87MG, DAOY and T98G), respectively. The mean absolute β2-AR mRNA level was 4.74 (range, −9.30 to 8.45) for tumor specimens and 7.64 (range, 5.85 to 8.88) for cell lines. These real-time quantitative (qRT)-PCR expression data were confirmed by immunohistochemical analysis. Our study evaluated the presence of β1- and β2-ARs in malignant pediatric brain tumors and brain tumor cell lines.

The involvement of matrix metalloproteinases (MMPs) has been suggested in cellular mechanisms leading to medulloblastoma (MB), the most common malignant brain tumor in children. A significant association of the expression levels of MMP-9 with survival and M stage suggests that patients with medulloblastoma metastatic disease at diagnosis may benefit from the anti-MMP therapy. Here, we have evaluated the tumorigenicity of medulloblastoma cells after infection with an adenovirus containing a 21 bp siRNA sequence of human MMP-9 gene (Ad-MMP-9). Infection of Daoy medulloblastoma cells with Ad-MMP-9 reduced MMP-9 activity and protein levels compared with parental and Ad-SV controls. Ad-MMP-9 decreased the number of viable Daoy cells in a concentration-dependent manner. FACS analysis indicated Ad-MMP-9 infection caused a dose-dependent cell cycle arrest in the G0/G1 phase. Ad-MMP-9-induced cell cycle arrest appears to be mediated by the ERK MAPK pathway and the cell cycle inhibitors p16INK4 and is phenotypically indistinguishable from senescence. Ad-MMP-9 treatment inhibited medulloblastoma tumor growth in an intracranial model and was mediated by upregulation of p16 expression. These studies validate the usefulness of targeting MMP-9 and provide a novel perspective in the treatment of medulloblastoma.

Methionine (MET)-dependent cell lines require MET to proliferate, and homocysteine (HCY) does not act as a substitute for this requirement. From six O6-methylguanine-DNA methyltransferase (MGMT)-efficient (mer+) cell lines tested, two medulloblastomas (Daoy and D-341) and a lung non-small-cell adenocarcinoma with metastatic potential (H-1623) were most sensitive to MET deprivation, while two glioblastomas (U-138, D-263) and a small-cell lung carcinoma H-1944 were moderately to weakly dependent. Regardless of the degree of MET dependence, all of these lines down-regulated their MGMT activity within 48-72 h of transfer from MET+HCY- to MET-HCY+ media, long before the eradication of the culture. Reduction of MGMT activity was due to a decline of both MGMT mRNA and protein levels. However, the reduction was not related to the methylation status of the MGMT promoter at the SmaI site or the HpaII sites in the body of the gene; such sites have been shown to be associated in MGMT regulation and in defining the mer phenotype. MET-dependent, mer+ tumour cells cultured in MET-HCY+ were more sensitive to BCNU (IC50 = 5-10 microM) than those cultured in MET+HCY-(IC50 = 45-90 microM), while MET-independent or mer- cell lines were unaffected. This indicates that reduction of MGMT, imposed by the absence of MET, renders mer+ tumour cells more susceptible to alkylating agents. The relatively selective suppression of MGMT activity in mer+ MET-dependent tumour cells, in combination with the inability of such cells to proliferate in the absence of MET, may lead to the development of more effective treatment strategies for mer+ MET-dependent tumours.

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Degradation of mRNA in bacteria is a regulatory mechanism, providing an efficient way to fine-tune protein abundance in response to environmental changes. While the mechanisms responsible for initiation and subsequent propagation of mRNA degradation are well studied, the mRNA features that affect its stability are yet to be elucidated. We calculated three properties for each mRNA in the E. coli transcriptome: G+C content, tRNA adaptation index (tAI) and folding energy. Each of these properties were then correlated with the experimental transcript half life measured for each transcript and detected significant correlations. A sliding window analysis identified the regions that displayed the maximal signal. The correlation between transcript half life and both G+C content and folding energy was strongest at the 5′ termini of the mRNAs. Partial correlations showed that each of the parameters contributes separately to mRNA half life. Notably, mRNAs of recently-acquired genes in the E. coli genome, which have a distinct nucleotide composition, tend to be highly stable. This high stability may aid the evolutionary fixation of horizontally acquired genes.

We have characterized an mRNA that increases in abundance after serum stimulation of quiescent mouse fibroblasts. This mRNA, designated 18A2, encodes a predicted polypeptide of 101 amino acids with homology to known calcium binding proteins. A variety of mouse tissues express the 18A2 mRNA, with the highest levels detected in the non-pregnant uterus and in the placenta. The concentration of 18A2 mRNA in total placental RNA decreases from day 8 to day 10 of pregnancy, and is below detection throughout the latter half of gestation. In serum-stimulated fibroblasts, the increase in 18A2 mRNA is dependent on protein synthesis. The 18A2 mRNA is similar in size, serum-inducibility, and sequence to the 2A9 mRNA (1), but these mRNAs are derived from distinct genes. This suggests that the mouse genome harbors a family of serum-inducible genes encoding proteins predicted to bind calcium.

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Background

The availability of high throughput experimental methods has made possible to observe the relationships between proteome and transcirptome. The protein abundances show a positive but weak correlation with the concentrations of their cognate mRNAs. This weak correlation implies that there are other crucial effects involved in the regulation of protein translation, different from the sole availability of mRNA. It is well known that ribosome and tRNA concentrations are sources of variation in protein levels. Thus, by using integrated analysis of omics data, genomic information, transcriptome and proteome, we aim to unravel important variables affecting translation.

Results

We identified how much of the variability in the correlation between protein and mRNA concentrations can be attributed to the gene codon frequencies. We propose the hypothesis that the influence of codon frequency is due to the competition of cognate and near-cognate tRNA binding; which in turn is a function of the tRNA concentrations. Transcriptome and proteome data were combined in two analytical steps; first, we used Self-Organizing Maps (SOM) to identify similarities among genes, based on their codon frequencies, grouping them into different clusters; and second, we calculated the variance in the protein mRNA correlation in the sampled genes from each cluster. This procedure is justified within a mathematical framework.

Conclusions

With the proposed method we observed that in all the six studied cases most of the variability in the relation protein-transcript could be explained by the variation in codon composition.

Summary

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs and rRNAs. Here we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA, and provides a resource for future studies of mitochondrial function (accessed at mitochondria.matticklab.com).

We have shown previously that when the Escherichia coli chromosomal lacZ gene is put under the control of an extended Shine-Dalgarno (SD) sequence (10 or 6 nucleotides in length), the translation efficiency can be highly variable, depending on the presence of AU-rich targets for ribosomal protein S1 in the mRNA leader. Here, the same strains have been used to examine the question of how strong ribosome binding to extended SD sequences affects the stability of lacZ mRNAs translated with different efficiencies. The steady-state concentration of the lacZ transcripts has been found to vary over a broad range, directly correlating with translation efficiency but not with the SD duplex stability. The observed strain-to-strain variations in lacZ mRNA level became far less marked in the presence of the rne-1 mutation, which partially inactivates RNase E. Together, the results show that (i) an SD sequence, even one that is very long, cannot stabilize the lacZ mRNA in E. coli if translation is inefficient; (ii) inefficiently translated lacZ transcripts are sensitive to RNase E; and (iii) AU-rich elements inserted upstream of a long SD sequence enhance translation and stabilize mRNA, despite the fact that they constitute potential RNase E sites. These data strongly support the idea that the lacZ mRNA in E. coli can be stabilized only by translating, and not by stalling, ribosomes.

The monoclonal antibody ONS-M21 recognizes an antigen found on the surface of glioma and medulloblastoma cells but does not react with the antigens of normal brain tissue. We purified and identified the 140-kDa protein by means of an antibody-binding affinity column. This 140-kDa antigen has sequences homologous to the amino-terminal region and five parts of the internal domain of integrin α3. When the integrin α3-related sequences was amplified and used to analyse the mRNA of glioma and medulloblastoma surgical specimens, the transcription level of integrin α3 mRNA appeared to be quantitatively correlated with the grade of malignancy. These findings suggest that the ONS-M21 antibody, which reacts with integrin α3, might be useful in the diagnosis of gliomas and medulloblastomas. © 1999 Cancer Research Campaign

Background:

Secreted protein acidic and rich in cysteine (SPARC), a matricellular glycoprotein, modulates cellular interaction with the extracellular matrix and is capable of altering the growth of various cancers. We therefore sought to determine the effect of SPARC expression on medulloblastoma tumour growth and angiogenesis.

Methods:

To this extent, we selected three SPARC full-length cDNA overexpressed clones (Daoy-SP). Consequences of SPARC overexpression were studied in terms of cell growth, angiogenesis using co-culture assay in vitro, dorsal skin-fold chamber assay in vivo, PCR Array for human angiogenic genes, as well as western blotting for angiogenic molecules and tumour growth, in an orthotopic tumour model.

Results:

The SPARC protein and mRNA levels were increased by approximately three-fold in Daoy-SP cells compared with parental (Daoy-P) and vector (Daoy-EV) controls. Daoy-SP clones reduced tumour cell-induced angiogenesis in vitro and in vivo, and formed small tumours with fewer blood vessels when compared with controls. Matrix metalloprotease-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression were decreased in Daoy-SP clones. Further, inhibition of MMP-9 expression caused SPARC-mediated inhibition of angiogenesis and tumour growth as MMP-9 rescued SPARC-mediated anti-angiogenic effect in vitro and tumour growth inhibition in vivo.

Conclusion:

Overexpression of SPARC decreases angiogenesis, which leads to decreased tumour growth. Further, the role of MMP-9 could be attributed to the anti-angiogenic effect of SPARC.

The intrinsic stochasticity of gene expression leads to cell-to-cell variations, noise, in protein abundance. Several processes, including transcription, translation, and degradation of mRNA and proteins, can contribute to these variations. Recent single cell analyses of gene expression in yeast have uncovered a general trend where expression noise scales with protein abundance. This trend is consistent with a stochastic model of gene expression where mRNA copy number follows the random birth and death process. However, some deviations from this basic trend have also been observed, prompting questions about the contribution of gene-specific features to such deviations. For example, recent studies have pointed to the TATA box as a sequence feature that can influence expression noise by facilitating expression bursts. Transcription-originated noise can be potentially further amplified in translation. Therefore, we asked the question of to what extent sequence features known or postulated to accompany translation efficiency can also be associated with increase in noise strength and, on average, how such increase compares to the amplification associated with the TATA box. Untangling different components of expression noise is highly nontrivial, as they may be gene or gene-module specific. In particular, focusing on codon usage as one of the sequence features associated with efficient translation, we found that ribosomal genes display a different relationship between expression noise and codon usage as compared to other genes. Within nonribosomal genes we found that sequence high codon usage is correlated with increased noise relative to the average noise of proteins with the same abundance. Interestingly, by projecting the data on a theoretical model of gene expression, we found that the amplification of noise strength associated with codon usage is comparable to that of the TATA box, suggesting that the effect of translation on noise in eukaryotic gene expression might be more prominent than previously appreciated.

Author Summary

The stochastic nature of gene expression leads to cell-to-cell differences in protein level referred to as noise. Expression noise can be disadvantageous, by affecting the precision of biological functions, but it may also be advantageous by enabling heterogeneous stress-response programs to environmental changes. Therefore various genes and gene groups might display various levels of expression noise. Importantly, gene expression is a multi-step process and the stochasticity of its individual steps, including transcription and translation, contributes to the resulting variability. Recent single cell analyses of gene expression in yeast have confirmed the theoretically predicted general trend where expression noise scales with protein abundance. However, some deviations from this basic trend have also been observed, prompting questions about the contribution of gene-specific features to such deviations. Accounting for noise heterogeneity in different gene groups, we revealed a clear relationship between noise and translation-related genomic features, specifically codon usage and 5′ UTR secondary structure. Our results suggest that the effect of translation on these deviations might be more prominent than previously appreciated, and provide important clues towards understanding expression stochasticity in yeast.

Background

Gene transcription is regulated by DNA transcriptional regulatory elements, promoters and enhancers that are located outside the coding regions. Here, we examine the characteristic 3-base periodicity of the coding sequences and analyse its correlation with the genome-wide transcriptional profile of yeast.

Principal Findings

The analysis of coding sequences by a new class of indices proposed here identified two different sources of 3-base periodicity: the codon frequency and the codon sequence. In exponentially growing yeast cells, the codon-frequency component of periodicity accounts for 71.9% of the variability of the cellular mRNA by a strong association with the density of elongating mRNA polymerase II complexes. The mRNA abundance explains most of the correlation between the codon-frequency component of periodicity and protein levels. Furthermore, pyrimidine-ending codons of the four-fold degenerate small amino acids alanine, glycine and valine are associated with genes with double the transcription rate of those associated with purine-ending codons.

Conclusions

We demonstrate that the 3-base periodicity of coding sequences is higher than expected by the codon usage frequency (CUF) and that its components, associated with codon bias and amino acid composition, are correlated with gene expression, principally at the level of transcription elongation. This indicates a role of codon sequences in maximising the transcription efficiency in exponentially growing yeast cells. Moreover, the results contrast with the common Darwinian explanation that attributes the codon bias to translational selection by an adjustment of synonymous codon frequencies to the most abundant isoaccepting tRNA. Here, we show that selection on codon bias likely acts at both the transcriptional and translational level and that codon usage and the relative abundance of tRNA could drive each other in order to synergistically optimize the efficiency of gene expression.

By characterizing dynamic changes in yeast protein abundance following osmotic shock, this study shows that the correlation between protein and mRNA differs for transcripts that increase versus decrease in abundance, and reveals physiological reasons for these differences.

The correlation between protein and mRNA change is very high at transcripts that increase in abundance, but negligible at reduced transcripts following NaCl shock.Modeling and experimental data suggest that reducing levels of high-abundance transcripts helps to direct translational machinery to newly made transcripts.The transient burst of transcript increase serves to accelerate changes in protein abundance.Post-transcriptional regulation of protein abundance is pervasive, although most of the variance in protein change is explained by changes in mRNA abundance.

Natural microenvironments change rapidly, and living creatures must respond quickly and efficiently to thrive within this flux. At all cellular levels—signaling, transcription, translation, metabolism, cell growth, and division—the response is dynamic and coordinated. Some aspects of this response, such as dynamic changes of the transcriptome, are well understood. But other aspects, like the response of the proteome, have remained obscured primarily because of previous limitations in technology. Without coordinated time-course data, it has remained impossible to correctly characterize the correlations and dependencies between these two essential levels of cell biology.

This work presents an extended picture of the coordinated response of the transcriptome and proteome as cells respond to an abrupt environmental change. To assay proteomic dynamics, we developed a strategy for large-scale, multiplexed quantitation using isobaric tags and high mass accuracy mass spectrometry. This sensitive yet efficient platform allows for the expedient collection of quantitative time-course proteomic data at six time points, sufficiently reproducible to permit meaningful interpretation of variation across biological replicates. Time-course transcriptome data were generated from paired biological samples, allowing us to examine the relationships between changes in mRNA and protein for each gene in terms of direction and intensity, as well as the characteristics of the temporal profiles for each gene.

It was immediately obvious that a single measure of correlation across the entire data set was a meaningless metric. We therefore analyzed relationships between mRNA and protein for different subsets of data. In response to osmotic shock, hundreds of transcripts are highly induced, and their temporal pattern reveals a transient peak of maximal induction, which resolves into a new elevated level as cells acclimate (Figure 2). For this group of genes, there is extremely high correlation between peak mRNA change and protein change (R2∼0.8). But the dynamics of the molecules differ: while mRNA levels transiently overshoot their final levels, proteins gradually rise in abundance toward their new, elevated state. We observed, however, that a measure of efficiency connects the two profiles. The time it takes for a protein to acclimate to its new state correlates with the magnitude of the excess mRNA induction. Thus, the cell imparts an urgency to protein induction by transiently producing excess transcript.

The most surprising result, however, involves transcripts that decrease in abundance. In response to osmotic shock, the cell transiently reduces over 600 transcripts, many of which are among the most highly expressed in unstressed cells. But protein levels for these genes remain, for the most part, almost completely unchanged. The stark absence of protein repression is independent of basal protein abundance, independent of reported protein half-lives, reproducible across biological replicates, and validated by quantitative western blots. Furthermore, since we do detect a handful of proteins whose abundance is significantly reduced, our technology is capable of identifying protein loss. Thus, we conclude that transcript reduction serves another purpose besides reducing protein levels.

To explore alternate interpretations of the consequence of transcriptional repression, we devised a mass-action kinetic model, which describes protein changes based on mRNA dynamics in the context of transient changes in the rates of cell division. The model successfully recapitulated the observed data, allowing us to alter modeling parameters to test various hypotheses.

In response to osmotic shock, overall rates of translation temporarily decrease and cell growth transiently arrests before resuming at a slower rate. We reasoned that mRNA reduction might lower the rate of new protein synthesis, but that retarded production is balanced by reduced cell division. We explored both aspects of this logic with our model.

As expected, removing cell division from our model led to a calculated decrease of protein levels, indicating that reduced growth is necessary for maintaining protein levels. However, when we computationally held mRNA levels stable and calculated protein levels in the absence of mRNA repression, we did not find the expected increase in protein abundance.

We then considered the possibility that one function of the regulated repression of these highly abundant transcripts was to liberate proteins essential for translation, such as ribosomes or translation initiation factors. To explore this, we examined a mutant lacking the Dot6p/Tod6p transcriptional repressors, which fails to properly repress ∼250 genes in response to osmotic shock. In the wild type, the mRNA for a Dot6p/Tod6p target (ARX1) decreased seven-fold, and the remaining transcript was generally unassociated with poly-ribosomes. In the mutant, however, the mRNA levels were reduced only two-fold, while the remaining transcript continued to bind ribosomes. Therefore, failure to reduce transcript levels led to a persistent association with poly-ribosomes, thereby consuming translational machinery.

Our hypothesis is, therefore, that widespread changes in the transcriptome promote efficient translation of new proteins. Transcript increase serves to increase abundance of the encoded proteins, while reduction of some of the most abundant and highly translated mRNAs supports this project by liberating translational capacity. While it is not clear what factors are the limiting elements, it is clear that a full picture of cellular biology requires exploring the dynamics of the cellular response.

The transcriptome and proteome change dynamically as cells respond to environmental stress; however, prior proteomic studies reported poor correlation between mRNA and protein, rendering their relationships unclear. To address this, we combined high mass accuracy mass spectrometry with isobaric tagging to quantify dynamic changes in ∼2500 Saccharomyces cerevisiae proteins, in biological triplicate and with paired mRNA samples, as cells acclimated to high osmolarity. Surprisingly, while transcript induction correlated extremely well with protein increase, transcript reduction produced little to no change in the corresponding proteins. We constructed a mathematical model of dynamic protein changes and propose that the lack of protein reduction is explained by cell-division arrest, while transcript reduction supports redistribution of translational machinery. Furthermore, the transient ‘burst' of mRNA induction after stress serves to accelerate change in the corresponding protein levels. We identified several classes of post-transcriptional regulation, but show that most of the variance in protein changes is explained by mRNA. Our results present a picture of the coordinated physiological responses at the levels of mRNA, protein, protein-synthetic capacity, and cellular growth.

Background

With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis.

Methods

To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425).

Results

siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector.

Conclusion

In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.

Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression datasets from seven different species: two bacteria, yeast, nematode, fly, human, and plant. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge.

This paper connects Alu repeats, the most abundant repetitive elements in the human genome and microRNAs, small RNAs that alter gene expression at the post-transcriptional level. Base-pair complementarity could be demonstrated between the seed sequence of a subset of human microRNAs and Alu repeats that are integrated parallel (sense) in mRNAs. The most common target site coincides with the evolutionary most conserved part of Alu. A primate-specific gene cluster on chromosome 19 encodes the majority of miRNAs that target the most conserved sense Alu site. The individual miRNA genes within this cluster are flanked by an Alu-LINE signature, which has been duplicated with the clustered miRNA genes. Gene duplication events in this locus are supported by comparing repeat length variations of the LINE elements within the cluster with those in the rest of the chromosome. Thus, a dual relationship exists between an evolutionary young miRNA cluster and their Alu targets that may have evolved in the same time window. One hypothesis for this dual relationship is that these miRNAs could protect against too high rates of duplicative transposition, which would destroy the genome.

An in vitro autophosphorylation assay has been used to demonstrate that there is considerable variation in met associated protein kinase among human tumour cell lines. Of particular note was the very high level of autophosphorylation of the 140 kD met protein (p140met) in experiments with A431 human cervical carcinoma cells. In contrast in experiments with Daoy human medulloblastoma cells we failed to detect phosphorylation of p140met; instead a high level of phosphorylation of a 132 kD protein was observed. To help understand the basis for the variation in kinase activity and to learn more about the structure of the mature met protein we have analysed p140met in SDS-polyacrylamide gels under non-reducing conditions. Under these conditions the met protein had an apparent molecular weight of 165,000 indicating that the mature met protein may exist as an alpha beta complex in which p140met (designated the beta subunit) is joined by disulphide bonds to a smaller, 25 kD, alpha-chain. We have identified a potential proteolytic cleavage site with the sequence Lys-Arg-Lys-Lys-Arg-Ser at amino acids 303-308 in the human met protein that may account for cleavage of the met protein into alpha and beta subunits.

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Background

Musashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer.

Methods

We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells.

Results

We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

Conclusion

Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

Alpha-foetoprotein (AFP) synthesis, although repressed in normal adults, is increased in the majority of patients with hepatocellular carcinoma (HCC). We have investigated whether active transcription of the AFP gene may explain raised serum AFP concentrations in patients with HCC and hepatoblastoma by assaying human tumour and non-neoplastic tissue by molecular hybridization for the presence of mRNA encoding AFP. Ten operative HCC and six autopsy HCC specimens, two HCC cell lines, and one hepatoblastoma specimen were examined. Total cellular RNA and poly-(A)+ RNA were extracted and AFP mRNA sequences sought by dot-blot and Northern blot hybridisation to a human cDNA AFP probe. Cellular AFP was localised by avidin-biotin staining. AFP mRNA was detected in 8/10 operative specimens, as well as PLC/PRF/5 nude mouse tumours. Weaker hybridization was detected in 4/6 autopsy specimens. Signals of comparable intensity to that in operative tumours were detected in non-neoplastic tissue of 6 patients. AFP mRNA from nude mouse tumours migrated as a 20S discrete band on agarose gel electrophoresis, whereas a more complex hybridization pattern was evident in human tumours. Positive cytoplasmic immuno-staining for AFP was observed in 4 tumours and 2 corresponding non-neoplastic specimens and in a HCC cell line. In non-neoplastic liver, AFP was localised in cells that appeared dysplastic. Thus steady-state levels of AFP mRNA are detectable in human HCC tissue and surrounding non-neoplastic liver. These findings may prove pertinent to an understanding of the genetic expression of AFP in malignant hepatocytes, and the sequence of events leading to uncontrolled cellular proliferation.

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miRNAs are small RNA molecules (′ 22nt) that interact with their corresponding target mRNAs inhibiting the translation of the mRNA into proteins and cleaving the target mRNA. This second effect diminishes the overall expression of the target mRNA. Several miRNA-mRNA relationship databases have been deployed, most of them based on sequence complementarities. However, the number of false positives in these databases is large and they do not overlap completely. Recently, it has been proposed to combine expression measurement from both miRNA and mRNA and sequence based predictions to achieve more accurate relationships. In our work, we use LASSO regression with non-positive constraints to integrate both sources of information. LASSO enforces the sparseness of the solution and the non-positive constraints restrict the search of miRNA targets to those with down-regulation effects on the mRNA expression. We named this method TaLasso (miRNA-Target LASSO).

We used TaLasso on two public datasets that have paired expression levels of human miRNAs and mRNAs. The top ranked interactions recovered by TaLasso are especially enriched (more than using any other algorithm) in experimentally validated targets. The functions of the genes with mRNA transcripts in the top-ranked interactions are meaningful. This is not the case using other algorithms.

TaLasso is available as Matlab or R code. There is also a web-based tool for human miRNAs at http://talasso.cnb.csic.es/.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.

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Background

Genome architecture is profoundly influenced by transposable elements (TEs), and natural selection against their harmful effects is a critical factor limiting their spread. Genome defense by the piRNA silencing pathway also plays a crucial role in limiting TE proliferation. How these two forces jointly determine TE abundance is not well understood. To shed light on the nature of factors that predict TE success, we test three distinct hypotheses in the Drosophila genus. First, we determine whether TE abundance and relaxed genome-wide purifying selection on protein sequences are positively correlated. This serves to test the hypothesis that variation in TE abundance in the Drosophila genus can be explained by the strength of natural selection, relative to drift, acting in parallel against mildly deleterious non-synonymous mutations. Second, we test whether increasing TE abundance is correlated with an increased rate of amino-acid evolution in genes encoding the piRNA machinery, as might be predicted by an evolutionary arms race model. Third, we test whether increasing TE abundance is correlated with greater codon bias in genes of the piRNA machinery. This is predicted if increasing TE abundance selects for increased efficiency in the machinery of genome defense.

Results

Surprisingly, we find neither of the first two hypotheses to be true. Specifically, we found that genome-wide levels of purifying selection, measured by the ratio of non-synonymous to synonymous substitution rates (ω), were greater in species with greater TE abundance. In addition, species with greater TE abundance have greater levels of purifying selection in the piRNA machinery. In contrast, it appears that increasing TE abundance has primarily driven adaptation in the piRNA machinery by increasing codon bias.

Conclusions

These results indicate that within the Drosophila genus, a historically reduced strength of selection relative to drift is unlikely to explain patterns of increased TE success across species. Other factors, such as ecological exposure, are likely to contribute to variation in TE abundances within species. Furthermore, constraints on the piRNA machinery may temper the evolutionary arms race that would drive increasing rates of evolution at the amino acid level. In the face of these constraints, selection may act primarily by improving the translational efficiency of the machinery of genome defense through efficient codon usage.

Protein concentrations depend not only on the mRNA level, but also on the translation rate and the degradation rate. Prediction of mRNA's translation rate would provide valuable information for in-depth understanding of the translation mechanism and dynamic proteome. In this study, we developed a new computational model to predict the translation rate, featured by (1) integrating various sequence-derived and functional features, (2) applying the maximum relevance & minimum redundancy method and incremental feature selection to select features to optimize the prediction model, and (3) being able to predict the translation rate of RNA into high or low translation rate category. The prediction accuracies under rich and starvation condition were 68.8% and 70.0%, respectively, evaluated by jackknife cross-validation. It was found that the following features were correlated with translation rate: codon usage frequency, some gene ontology enrichment scores, number of RNA binding proteins known to bind its mRNA product, coding sequence length, protein abundance and 5′UTR free energy. These findings might provide useful information for understanding the mechanisms of translation and dynamic proteome. Our translation rate prediction model might become a high throughput tool for annotating the translation rate of mRNAs in large-scale.

Events leading to cAMP accumulation after partial hepatectomy (PH) and effects of cAMP on hormonal induction of DNA synthesis in hepatocytes were characterized. Hepatic cAMP peaked biphasically post-PH and paralleled changes in adenylyl cyclase activity. Fluctuations in cyclase activity were not explained by variations in glucagon receptor kinetics, but reflected altered G-protein expression. Membrane levels of the stimulatory G-protein, Gs alpha, increased early after PH and were sustained. Levels of the inhibitory G-protein, Gi2 alpha, increased more slowly, peaked later, and quickly fell. Levels of both G-proteins correlated poorly with levels of their mRNAs, suggesting posttranscriptional factors modify their membrane concentrations. When growth factor-induced DNA synthesis was compared in hepatocyte cultures grown with or without agents that increase intracellular cAMP, DNA synthesis was inhibited by sustained high levels of cAMP but was enhanced when high cAMP levels fell. In both regenerating liver and hepatocyte cultures, the expression of a "differentiated" hepatocyte gene, phosphoenolpyruvate carboxykinase, correlated with elevated cAMP levels. These data suggest that the differential expression of G-proteins integrates signals initiated by several growth factors so that the accumulation of cAMP is tightly regulated post-PH. The ensuing variations in cAMP levels modulate both growth and differentiated functions during liver regeneration.

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