Genomics of mRNA turnover

Most studies on eukaryotic gene regulation have focused on mature mRNA levels. Nevertheless, the steady-state mRNA level is the result of two opposing biological processes: transcription and degradation, both of which can be important points to regulate gene expression. It is now possible to determine the transcription and degradation rates (TR and DR), as well as the mRNA amount, for each gene using DNA chip technologies. In this way, each individual contribution to gene expression can be analysed. This review will deal with the techniques used for the genomic evaluation of TR and DR developed for the yeast Saccharomyces cerevisiae. They will be described in detail and their potential drawbacks discussed. I will also discuss the integration of the data obtained to fully analyse the expression strategies used by yeast and other eukaryotic cells

Nucleo-cytoplasmic shuttling of RNA-binding factors: mRNA buffering and beyond

Gene expression is a highly regulated process that adapts RNAs and proteins content to the cellular context. Under steady-state conditions, mRNA homeostasis is robustly maintained by tight controls that act on both nuclear transcription and cytoplasmic mRNA stability. In recent years, it has been revealed that several RNA-binding proteins (RBPs) that perform functions in mRNA decay can move to the nucleus and regulate transcription. The RBPs involved in transcription can also travel to the cytoplasm and regulate mRNA degradation and/or translation. The multifaceted functions of these shuttling nucleo-cytoplasm RBPs have raised the possibility that they can act as mRNA metabolism coordinators. In addition, this indicates the existence of crosstalk mechanisms between the enzymatic machineries that drive the different mRNA life-cycle phases. The buffering of the mRNA concentration is the best known consequence of a transcription-degradation crosstalk counteraction, but alternative ways of RBP action can also imply enhanced gene regulation

The transcriptional inhibitor thiolutin blocks mRNA degradation.

Thiolutin is commonly used as a general inhibitor of transcription in yeast. It has been used to calculate mRNA decay rates by stopping the transcription and then determining the relative abundance of individual mRNAs at different times after inhibition. We report here that thiolutin is also an inhibitor of mRNA degradation, and thus its use can lead to miscalculations of mRNA half-lives. The inhibition of mRNA decay seems to affect the mRNA degradation pathway without impeding poly(A) shortening, given that the decay rate of total poly(A) amount is not reduced by thiolutin. Moreover, the thiolutin-dependent inhibition of mRNA degradation has variable effects on different functional groups of genes, suggesting that they use various degradation pathways for their mRNAs

A genomic study of the Inter-ORF distances in Saccharomyces cerevisiae

The genome of eukaryotic microbes is usually quite compacted. The yeast Saccharomyces cerevisiae is one of the best-known examples. Open reading frames (ORFs) occupy about 75% of the total DNA sequence. The existence of other, non-protein coding genes and other genetic elements leaves very little space for gene promoters and terminators. We have performed an in silico study of inter-ORF distances that shows that there is a minimum distance between two adjacent ORFs that depends on the relative orientation between them. Our analyses suggest that different kinds of promoters and terminators exist with regard to their length and ability to overlap each other. The experimental testing of some putative exceptions to the minimum length model in tandemly orientated ORF pairs suggests that, in those cases, defects in promoter or terminator functionality exist that provoke transcription of polycistronic mRNAs

Saccharomyces cerevisiae glutaredoxin 5-deficient cells subjected to constitutive oxidizing conditions are affected in the expression of specific sets of genes

The Saccharomyces cerevisiae GRX5 gene codes for a mitochondrial glutaredoxin involved in the synthesis ofiron/sulfur clusters. Its absence prevents respiratory growth and causes the accumulation of iron inside cellsand constitutive oxidation of proteins. Null Δgrx5 mutants were used as an example of continuously oxidizedcells, as opposed to situations in which oxidative stress is instantaneously caused by addition of external oxidants.Whole transcriptome analysis was carried out in the mutant cells. The set of genes whose expression wasaffected by the absence of Grx5 does not significantly overlap with the set of genes affected in respiratorypetite mutants. Many Aft1-dependent genes involved in iron utilization that are up-regulated in a frataxin mutantwere also up-regulated in the absence of Grx5. BIO5 is another Aft1-dependent gene induced both upon irondeprivation and in Δgrx5 cells; this links iron and biotin metabolism. Other genes are specifically affected underthe oxidative conditions generated by the grx5 mutation. One of these is MLP1, which codes for a homologueof the Slt2 kinase. Cells lacking MLP1 and GRX5 are hypersensitive to oxidative stress caused by externalagents and exhibit increased protein oxidation in relation to single mutants. This in turn points to a role forMlp1 in protection against oxidative stress. The genes of the Hap4 regulon, which are involved in respiratorymetabolism, are down-regulated in Δgrx5 cells. This effect is suppressed by HAP4 overexpression. Inhibition ofrespiratory metabolism during continuous moderately oxidative conditions could be a protective response bythe cell

A genomic view of mRNA turnover in yeast

The steady-state mRNA level is the result of two opposing processes: transcription and degradation; both of which can provide important points to regulate gene expression. In the model organism yeast Saccharomyces cerevisiae, it is now possible to determine, at the genomic level, the transcription and degradation rates, as well as the mRNA amount, using DNA chip or parallel sequencing technologies. In this way, the contribution of both rates to individual and global gene expressions can be analysed. Here we review the techniques used for the genomic evaluation of the transcription and degradation rates developed for this yeast, and we discuss the integration of the data obtained to fully analyse the expression strategies used by yeast and other eukaryotic cells. Le taux de l"ARNm est maintenu à l"équilibre grâce à deux processus antagonistes : la transcription et la dégradation. Ces deux mécanismes sont cruciaux pour réguler l"expression des gènes. Dans l"organisme modèle Saccharomyces cerevisiae, il est maintenant possible de déterminer, au niveau génomique, les taux respectifs de transcription et de dégradation, ainsi que la quantité d"ARNm présente, en utilisant les puces à ADN ou le séquençage en parallèle. De cette manière, il est possible de connaître la contribution de chacun de ces processus en analysant le niveau d"expression des gènes individuellement et globalement. Nous présentons et comparons dans cet article les techniques utilisées pour évaluer les taux respectifs de transcription et de dégradation des transcrits de cette levure. Nous discutons la possibilité de l"utilisation des données obtenues pour analyser en profondeur les stratégies d"expression employées par la levure ainsi que par d"autres cellules eucaryotes. -------------------------------------------------------------------------------

Eukaryotic RNA Polymerases: the many ways to transcribe a gene

In eukaryotic cells, three nuclear RNA polymerases (RNA pols) carry out the transcription from DNA to RNA, and they all seem to have evolved from a single enzyme present in the common ancestor with archaea. The multiplicity of eukaryotic RNA pols allows each one to remain specialized in the synthesis of a subset of transcripts, which are different in the function, length, cell abundance, diversity, and promoter organization of the corresponding genes. We hypothesize that this specialization of RNA pols has conditioned the evolution of the regulatory mechanisms used to transcribe each gene subset to cope with environmental changes. We herein present the example of the homeostatic regulation of transcript levels versus changes in cell volume. We propose that the diversity and instability of messenger RNAs, transcribed by RNA polymerase II, have conditioned the appearance of regulatory mechanisms based on different gene promoter strength and mRNA stability. However, for the regulation of ribosomal RNA levels, which are very stable and transcribed mainly by RNA polymerase I from only one promoter, different mechanisms act based on gene copy variation, and a much simpler regulation of the synthesis rate

The relative importance of transcription rate, cryptic transcription and mRNA stability on shaping stress responses in yeast

It has been recently stated that stress-responding genes in yeast are enriched in cryptic transcripts and that this is the cause of the differences observed between mRNA amount and RNA polymerase occupancy profiles. Other studies have shown that such differences are mainly due to modulation of mRNA stabilities. Here we analyze the relationship between the presence of cryptic transcripts in genes and their stress response profiles. Despite some of the stress-responding gene groups being indeed enriched in specific classes of cryptic transcripts, we found no statistically significant evidence that cryptic transcription is responsible for the differences observed between mRNA and transcription rate profiles

A trans‐omics comparison reveals common gene expression strategies in four model organisms and exposes similarities and differences between them

The ultimate goal of gene expression regulation is on the protein level. However, because the amounts of mRNAs and proteins are controlled by their synthesis and degradation rates, the cellular amount of a given protein can be attained by following different strategies. By studying omics data for six expression variables (mRNA and protein amounts, plus their synthesis and decay rates), we previously demonstrated the existence of common expression strategies (CESs) for functionally related genes in the yeast Saccharomyces cerevisiae. Here we extend that study to two other eukaryotes: the yeast Schizosaccharomyces pombe and cultured human HeLa cells. We also use genomic data from the model prokaryote Escherichia coli as an external reference. We show that six-variable profiles (6VPs) can be constructed for every gene and that these 6VPs are similar for genes with similar functions in all the studied organisms. The differences in 6VPs between organisms can be used to establish their phylogenetic relationships. The analysis of the correlations among the six variables supports the hypothesis that most gene expression control occurs in actively growing organisms at the transcription rate level, and that translation plays a minor role. We propose that living organisms use CESs for the genes acting on the same physi-ological pathways, especially for those belonging to stable macromolecular complexes, but CESs have been modeled by evolution to adapt to the specific life circumstances of each organism