Project description:Intrinsic RNA-Binding Preferences of Eukaryotic Translation Initiation Factor eIF4G Contribute to Competitive Discrimination of Different mRNAs
Project description:Microarray comparisons of transcript level in wild-type Arabidopsis and eif3h mutant plants. Goal:; To detect any change in transcript level between WT and eif3h mutant. BACKGROUND: The eukaryotic translation initiation factor eIF3 has multiple roles during the initiation of translation of cytoplasmic mRNAs. However, the contributions of individual subunits of eIF3 to the translation of specific mRNAs remain poorly understood. RESULTS: Working with stable reporter transgenes in Arabidopsis thaliana it was demonstrated that the h subunit of; eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5â leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant plants revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. Independently, eIF3h also boosted polysome loading of mRNAs with long coding sequences. Moreover, the lesion in eIF3h revealed a concerted upregulation of translation for specific functional subgroups of mRNAs, including ribosomal proteins and proteins involved in photosynthesis. CONCLUSIONS: The intact eIF3h protein contributes to efficient translation initiation on 5â leader sequences harboring multiple uORFs, although mRNA features independent of uORFs were also implicated. Moreover, our data suggest that regulons of translational control can be revealed by mutations in generic translation initiation factors. Experiment Overall Design: Total RNA samples were isolated from 10-day-old wild-type and eif3h mutant plants
Project description:Microarray comparisons of transcript level in wild-type Arabidopsis and eif3h mutant plants. Goal: To detect any change in transcript level between WT and eif3h mutant. BACKGROUND: The eukaryotic translation initiation factor eIF3 has multiple roles during the initiation of translation of cytoplasmic mRNAs. However, the contributions of individual subunits of eIF3 to the translation of specific mRNAs remain poorly understood. RESULTS: Working with stable reporter transgenes in Arabidopsis thaliana it was demonstrated that the h subunit of eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5’ leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant plants revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. Independently, eIF3h also boosted polysome loading of mRNAs with long coding sequences. Moreover, the lesion in eIF3h revealed a concerted upregulation of translation for specific functional subgroups of mRNAs, including ribosomal proteins and proteins involved in photosynthesis. CONCLUSIONS: The intact eIF3h protein contributes to efficient translation initiation on 5’ leader sequences harboring multiple uORFs, although mRNA features independent of uORFs were also implicated. Moreover, our data suggest that regulons of translational control can be revealed by mutations in generic translation initiation factors. Keywords: mutant, total RNA
Project description:The reduction of T-cell intracellular antigen (TIA) proteins in transformed cells leads to the acquisition of aberrant cellular phenotypes promoting uncontrolled cell proliferation and tumor growth. Here we show that global and specific translational rates are regulatory gene events that contribute markedly to the acquisition of above cellular phenotypes. For example, we observe a significant increase of ribosomal population and translational machinery components in TIA-reduced HeLa cells. Polysomal microarray analysis shows specific changes at both the transcript and translational level following TIA reduction, identifying translationally regulated mRNAs that are not transcriptionally regulated which seem to be prevalent for the adaptation to the new environmental conditions. Validation of microarray data using RT-QPCR and immunological analysis for representative genes were carried out. Up-regulated in this class of mRNA are those involved in cell-cycle progression and DNA replication/repair, including several mRNAs with specific sequences to bind TIA proteins. Our data support a hypothesis that a concerted activation of both global and selective translational rates is relevant for the transition from quiescent to proliferative status in TIA-depleted HeLa cells. Two independent replicates were performed for each experimental condition and hybridized to SurePrint G3 Human GE 8x60K Agilent microarrays.
Project description:Long Undecoded Transcript Isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed some important aspects of LUTI-based repression, how these features impact gene regulation at a global scale is unknown. By using transcript leader and direct RNA sequencing, here we identify 74 LUTI candidates that are expressed specifically during meiotic prophase. Translational repression of these candidates is ubiquitous and dependent on upstream open reading frames. However, LUTI-based transcriptional repression is highly variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression.
Project description:Cellular signaling controls translation through cis regulatory elements found in mRNAs. Gcn2 is the master regulator of translation during nutrient limitation in normal and cancer cells. Activated Gcn2 phosphorylates eIF2a, thereby repressing general translation while activating translation of specific mRNAs with upstream ORFs (uORFs) in its leader regions. Here we performed genome-wide measurement of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysomal microarray hybridization experiments identified a group of 1779 genes whose translation is up-regulated in Gcn2-dependent manner. We find that translation is reprogrammed to enhance organellar synthesis and transcription and repress ribosome synthesis. The 1779 genes included gcn5 and hri2 shown to promote growth under histidine starvation. They encode histone acetyl transferase and heme-binding eIF2a kinase activated by oxidative stress, and their 5’-leader contains 3 or 4 uORFs, respectively. Our reporter studies show that uORFs in gcn5 and hri2 operate similarly to those found in S. cerevisiae GCN4, the founding case for uORF-dependent regulation. Moreover, motif analysis identified 5’-UGA(C/G)GG-3’ as a motif promoting translation during starvation. Using hrd1 5’UTR with such a motif, we demonstrated its requirement in Gcn2-dependent translational control. To our knowledge, this is the first nucleotide motif found to be responsible for translational control by Gcn2. We propose that Gcn2 mediates translational control of more specific mRNAs than previously anticipated
Project description:The reduction of T-cell intracellular antigen (TIA) proteins in transformed cells leads to the acquisition of aberrant cellular phenotypes promoting uncontrolled cell proliferation and tumor growth. Here we show that global and specific translational rates are regulatory gene events that contribute markedly to the acquisition of above cellular phenotypes. For example, we observe a significant increase of ribosomal population and translational machinery components in TIA-reduced HeLa cells. Polysomal microarray analysis shows specific changes at both the transcript and translational level following TIA reduction, identifying translationally regulated mRNAs that are not transcriptionally regulated which seem to be prevalent for the adaptation to the new environmental conditions. Validation of microarray data using RT-QPCR and immunological analysis for representative genes were carried out. Up-regulated in this class of mRNA are those involved in cell-cycle progression and DNA replication/repair, including several mRNAs with specific sequences to bind TIA proteins. Our data support a hypothesis that a concerted activation of both global and selective translational rates is relevant for the transition from quiescent to proliferative status in TIA-depleted HeLa cells.
Project description:Protein synthesis is an energy-demanding process essential for cell proliferation and survival. Balancing the cost of protein synthesis with available resources has driven the evolution of its nutrient-dependent regulation. A central mechanism in this regulation is the repression of translation of the protein synthesis machinery during unfavorable growth conditions. This is mediated via mammalian target of rapamycin (mTOR), a master regulator of growth conserved from yeast to human. Despite extensive research, and the elucidation of a number of important factors, how mRNAs are translationally regulated by mTOR is still unclear. Repression depends on a 5’ Terminal Oligo Pyrimidine (TOP) motif which is conserved across vertebrates and present in Drosophila melanogaster. In Caenorhabditis elegans and the marine chordate Oikopleura dioica most TOP mRNAs are trans-spliced to a spliced leader. This results in the removal of the originally transcribed 5’ end and its replacement with a common short RNA sequence. In both species the 5’ end of the spliced leader is pyrimidine-enriched but does not meet strict requirements for a canonical TOP motif. How this affects the translational control of TOP mRNAs is unknown. Here, using transcriptome-wide ribosome profiling on whole animals treated with the mTOR inhibitor Torin 1, we show that trans-spliced TOP mRNAs in O. dioica are subject to mTOR-dependent translational control. We also show, using existing data, that trans-spliced transcripts in C. elegans are differentially translated upon recovery from starvation-induced developmental diapause. Together our results demonstrate that spliced leaders in metazoans are targets for mTOR-dependent translational control in response to nutrient availability. This indicates that trans-splicing in metazoans, the function of which has remained largely enigmatic, plays a key role in the coordinated translational regulation of growth-related genes. Moreover, our results reveal an innovative strategy for rapid evolution and developmental control of downstream targets of the ancient mTOR pathway.
Project description:Deregulation of translational control is frequently implicated in the establishment and maintenance of the cancer phenotype. Anti-cancer drugs targeting protein synthesis are confronted by the problem that genes can exploit alternative translational mechanisms. To better understand the molecular mechanism behind these alternative translational routes we have exploited an ex-vivo assay that follows a competitive re-recruitment of cellular mRNAs onto polysomes in cells treated or not with rapamycin.