Project description:The goals of this study are to compare NGS-derived transcriptomes (RNA-Seq) derived from ded1-ref mutant and normal maize endosperm tissue. Homozygous ded1-ref mutants exhibit a seed defect. Maize Ded1 encodes a transcription factor. This study characterizes DEGs between ded1-ref and normal endosperm and serves to identify direct target genes together with DAP-seq data.
Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling.The study includes 32 samples, comprised of 16 mRNA-Seq samples and 16 ribosome footprint profiling samples, derived from biological replicates of 3 mutant strains, ded1-cs, ded1-ts and tif1-ts, and the corresponding wild-type strains. The tif1-ts mutant and its wild-type counterpart were analyzed at 30°C and 37°C.
Project description:We replaced the DED1 ORF with DBP1 and inserted this either twice or four times in a diploid cell. We determined that with twice as much DBP1 expressable, the same amount of protein was made as for DED1, and there were few changes in gene expression. The transcripts that were translated better with DED1 were ones with highly structured 5' leaders, consistent with Dbp1 acting as a less effective initiation helicase than Ded1.
Project description:DAP-seq was used to generate genome-wide DNA:TF interaction maps for fourteen maize ARFs from the evolutionarily conserved class A ‘activator’ and class B ‘repressor’ clades. Among the distinct binding sites that were identified, we observed a high degree of overlap for ARFs of the same class, but found substantial differences in motif sequence, spacing, site preference, and association with auxin induced genes among clade A and clade B ARFs.
Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs.
Project description:DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP) to crosslink Ded1 to 4-thiouridine-incorported RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on a relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.
Project description:The role of Ded1 helicase is pivotal in reducing ribosomal biogenesis and inducing autophagy under TORC1 inhibition. Incorporating mutations in Ded1 rescues these effects, leading to rapamycin resistance.
Project description:As 5-15% of higher eukaryotes genes are transcription factors (TFs), the lack of transcription factor binding site (TFBS) information for most factors in most organisms limits the study of gene regulation. Here we describe a next-generation sequencing method, DNA affinity purification (DAP-Seq), an in vitro gDNA/TF interaction assay that produces whole-genome TFBS annotation for any factor from any organism. Like ChIP-Seq, DAP-Seq resolves TFBS as discrete peaks at genomic locations which allows for accurate motif prediction direct assignment of functionally relevant target genes, and shows better overlap with ChIP-Seq peaks than indirect motif assignment approaches. We applied DAP-Seq to a set of 50 transcription factors in eight Arabidopsis thaliana and one Zea Mays families to gain novel biological insight into TFBS architectures, functions, evolution and methylation-sensitivity. Overall, DAP-Seq offers a low-cost high-throughput approach to identify TFBS in native sequence context for any organism complete with all DNA chemical modifications.
Project description:We replaced the DED1 ORF with DBP1 and tested vegetative growth at 30C or 37C to determine helicase specificity and temperature-dependent effects. We found that DBP1 expressing cells do not downregulate all of the housekeeping genes that DED1 expressing cells normally down regulate at high temperature