Project description:RNAseq was performed in siJARID2 and control siRNA-treated human adipose tissue derived stem cells (hASC). The tretment of siRNA was performed by electroporation one day before induction of differentiation in vitro. The cells were lyzed and RNA was purified on day 6 (mid differentiation) and day 13 (full differentiation) from differentiation start.
Project description:Purpose: Identify genes regulated by ALOX15 in A549 cells that were treated with +/- IL4 and +/- ALOX15 siRNA by Next-gen sequencing
Project description:Purpose: Identify genes regulated by ALOX15 in Normal human bronchial epithelial (NHBE) cells that were treated with +/- IL4 and +/- ALOX15 siRNA by Next-gen sequencing
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3B knockout human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype and UPF3B KO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3A.
Project description:In order to systematically assess the frequency and origin of stop codon recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon recoding events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon recoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon recoding in E. coli. Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.
Project description:A ELMSeq reporter cassette was created to monitor Dam levels by methylation, and introduced in the genome. The regions of 6 nt upstream and 6 nt downstream the stop codon were randomized to study their effect on gene expression. The ELMSeq reporter cassette was composed of: promoter - dam - random N6 - stop codon TAA - random N6 - spacer - 4xGATC. The amplicon was spanning the C-terminal region. The cassette was introduced in Mycoplasma pneumoniae.
Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B and UPF3A in NMD, we have generated UPF3B knockout (KO) and UPF3A-UPF3B double KO (dKO) human Flp-In T-REx 293 cells using CRISPR-Cas9. We generated RNA-Sequencing data for wildtype, UPF3B KO and UPF3A-UPF3B dKO cells with additional siRNA-mediated knockdown of Luciferase (Luc) as control or UPF3B.
Project description:Stop codon recoding events give rise to longer proteins, which may alter the proteins function and thereby generate short-lasting phenotypic variability from a single gene.
In order to systematically assess the frequency and origin of recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E.coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur as high as 80 percent of the time, depending on the genetic context, suggesting that evolution frequently samples stop codon recoding events. Targeted mass spectrometry and RNA-seq analyses showed that not only translational but also transcriptional errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide mass -spectrometry revealed that temperature regulates the expression of cryptic peptides generated by stop codon recoding in E.coli.
Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.
Project description:Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.ΔA.exp lacking all UAG stop codons was created, freeing this ‘amber’ stop codon for other purposes. An engineered ‘amber initiator’ that activates translation at UAG codons is available, but little is known about amino acid it assigns to the amber start codon. Here, we combine for the first time the amber initiator tRNA in C321.ΔA.exp and measure its cellular effects. Using, targeted proteomics, we found that the amber initiator tRNA initiates proteins exclusively with methionine.
