Rnmt cKO vs control activated CD4 T cell ribo Mega-SEC
Ontology highlight
ABSTRACT: We aimed to identify how loss of the cap methyltransferase RNMT affected the ribosomes and ribosome-associated proteins in activated CD4 T cells.
Project description:We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC, which was achieved within 15 min from sample injection to fraction collection. Ribo Mega-SEC reproducibly shows translating ribosomes exist predominantly in polysome complexes in extracts isolated from human cell lines and mouse liver tissue, which alter in response to starvation. Ribo Mega-SEC provides a rapid, efficient, convenient and highly reproducible method for studying functional translation complexes and is easily combined with high-through put analysis such as proteomics and RNA-Seq, or with structural analysis using electron microscopy. We propose that Ribo Mega-SEC analysis is an accessible alternative to traditional polysome profiling using sucrose density gradients.
Project description:We investigated the role of RNMT in T cells using an Rnmt conditional knockout mouse model. We report that the mRNA cap methyltransferase, RNMT, supports naïve T cell survival and activation-induced proliferation. We demonstrate that RNMT has gene-specific impacts in T cells, selectively regulating expression of terminal polypyrimidine tract (TOP) mRNAs which are targets of the m7G-cap binding protein LARP1. These ribosome footprinting experiments investigate the effect of Rnmt cKO on the activated CD4 T cell transcriptome and translatome.
Project description:We investigated the role of RNMT in T cells using an Rnmt conditional knockout mouse model. We report that the mRNA cap methyltransferase, RNMT, supports naïve T cell survival and activation-induced proliferation. We demonstrate that RNMT has gene-specific impacts in T cells, selectively regulating expression of terminal polypyrimidine tract (TOP) mRNAs and small RNAs including snoRNAs. To deterine if had any effect on ribosomal RNA modification we carried out pseudoridine sequencing.
Project description:We present a genome-wide assessment of small open reading frames (smORF) translation by ribosomal profiling of polysomal fractions in Drosophila S2 cell. In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits. Ribosomal profiling of large and small polysomal fractions in Drosophila S2 cells to assess translation of smORFs
Project description:The AF4/FMR2 proteins AFF1 and AFF4 act as a scaffold to assemble the Super Elongation Complex (SEC) that strongly activates transcriptional elongation of HIV-1 and cellular genes. Although they can dimerize, it is unclear whether the dimers exist and function within a SEC in vivo. Furthermore, it is unknown whether AFF1 and AFF4 function similarly in mediating SEC-dependent activation of diverse genes. Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities. While the AFF1-SEC is more potent in supporting HIV-1 transactivation by the viral Tat protein, the AFF4-SEC is more important for HSP70 induction upon heat shock. The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency. Finally, genome-wide analysis confirms that the genes regulated by AFF1- and AFF4-SEC are largely non-overlapping and perform distinct functions. Thus, the SEC represents a family of related complexes that exist to increase the regulatory diversity and gene control options during transactivation of diverse cellular and viral genes. RNA-seq in HeLa cells of wild-type and after RNAi of AFF1 or AFF4.
Project description:The mRNA cap recruits factors essential for transcript processing and translation initiation. We report that regulated mRNA cap methylation is a feature of embryonic stem cell (ESC) differentiation. Expression of the mRNA cap methyltransferase activating subunit, RAM, is elevated in ESCs resulting in high levels of mRNA cap methylation and expression of Oct4 and Sox2 and other pluripotency-associated factors. During neural differentiation, RAM is suppressed which is required for loss of Oct4 and Sox2 and correct expression of neural markers. Cells were treated with control or RAM siRNA and cytosolic RNA or polysome RNA (actively translated) was sequenced.
Project description:We aim to find the gene-specific effects of Rnmt KO in mouse CD4 T cells. TMT proteomics datasets were generated to find out which proteins are dependent on RNMT for their expression.
Project description:We aim to find the gene-specific effects of Rnmt KO in mouse CD4 T cells. TMT proteomics datasets were generated to find out which proteins are dependent on RNMT for their expression.
Project description:To evaluate the genome-wide changes in gene translational efficiency during the development of heart failure, we performed transverse aortic constriction(TAC) in male C57BL/6 mice. According to our experience, hypertrophy of the left ventricle was observed 2 weeks after TAC. Cardiac decompensation was observed at 5 weeks. We collected left ventricular tissues at 0, 2, 5 weeks after TAC and then performed ribosome footprinting and sequencing.