Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation
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ABSTRACT:
PROVIDER: PRJNA638017 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:This SuperSeries is composed of the SubSeries listed below.
2022-01-20 | GSE151989 | GEO
Project description:We treated HEK293T cells with different doses of RNase and sequence RNA fragements after the treatments. Furthermore, we developed a low-input RNase footprinting approach to quantify genome-wide RNA translation using 50,000 and 1,000 HEK293T and K562 cells. The ribosome profiling and RNA-seq data were used as the control.
2022-01-20 | GSE151986 | GEO
Project description:Low-input RNase footprinting to profile RNA translation
| PRJNA638018 | ENA
Project description:The roles of RNA-binding proteins as chaperones in the lifecycles of mRNAs are not well understood. The mammalian mitochondrial genome has been compressed over evolution to a size of just 16 kb, nevertheless the expression of its genes requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal systems, providing an opportunity to use it as a model system to understand the fundamental aspects of gene expression. Here we combine RNase footprinting with PAR-CLIP at unprecedented depth to reveal the importance of RNA-protein interactions guided by the LRPPRC/SLIRP complex in dictating RNA folding within the mitochondrial transcriptome. We show that LRPPRC, in complex with its protein partner SLIRP, binds throughout the mitochondrial transcriptome, with a preference for mRNAs, and its loss affects the entire secondary structure and stability of the transcriptome. We demonstrate that the LRPPRC/SLIRP complex is a global RNA chaperone that stabilizes RNA structures to expose the required sites for translation, stabilization and polyadenylation. Our findings reveal a general mechanism where extensive RNA-protein interactions ensure that RNA is accessible for its biological functions.
2017-11-21 | GSE100729 | GEO
Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage.
2015-04-29 | GSE62102 | GEO
Project description:RNase P is the essential activity that performs the 5’ maturation of tRNA precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to understand their integration with gene expression pathways. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/B localize in the nucleus and find that their double knock out mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome wide tRNAseq approach, we observe that TRM1A/B are responsible for the m2,2G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/B mutants for specific tRNAs, in particular, tRNAs containing a m2,2G modification at position 26 that are strongly down-regulated in TRM1A/B mutants. Altogether, results indicate that the m2,2G adding enzymes TRM1A/B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNAs biogenesis.
2023-09-04 | GSE223876 | GEO
Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.
2015-04-29 | E-GEOD-62102 | biostudies-arrayexpress
Project description:Two types of RNA:DNA associations can lead to genome instability: the formation of R-loops during transcription and the incorporation of ribonucleotide monophosphates (rNMPs) into DNA during replication. Both ribonuclease (RNase) H1 and RNase H2 degrade the RNA component of R-loops, whereas only RNase H2 can remove one or a few rNMPs from DNA. We performed high-resolution mapping of mitotic recombination events throughout the yeast genome in diploid strains of Saccharomyces cerevisiae lacking RNase H1 (rnh1Δ), RNase H2 (rnh201Δ), or both RNase H1 and RNase H2 (rnh1Δ rnh201Δ). We found little effect on recombination in the rnh1Δ strain, but elevated recombination in both the rnh201Δ and the double-mutant strains; levels of recombination in the double mutant were about 50% higher than in the rnh201 single-mutant strain. An rnh201Δ mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase ε (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Polε. This result suggests that the elevated recombination observed in the absence of only RNase H2 is primarily a consequence of R loops rather than misincorporated rNMPs.
2015-09-23 | GSE73334 | GEO
Project description:RNase P is essential to perform the 5’ maturation of tRNA precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to understand their integration with gene expression pathways. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 makes a complex with the tRNA methyl transferases TRM1A and B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/B localize in the nucleus and find that their double knock out mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome wide tRNAseq approach, we observe that TRM1A/B are responsible for the m2,2G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/B mutants for specific tRNAs, in particular, tRNAs containing a m2,2G modification at position 26 that are strongly down-regulated in TRM1A/B mutants. Altogether, results indicate that the m2,2G adding enzymes TRM1A/B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNAs biogenesis.
2023-10-20 | PXD040022 | Pride
Project description:We applied ribosomal footprinting experiment followed by RNA-seq in order to detect circular RNA associated with ribosomes in fly heads.
2017-03-24 | GSE79626 | GEO