A tRNA modifying enzyme facilitates RNase P activity in Arabidopsis nuclei
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ABSTRACT:
PROVIDER: PRJNA928711 | ENA |
REPOSITORIES: ENA
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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: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:tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of tRFs population. By comparing wild-type and knock-out macrophage cell lines our previous work (Lu L, et al. CMLS, 2022, 79: 209) revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening synthetic tRNAs, single-mutant variants and anticodon-loop DNA/RNA hairpins. By sequencing the tRFs products, we identified the cleavage selectivity by recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Knowledge of RNase 2 specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.
2024-05-29 | GSE250347 | GEO
Project description:In this study, we use small RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. Complemented and extended by the use of northern blots, our results demonstrate that tRNAs and Y RNAs in the vesicle-depleted extracellular compartment are released from cells as full-length precursors. Following their release, tRNAs and Y RNAs are processed by RNase 1 into distinct fragments. In addition, our findings show that standard sequencing methods employed to detect tRNA fragments leave many of such fragments undetected and that a combination of end healing, 3’ deacylation and RNA modification removal before library preparation can substantially improve detection of tRNA halves and reduce biases.
2020-06-30 | GSE148516 | GEO
Project description:RNase III is an important and highly conserved endoribonuclease known to impact rRNA, mRNA and ncRNA abundances by RNA processing. In this study we analyzed the effects of an inactivation of RNase III (inactivated through substitution of two strictly conserved amino acids within the active enzyme center) on the transcriptome of the facultative phototrophic model organism Rhodobacter sphaeroides.
2024-02-19 | GSE236804 | GEO
Project description:During transcription the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region transcribed by Pol I and over the 5S rDNA transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5?-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on highly expressed protein-coding genes in the wild-type, notably over the second exon of spliced ribosomal protein genes. ChIP-seq of RNA-DNA hybrids using antibody S9.6
2014-11-03 | E-GEOD-53420 | biostudies-arrayexpress
Project description:Structural determinants for tRNA selective cleavage by RNase 2/EDN
| PRJNA1053393 | ENA
Project description:Since numerous RNAs and RBPs prevalently localize to active chromatin regions, many RNA binding proteins (RBPs) may be potential transcriptional regulators. RBPs are generally thought to regulate transcription via noncoding RNAs. Here, we describe a distinct, dual mechanism of transcriptional regulation by the previously uncharacterized tRNA-modifying enzyme, hTrmt13. On one hand, hTrmt13 acts in the cytoplasm to catalyze 2'-O-methylation of tRNAs, thus regulating translation in a manner depending on its tRNA-modification activity. On the other hand, nucleus-localized hTrmt13 directly binds DNA as a transcriptional co-activator of key epithelial mesenchymal transition factors, thereby promoting cell migration independent of tRNA-modification activity. These dual functions of hTrmt13 are mutually exclusive, as it can bind either DNA or tRNA through its CHHC zinc finger domain. Finally, we find that hTrmt13 expression is tightly associated with poor prognosis and survival in diverse cancer patients. Our discovery of the noncatalytic roles of an RNA modifying enzyme provides a new perspective for understanding epitranscriptomic regulation.
2021-10-29 | GSE166276 | GEO
Project description:Since numerous RNAs and RBPs prevalently localize to active chromatin regions, many RNA binding proteins (RBPs) may be potential transcriptional regulators. RBPs are generally thought to regulate transcription via noncoding RNAs. Here, we describe a distinct, dual mechanism of transcriptional regulation by the previously uncharacterized tRNA-modifying enzyme, hTrmt13. On one hand, hTrmt13 acts in the cytoplasm to catalyze 2'-O-methylation of tRNAs, thus regulating translation in a manner depending on its tRNA-modification activity. On the other hand, nucleus-localized hTrmt13 directly binds DNA as a transcriptional co-activator of key epithelial mesenchymal transition factors, thereby promoting cell migration independent of tRNA-modification activity. These dual functions of hTrmt13 are mutually exclusive, as it can bind either DNA or tRNA through its CHHC zinc finger domain. Finally, we find that hTrmt13 expression is tightly associated with poor prognosis and survival in diverse cancer patients. Our discovery of the noncatalytic roles of an RNA modifying enzyme provides a new perspective for understanding epitranscriptomic regulation.
2021-10-29 | GSE166274 | GEO
Project description:Since numerous RNAs and RBPs prevalently localize to active chromatin regions, many RNA binding proteins (RBPs) may be potential transcriptional regulators. RBPs are generally thought to regulate transcription via noncoding RNAs. Here, we describe a distinct, dual mechanism of transcriptional regulation by the previously uncharacterized tRNA-modifying enzyme, hTrmt13. On one hand, hTrmt13 acts in the cytoplasm to catalyze 2'-O-methylation of tRNAs, thus regulating translation in a manner depending on its tRNA-modification activity. On the other hand, nucleus-localized hTrmt13 directly binds DNA as a transcriptional co-activator of key epithelial mesenchymal transition factors, thereby promoting cell migration independent of tRNA-modification activity. These dual functions of hTrmt13 are mutually exclusive, as it can bind either DNA or tRNA through its CHHC zinc finger domain. Finally, we find that hTrmt13 expression is tightly associated with poor prognosis and survival in diverse cancer patients. Our discovery of the noncatalytic roles of an RNA modifying enzyme provides a new perspective for understanding epitranscriptomic regulation.
2021-10-29 | GSE166270 | GEO