Project description:Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S rRNA 3’ end maturation during late-40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the effects of eIF5B have not been studied at the genome-wide level in any organism, and 18S rRNA 3’ end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat-stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3’ end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3’ end maturation or metabolism. We quantitatively defined new processing hotspots and identified adenylation as the prevalent non-templated RNA modification at the 3’ ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNAi to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3’ portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late-40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, mRNA translation initiation, and siRNA biogenesis in plants.
Project description:Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S rRNA 3’ end maturation during late-40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the effects of eIF5B have not been studied at the genome-wide level in any organism, and 18S rRNA 3’ end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat-stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3’ end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3’ end maturation or metabolism. We quantitatively defined new processing hotspots and identified adenylation as the prevalent non-templated RNA modification at the 3’ ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNAi to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3’ portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late-40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, mRNA translation initiation, and siRNA biogenesis in plants.
Project description:Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S rRNA 3’ end maturation during late-40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the effects of eIF5B have not been studied at the genome-wide level in any organism, and 18S rRNA 3’ end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat-stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3’ end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3’ end maturation or metabolism. We quantitatively defined new processing hotspots and identified adenylation as the prevalent non-templated RNA modification at the 3’ ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNAi to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3’ portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late-40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, mRNA translation initiation, and siRNA biogenesis in plants.
Project description:Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S rRNA 3’ end maturation during late-40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the effects of eIF5B have not been studied at the genome-wide level in any organism, and 18S rRNA 3’ end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat-stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3’ end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3’ end maturation or metabolism. We quantitatively defined new processing hotspots and identified adenylation as the prevalent non-templated RNA modification at the 3’ ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNAi to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3’ portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late-40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, mRNA translation initiation, and siRNA biogenesis in plants.
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately. Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA. Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2. Examination of targets for pre-rRNA endonucleases in yeast cells.