Project description:Ribosome small subunit (SSU) is assembled by the SSU processome which contains approximately 70 non-ribosomal protein factors. The biochemical mechanism for the SSU processome in 18S rRNA processing and maturation has been extensively studied, however, how the SSU processome components enter to the nucleolus has not been systematically investigated. Here we checked the nucleolar localization of 50 human SSU processome components and find that UTP3 and other 24 proteins enter to the nucleolus autonomously. For the remaining 25 proteins we find that UTP3/SAS10 assists the nucleolar localization of five proteins, namely MPP10, UTP25, EMG1 and two UTP-B components UTP12 and UTP13, and this ferry function of UTP3 is conserved in zebrafish. We also find that knockdown of human UTP3 impairs the cleavage at A0-site while loss-of-function of either utp3/sas10 or utp13/tbl3 in zebrafish causes an accumulation of the processed products containing the 5′ETS, supporting the crucial role of UTP3 in mediating the 5′ETS processing and degradation. Moreover, UTP3 directly interacts with and delivers EXOSC10 into the nucleolus, suggesting that UTP3 may play a direct role in recruiting the nuclear exosome to the SSU processome for degradation of the processed 5′ETS. These findings lay the ground for studying the mechanism of cytoplasm-to-nucleolus trafficking of the SSU processome components and the multifaceted roles of UTP3 during pre-rRNA processing.
Project description:Iron-rich pelagic aggregates (iron snow) were collected directly onto silicate glass filters using an electronic water pump installed below the redoxcline. RNA was extracted and library preparation was done using the NEBNext Ultra II directional RNA library prep kit for Illumina. Data was demultiplied by GATC sequencing company and adaptor was trimmed by Trimgalore. After trimming, data was processed quality control by sickle and mRNA/rRNA sequences were sorted by SortmeRNA. mRNA sequences were blast against NCBI-non redundant protein database and the outputs were meganized in MEGAN to do functional analysis. rRNA sequences were further sorted against bacterial/archeal 16S rRNA, eukaryotic 18S rRNA and 10,000 rRNA sequences of bacterial 16S rRNA, eukaryotic 18S rRNA were subset to do taxonomy analysis.
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.