Abstract
ABSTRACTRibosome production is an essential cellular process involving a plethora of trans-acting factors, such as nucleases, methyltransferases, RNA helicases and kinases that catalyse key maturation steps. Precise temporal and spatial regulation of such enzymes is essential to ensure accurate and efficient subunit assembly. Here, we focus on the maturation of the 3ʹ end of the 18S rRNA in human cells. We reveal that human RIO2 is an active kinase that phosphorylates both itself and the rRNA methyltransferase DIM1 in vitro. In contrast to yeast, our data confirm that human DIM1 predominantly acts in the nucleus and we further demonstrate that the 21S pre-rRNA is the main target for DIM1-catalysed methylation. We show that the PIN domain of the endonuclease NOB1 is required for site 3 cleavage, while the zinc ribbon domain is essential for pre-40S recruitment. Furthermore, we also demonstrate that NOB1, PNO1 and DIM1 bind to a region of the pre-rRNA encompassing the 3ʹ end of 18S and the start of ITS1, in vitro. Interestingly, NOB1 is present in the cell at higher levels than other pre-40S factors. We provide evidence that NOB1 is multimeric within the cell and show that NOB1 multimerisation is lost when ribosome biogenesis is blocked. Taken together, our data indicate a dynamic interplay of key factors associated with the 3ʹ end of the 18S rRNA during human pre-40S biogenesis and highlight potential mechanisms by which this process can be regulated.
Highlights
The assembly of eukaryotic ribosomes, which are composed of 4 ribosomal RNAs and ~80 ribosomal proteins, is a highly complex and energy-consuming cellular process [1,2]
To confirm the requirement for the human counterparts of several yeast proteins in 18S ribosomal RNAs (rRNAs) maturation in human cells and gain insight into the precise stages at which these proteins act, we used RNAi to deplete key factors predicted to be involved in the late stages of human 18S rRNA maturation and examined the effects on pre-rRNA processing. siRNAs targeting the ribosome biogenesis factors DIM1 (DIMT1L), NOB1, PNO1 (DIM2), RIO2 (RIOK2), ENP1 (BYSL) and PRP43 (DHX15), or control siRNAs targeting firefly luciferase, were transfected into HEK293 cells and, 60 h later, RNA was extracted, separated by denaturing agarose gel electrophoresis and analysed by northern blotting using a probe hybridising to the 5ʹ end of ITS1 (Figure 1(A))
The efficiency of each knockdown was determined by western blotting using antibodies against the endogenous proteins, except in the case of DIM1, where due to the lack of a functional DIM1 antibody, we analysed the ability of the siRNAs to deplete FLAG-tagged human DIM1 stably expressed in HEK293 cells (Supplementary Figure S1)
Summary
The assembly of eukaryotic ribosomes, which are composed of 4 ribosomal RNAs (rRNAs) and ~80 ribosomal proteins, is a highly complex and energy-consuming cellular process [1,2]. The fourth rRNA, 5S, is transcribed by RNA polymerase III in the nucleoplasm from where it is imported into the nucleolus and integrated into the large ribosomal subunit (LSU) as part of the 5S RNP [2]. An early pre-ribosomal complex (90S) is assembled onto the nascent pre-rRNA, which is separated into precursors of the small subunit (SSU; pre-40S) and large subunit (pre-60S) [2]. These complexes undergo extensive maturation in the nucleolus prior to nucleoplasmic remodelling events that generate export-competent particles, which are transported to the cytoplasm where final maturation and quality control steps occur
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