Interactions and activities of factors involved in the late stages of human 18S rRNA maturation.

Ribosome 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 3' 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 3' 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 3' end of the 18S rRNA during human pre-40S biogenesis and highlight potential mechanisms by which this process can be regulated.

Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing.

2'-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C'/D' motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA-protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C'/D' motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA-rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.