Rcl1 protein, a novel nuclease for 18 S ribosomal RNA production

Abstract

In all forms of life, rRNAs for the small and large ribosomal subunit are co-transcribed as a single transcript. Although this ensures the equimolar production of rRNAs, it requires the endonucleolytic separation of pre-rRNAs to initiate rRNA production. In yeast, processing of the primary transcript encoding 18 S, 5.8 S, and 25 S rRNAs has been studied extensively. Nevertheless, most nucleases remain to be identified. Here, we show that Rcl1, conserved in all eukaryotes, cleaves pre-rRNA at so-called site A(2), a co-transcriptional cleavage step that separates rRNAs destined for the small and large subunit. Recombinant Rcl1 cleaves pre-rRNA mimics at site A(2) in a reaction that is sensitive to nearby RNA mutations that inhibit cleavage in vivo. Furthermore, mutations in Rcl1 disrupt rRNA processing at site A(2) in vivo and in vitro. Together, these results demonstrate that the role of Rcl1 in eukaryotic pre-rRNA processing is identical to that of RNase III in bacteria: to co-transcriptionally separate the pre-rRNAs destined for the small and large subunit. Furthermore, because Rcl1 has no homology to other known endonucleases, these data also establish a novel class of nucleases.

FIGURE 1.

A, schematic of the pre-RNA processing pathway. Endonucleolytic and exonucleolytic cleavage sites are highlighted with red and blue arrows, respectively, with the name of the nuclease involved if known or hypothesized. The 21 S rRNA is shown. It is not converted to 18 S rRNA. The locations of Northern probes are indicated with bars. B, Rcl1 structure (adapted from Ref. 12). For illustration purposes, a single-stranded RNA substrate is modeled into the suggested 3′-substrate binding site. The R327A/D328A/K330A residues and putative active site are indicated.

FIGURE 2.

Rcl1 cleaves at site A₂. A, schematic view of ITS1. Start (H44 and H45) and end sites (A₂ and ITS1) of pre-rRNA constructs, and substitution and deletion mutations around the A₂ site are indicated. B, 1 μm Rcl1 was incubated with H44-ITS1 substrate, and samples were collected after 0, 15, 60, 90, and 120 min. Samples incubated without Rcl1 were collected at 0 and 120 min. C, 1 μm Rcl1 was incubated with H45-ITS1 substrate, and samples were collected as described above. D, plots of the reactions with H44-ITS1 (●) and H45-ITS1 (■) were fit to Equation 1. E, increasing concentrations of Rcl1 were incubated with H44-ITS1 substrate, and samples were removed at 0, 15, 30, and 60 min. F, the Rcl1 concentration dependence was fit with the Michaelis-Menten equation to yield k_cleav = 0.049 min⁻¹ and K½ = 1.5 μm.

FIGURE 3.

Rcl1 cleavage of mutant pre-rRNA substrates. A, H44-ITS1 WT, sub2, and Δ6–3′ substrates were incubated for 0, 30, or 60 min in the absence or presence of 3 μm Rcl1. B, data were fit to Equation 1 to yield rate constants of 0.020, 0.0061, and 0.0057 min⁻¹ for WT (♦), sub2 (■), and Δ6–3′ (●), respectively. Fig. 2 shows nucleotide changes.

FIGURE 4.

Rcl1 mutants are impaired in A₂ site cleavage in vivo and in vitro. A, 10-fold serial dilutions of S. cerevisiae strain YKK55 harboring genomic Rcl1 under the control of a galactose-inducible promoter transformed with plasmids carrying no insert, wild type Rcl1, truncations at amino acids 309 and 341, or point mutations at R327A, D328A, and K330A (RDK) were plated on galactose and glucose solid media (YPGal and YPD, respectively) and incubated for 2 days. B, Northern analysis of pre-rRNAs from the strains in 0, 6, and 9 h (A) after depletion of endogenous Rcl1 by switching to glucose media. U2 RNA was used as a loading control. Probes are indicated next to the panels, and their locations are noted in Fig. 1A. 21 S rRNA was detected with probe c, but not probe z, indicating processing had occurred at site A₁. C (left), Rcl1 cleaves an RNA oligonucleotide (ACAACACACU) containing the A₂ cleavage site at the three CA sites observed with longer transcripts. Right, cleavage of the same RNA by purified MBP-tagged WT Rcl1 and Rcl1-R327A/D328A/K330A. Bottom, model for the interaction of Rcl1 and MBP-tagged Rcl1 with RNA.