U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production

Abstract

Saccharomyces cerevisiae snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) required for the processing of 18S rRNA. Here, we show that the previously characterized human, reptilian, amphibian, and fish U17 snoRNAs represent the vertebrate homologues of yeast snR30. We also demonstrate that U17/snR30 is present in the fission yeast Schizosaccharomyces pombe and the unicellular ciliated protozoan Tetrahymena thermophila. Evolutionary comparison revealed that the 3'-terminal hairpins of U17/snR30 snoRNAs contain two highly conserved sequence motifs, the m1 (AUAUUCCUA) and m2 (AAACCAU) elements. Mutation analysis of yeast snR30 demonstrated that the m1 and m2 elements are essential for early cleavages of the 35S pre-rRNA and, consequently, for the production of mature 18S rRNA. The m1 and m2 motifs occupy the opposite strands of an internal loop structure, and they are located invariantly 7 nucleotides upstream from the ACA box of U17/snR30 snoRNAs. U17/snR30 is the first identified box H/ACA snoRNA that possesses an evolutionarily conserved role in the nucleolytic processing of eukaryotic pre-rRNA.

FIG. 1.

Processing of pre-rRNA in yeast S. cerevisiae. (A) The major processing pathway of pre-rRNA. The primary rRNA transcript (pre-rRNA) contains externally (ETS) and internally (ITS) transcribed spacers. Arrows, sites of endo- and exonucleolytic cleavage (12, 27, 52). (B) Processing of 35S pre-rRNA in snR30-depleted yeast cells.

FIG. 2.

Predicted two-dimensional structure of yeast snR30. The nucleotide sequence of snR30 was first published by Bally et al. (2) and later modified by Balakin et al. (1). The box ACA and the putative box H motifs are boxed. Arrows, regions deleted in R30-d5′ and R30-dIH RNAs. The nucleotide sequence of the altered box H motif of R30-H is shown.

FIG. 3.

Determination of the functionally essential regions of yeast snR30. (A) Schematic structure of the construct used to express the wild-type and mutant versions of snR30. The coding region of the yeast SNR30 gene (open arrow) was placed between the promoter (SNR5-P) and terminator (SNR5-T) of the yeast SNR5 gene (5). Relevant restriction sites are indicated (H, HindIII; K, KpnI; X, XhoI; B, BamHI). (B) Proposed secondary structure of the snR5-snR30 chimeric RNA (R5-R30S). Nucleotides are numbered according to the wild-type snR5 and snR30 snoRNAs. The box H/ACA elements and the evolutionarily conserved m1 and m2 sequence motifs are boxed. (C) Growth properties of yeast GAL::snR30 cells transformed with the pFL45/SNR, pFL45/SNR/R30, pFL45/SNR/R30-H, pFL45/SNR/R30-dIH, pFL45/SNR/R30-d5′, pFL45/SNR/R5-R30S, or pFL45/SNR/R5-R30L expression plasmid on galactose- (GAL) and glucose-containing (GLU) solid medium. (D) Northern blot analysis of the expression of wild-type and mutant snR30 RNAs. Total RNAs extracted from GAL::snR30 cells transformed with the pFL45/SNR, pFL45/SNR/R30, pFL45/SNR/R30-H, pFL45/SNR/R30-dIH, pFL45/SNR/R30-d5′, pFL45/SNR/R5-R30S, or pFL45/SNR/R5-R30L expression construct were separated on a 6% sequencing gel, electroblotted onto a nylon membrane, and probed with a terminally labeled oligodeoxynucleotide complementary to the 18 3′-terminal nucleotides of snR30. Lane M, size markers (terminally labeled HaeIII- and TaqI-digested pBR322).

FIG. 4.

Evolutionary conservation of U17/snR30. (A) Proposed structures of the 3′-terminal hairpins of vertebrate U17 snoRNAs. Sequences (with GenBank accession numbers in parentheses) of the human U17a (26) (L16791), C. caretta U17f (9) (AJ306558), X. laevis U17f (8) (X71081), and F. rubripes U17f (7) (X94942) snoRNAs have been reported. The conserved m1 and m2 sequence motifs are boxed. The ACA boxes are underlined. (B) Structure of the 3′-terminal hairpin of putative U17 snoRNAs obtained from S. pombe (AJ544685) and T. thermophila (AJ544686). (C) Expression of S. pombe and T. thermophila U17 snoRNAs. Total cellular RNAs were separated on a 6% sequencing gel, electroblotted onto nylon membranes, and hybridized with sequence-specific oligodeoxynucleotide probes. Lane M, size markers.

FIG. 5.

Functional characterization of S. pombe U17 snoRNA. (A) Proposed secondary structure of S. pombe U17. The conserved sequence elements are boxed. (B) Northern analysis. Total RNAs extracted from GAL::snR30 cells transformed with either the pFL45/SNR (FL45/SNR) or the pFL45/SNR/U17pombe (U17pombe) expression vector were separated on a 6% sequencing gel, transferred onto a nylon membrane, and probed with an oligonucleotide probe specific for S. pombe U17. Lane M, size markers. (C) Growth properties of yeast FL45/SNR, R30, and U17pombe strains on galactose- (GAL) and glucose-containing (GLU) media.

FIG. 6.

The conserved m1 and m2 elements of snR30 are essential for cell viability. (A) Schematic structure of the mutant snR30m1 and snR30m2 RNAs. The wild-type (boxed) and mutant sequences of the m1 and m2 motifs of snR30 are indicated. The predicted lengths of antisense RNA probes protected by snR30m1 (417 nucleotides [nt]), snR30m2 (466 nt), and snR30 (486 nt) are indicated. (B) RNase A/T1 mapping. RNAs obtained from the GAL::snR30, FL45/SNR, R30, R30m1, and R30m2 strains were annealed with an internally labeled antisense RNA probe complementary to the 3′-terminal part of snR30. Upon incubation with a mixture of RNase A and T1, protected RNAs were separated on a 6% denaturing polyacrylamide gel. The control lane shows a mapping reaction with E. coli RNA. Lane M, size markers. (C) Growth of yeast FL45/SNR, R30, R30m1, and R30m2 strains on galactose- (GAL) and glucose-containing (GLU) media.

FIG. 7.

Alteration of the m1 and m2 motifs of snR30 disrupts the production of 18S rRNA. The steady-state levels of the 20S and 23S processing intermediates and the mature 18S and 25S rRNAs were determined by Northern analyses. Total RNA isolated from the R30, FL45/SNR, R30m1, R30m2, and U17pombe cells grown for 0, 6, 12, 18, 24, and 36 h on glucose was separated on agarose-formaldehyde gels and transferred onto nylon membranes. The RNA blots were probed with terminally labeled oligonucleotides complementary to 18S (b), 25S (c), or ITS-1 (a) sequences.