The putative nucleic acid helicase Sen1p is required for formation and stability of termini and for maximal rates of synthesis and levels of accumulation of small nucleolar RNAs in Saccharomyces cerevisiae

Abstract

Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of approximately 1,400-nt 3'-extended forms (snR13R) and a 108-nt 5'-truncated form (snR13T) that is missing 16 nt at the 5' end. A subpopulation of snR13R contains the same 5' truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5' end and promotes maturation of the 3' end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5' end of snR13T and the 3' end of snR13F reside within C2U4 sequences that immediately flank the C and D boxes. A mutation in the 5' C2U4 repeat causes underaccumulation of snR13F, whereas mutations in the 3' C2U4 repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3' C2U4 repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3' C2U4 sequence act in a common pathway to facilitate 3' end formation. Based on these findings, we propose that Sen1p and the C2U4 repeats that flank the C and D boxes promote maturation of the 3' terminus and stability of the 5' terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.

FIG. 1

(A) Schematic representation of the SNR13 chromosomal region on chromosome IV showing the linear relationships between snR13R, snR13F, and snR13T. The upper line represents SNR13 DNA. YDR472w (shown 5′ to 3′) is an ORF contained within the snR13R 3′ tail. The positions where oligonucleotides used in this study anneal are shown below the chromosome (PCR1, U13A, 13BIO, 13A through 13G, and PCR2). snR13R, -F, and -T are drawn to scale in the 5′-to-3′ direction and indicated by bold horizontal lines. The positions of primer extension stops and the positions of four C₂U₄ repeats are shown. (B) Structure of the null allele snR13-Δ1. (C) DNA from the SNR13 region contained in centromeric plasmid pTR48 (see Materials and Methods).

FIG. 2

Detection of TMG-capped RNAs by Midwestern and Northern blotting. (A) Midwestern blot. Total RNA was extracted from strain 1971 (SEN1) (lanes 1, 3, 5, and 7) and FWY1 (sen1-1) (lanes 2, 4, 6, and 8) at the times indicated following a temperature shift from 25 to 37°C. RNA was fractionated on a 9% polyacrylamide gel, transferred to nylon, and detected with anti-TMG antibodies (28). snR11 and snR31 decrease in abundance as a consequence of thermal inactivation of Sen1p. RNA species b shows a similar sen1-1-dependent decrease in abundance but has not yet been positively identified (28). (B) The blot shown in panel A was reprobed with ³²P-end-labeled oligonucleotides complementary to snR11 and snR31. A probe complementary to SUF4, a glycine tRNA derived from an intronless precursor (24), was used for a loading control (not shown).

FIG. 3

Accumulation of snR13-related RNAs in a strain carrying sen1-1. (A) Strains 1971 (wild type) and FWY1 (sen1-1) were grown at 25°C and then shifted to 37°C. Total RNA was extracted at the time intervals indicated and analyzed by Northern blotting using oligonucleotide 13A (Fig. 1) as the probe. SUF4 tRNA was used as a loading control (not shown). Three RNAs were detected: snR13R (retarded mobility), snR13F (final form), and snR13T (truncated form). Size standards are indicated in nucleotides on the left. (B) The blot shown in panel A was stripped and reprobed with oligonucleotide 13D, which anneals downstream from the 3′ end of snR13F (Fig. 1). This probe detects only snR13R RNA.

FIG. 4

Kinetics of accumulation of snR13 RNAs following inactivation of Sen1p. Data from the Northern blot shown in Fig. 3A and from a duplicate experiment were quantitated with a PhosphorImager, averaged, and plotted. RNA accumulation levels were normalized by using SUF4 tRNA as a loading control. The data are expressed as a proportion of snR13F accumulation at time zero. (A) Accumulation of snR13F (□) at time intervals following a shift from 25 to 37°C in strain 1971 (SEN1). The abundances of snR13R and snR13T were too low for reliable quantitation. (B) Accumulation of snR13R (○), snR13F (□), and snR13T (◊) at time intervals following a shift from 25 to 37°C in strain FWY1 (sen1-1).

FIG. 5

RNase H mapping of snR13R RNA. RNA was extracted from strain FWY1 (sen1-1) 4 h after temperature shift. Lanes are labeled with the names of the oligonucleotides used for each RNase H cleavage reaction. The positions to which they anneal are shown in Fig. 1. Samples were gel fractionated and transferred to a nylon membrane. Numbers at the right indicate the nucleotide positions of RNA size standards. (A) RNase H cleavage products were probed with oligonucleotide 13A, which detects RNA fragments 5′ of the cleavage site. The reaction in the left lane (no oligonucleotide [oligo]) shows the positions of full-length RNAs resulting when the cleavage reaction was performed in the absence of any oligonucleotide. (B) The blot shown in (A) was stripped and reprobed with oligonucleotide 13H (Fig. 1), which detects RNA fragments 3′ of the cleavage site.

FIG. 6

Primer extension of snR13 RNAs. (A) Primer extension of total RNA from strains 1971 (SEN1) and FWY1 (sen1-1) grown for 4 h at 37°C. Oligonucleotide 13A (Fig. 1) was end labeled and used for both primer extension mapping and dideoxy sequencing from plasmid pTR43 (Materials and Methods). cDNA band U₁₅ is due to internal termination within snR13F. U₁₇ arises from the 5′ end found in snR13T and a subpopulation of snR13R RNA. (B) Primer extension of total RNA from strain FWY1 (sen1-1) grown for 4 h at 37°C, using an end-labeled oligonucleotide 13B (Fig. 1). Stops A₁ and U₁₇ correspond to two distinct 5′ ends of snR13R RNA. Stops U₁₅ and U₁₀₄ probably arise due to internal termination of reverse transcriptase within snR13R RNA.

FIG. 7

TLC of snR13F purified from strain 1971 (SEN1) (see Materials and Methods). In each chromatogram, the loading origin is in the lower left corner, with the first dimension developed from bottom to top and the second dimension developed from left to right. Quantitative analysis of each chromatogram is provided in Table 2. (A) Chromatogram of the products of RNase T2 digestion of snR13F RNA. Spots correspond to nucleoside 3′ monophosphates and the presumed cap dinucleotide. (B) Chromatogram of the products of RNase T2 digestion of RNA fragment T1_a fragment (U₇ to G₂₄). (C) Chromatogram of the products of RNase T2 digestion of RNA fragment T1_b (A₈₈ to G₁₂₁).

FIG. 8

In vivo pulse-labeling of snR13 RNAs. Following labeling, RNAs were purified by using the biotinylated oligonucleotide 13BIO (Fig. 1; Materials and Methods). Lane 1, RNA from strain 1971 (SEN1) labeled for 1 h at 25°C; lane 2, RNA from strain 1971 shifted to 37°C for 1 h and then ³²P labeled for 1 h at 37°C; lane 3, RNA from strain FWY1 (sen1-1) labeled for 1 h at 25°C; lane 4, RNA from strain FWY1 shifted to 37°C for 1 h and then ³²P labeled for 1 h at 37°C. The identities of the RNAs are indicated on the right; RNA size standards are indicated in nucleotides on the left.

FIG. 9

Effects of mutations in snR13 on RNA accumulation. (A) Predicted secondary structure of wild-type snR13F RNA, generated by using the Genetics Computer Group Fold program (11). The locations of the C and D boxes, the primer extension stops, and the mutations R1AA, R3SUB, R3AA, and RNEW are shown (see text). The 3′ end of wild-type snR13F is formed between residues C₁₂₄ and U₁₂₅. (B) Effects of snR13 mutations. Strain TR110, which carries snR13-Δ1, was transformed with plasmids as indicated below. Transformants were grown at 30°C followed by RNA extraction and Northern blotting using oligonucleotide 13A (Fig. 1) as the probe. Lane 1, pTR48 (wild-type SNR13); lane 2, pTR51 (snR13-R3AA); lane 3, pTR53 (snR13-R3SUB); lane 4, pTR52 (snR13-R1AA); lane 5, pTR56 (snR13-RNEW); lane 6, pRS315 (vector only). Arrows to the right indicate the mobilities of the novel RNAs. Size standards are indicated in nucleotides to the right. (C) A shorter exposure of the Northern blot shown in panel B shows that snR13F is decreased in abundance in lane 4 (see text).

FIG. 10

(A) Epistatic interactions between sen1-1 and mutations in SNR13. RNA was extracted from strain TR116 (sen1-1 snR13-Δ1) transformed with pTR48 (wild type) and with plasmids containing the snR13 mutations R3AA, R3SUB, R1AA, and RNEW RNA was (see Materials and Methods). Probe 13A (Fig. 1) was used to probe a Northern blot. RNA was extracted from cells grown at 25°C, a permissive temperature for sen1-1 (P), and from cells grown at 25°C followed by a shift to 37°C, a nonpermissive temperature for sen1-1, for 4 h (N). Arrows to right indicate positions of the novel RNAs. (B) Analysis of novel doublet RNA mobility by Northern blotting of RNA extracted from TR116 transformed with pTR51 (sen1-1 snR13-R3SUB) grown at 25°C (P) and then shifted to 37°C (N). The blots were probed with oligonucleotides U13A, 13A, 13B, and 13H (Fig. 1). Sizes are indicated in nucleotides on the right.