INTRODUCTION

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snoRNAs (small nucleolar RNAs) are required for processing and posttranscriptional modification of rRNA (22, 23, 36). Several snoRNAs facilitate endonucleolytic cleavages in pre-rRNA. In addition, numerous snoRNAs that contain C and D boxes, short conserved sequences found near snoRNA termini, are involved in the formation of 2'-O-methylribose residues in rRNA (7, 8, 17, 35, 37). Other snoRNAs that contain the sequence ACA near their 3' ends facilitate the formation of pseudouridine in rRNA (25). Many yeast snoRNAs contain trimethylguanosine (TMG) caps at their 5' ends (23). TMG caps are hypermethylated forms of monomethylguanosine caps that are added cotranscriptionally to the 5' end of RNA polymerase II primary transcripts. The presence of a TMG cap may function to protect the 5' ends of snoRNAs from exonucleolytic decay.

Most snoRNAs are synthesized via RNA maturation pathways that convert pre-snoRNAs to mature snoRNAs. Much is now known about the cis-acting requirements of snoRNA processing. C/D box snoRNAs contain C boxes near their 5' termini and D boxes near their 3' termini. These RNAs bind to fibrillarin, an abundant nucleolar protein, and fold into secondary structures containing a base-paired stem that forms between sequences immediately 5' to C boxes and 3' to D boxes. The C/D box-fibrillarin snoRNP complex is required for the formation of 5' and 3' termini of mature snoRNAs. Mutations in the yeast and vertebrate U14 C and D boxes that perturb base pairing cause underaccumulation of U14 (16, 41, 43). Compensatory mutations that restore base pairing also restore normal accumulation. These results indicate that the C/D box-fibrillarin snoRNP complex is important for the formation and stability of C/D box snoRNAs.

In addition to fibrillarin, other trans-acting factors are required for the maturation and stability of snoRNAs. The C/D box snoRNAs U14, U16, and U18 undergo endonucleolytic cleavages and/or end-trimming steps that convert the primary transcripts into mature snoRNAs (4, 5, 41). Competition experiments suggest the existence of common trans-acting factors that facilitate the processing of these snoRNAs (6, 41). The D box of Xenopus U3, U8, and U14 snoRNAs is required for nuclear retention, cap hypermethylation, and stability (34). Nuclear retention of U3, U8, and U14 is saturable, suggesting the existence of shared trans-acting factors that bind D boxes. Furthermore, maturation of yeast U14 and snR190, which are derived from a shared polycistronic precursor, requires the 5'3' exonucleases Rat1p and Xrn1p (19, 27). Some snoRNAs contain near their 3' ends the sequence ACA which facilitates 3' end formation. When the ACA box of yeast snR11 is mutated, underaccumulation of mature snR11 results. ACA snoRNAs associate with the protein Gar1p, which may be involved in stability or 3' end formation (2). Yeast ACA pre-snoRNAs are processed in human cell extracts, suggesting that snoRNA processing enzymes are conserved between yeast and human genomes (14).

In this report, we show that the putative nucleic acid helicase Sen1p is required for the maturation and stability of snoRNAs in yeast. The SEN1 gene was cloned by complementation of the temperature-sensitive mutation sen1-1, which causes a 10-fold increase in the accumulation of pre-tRNAs at both permissive and restrictive temperatures and a 90% reduction in the in vitro activity of tRNA-splicing endonuclease (42). 252 kDa Sen1p localizes to the nucleus and contains a consensus P-loop nucleoside triphosphate binding motif and an RNA helicase motif similar to that of the mouse protein Mov10 and yeast Upf1p (10, 18, 20, 21, 38). Upf1p has ATP-dependent nucleic acid helicase activity in vitro (9). The sen1-1 mutation causes a GA change at a position that is conserved between the Sen1p and Upf1p helicase motifs (10).

The effects of loss of Sen1p function are not limited to tRNA splicing. The sen1-1 mutation causes mislocalization of nucleolar proteins and defects in several steps in pre-rRNA processing (38, 39). Recently, it was shown that Sen1p forms RNP complexes with snoRNAs since it coimmunoprecipitates with more than 20 snoRNA species (39). In addition, chimeric mRNAs containing a portion of antisense U6 snRNA accumulate in excess in cells carrying a mutation in the SEN1 gene (33). It therefore appears likely that Sen1p performs a function that affects multiple RNA biosynthetic pathways. In this report, we show that yeast snR13, a TMG-capped snoRNA that contains C and D boxes (23), exists in wild-type cells in three forms, including an abundant RNA that is presumed to be the functional snR13 snoRNA as well as much less abundant 3' extended and 5' truncated forms. Our results suggest that Sen1p is required for the maturation and stability of snR13.

	MATERIALS AND METHODS

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Strains and media. The strains used in this study were as follows: 1971 (MAT ura3-52 leu2-3,112 pep4-3), FWY1 (MAT ura3-52 leu2-3,112 sen1-1 pep4-3), TR110 (MATa ura3 leu2 snR13-1), and TR116 (MAT ura3 leu2 his3 snR13-1 sen1-1). Strain FWY1 is an isogenic derivative of strain 1971 that was created by gene replacement (30). snR13-1 was substituted for the wild-type chromosomal locus by gene replacement. Strain TR116 was derived from a cross between an snR13-1 strain and strain FWY1. Standard yeast media were used for cell culture except for in vivo labeling with orthophosphate, where low-phosphate YEPD was used (29). Temperature shift experiments were performed by adding an equal volume of medium prewarmed to 49°C to cultures grown at 25°C. In strains carrying sen1-1, 25°C is a permissive temperature and 37°C is a restrictive temperature for growth (42).

Plasmids. pTR48 (snR13 LEU2 CEN) was constructed by PCR amplification of a region of wild-type genomic DNA with oligonucleotide primers PCR1 and PCR2 (Table 1; Fig. 1), using Pfu DNA polymerase (Stratagene Corp.). The resulting PCR product was ligated into the SmaI site of pRS315, which carries a centromere and the LEU2 gene. pTR48 contains the entire snR13 transcriptional unit. The 5' end of cloned DNA present in pTR48 begins 260 bases of DNA upstream from the snR13 +1 position and extends approximately 450 bases past the 3' end of the reduced-mobility form. pTR43 was constructed by PCR amplification of wild-type DNA, using the primers PCR1 and 13D (Table 1; Fig. 1). The product was subcloned into the SmaI site of the yeast 2µm vector Yep351 (15). The gene disruption snR13-1 was constructed by replacing a Bst1107I/BclI restriction fragment containing snR13F DNA with a restriction fragment containing the URA3 gene. snR13-1 was substituted for the wild-type chromosomal locus by standard one-step gene replacement (30). Strain TR116 was derived from a cross between an snR13-1 strain and strain FWY1. DNA fragments carrying mutant alleles of snR13 were inserted into a CEN LEU2 vector as follows: pTR51 (snR13-R3AA), pTR52 (snR13-R1AA), pTR53 (snR13-R3SUB), and pTR56 (snR13-RNEW).

View larger version (19K): [in this window] [in a new window]   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 snR13-1. (C) DNA from the SNR13 region contained in centromeric plasmid pTR48 (see Materials and Methods).

General RNA methods. RNA was prepared by hot phenol extraction (20). Midwestern blotting was performed as described previously (28). Oligonucleotide-directed RNase H digests were performed by adding 1 pmol of oligonucleotide to samples containing 10 µg of total yeast RNA in 40 mM Tris HCl (pH 7.7)-4 mM MgCl₂-1 mM dithiothreitol-30 µg of bovine serum albumin per ml. Samples were denatured for 5 min at 65°C and allowed to cool slowly to 25°C; then 0.1 U of RNase H was added, and samples were digested for 30 min. Reactions were stopped by adding an equal volume of 95% formamide-20 mM EDTA-0.05% bromophenol blue-0.05% xylene cyanol. Primer extension was performed using with avian myeloblastosis reverse transcriptase (Promega Corporation) at 41°C. Secondary structure predictions were performed with the Genetics Computer Group Fold program, using default parameters (11). Oligonucleotides used in this study are described in Table 1; the locations in snR13 to which oligonucleotides anneal are shown in Fig. 1.

Analysis of in vivo-labeled RNAs. snR13F RNA was prepared for thin-layer chromatography (TLC) by labeling for 7 h at 25°C with 1 mCi of [³²P]orthophosphate. snR13 RNAs were purified from hot phenol-extracted total RNA by hybridization to the 5'-biotinylated oligonucleotide 13BIO (Table 1; Fig. 1). 13BIO-snR13 hybrids were bound to streptavidin-conjugated magnetic beads (Promega) in 0.5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and washed four times in 0.1× SSC, using a magnet for retention of the DNA-RNA hybrids. Elution was accomplished by resuspension in water. snR13F RNA was purified from the hybrid-selected RNAs by elution from a preparatory polyacrylamide gel in 0.3 ml of 0.5 M ammonium acetate-1 mM EDTA-0.2% SDS. Conditions for RNase T1 and T2 digestion were described previously (3). Digestion products were separated by two-dimensional cellulose TLC. The solvent system for the first dimension was isobutyric acid-0.5 M NH₄OH in a 5:3 (vol/vol) ratio. After drying in a cool stream of air, chromatography in the second dimension was accomplished with isopropanol-concentrated HCl-water in the ratio 14:3:3 (vol/vol/vol) as the solvent system (26).

Mutagenesis. Site-directed mutagenesis was performed on pTR48 (Fig. 1), using an oligonucleotide-directed method that extends 5'-phosphorylated primers containing desired mutations with Klenow enzyme followed by ligation (Chameleon site-directed mutagenesis system; Stratagene). Phosphorylated oligonucleotides R3AA, R1AA, RNEW, and R3SUB were used as primary primers, and CMUT was used as the secondary primer (Table 1; Fig. 1). CMUT is complementary to a polylinker and changes a unique SacI restriction site present on pTR48 to an SphI site. The primers were annealed, extended, and ligated, and the resulting products were transformed into a mutS mismatch repair-deficient Escherichia coli strain. Plasmids were isolated and transformed into E. coli DH5. Plasmids prepared from DH5 that contained a new SphI site and displayed correct restriction digest patterns were sequenced through the entire snR13F region to identify plasmids carrying a mutant snR13 gene.

	RESULTS

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Inactivation of Sen1p affects the accumulation of TMG-capped snoRNAs. Midwestern blotting was used to assess whether specific TMG-capped RNAs change in abundance when Sen1p is inactivated. RNA was extracted from two strains with isogenic genetic backgrounds, FWY1 (sen1-1) and 1971 (SEN1) (Materials and Methods). Samples were taken at 4-h time intervals following a shift of each strain from 25 to 37°C. Antibodies against TMG caps were used to probe total RNA transferred to a nylon membrane (28). The previously described array of TMG-capped RNAs detected by Midwestern blotting include four of the five spliceosomal snRNAs and a substantial number of snoRNAs.

View larger version (59K): [in this window] [in a new window]   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).

View larger version (86K): [in this window] [in a new window]   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.

View larger version (12K): [in this window] [in a new window]   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).

snR13-1

Novel forms of snR13 RNA consist of 5' truncations and 3' extensions of snR13F. Since forms R and T appeared to be related to snR13F, we considered several possible causes for the slow mobility observed for band R, including a 5' extension, a 3' extension, or possibly an unusual topological structure. We reprobed the blot shown in Fig. 3A with oligonucleotide 13D (Fig. 1A; Table 1), which anneals downstream from the normal 3' end of snR13F and which should not anneal with the F or the T form. Since this probe detected only form R (Fig. 3B), we performed further experiments to delineate the extent of the 3' extension.

View larger version (71K): [in this window] [in a new window]   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.

View larger version (71K): [in this window] [in a new window]   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.

View larger version (46K): [in this window] [in a new window]   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 T1a fragment (U7 to G24). (C) Chromatogram of the products of RNase T2 digestion of RNA fragment T1b (A88 to G121).

Newly synthesized snR13 RNAs. To visualize newly synthesized RNA, we examined in vivo pulse-labeled RNA from strains 1971 (SEN1) and FWY1 (sen1-1). Pulse-labeling at 25°C was accomplished by adding 1 mCi of [³²P]orthophosphate for 1 h to SEN1 or sen1-1 cells growing in low-phosphate YEPD at 25°C. Pulse-labeling at 37°C was accomplished by shifting the cells from 25 to 37°C for 1 h followed by an additional hour of labeling at 37°C with 1 mCi of [³²P]orthophosphate. Total labeled RNA was extracted, and the snR13 RNAs were purified by hybrid selection using biotinylated oligonucleotide 13BIO (Fig. 1A).

View larger version (51K): [in this window] [in a new window]   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.

Identification of a sequence important in snR13 RNA synthesis. Inspection of the snR13 primary sequence revealed that the sequence C₂U₄ is reiterated three times in snR13F (Fig. 9A). The normal 3' end reported for snR13 (23) (GenBank entry SCU16692) is embedded within C₂U₄ repeat 3. The premature primer extension stop at position U₁₅ and the 5' truncation at U₁₇ map within C₂U₄ repeat 1. 

View larger version (33K): [in this window] [in a new window]   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 snR13-1, 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).

snR13-1

Epistatic relationship between sen1-1 and mutations in snR13. We assessed the consequences of combining mutations in C₂U₄ repeats with sen1-1. To accomplish this, plasmids carrying each of the snR13 mutant alleles were transformed into the haploid strain TR116, which carries the chromosomal mutations sen1-1 and snR13-1 (Materials and Methods). Strain TR116 was also transformed with plasmid pTR48, which carries DNA coding for the entire wild-type snR13 transcriptional unit (Fig. 1C). Transformants were grown at the permissive temperature of 25°C in selective medium to maintain the plasmids. In addition, transformants were grown to mid-log phase in selective medium at 25°C and then shifted to the nonpermissive temperature of 37°C for 4 h.

View larger version (77K): [in this window] [in a new window]   FIG. 10.   (A) Epistatic interactions between sen1-1 and mutations in SNR13. RNA was extracted from strain TR116 (sen1-1 snR13-1) 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.

	DISCUSSION

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In this report, we describe a family of RNAs that are derived from the snR13 locus of S. cerevisiae. The most abundant of these, snR13F, is a TMG-capped, C/D box-containing snoRNA that most likely functions in rRNA maturation in the nucleolus (23). Three overlapping RNAs, snR13R, snR13F, and snR13T, were detected in wild-type cells by Northern blotting. The steady-state levels of these RNAs changed when strains carrying the temperature-sensitive mutation sen1-1 were shifted to the restrictive temperature. snR13R and snR13T increased in abundance with similar kinetics. The level of snR13F declined in both wild-type and sen1-1 cells at the restrictive temperature, obscuring any potential change due to the sen1-1 mutation itself. The reason for this decline at an elevated temperature is unknown.

When in vivo-labeled, nascent snR13 RNA was analyzed in both wild-type and temperature-shifted sen1-1 cells, similar changes were observed except that the amount of snR13F RNA synthesized was severely reduced in sen1-1 cells shifted to the restrictive temperature. This result indicates that the rate of synthesis is significantly reduced. We identified in wild-type and temperature-shifted sen1-1 cells several additional nascent RNAs that were not detected on Northern blots. Two long forms of snR13, called R_L and R_S, were detected. R_L is probably equivalent to the snR13R RNAs detected on Northern blots, because it responds to inactivation of Sen1p in the same way as snR13R. Similar amounts of R_S were detected at the permissive and restrictive temperature. The relationship of R_S to R_L is not yet known. Our results indicate that a function provided by Sen1p, which is most likely an RNA unwinding activity, is required for the synthesis of snR13F. This is consistent with an interpretation in which snR13F is derived from R_L by a posttranscriptional pathway leading to 3' end formation.

We used RNase H mapping and primer extension to characterize the three stable RNAs that accumulate on Northern blots. These RNAs are distinguished by a 5' truncation, a 3' extension, or both. snR13R is a mixed population of RNAs that share in common a ~1,300-nucleotide extension of the 3' end of snR13F. RNase H mapping indicates that R form RNAs contain two discrete 5' ends and two discrete 3' ends. Depending on the distribution of 5' and 3' ends, there are two to four species of snR13R. Using primer extension, we showed that one of the 5' ends of snR13R is identical to the 5' end of snR13F (residue A₁ in Fig. 1 and 6). Since Midwestern blotting and TLC analysis indicate that snR13F contains a TMG cap, it seems likely that a subpopulation of snR13R may also contain a TMG cap. We could not detect a TMG-capped R form on Midwestern blots, but this could be because of low abundance, poor transfer to nylon membranes, or comigration with other TMG-capped RNAs.

The changes in synthetic rates and levels of accumulation of the snR13 RNAs result from a requirement of Sen1p to both stabilize the 5' terminus and to promote formation of the 3' terminus from a 3'-extended RNA. The truncated 5' end found among snR13R RNAs begins at nucleotide U₁₇ and is therefore missing the first 16 nucleotides found in snR13F RNA. snR13T also begins at U₁₇. snR13T RNA was detected on Northern blots but not on Midwestern blots and is therefore not likely to contain a TMG cap. Based on these findings, we propose that snR13F, snR13T, and the 5'-truncated version of snR13R are all derived from a common monomethyl-G-capped primary transcript that becomes hypermethylated to form a single TMG-capped RNA.

The 5' truncation of snR13R and snR13T may result from exonucleolytic degradation or endonucleolytic cleavage of a single primary transcript that begins at nucleotide A₁, producing a capless, 5'-truncated RNA. If the two 5' ends were derived from two primary transcripts initiating at two different transcriptional start sites, all of the RNAs might be expected to have a TMG cap which is synthesized in conjunction with transcription by RNA polymerase II. The lack of a TMG cap in snR13T is inconsistent with this model. Our interpretation of the data implies that Sen1p is required for maintenance of the 5' end. In absence of Sen1p function, the 5' end is removed up to a position approaching the location of the C/D base-paired stem.

Primer extension stops were detected at positions U₁₅ and U₁₀₄. Both stops map within the sequence C₂U₄ that is repeated three times in snR13F and snR13T and once more in snR13R. The truncated 5' end beginning at U₁₇ in a subpopulation of snR13R and in snR13T maps within C₂U₄ repeat 1. The 3' ends of snR13F and snR13T are located within C₂U₄ repeat 3. Because of the apparent significance of C₂U₄ repeats, we analyzed a substitution of CA₂U₃ for C₂U₄ repeat 1, which contains the U₁₅ and U₁₇ primer extension stops, and substitutions of CA₂U₃ and A₆ for C₂U₄ repeat 3, which contains the 3' terminus of the snR13F and snR13T RNAs (Fig. 1 shows the locations of these repeats).

If C₂U₄ sequence repeats 1 and 3 were to function as signals for transcription initiation and termination, respectively, we would expect that mutations that impair the function of these sequences might cause increased accumulation of RNAs containing extended 5' and/or 3' ends. Our results show that this is not the case. The mutation in repeat 1 caused decreased accumulation of snR13F RNA without a concomitant increase in 5'-truncated snR13T and 3'-extended snR13RR. Both of the mutations in repeat 3 failed to cause detectable increases in the accumulation of snR13T and snR13R RNAs. Instead, these mutations resulted in the accumulation of a pair of novel RNAs that differ markedly from R forms. The novel doublet RNAs failed to migrate according to size and may not contain a significant 3' extension, indicating a potential level of structural complexity not expected for simple 3'-extended transcripts. These results make it unlikely that C₂U₄ repeats function in transcription initiation or termination. Instead, they are likely to play a role in maturation.

The predicted secondary structure of snR13F (Fig. 9) suggests that C₂U₄ repeats 1 and 3 may reside near each other in three-dimensional space. The two repeats flank a base-paired region that overlaps with the C and D boxes. In our studies we detected 5' and 3' ends within repeats 1 and 3, respectively, but failed to detect RNAs with an endpoint in repeat 2 or in a new repeat created by the snR13-RNEW mutation. These results indicate that the presence of a C₂U₄ repeat is not sufficient to specify a site for end formation. According to this interpretation, C₂U₄ repeats may play a role in maturation by providing a preferred context to the neighboring base-paired C/D box. Additional evidence supporting this view comes from a comparison of sequences surrounding C and D boxes in other snoRNAs. When the sequences of other snoRNAs were examined, we found that several contained either an exact C₂U₄ match or a close match differing by a single nucleotide (Table 3). However, some snoRNAs such as snR10 and snR31 do not possess any sequences resembling C₂U₄ (2, 36). These observations indicate that C₂U₄ is most likely a preferred sequence but not one that is globally required in all snoRNAs.

The phenotypes conferred by sen1-1 and by mutations in C₂U₄ repeats 1 and 3 resemble the phenotypes of C/D box mutations described by others (6, 13, 41). For example, deletion of a short stem that immediately flanks the C and D boxes in human U14 snoRNA results in maturation defects. Also, Xenopus U16 and U18 RNAs require intact C and D boxes for end maturation. The 3' end of Xenopus U16 is formed within a sequence context identical to that found for snR13 and proceeds by a reaction requiring protein factors that have been partially purified. We speculate that one of the factors could be Sen1p in yeast and its homologue in higher eukaryotes.

We tested these ideas further by analyzing double mutants carrying sen1-1 and each of the mutations in C₂U₄ repeats 1 and 3. When mutations in repeat 1 were combined in the same haploid strain with the sen1-1 mutation, the abundance of snR13F was significantly reduced at the restrictive temperature. We have not yet determined whether this is due to a reduced rate of formation of snR13F or to an increased rate of decay of the mature RNA. When mutations in repeat 3 were combined with the sen1-1 mutation, R forms accumulated at the expense of the novel doublet RNAs upon a shift from permissive to restrictive temperature. This finding indicates that an epistatic relationship exists between Sen1p and C₂U₄ repeat 3. Based on this finding, we propose that Sen1p is required prior to repeat 3. Furthermore, the epistatic relationship suggests a connection between Sen1p, C₂U₄ repeat 3, and the C/D box-fibrillarin snoRNP complex. Repeat 3 may provide a good sequence context that influences the binding of C/D box proteins that both protect snR13F from degradation and promote 3' end maturation. Sen1p might be recruited into the snoRNP complex to facilitate 3' end formation. Alternatively, Sen1p might promote the assembly of a snoRNP complex.

There are two reasons for suspecting that Sen1p may play a direct role in the formation of RNA termini and may be recruited into or assist in the assembly of snoRNP complexes. The changes in synthetic rates and levels of accumulation caused by loss of Sen1p function commence immediately upon temperature shift, which would be expected if Sen1p plays a direct role in snoRNA maturation. By contrast, the accumulation of tRNA precursors observed in strains carrying sen1-1 does not correlate with growth temperature, indicating that an indirect effect of Sen1p on tRNA splicing is likely (42). Most importantly, snR13F coimmunoprecipitates with wild-type Sen1p, suggesting that an RNA-protein physical interaction is likely (39). It remains to be determined whether snR13R and snR13T RNAs are also present in precipitates because their abundance in wild-type strains is very low.

Direct evidence that the novel doublet RNAs represent trapped intermediates in a maturation pathway is still lacking. We were unable to detect newly synthesized RNAs corresponding to the novel doublet RNAs in wild-type cells, but they may still exist as extremely labile RNAs. If they correspond to intermediates in 3' end formation, their existence suggests that 3' end formation involves more than one step. We are currently investigating the structures of these RNAs to determine why they exhibit anomalous migration on gels and whether they differ in length at the 5' end in a manner similar to snR13F, snR13T, and the subpopulations of the snR13R RNAs.

Inactivation of Sen1p caused time-dependent changes in the accumulation of the snoRNAs snR10, snR11, snR13, snR31, snR40, and snR42, which include both C/D box and ACA snoRNAs, and the snRNA U5 (28, 39). In addition, other RNAs have recently been shown to exhibit altered accumulation upon inactivation of Sen1p, including RNAs of unexpected size containing U5, snR40, and snR45 sequences (39). Since loss of Sen1p function affects multiple RNAs and since it is recruited into RNP complexes containing as many as 20 snoRNAs (39), we suspect that Sen1p plays a role in the maturation of numerous snoRNAs.

The sen1-1 mutation causes pleiotropic defects in RNA metabolism, including effects on tRNA and rRNA processing (39, 42). Also, temperature-shifted sen1-1 cells exhibit altered localization of nucleolar proteins such as fibrillarin (38). Since changes in rRNA processing and nucleolar organization do not appear until several hours after temperature shift of sen1-1 cells but perturbations of snR13 synthesis commence immediately, it seems likely that global perturbations in snoRNA synthesis are primary defects in sen1-1 cells. The nucleolar and rRNA defects probably develop as a secondary consequence because rRNA maturation requires snoRNAs. The primary defect responsible for the accumulation of intron-containing pre-tRNAs in sen1-1 cells is still unknown. Our studies raise the possibility that a small RNA whose synthesis depends on Sen1p could be required for tRNA synthesis.

snR13R RNAs extend into and include all of the downstream ORF YDR472w. At present, we are unable to interpret the significance of this finding. Recent evidence indicates that YDR472w produces a polyadenylated mRNA that is likely to be translated into a protein product (21a). Studies are in progress to determine whether the snR13R RNAs encode a protein in addition to producing a snoRNA.