Nucleic Acids Research

Oligonucleotides

U1-2, 5[prime]-CCC GAA TTC ATA CTT ACC TTA AGA TAT CA-3[prime]; U1-3, 5[prime]-GGG AAG CTT AAA ATA AAT CAA AAA TTA TAA G-3[prime]; U1-6, 5[prime]-ATC AGT AGG ACT TCT TGA TC-3[prime]; U1-9, 5[prime]-CGT ATG TGT GTG TGA CCA AG-3[prime]; U1-10, 5[prime]-AAC AAG GGA ATG GAA ACG TC-3[prime]; U1-16, 5[prime]-GAT CAG TAG GAC TTC TTG ATC-3[prime]; U1-22, 5[prime]-GGG TGG ATC TTA TAA TTC ACA CTT TAT TTT CAG AAA TAA ATG G-3[prime]; U1-23, 5[prime]-CCA TTT ATT TCT GAA AAT AAA GTG TGA ATT ATA AGA TCC ACC C-3[prime]; U1-24, 5[prime]-GGG CAA GAA GCT TGA TAT ACA ACA C-3[prime]; U1-25, 5[prime]-GGA ACA TTT TTC TAT TTA TTT C-3[prime]; pTZ-80, 5[prime]-ACG CCA GCT GGC GAA AGG GGG-3[prime]; SPP381-8, 5[prime]-ATG AGT TTT AGA CAT TTC AAG AGG-3[prime].

Yeast strains

PSU1[Delta]192-507, MATa [Delta]trp1 [Delta]his3 ura3-52 lys2-801 snr19::LYS2, pSE358(TRP1 snr19[Delta]192-507); XLT10-pXl46, MATa trp1-289 ura3-52 leu2-3,112 arg4(RV^-) ade2 snr19[Delta]::ADE2, pXL46(URA3 GAL::U1) (27); XLT10-pXl46T, MATa ura3-52 leu2-3,112 arg4(RV^-) ade2 snr19[Delta]::ADE2, pXL46(URA3 GAL::U1); RSY1, MATa trp1-289 ura3-52 leu2-3,112 arg4(RV^-) ade2 snr19[Delta]::ADE2, pXL46(URA3 GAL::U1), YEplac112(TRP1 snr19[Delta]192-507Sm); RSY2, MATa trp1-289 ura3-52 leu2-3,112 arg4(RV^-) ade2 snr19[Delta]::ADE2, pXL46(URA3 GAL::U1), YEplac112(TRP1 snr19Sm); BZY0-14, MATa trp1-289 ura3-52 leu2-3,112 arg4(RV^-) ade2 snr19[Delta]::ADE2, pXL46(URA3 GAL::U1), YIplac128(LEU2 SMD1HA); RSY3, MATa trp1-289 ura3-52 leu2-3,112 arg4(RV^-) ade2 snr19[Delta]::ADE2, pXL46(URA3 GAL::U1), YIplac128(LEU2 SMD1HA) YEplac112(TRP1 snr19Sm); PAY4[Delta]192-507, MATa trp1 ura3-52 leu2-3,112 arg4(RV^-) ade2 snr19[Delta]::ADE2, pXL46(URA3 GAL::U1) PRP39HA(integrated) YEplac112(TRP1 snr19[Delta]192-507); BZY1-16d, MAT[alpha] trp1 ura3-52 leu2-3,112 smd1::LEU2 snr19::LYS2, pBM150(URA3 GAL1::SMD1HA) (5); BZY2-51a, MATa trp1 ura3-52 smd1::LEU2 snr19::LYS2, pBM150(URA3 GAL1::SMD1HA), pSE358(TRP1 SNR19[Delta]192-507); MA52, MATa leu2-3,112 ura3-52 his3-d200 lys2[Delta] trp1 pep4-3 prb1 prc1 rnt1::HIS3 (28); MA6, MATa leu2-3,112 ura3-52 his3-d200 lys2[Delta] trp1 pep4-3 prb1 prc1 rnt1::HIS3, pRS316-RNT1 (28); TS192, MAT[alpha] trp1-289 ura3-52 leu2-3,112 his3 cyh^r prp38-1 (29).

Plasmid construction and analysis

The full-length SNR19 (U1) gene was subcloned as a 2.3 kb AccI-HindIII fragment (23) into the TRP1-marked Escherichia coli/yeast shuttle vector YEplac112 (US Biochemical; 30). Construction of the U1[Delta]192-507 functional deletion mutant was previously described (31). To be compatible with the yeast strains used, U1[Delta]192-507 was transferred as a 900 bp HindIII fragment from pSE358 (31) into vector YEplac112. Sm site mutations were introduced into SNR19 (U1) and U1[Delta]192-507 by site-specific mutagenesis (32,33) with oligonucleotides U1-3 and U1-24. The resulting mutant alleles, U1Sm and U1[Delta]192-507Sm, were confirmed by DNA sequence analysis prior to yeast transformation. Yeast strains XLT10-pXL46 (27) and BZYO-14 were used as hosts for the plasmid-expressed U1 genes. The GAL::SMD1HA-containing strain, BZY1-16d, has been described elsewhere (5). To place GAL::SMD1HA and U1[Delta]192-507 in the same genetic background, strain BZY1-16d was crossed with PSU1[Delta]192-507 and the diploid progeny selected on galactose-containing medium that lacked uracil and tryptophan. The diploid strain was sporulated and Trp-positive, galactose-dependent haploid progeny identified. Northern analysis with a U1-specific probe was then used to identify yeast that expressed U1[Delta]192-507 as the sole source of U1 snRNA.

RNA analysis

Total cellular RNA was extracted from logarithmically growing cultures by glass bead disruption followed by phenol extraction and ethanol precipitation (34). To metabolically deplete U1 snRNA or Smd1HAp, the appropriate yeast strain (XLT10-pXL46, RSY1, RSY2, BZY0-14, RSY3 or BZY2-51a) was initially cultured in 1% yeast extract, 2% Bactopeptone, 2% galactose broth (YPgal) (35) to mid-log phase at 30°C. The cells were then collected by centrifugation at 7000 g and resuspended in the same medium with 2% glucose substituted as the sugar (YPglu). All cultures were incubated with periodic dilution to maintain cell densities below 1 × 10⁷ cells/ml. RNA samples isolated from these cultures were fractionated on denaturing 1% agarose-0.22 M formaldehyde gels or on 7 M urea-5% polyacrylamide gels (29). Northern transfers were hybridized with random prime-labeled PCR fragments corresponding to RP51A intron plus exon sequences (29), mature U1 snRNA (primers U1-2 and U1-3) and U1 downstream sequences (primers U1-25 and pTZ-80). RNA from yeast that express GAL::U1, U1[Delta]192-507 (strain PAY4[Delta]192-507), GAL::U1, U1[Delta]192-507Sm (strain RSY1), U1[Delta]192-507 (strain PSU1[Delta]192-507), GAL::U1 (strain XLT10-pXL46), GAL::U1, U1Sm (strain RSY2), GAL::SMD1HA, U1[Delta]192-507 (strain BZY2-51a), rnt1::HIS3 (strain MA52) and RNT1 (strain MA6) were analyzed by this method.

Oligonucleotide-directed RNase H cleavage was done essentially as described (36). Ten micrograms of RNA was denatured at 70°C for 10 min in 19 µl of 1 mM EDTA, pH 7.4. Next, 1 µg of oligonucleotide U1-10, SPP381-8 or oligo(dT)₂₀ was added and the hybridization mixture incubated at 26°C for 15 min. One microliter of 4 M KCl was added and the incubation continued for 15 min at 26°C. To effect cleavage, 20 µl of TM buffer (40 mM Tris-HCl, pH 7.6, 60 mM MgCl₂) and 1 U of E.coli RNase H (Gibco BRL) were added for 30 min at 37°C. The RNAs were then phenol extracted, concentrated by ethanol precipitation and used for northern analysis. RNA from yeast that express GAL::SMD1HA, U1[Delta]192-507 (strain BZY2-51a) and GAL::U1, U1[Delta]192-507Sm (strain RSY1) were analyzed by this method.

For the RNase protection assays (36), the U1 snRNA genes described above were subcloned as HindIII-EcoRI fragments into the in vitro transcription vector pGEM-4Z (Promega). ³²P-labeled RNA complementary to U1 was prepared by T7 RNA polymerase transcription of SalI-cleaved DNA with [[alpha]-³²P]UTP. Aliquots of 5-10 µg of total yeast RNA were hybridized with 100 000-500 000 c.p.m. of uniformly labeled, gel-purified RNA probe for 1 h at 65°C in 20 µl of solution R1 (0.9 M NaCl, 7.5 mM EDTA, pH 7.4). Next, 105 µl of solution R2 (5 mM EDTA, pH 7.4, 0.5 U RNase One[trade]; Promega) was added and the sample digested at room temperature for 1 h. After phenol extraction, the protected RNA fragments were resolved on a denaturing 7 M urea-6% polyacrylamide gel together with RNA molecular weight markers. The markers used included in vitro transcripts of EcoRI-cleaved pTZ19U (61 nt), PvuII-cleaved pTZ19U (149 nt), HindIII-cleaved U1 pGEM-4Z (769 nt), HindIII-cleaved U1[Delta]192-507 (462 nt) and BamHI-cut 25S rDNA (228 nt; S.Abou Elela and M.Ares, personal communication). One nucleotide has been added to the size of the markers to account for the inclusion of a CAP analog, diguanosine triphosphate (Pharmacia), in the reaction mixture. To more precisely map the RNase digestion products, the RNAs were also resolved adjacent to an RNA sequence (37) of 3[prime]-end-labeled U1[Delta]192-507 RNA. RNA from yeast that express GAL::U1 (strain XLT10-pXL46), GAL::U1, U1Sm (strain RSY2), U1[Delta]192-507 (strain PSU1[Delta]192-507), GAL::U1, U1[Delta]192-507Sm (strain RSY1) and U1 (strain TS192) were analyzed by this method.

The 5[prime]-ends of the various U1 transcripts were mapped by primer extension with 5[prime]-end-labeled oligonucleotide U1-16, complementary to nucleotides +26 to +45 of U1. For each reaction, 10 µg of total yeast RNA and 3 U of avian myeloblastosis virus reverse transcriptase were used (Life Sciences Inc., St Petersburg, FL) under conditions previously described (38). A DNA sequence generated with the same primer and the U1[Delta]192-507 subcloned gene was run in parallel to determine the lengths of the extension products. RNA from yeast that express GAL::U1 (strain XLT10-pXL46), U1[Delta]192-507 (strain PSU1[Delta]192-507) and GAL::U1, U1[Delta]192-507Sm (strain RSY1) were analyzed by this method.

In vitro RNase III reactions

RNase III digestions were done in yeast extract by incubating the synthetic RNA precursor substrate (~100 000 c.p.m.) with 30% (v/v) yeast splicing extract (39), 2.5 mM MgCl₂, 3% polyethylene glycol (average mol. wt 8000), 100 mM KHPO₄ (pH 7.2) and 2 mM ATP in a final volume of 10 µl. After 1-10 min at 30°C the reactions were stopped by the addition of 100 µl of PK buffer (100 mM Tris-HCl, pH 7.5, 12.5 mM EDTA, 150 mM NaCl, 1% SDS, 2 mg/ml proteinase K; Gibco BRL) for 5 min at 37°C. The RNAs were recovered by phenol extraction and ethanol precipitation and then resolved by denaturing gel electrophoresis with the untreated substrate and the molecular weight standards described above. The RNAs used for RNase III digestion included the T7 RNA polymerase transcribed BamHI-cut 25S rDNA control substrate and the SP6 RNA polymerase transcribed HindIII-cut U1 pGEM-4Z derivatives. In some experiments 5[prime]-end-labeled RNA (40) or 3[prime]-end-labeled RNA (38) was substituted for the uniformly labeled RNA.

Purified GST-Rnt1p, a kind gift of M.Ares and S.Abou Elela (26), was also used to map RNase III cleavage sites in the synthetic U1 snRNA precursors. Here, 0.5 µl of Rnt1p was added to a 10 µl reaction composed of the RNA substrate and R3 buffer (30 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 5 mM spermidine, 30 mM KCl, 0.1 mg/ml wheat germ tRNA) (26). After 1-10 min the reactions were terminated as described for the yeast extract cleavages and the samples analyzed by denaturing gel electrophoresis.

The 3[prime]-terminal U1 Sm site is essential in vivo

The yeast U1 snRNA has three sequence elements that match the Sm site consensus. Two of the three sites are found within a double-stranded region of the internal yeast-specific domain (positions 224-230 and 241-247) and can be deleted without obvious loss of U1 function (31,41). The third resides at the more characteristic Sm site location near the 3[prime]-end of the transcript (position 553-559). To test whether the 3[prime]-terminal site was essential for U1 snRNA activity, we replaced the natural Sm sequence, AUUUUUGA, with a different sequence of equivalent length, AUUCACAC. This mutation was introduced into a plasmid-borne copy of the natural full-length SNR19 gene and into a functional deletion derivative that lacked the first two Sm sites (U1[Delta]192-507; 31). The shorter derivative, U1[Delta]192-507Sm, allowed for clean resolution of the mutant RNA from a co-expressed wild-type U1 RNA. In addition, the parallel use of U1Sm and U1[Delta]192-507Sm mutants permitted assessment of the 3[prime]-terminal Sm site activity in the presence and absence of the potentially compensatory upstream sites.

The U1 derivatives were transformed into a yeast strain that contained a chromosomal null allele of SNR19 (27) and a plasmid-borne U1 gene allele under the transcriptional control of the GAL10 promoter (GAL::U1; 42). While natural promoters are required for proper U snRNA processing in vertebrates (43-45), GAL10-driven yeast snRNAs are functional and appear to be processed normally (6,46-49; below). Neither the full-length mutant, U1Sm, nor the deletion derivative, U1[Delta]192-507Sm, produced a dominant negative phenotype when co-expressed with GAL::U1 (Fig. 1A, -tryptophan +galactose). To test for function, the transformants were streaked on medium that repressed GAL::U1 transcription and selected against the physically linked URA3 gene (Fig. 1A, +5-FOA, +glucose). As expected, the U1[Delta]192-507 allele compensated for the loss of GAL::U1 expression and colonies formed on the 5-FOA plates. In contrast, the U1Sm and U1[Delta]192-507Sm mutants did not form colonies and appeared, therefore, to be defective in U1 function. Consistent with a primary defect in pre-mRNA splicing, transcriptional repression of GAL::U1 caused cellular mRNA to decrease and pre-mRNA to accumulate in yeast transformed with U1Sm or U1[Delta]192-507Sm (Fig. 1B and data not shown). Thus, the terminal Sm site of the U1 snRNA gene is both necessary and sufficient to support in vivo pre-mRNA splicing.

Figure 1. Effect of 3[prime]-terminal Sm site mutation on cell viability and pre-mRNA splicing. (A) Growth of yeast strains transformed with GAL::U1 alone (strain XLT10-pXL46T) or GAL::U1 and U1[Delta]192-507Sm (strain RSY1), U1[Delta]192-507 (strain PAY4[Delta]192-507) or U1Sm (strain RSY3). Yeast were cultured in parallel under conditions permissive (-tryptophan +galactose) or restrictive (+5-FOA +glucose) for GAL::U1 expression. (B) Northern blot of RNA isolated from yeast transformed with GAL::U1, U1[Delta]192-507Sm before (lane 1) and after 4, 8 and 12 h of GAL::U1 repression (lanes 2-4). The intron-containing gene RP51A was used as a probe. The positions of the pre-mRNA and mRNA are noted by arrows.

Interference with Sm site-protein interactions causes the accumulation of lengthened forms of U1 snRNA

GAL::U1 yeast co-transformed with U1[Delta]192-507 accumulated RNAs corresponding to the predicted lengths of the fully processed 568 nt U1 and 258 nt U1[Delta]192-507 species (Fig. 2A, termed U1[alpha] and U1[Delta][alpha], respectively). In contrast, U1[Delta][alpha] was not detected in yeast co-transformed with the U1[Delta]192-507Sm mutant. Rather, a prominent RNA band ~75 nt longer, U1[Delta][beta], accumulated in the Sm mutant (compare lane 1 with lanes 2-4). A variable amount of a third, more diffuse RNA, U1[Delta][gamma], was also observed in the U1[Delta]192-507Sm transformant. Transcriptional repression of GAL::U1 reduced U1[alpha] levels but not U1[Delta][beta] or U1[Delta][gamma], showing that these alternative snRNA forms were not transcribed from the GAL10 fusion construct. Equivalent results were obtained when U1Sm was introduced as a mutant construct, although a prominent U1[gamma] form was not observed (Fig. 2B). Thus, for both U1 and U1[Delta]192-507, mutation of the terminal Sm site was lethal and caused the accumulation of extended U1 snRNA derivatives.

Figure 2. Analysis of U1 snRNA upon perturbation of Sm site-Sm protein interactions. (A) Northern blot of U1 snRNA from the GAL::U1, U1[Delta]192-507Sm transformant before (lane 2) and after (lanes 3-6) GAL::U1 repression. Control RNA from the yeast transformant bearing a functional short U1 (i.e. GAL::U1, U1[Delta]192-507) is presented in lane 1. (B) Northern blot of U1 snRNA from the GAL::U1, U1Sm transformant before (lane 2) and after (lanes 3-6) GAL::U1 repression. Lanes 1 and 7 contain RNA from the singly transformed GAL::U1 culture. (C) Analysis of U1 RNA upon metabolic depletion of the Sm protein, Smd1p. Yeast expressing the GAL::SMD1HA plasmid and U1[Delta]192-507 were grown under conditions which induce (lane 1) or repress (lane 2) GAL::SMD1HA gene expression for 14 h. RNA was isolated and analyzed by northern blot probed with the U1 gene body. In each panel, U1 denotes full-length U1 RNA, while U1[Delta] represents the U1[Delta]192-507 derivative. The symbol [alpha] marks the fully mature RNA species from each gene; [beta] and [gamma] represent the 3[prime]-lengthened snRNA forms.

The Sm sequence might contribute directly to the processing of the U1 snRNA or act indirectly through the association of the Sm-bound common snRNP polypeptides. To address this question, we asked whether an alternative means to perturb the Sm RNP structure produced a similar pattern of U1 snRNA forms. Yeast were assayed for U1 snRNA length both before and after transcriptional repression of the nutritionally regulated GAL::SMD1HA fusion gene which encodes the essential Smd1p common snRNP protein tagged with a hemagglutinin epitope (5,8; Fig. 2C). Although less complete than what was observed with the Sm site change, U1[Delta][alpha] levels decreased and U1[Delta][beta] and U1[Delta][gamma] levels increased with depletion of the Sm-associated Smd1HAp protein (Fig. 2C). Thus, the RNP structure of the Sm site plays an important role in U1 snRNA 3[prime]-end formation.

Extended forms of U1 snRNA have additional 3[prime]-sequence

It is of interest that while lower in abundance, the U1[Delta][beta] and U1[Delta][gamma] RNAs are present in yeast transformed with the functional U1[Delta]192-507 construct (Fig. 3, lanes 1 and 3). Oligonucleotide-directed RNase H digestion was used to confirm that these RNAs were truly derived from the U1 gene. An oligo complementary to U1 nt 170-189 directed RNase H cleavage of the U1[Delta][alpha], U1[Delta][beta] and U1[Delta][gamma] RNAs (lane 2). Each U1 band was also cleaved with oligonucleotides directed against U1 nt 26-45 and 130-148 but not with the control oligonucleotide, 381-1 (lane 1 and data not shown). Equivalent results were obtained with the U1-specific oligonucleotides when U1[Delta]192-507Sm-derived RNA was used as an enriched source of the U1[Delta][beta] and U1[Delta][gamma] RNAs (data not shown). When oligo(dT)₂₀ was used to direct RNase H cleavage, the U1[Delta][gamma] band was selectively lost (compare lanes 3 and 4). U1[Delta][gamma] was also selectively enriched by oligo(dT) chromatography (data not shown). Since no extended poly(A) tracts flank the U1 gene (23,24) the U1[gamma] oligo(dT)₂₀ sensitivity is most likely due to post-transcriptional modification. RNase H treatment of the U1[Delta]192-507Sm-derived RNA with oligo(dT)₂₀ likely shifts U1[Delta][gamma] to the U1[Delta][beta] position since no novel product was generated by this treatment (Fig. 3B). Taken together, the RNase H digestion results indicate that U1[Delta][beta] and U1[Delta][gamma] are transcribed from the U1[Delta]192-507 allele and that U1[Delta][gamma] is a polyadenylated U1 derivative of U1[Delta][beta].

Figure 3. Oligonucleotide-directed RNase H cleavage of extended U1 snRNAs. (A) A northern blot of U1 snRNA isolated from galactose grown U1[Delta]192-507 transformed yeast. The RNA was hybridized with a non-specific oligonucleotide (381-8, lane 1), a U1-specific oligonucleotide complementary to U1 at nt 170-189 from the 5[prime]-end (U1-10, lane 2) or oligo(dT) (lane 4) and then digested with RNase H. Undigested RNA is shown in lane 3. (B) RNase H analysis of U1 snRNA isolated from cells that express GAL::U1 and U1[Delta]192-507Sm. RNA was hybridized with oligo(dT) (lane 1) or a non-specific oligo (381-8, lane 2) and digested with RNase H.

While not definitive, the lengths of the RNase H products relative to the oligonucleotide binding sites suggested that U1[Delta][alpha], U1[Delta][beta] and U1[Delta][gamma] RNAs shared a common 5[prime]-end. Primer extension analysis confirmed that GAL::U1, U1[Delta]192-507 and U1[Delta]192-507Sm all initiated transcription at the previously reported 5[prime] adenosine (Fig. 4A, lanes 1-4; 23,24). In the case of U1[Delta]192-507Sm, the RNA was assayed both in the presence of the wild-type U1 (lane 3) and after transcriptional repression depleted U1[alpha] to nearly undetectable levels (lane 4). Since the cDNA produced was the same length, U1[Delta][beta] must differ from U1[alpha] due to heterogeneity 3[prime] of the primer binding site.

Figure 4. Mapping the ends of lengthened U1 transcripts. (A) Primer extension analysis of U1 snRNA 5[prime]-ends from yeast that express GAL::U1 (lane 1), U1[Delta]192-507 (lane 2), GAL::U1 and U1[Delta]192-507Sm (lane 3) and GAL::U1, U1[Delta]192-507Sm after GAL::U1 repression. The end-labeled cDNA products were fractionated alongside a U1 gene sequencing reaction done with the same oligonucleotide (lanes 5-8). (B) RNase protection of the U1 snRNA 3[prime]-ends. Uniformly labeled RNA probes complementary from U1 nt 508 to the HindIII site located 190 nt downstream of the natural U1 3[prime]-end were used for hybridization. A U1[Delta]192-507-derived probe was used in reactions shown in lanes 2, 5 and 8. A U1[Delta]192-507Sm-derived probe, which contains an additional 24 nt at its 5[prime]-end, was used in reactions 2, 4, 6, 7 and 9. The RNA sources were GAL::U1 (lane 2), GAL::U1, U1Sm in galactose medium (lane 3) and after 4 h in glucose medium (lane 4), U1[Delta]192-507 (lane 5) and GAL::U1, U1[Delta]192-507Sm in galactose medium (lane 6) and after 4 h in glucose medium (lane 7). Lanes 8 and 9 contain tRNA controls for probe self-annealing. RNA markers are shown in lane 1 with sizes noted on the right. The relevant protected products are indicated by arrows at the left as U1[Delta][alpha], U1[alpha], U1[Delta][beta] and U1[beta], respectively. A 45 nt GAL::U1-derived fragment protected by the Sm probe is noted with an asterisk. RNA fragment lengths were determined by fractionating the protected fragments on high resolution sequencing gels adjacent to an RNA sequencing reaction (data not shown). (C) Comparison of the 3[prime]-end RNase protection of endogenous U1 snRNA (strain TS192, lane 4) and GAL::U1 (lane 2). Lane 3 contains RNA from the shortened functional U1 gene, U1[Delta]192-507. The 61 nt RNA marker band is also shown (lane 1).

An RNase protection assay (36) was used to map the 3[prime]-ends of the U1 derivatives (Fig. 5B). Cellular RNA was hybridized to a uniformly labeled probe that extended from U1 snRNA nt 508 to a position 191 nt downstream of the natural U1 3[prime]-end (23,24). The cellular U1[Delta]192-507 RNA protected probe fragments of between 61 and 69 nt (Fig. 4B, lane 4, and C, lane 3); a 66 nt product corresponds to the published 3[prime]-end of U1[alpha] snRNA (23,24). The high A+U content of the 3[prime]-end likely contributes to the minor heterogeneity of the RNase digestion products. GAL::U1 and wild-type U1 produced equivalent fragments, foreshortened by 6 nt due to a linker inserted in the 3[prime]-end of the U1[Delta]192-507 probe (31; Fig. 4B, lane 2, and C, lanes 2 and 4; Materials and Methods). GAL::U1-containing yeast that simultaneously expressed U1Sm or U1[Delta]192-507Sm also produced the U1[alpha] RNA fragment (45 nt, *) but this band was much reduced after glucose repression of GAL::U1 (compare lane 2 with 3 and 5 with 6). In addition, yeast with Sm mutant genes produced a large amount of RNA that is 64-78 nt longer than the mature U1 snRNA, termed U1[Delta][beta] (lanes 5 and 6) and U1[beta] (lanes 2 and 3). Together, the RNase mapping data show that unlike the vertebrate system (43-45,50) a major step in yeast U1 snRNA 3[prime]-end formation requires the Sm site and is not promoter dependent.

Figure 5. Synthetic pre-U1 snRNA cleavage in cell-free extracts. (A) Synthetic U1 RNAs which extend 190 (U1[Delta]192-507, lanes 2-10) or 188 nt (U1, lane 11) downstream of the natural U1 3[prime]-end were incubated in yeast splicing extract for the indicated times and then resolved on a denaturing 5% polyacrylamide gel. The RNAs were 5[prime]-end-labeled (lanes 2-4), uniformly labeled (lanes 5-7 and 11) or 3[prime]-end-labeled (lanes 8-10) prior to processing. The lengths, in nucleotides, of the molecular size standards (lane 1) are indicated on the left. The processing precursors (A and A[prime]) and products (B-F) are indicated by arrows on the right. (B) Schematic of processing products from in vitro cleavage reactions. The open box denotes the U1 gene body and lines represent the sequence located downstream of the mature U1 3[prime]-end. Products are diagrammed beside each fragment name (bands B-F) along with the precursors (bands A and A[prime]). The length of each RNA is indicated in nucleotides; the size for full-length U1 substrate (A[prime]) is noted in parentheses.

RNase III cleaves U1 snRNA precursors

Synthetic U1 snRNAs were produced to investigate whether extended U1 snRNA could be processed by yeast splicing extract. These uniformly radiolabeled RNAs consisted of the U1[Delta]192-507 or U1 mature RNA sequences followed by 190 and 188 nt of downstream sequence, respectively. Both pre-U1[Delta]192-507 (Fig. 5A, lanes 2-10) and pre-U1 (lane 11) RNAs were efficiently cleaved to produce five additional bands (Fig. 5A, labeled B-F). When the pre-U1[Delta]192-507 RNA was 5[prime]-end-labeled only bands A-C were visible. Band A is the uncut precursor while bands B and C correspond to stable RNA products that end 80 and 115 nt, respectively, downstream of the U1[alpha] 3[prime]-end observed in vivo (Fig. 5B). Band B was present only in the earliest time points suggesting that it is an intermediate in the processing reaction. When the pre-U1[Delta]192-507 snRNA was 3[prime]-end-labeled before processing, bands D and E were present in the final reaction products. The 3[prime]-end-labeled D and E RNAs were cleaved at positions 75 and 110 upstream of the 3[prime]-end of the synthetic precursor (Fig. 5B). Of the six RNA forms observed, the 35 nt F band was the only internally derived fragment (i.e. was not found as a 5[prime]- or 3[prime]-end-labeled species) and was derived from a double cleavage at positions 75 and 110 upstream of the 3[prime]-end. Pre-U1 snRNA was processed at precisely the same nucleotides as the pre-U1[Delta]192-507 derivative, showing that the processing reaction was not significantly influenced by the large internal deletion (compare lanes 5 and 11).

The electrophoretic pattern of 5[prime]- and 3[prime]-labeled products was consistent with endonucleolytic cleavages of the synthetic U1 pre-snRNA. Recently, the yeast RNT1 gene product, Rnt1p (RNase III), was shown to cleave the 3[prime]-ends of synthetic U2 and U5 snRNA precursors (25,26). Two experiments were performed to determine whether RNase III was responsible for the U1 snRNA cleavages. First, processing in the wild-type extract was compared with that observed in an extract prepared from an rnt1::HIS mutant strain (Fig. 6, RNT1 and rnt1::HIS3, respectively). The RNT1 extract efficiently cleaved a 228 nt 25S rRNA control substrate at the expected positions, 15 and 50 nt from the 3[prime]-end (lanes 2, 4 and 6-14; S.Abou Elela and M.Ares, personal communication; 28). The synthetic U1[Delta]192-507 precursor was cleaved by the RNT1 extract in the presence or absence of the control substrate (Fig. 6, lanes 3 and 5) (6-8). In contrast, neither RNA was processed by the rnt1::HIS3 extract (lanes 9-11). Lack of cleavage was not due to a general poor quality of the extract since the RNT1 and rnt1::HIS3 extracts had equivalent RNA and protein contents and both efficiently spliced pre-mRNA (data not shown).

Figure 6. In vitro Rnt1p cleavage of the synthetic pre-U1. Uniformly labeled pre-U1[Delta]192-507 snRNA, U1[Delta]192-507Sm snRNA and a 25S rRNA control substrate were incubated for the indicated times with a wild-type yeast extract (RNT1, lanes 4-8; 15), the rnt1::HIS3 mutant extract (lanes 9-11) or with purified GST-Rnt1p (lanes 12-14). Lane 1 contains the molecular weight markers and lanes 2 and 3 the unreacted rRNA control and U1[Delta]192-507 substrates, respectively. An asterisk in lane 4 indicates the major resolvable rRNA cleavage product; the U1[Delta]192-507-derived substrates and products are indicated by arrows.

As a second test of Rnt1p activity in U1 processing, recombinant Rnt1p, GST-Rnt1p, was incubated directly with U1 snRNA (lanes 12-14). The pattern of cleavage was identical to that observed with the yeast extract except that band D, a possible intermediate in processing, was not found. The concordance of the RNA cleavage patterns adds support for endogenous Rnt1p being the U1 cleavage activity in the yeast extracts.

The Rnt1p cleavages are shown in Figure 7 in the context of a putative secondary structure downstream of the U1 mature 3[prime]-end. The structure was predicted by RNAdraw with a [Delta]G of -28.01 kcal (51). Similar structures were also generated by Mfold (52). The cleavages are staggered by 2 nt on either side of a stem in a stem-loop structure. While we have no direct evidence that this structure forms in vivo or in vitro, similar structures and RNase III cleavage patterns have been presented for yeast U2 snRNA (25), yeast U5 snRNA (26), yeast snr190-U14 RNA (53), yeast 25s rRNA (28), E.coli 16S rRNA, E.coli 23 rRNA, E.coli rnc RNA, E.coli rpsO-pnp RNA, E.coli met-orf15A-nusA RNA, E.coli sib3 RNA, the bacteriophage [lambda] N-leader RNA and T7 bacteriophage RNA (reviewed in 54).

While GST-Rnt1p is able to cleave synthetic U1 snRNA precursors, mutation of this gene does not appreciably decrease the intracellular abundance of fully mature U1[alpha] snRNA (Fig. 8; 26). However, another RNA species, U1[epsis], was much increased in the rnt1::HIS3 mutant yeast. U1[epsis] migrates above U1[beta] as a closely spaced doublet ~750 nt in length. U1[epsis] is a 3[prime]-extended form as this RNA hybridized both with a probe restricted to the U1[alpha] sequence as well as with a probe that extended from the U1[alpha] 3[prime]-end to the HindIII site located 192 nt downstream (Fig. 8). U1[epsis] may not be polyadenylated since its electrophoretic mobility is insensitive to RNase H digestion with oligo(dT)₂₀ (data not shown). However, given the large size of U1[epsis], a short poly(A) tail might not be detectable by this method. Since the levels of U1[epsis] were reduced when the rnt1::HIS3 mutant was complemented by a plasmid-borne copy of RNT1 (Fig. 8, compare lanes 3 and 4), U1[epsis] abundance directly relates to the endogenous levels of RNase III. Based on this, we conclude that Rnt1p, while not required for U1 snRNA maturation, does influence steady-state levels of 3[prime]-extended U1 snRNAs.

Figure 7. U1 snRNA extended 3[prime]-ends and Rnt1p cleavage sites presented in the context of a putative RNA secondary structure. The sequence for yeast U1 snRNA extending from the 3[prime]-terminal Sm site (boxed) to downstream of the natural 3[prime]-end (U1[alpha]). The 3[prime]-ends of RNAs which accumulate in vivo with the Sm mutation are noted by underlining. Large arrows show the location of the major Rnt1p cleavage sites observed in vitro; small arrows show the position of weaker cleavage sites. The secondary structure prediction was done with RNADraw (51). ([Delta]G = -28.01 kcal).

DISCUSSION

Although three sites corresponding to the Sm consensus are present in yeast U1 snRNA, only the 3[prime]-terminal Sm site is required for splicing. This result is consistent with a proposed secondary structure of yeast U1 snRNA (55) in which only the 3[prime]-terminal Sm element is present as a characteristic single-stranded element (3,4). Both upstream Sm-like sites are predicted to be partially or fully base paired with other regions of the yeast-specific core (55). The location of the final site is directly downstream of a base paired segment joining the two ends of the snRNA (the closure sequence; 23,56), is within 8 nt of the 3[prime]-end of the mature transcript and is well placed for an element that participates in 3[prime]-end maturation. Here we demonstrate a pivotal role for the Sm site and its associated proteins in the biosynthesis of yeast U1 snRNA.

Two prominent forms of 3[prime]-extended U1 snRNA are found in yeast, a non-polyadenylated snRNA extended 64-78 nt (U1[beta]) and a related polyadenylated form (U1[gamma]). Several lines of evidence suggest that one or both of these snRNAs may be precursors to U1 snRNA. First, 3[prime]-extended biosynthetic precursors are the rule rather than the exception for pol II transcripts (57) and recent studies have suggested that 3[prime]-extended precursor forms of U2 and U5 snRNAs exist in yeast (6,25,26,58). Second, the 3[prime]-extended U1 snRNA species are observed in low abundance in wild-type yeast but increase in abundance with experimental manipulation of the Sm site or the Smd1p core protein (Fig. 2 and data not shown). This impact of Smd1HAp depletion is consistent with a build-up of precursors (U1[Delta][beta] and U1[Delta][gamma]) resulting from a kinetic barrier to product formation (U1[Delta][alpha]). Inactivation of the common snRNP protein Brr1p slows conversion of the putative U2 precursor to its mature form (6). Although Brr1p has not been shown to be part of the Sm core particle, this protein interacts genetically with Smd1p and the removal of either activity produces similar defects in snRNA stability and cap modification (5,6,8). Third, like the U2 and U5 snRNAs (25,26), U1 has a pair of Rnt1p cleavage sites located downstream of the mature snRNA (Figs 5 and 6). In each case the rnt1::HIS3 mutation changes the wild-type level of the snRNA. The steady-state levels of mature U2 and U5L snRNA, but not U1 snRNA, decrease in the rnt1::HIS3 background. For U2, but not U1, the rnt1::HIS3 mutation also results in a large increase in polyadenylated snRNA. Taken together, the available data support the view that the pol II spliceosomal snRNAs are matured at their 3[prime]-ends through similar though not necessarily identical means.

Figure 8. U1 snRNA in yeast mutant for RNT1. Endogenous U1 snRNA from an RNase III mutant (lanes 2 and 4) and the same strain carrying a plasmid-borne RNT1 gene (lanes 1 and 3) was analyzed by northern blot. The membrane was probed with the 568 nt U1 gene body (probe A) and with a PCR probe prepared from the sequences beginning at the natural U1 3[prime]-end and extending downstream 180 nt (probe B). U1[alpha] represents mature U1; U1[epsis] represents a U1-derived RNA ~750 nt long. On longer exposure, the low abundance U1[epsis] band can also be observed with probe A (data not shown). The low level of U1[alpha] observed with probe B results from a trace amount of PCR template present in this preparation. U1[alpha] is much more abundant than U1[epsis]; the U1[epsis]:U1[alpha] ratio with probe B does not reflect the relative intracellular abundance of these RNAs.

Xenopus laevis, human and mouse U1 molecules are synthesized as nuclear precursors with 10-12 nt extensions (59-62) that are trimmed to having one or two extra nucleotides in the cytoplasm (59,60). A nuclear activity has been identified that specifically removes the remaining nucleotide of the U1 precursor (50), consistent with the observation that mature U1 snRNAs are exclusively nuclear (50,60). Since in vertebrates the Sm site is not required for nuclear export (63), the initial step of snRNA processing is Sm site-independent. In contrast, the general inhibition of nucleocytoplasmic exchange by vesicular stomatitis viral infection (64,65) or viral M protein expression (66) inhibits the processing reaction and results in the accumulation of nuclear snRNA precursors. If yeast behaves similarly, the Sm site mutation would trap U1 precursors in the cytoplasm. In this case, the 3[prime]-extended U1[beta] RNA might be viewed as a much longer version of the vertebrate +1 cytoplasmic species. It is not clear why the 3[prime]-extensions of the yeast Sm mutant are not processed more closely to the natural 3[prime]-end, as in vertebrates. It is possible, however, that in yeast the Sm site mutation inhibits nuclear export and the extended RNAs are completely nuclear and thus unavailable to a cytoplasmic processing activity. Enhanced nuclear retention might also lead to promiscuous polyadenylation and, hence, the observed increased levels of U1[Delta][gamma] observed with the Sm site change. However, since depletion of Smd1p also results in increased U1[Delta][gamma] levels (Fig. 2C), one would need to argue that: (i) Smd1p binds prior to nuclear export (in contrast to vertebrates; 67,68); (ii) Smd1p assists in the removal of polyadenylated tails; or (iii) that U1[Delta][gamma] levels may increase due to an indirect consequence of splicing inhibition that occurs with Smd1p depletion.

U1 snRNA is an efficient substrate for Rnt1p and is processed almost identically in yeast extract and with the purified enzyme. Thus, it seems almost certain that Rnt1p, not an Rnt1p-cleaved catalytic RNA, contains the observed U1 processing activity in yeast. A minor difference noted between the extract and Rnt1p processing results was that band D, generated in the yeast extract, was not observed with purified Rnt1p. However, band D is a singly cleaved precursor RNA form (Fig. 6) and this difference may simply reflect a more efficient processing by the purified enzyme into the doubly cleaved E and F RNAs. U5 snRNA is also readily cleaved by Rnt1p at sites located 26-27 (pre-U5L) and 90-91(pre-U5S) nt downstream of the mature ends (26). In this case, however, the cleaved products are then processed further by an uncharacterized nuclease to 3-4 nt downstream of the natural U5 3[prime]-ends (26). In vitro, U1 snRNA is a much less favored substrate for the second nuclease activity since little or no U1 is processed beyond the Rnt1p cleavage sites (Figs 5 and 6 and data not shown). It remains to be determined whether cis-inhibitory elements or missing trans-acting factors account for this difference.

Model of U1 biosynthesis

The following model accounts for our experimental observations and offers a framework for future experimentation (Fig. 9). In the rnt1::HIS3 mutant we observe a U1 snRNA form extended by at least 180 nt, U1[epsis]. We propose that U1[epsis] is a U1 snRNA precursor, perhaps formed by transcriptional termination, and that Rnt1p cleaves U1[epsis] at positions 80 and 115 from the mature 3[prime]-end to form U1[beta]. U1[beta] may then be processed through an Sm-dependent pathway to mature U1[alpha] RNA. Alternatively, U1[beta] may be polyadenylated to form U1[gamma]. Since polyadenylated spliceosomal snRNAs have not been observed (23,24,69-71), U1[gamma] may reflect promiscuous polyadenylation enhanced by kinetic impairment of the normal processing pathway. Alternatively, as speculated for an unexpected form of polyadenylated telomerase RNA (72), snRNAs may normally cycle between polyadenylated and deadenylated species.

Figure 9. Model for U1 snRNA 3[prime]-end formation. U1 snRNA appears to be processed along two alternative pathways. One pathway uses Rnt1p (yeast RNase III) to process the RNA to an intermediate state, U1[beta], which may become polyadenylated. An unknown activity, likely an exonuclease, is required to process U1[beta] to the mature U1[alpha] form. This activity may also be able to process the putative U1 precursor, U1[epsis], directly to U1[alpha]. However, in both cases, U1[alpha] formation in vivo passes through an Sm-dependent step. The closed circle represents the U1 gene body, while the sequence/structure represents the RNA including and downstream of the mature U1 3[prime]-end.

In the absence of Rnt1p, mature U1[alpha] still forms as long as the U1 Sm site is intact. Consequently, U1[epsis] or a related molecule must be processed directly to the Sm-dependent (U1[beta]) form. While yeast U2 snRNA maturation is fully Rnt1p-dependent (25), an Rnt1p bypass pathway has been suggested to account for U5S formation (26). The cellular benefit of dual pathways is unknown, but not unreasonable to contemplate. Perhaps the Rnt1p-dependent (or -independent) pathway contributes to the efficiency of U1 production at times of environmental stress or at certain times of the cell cycle. Alternatively, as shown for the dicistronic snoRNAs U14 and snr190, Rnt1p cleavage of U1 precursors may contribute to the formation of an uncharacterized co-transcribed downstream RNA (53).

Clearly, the pathway for yeast U1 snRNA biosynthesis is complex and has features that distinguish it from what has been observed for vertebrate U1 and for yeast U2 and U5 snRNAs. Future experiments will be required to test the validity of our dual pathway model and to identify the Sm-dependent and -independent enzymatic activities required for U1 3[prime]-end formation.

ACKNOWLEDGEMENTS

We thank Manny Ares Jr, Guillaume Chanfreau, Tom Cech and Thoru Pederson for their helpful comments and discussions. We are indebted to Manny Ares Jr and Sherif Abou Elela for the MA52 and MA6 yeast, purified GST-Rnt1p and the RNase III control substrate plasmid. This work was supported by National Institutes of Health grant GM42476 to B.C.R. R.L.S. is a 1998-1999 American Association of University Women Postdoctoral Fellow.

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*To whom correspondence should be addressed. Tel: +1 606 257 5530; Fax: +1 606 257 1717; Email: rymond@pop.uky.edu
⁺Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA

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