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Molecular and Cellular Biology, June 2002, p. 4101-4112, Vol. 22, No. 12
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.12.4101-4112.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Xenopus LSm Proteins Bind U8 snoRNA via an Internal Evolutionarily Conserved Octamer Sequence

Nenad Tomasevic and Brenda A. Peculis*

Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1766

Received 18 December 2001/ Returned for modification 5 February 2002/ Accepted 21 February 2002


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ABSTRACT
 
U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.


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INTRODUCTION
 
More than 100 snoRNAs have been shown to play various roles in ribosome biogenesis (26, 54). The majority of the snoRNAs are responsible for directing site-specific modification of the rRNA: the C/D box snoRNAs direct site-specific methylation of ribose, while the H/ACA snoRNAs identify uridines that will be converted to pseudouridine (reviewed in references 3, 34, 39, 54, and 59). In base pairing directly with the rRNA, most snoRNAs probably also play direct or indirect roles as RNA chaperones, facilitating and modulating the correct folding of the pre-rRNA (4, 47). Only seven snoRNAs have been shown to be involved in pre-rRNA cleavage events that produce mature rRNAs. U3, U14, U22, snR10, and snR30 snoRNAs are needed for maturation of 18S rRNA, the small ribosomal subunit RNA. Yeast RNase MRP affects the formation of the mature 5' terminus of 5.8S rRNA (26, 54).

U8 snoRNA is functionally unique; it is the only snoRNA demonstrated to be essential for processing of both 5.8S and 28S rRNAs (40). While the 5' end of U8 is required for an snoRNA-rRNA base-pairing interaction, this region of U8 RNA is not sufficient for in vivo function (35, 41). There are three well-conserved sequences in U8 RNA. Boxes C and D are present in all snoRNAs in the C/D class and are required for stability and core protein binding (41, 53). The loop sequence atop the third stem of U8 is conserved among vertebrate U8 RNAs (36). Experiments suggest that none of these sequences are sufficient for in vivo function of U8 ribonucleoprotein (RNP) (35, 41). Presumably, the unique role of U8 in ribosome biogenesis lies, in part, with the proteins constituting or recruited by U8 RNP to the processing complex to provide additional functional activities. Learning more about the U8-specific proteins could provide insight into additional roles for U8 snoRNP in ribosome biogenesis.

The proteins comprising RNP particles have been demonstrated to play a wide variety of functions. They may (i) provide enzymatic activity, (ii) have structural roles, (iii) be involved in subcellular localization of the RNP, and/or (iv) be responsible for posttranscriptional modification of the RNA upon which they assemble. Examples of proteins in RNPs playing enzymatic roles include Cbf5p, a shared core-binding protein in the H/ACA snoRNPs. Cbf5p is homologous to a family of pseudouridylases in Escherichia coli and may provide the pseudouridylase activity in the H/ACA snoRNPs (8, 20). Similarly, Nop1p/fibrillarin is an essential core-binding protein present in all C/D box snoRNPs (48, 54); it is believed to be the methyl transferase (32, 56).

Some proteins in RNPs provide structural roles and stabilize the RNAs to which they bind. In yeast, four core proteins are present in all H/ACA snoRNAs: Gar1p, Cbf5p, Nhp2p, and Nop10p (6, 15, 20, 31, 57). Depletion of any of these proteins results in destabilization of the H/ACA snoRNAs. Similarly, genetic depletion of the core proteins of the C/D box snoRNPs, Nop1/fibrillarin, Nop56p, Nop58p/Nop5p, and Snu13p/15.5 kDa, results in destabilization and loss of function of the C/D snoRNAs (18, 19, 50, 58, 60).

The Sm proteins are an example of proteins responsible for correct subcellular localization of certain snRNPs, as well as for posttranscriptional modification of the snRNAs and stability of the RNPs in vivo (22, 23, 25). After transcription in the nucleus, the snRNAs are transported to the cytoplasm, where the Sm proteins assemble in a stepwise process (44). Binding of Sm proteins triggers hypermethylation of the 5' cap; both Sm proteins and cap modification are required for subsequent import into the nucleus (13, 24, 43).

To gain a better understanding of the molecular mechanisms by which the U8 snoRNP facilitates pre-rRNA processing, we have focused on proteins unique to the U8 RNP as a strategy for identifying proteins conferring enzymatic or localization-specific activity. Here we report a 75-kDa protein complex present in Xenopus ovary extracts that bound with high specificity to U8 RNA. Analysis of this purified complex has identified six of these proteins as Xenopus homologues of the previously characterized LSm (like Sm) proteins (14, 33). We demonstrated that U8 RNA could be cross-linked in vitro to at least two of the LSm proteins in this purified complex. This purified complex did not bind the other C/D box snoRNAs tested here but did bind U6. Truncated U8 substrates and mutagenesis studies demonstrated that the sequence in the loop of the third stem of U8 RNA was required for binding of the Xenopus LSm complex. The binding site in Xenopus U8 snoRNA is different from the previously identified LSm-binding site on human U6 snRNA, implying the possible presence of a novel, nucleolus-specific protein present in this purified Xenopus LSm complex.


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MATERIALS AND METHODS
 
Protein purification. (i) Ammonium sulfate. Whole ovaries were surgically removed from mature female frogs, and crude extract was prepared as previously described (51). An aliquot of the cleared ovary extract (equivalent to approximately 10 g of ovary tissue) was brought to 40% saturation with saturated ammonium sulfate solution and mixed gently at 4°C for 3 h. The resulting slurry was centrifuged for 20 min at 12,000 x g. The 40% pellet was saved, and the resulting supernatant was brought to 60% saturation with ammonium sulfate. The ammonium sulfate pellet was resuspended in buffer I (20 mM Tris-HCl [pH 7.6], 1 mM EDTA, 1 mM dithiothreitol, 2% [wt/vol] glycerol). The conductivity in the sample was decreased by dilution to approximately 5 mS (approximately 15-fold dilution)

(ii) DEAE-Sepharose. The 40 to 60% ammonium sulfate pellet fraction was loaded onto a 50-ml DEAE-Sepharose CL-6B column (fast protein liquid chromatography system; Amersham Pharmacia Biotech) previously equilibrated with buffer I plus 60 mM NaCl. The column was washed with the same buffer until the A280 reached the baseline. Bound proteins were eluted with 150 mM NaCl in buffer I, followed by a 150-ml linear gradient (150 to 350 mM NaCl) in buffer I. Fractions (4 ml) were collected. Fractions that contained U8-binding activity were identified by gel shift analysis. Peak U8-binding activity eluted in fractions corresponding to an NaCl concentration of approximately 260 mM.

(iii) MonoQ HR. Fractions containing peak U8-binding activity from the DEAE-Sepharose column were pooled, diluted with 1 volume of buffer I, and then loaded onto a 1-ml MonoQ HR column (Bio-CAD system; PerSeptive Biosystems) pre-equilibrated with buffer I plus 60 mM NaCl. The column was washed with the same buffer and with 250 mM NaCl in buffer I. Bound proteins were eluted with a 15-ml linear gradient (250 to 380 mM NaCl) in buffer I. Fractions (0.5 ml) were collected and assayed for U8-binding activity by gel shift assay. Fractions typically containing maximal U8-binding activity eluted at an NaCl concentration of approximately 280 mM.

(iv) Heparin-Sepharose. Active fractions eluted from the MonoQ column were pooled (2.5-ml total volume), diluted with 2 volumes of buffer I, and loaded onto a 0.3-ml heparin-Sepharose CL-6B column equilibrated with buffer I plus 60 mM NaCl and assembled on an Amersham Pharmacia Biotech SMART system. Bound proteins were eluted with 150 mM NaCl in buffer I, followed by a 1.5-ml linear gradient (150 to 300 mM NaCl) in buffer I. Fractions (150 µl) were collected and assayed for U8-binding activity by gel shift assay. Fractions containing U8-binding activity typically eluted in fractions corresponding to an NaCl concentration of approximately 200 mM.

(v) MiniQ column. Fractions containing peak U8-binding activity from the heparin-Sepharose column were pooled (total volume of 1 ml), diluted with 1 volume of buffer I, and then loaded onto a 0.24-ml MiniQ HR column (SMART system) pre-equilibrated with buffer I plus 60 mM NaCl. The column was washed with 1.5 ml of the same buffer. Bound proteins were eluted with 150 mM NaCl in buffer I, followed by a 3.3-ml linear salt gradient (150 to 390 mM NaCl) in buffer I. Fractions (50 µl) were collected and assayed by gel shift assay. U8-binding activity typically eluted in fractions corresponding to an NaCl concentration of 290 mM.

(vi) Superdex S-200. The most active fractions eluted from the MiniQ column were pooled, concentrated to 20 µl with Microcon YM-10 concentrators (Amicon), and then applied to a Superdex S-200 column connected to a SMART chromatography system (Amersham Pharmacia Biotech). The column was pre-equilibrated in buffer I plus 250 mM NaCl. Fractions (50 µl) were collected and analyzed for both U8-binding activity by gel shift analysis and protein complexity by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).

Myc-tagged LSm constructs, coimmunoprecipitations, and lampbrush nuclear preparations. Two deoxyoligonucleotides complementary to the human LSm3 sequence (Table 1, primers 10 and 11) were generated; they were designed to have compatible cloning sites to maintain the reading frame with respect to the myc-tagged plasmid previously described (37). The 3' oligonucleotide (3'HLSm3) was used with total Xenopus RNA for reverse transcription, followed by PCR with both oligonucleotides (3'HLSm3 and 5'HLSm3). The resulting PCR product was cloned into the myc epitope-encoding vector and sequenced to confirm the accuracy of the LSm protein reading frame with respect to the tag. The plasmid was linearized and used for RNA transcription in vitro in the presence of a GpppG cap (Promega) analogue. Purified RNA (13 nl) was microinjected into the cytoplasm of hand-dissected Xenopus oocytes at a concentration of 40 ng/µl. After overnight incubation, the nuclei were hand dissected in 5:1 PO4 buffer supplemented with 2 mM MgSO4 (37). Nuclei from a single treatment (uninjected or injected with myc-XLSm3 or myc-NO38) were pooled in a microcentrifuge tube on ice containing 200 µl of NET2 (41). When all of the nuclei were collected, they were manually disrupted with a pipette tip and added to tubes containing aliquots of protein A-Sepharose resin (Pharmacia) that had previously been treated with the antibody used for immunoprecipitation: anti-myc antibody 9E10 (Chemicon) and anti LSm4 antibody (a generous gift from Reinhardt Lührmann). After 1 h at 4°C, the supernatant was removed and the resin was washed four times with NET2. Both the supernatant and the resin (pellet) were extracted with phenol, and the RNA was collected by precipitation with ethanol. RNAs were resolved on an 8% urea-acrylamide gel, transferred to nylon membrane (ZetaProbeGT; Bio-Rad), and hybridized with a full-length antisense U8, U1, or U6 riboprobe. Lampbrush chromosome preparations were prepared essentially as previously described (37). Antibodies used for immunofluorescence assay were against Lsm4, myc (9E10), or coilin (C236; a gift from Joe Gall).


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  		TABLE 1. Sequences of deoxyoligonucleotides used for PCR

Protein identification. A peptide sequence was obtained from samples from the pooled MiniQ eluate, resolved by 15% Tris-glycine SDS-PAGE, stained with Coomassie brilliant blue, and sent to the Wistar Protein Microchemistry/MassSpec Facility, Philadelphia, Pa., where both Edman degradation and mass spectrometry (MS)-MS analysis were done on bands resolved by SDS-PAGE. The protein sample and the gel were handled as recommended by the facility. Alternatively, an MS-MS sequence was obtained by Lewis Pannell (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health) on purified protein digested with trypsin and then subjected to time-of-flight MS (MS/TOF).

In vitro RNA transcription. [32P]UTP-labeled RNAs were transcribed in vitro with T7 RNA polymerase (Promega) and purified as previously described (41). U8 Lp3M RNA was transcribed as previously described (41). Templates for the truncated U8 constructs were generated by PCR with the deoxyoligonucleotides in Table 1 as primers and a wild-type U8 template. The oligonucleotides used for the truncated U8 constructs were as follows: 5'40, primer pairs 1 and 5; 3'100, primer pairs 2 and 9; 5'40CD, primer pairs 1 and 6; CSt3, primer pairs 2 and 7; St34, primer pairs 3 and 8; St4D, primer pairs 4 and 9. PCR was performed as previously described (41). Purified PCR products were used as templates for in vitro transcription (51).

Gel shift analysis. RNA-binding assays were performed (51) with a protein fraction aliquot (1 to 10 µl) and 60 fmol of [32P]UTP-labeled RNA. Supershift analyses were performed by incubation of 60 fmol of 32P-radiolabeled U8 RNA with 2 µl of purified 75-kDa protein eluted from the MiniQ column for 5 min, after which 1 µl of a 1:30 dilution of rabbit antiserum was added and the mixture was incubated for another 10 min. Resulting shifted complexes were resolved on a 4% native polyacrylamide gel. Rabbit antisera against human LSm4 and LSm1 were generously provided by T. Achsel and R. Lührmann. The control antiserum used (anti-X29) was not specific for this complex.

UV cross-linking. Cross-linking reaction mixtures were assembled and incubated as described above for the binding reactions and contained 100 ng of protein (MiniQ fractions) and 9 ng of [32P]UTP-labeled U8 RNA. After the binding incubation, the reaction mixture was exposed to 251-nm UV light (Stratalinker) at a distance of 3 cm for up to 15 min on ice. RNase T1 (1 U/ml; Roche Molecular Biochemicals) was added, and the reaction mixtures were incubated at 37°C for 30 min. The reaction mixtures were loaded onto a 4 to 20% gradient native gel. Gels were exposed to X-ray film at -80°C overnight. Radioactive bands were cut from the native gel, incubated in SDS Laemmli buffer for 10 min, and then heated for 5 min at 90°C before resolution of the proteins by SDS-18% PAGE. Gels were dried and exposed to X-ray film.

Native gel electrophoresis followed by SDS-PAGE. Protein from the MiniQ column was incubated with or without U8 RNA as described above. Samples were loaded onto a 4 to 20% native polyacrylamide gradient gel (Invitrogen). The gel was run for 2 h at 12 mA of constant current with Tris-glycine running buffer. The portion of the gel with labeled U8 RNA was excised, dried, and exposed to X-ray film. The rest of the gel was stained with Microwave Blue (Protiga Inc.) to visualize protein bands. All radioactive bands were cut, incubated in SDS Laemmli buffer for 10 min, and then heated for 5 min to 90°C, and proteins were resolved by SDS-18% PAGE and silver stained. For high-resolution separation of the Xenopus LSm proteins, an aliquot of protein was loaded onto a 12% NuPAGE gel (Invitrogen) run as recommended by the manufacturer.


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RESULTS
 
By using a gel mobility shift assay and competition binding assays, we have developed a means of screening Xenopus ovary extracts for proteins that bind U8 RNA (51). Because we are interested specifically in proteins that confer unique functional activities, we have focused on proteins that do not bind U3 or U14 snoRNA, thus eliminating from our consideration all C/D box core proteins. We report the identification and characterization of a complex of proteins present in a previously unexamined ammonium sulfate cut (see Materials and Methods). This protein complex binds U8 snoRNA in a highly specific manner.

Identification and purification of U complex-forming proteins. The active U8-binding fractions from MiniQ column chromatography, the final step in purification, were identified by gel mobility shift assays. The U8-binding activity and protein complexity of the eluate of the MiniQ column (fifth purification step) are shown in Fig. 1A and B, respectively. The binding activity profile correlates best with the elution profile of four major bands of approximately 18, 14, 13, and 12 kDa, labeled a, b, c, and d, respectively. In the most active U8-binding fractions, only these four bands could be detected by silver staining (lanes 4 through 6).



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  		FIG. 1. Examination of the protein composition of fractions containing peak U8-binding activity. (A) A gel shift analysis was performed with proteins from successive fractions eluted from a MiniQ column. Lane 1 contains U8 RNA alone. Lanes 2 to 11 contain equal volumes of consecutive fractions eluted from the MiniQ column. Free U8 RNA and the U complex are indicated. (B) Proteins from fractions identical to those analyzed in panel A were resolved by SDS-10 to 20% PAGE and visualized by silver staining. The proteins that corresponded best to U complex formation are indicated by the bracket and labeled a, b, c, and d. Positions of marker proteins and their molecular masses are indicated at the right. (C) An aliquot of protein from each step in the enrichment for U complex proteins was resolved by SDS-4 to 20% PAGE and visualized by Microwave Blue staining. Lanes: 1, total proteins from crude Xenopus ovary extract (7 µg); 2, 40 to 60% ammonium sulfate (AS) fraction (6 µg of protein); 3, DEAE-Sepharose fractions (DEAE Sep.) (2 µg of protein); 4, MonoQ fractions (MQ HR) (2 µg of protein); 5, heparin-Sepharose fractions (Heparin Sep.) (1 µg of protein); 6, MiniQ column fractions (0.2 µg of protein); 7, markers with molecular masses indicated at the right; 8 and 9, morpholineethanesulfonic acid-NuPAGE gel stained with Microwave Blue. The four proteins were resolved into at least six bands, and migration of markers in this gel system was as indicated to the left.

Figure 1C shows an SDS-PAGE analysis of protein complexity at each of the five steps of the purification protocol. This analysis indicated that four major bands and one or more minor bands (seen on various gels under different conditions) detected in the 12- to 18-kDa range form a complex and are purified by this procedure. Because Tris-glycine SDS-PAGE gave only limited resolution of proteins in this size range and we occasionally detected doublets for one or more of the bands, we used a morpholineethanesulfonic acid-NuPage gel (Invitrogen) to better resolve these proteins. This alternative gel system was able to cleanly resolve at least six bands, one of which was possibly a doublet (lane 9). Due to the differences in the buffering systems of the gels, it is not clear which combination of these six bands corresponds to bands a, b, c, and d identified by Tris-glycine SDS-PAGE. The role of these proteins in U8 binding was examined as described below.

Characterization of purified proteins. To estimate the mass of the U complex and determine which of the proteins detected in the MiniQ fractions were responsible for U8 binding, protein eluted from the MiniQ column was loaded onto a Superdex 200 column. Fractions were assayed for U8-binding activity. Only one sharp peak of absorption at 280 nm was observed on the chromatogram (Fig. 2A). This peak correlated with the elution profile of the U8-binding activity (Fig. 2B, lane 4). SDS-PAGE analysis of these fractions indicated that all four major bands (a, b, c, and d) were present in these fractions (data not shown). The elution profile of size standards indicated that the active binding fraction was approximately 75 kDa. This result suggested that the purified proteins may be incorporated into a 75-kDa complex in the absence of added RNA.



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  		FIG. 2. Determination of the mass of the U complex by gel filtration. (A) Elution of proteins from a Superdex 200 analytical column, as determined by A280 measurement, is indicated by the solid line. The sizes and elution of molecular mass standards are indicated and graphed. The binding activity resulting in formation of the U complex was assayed by gel shift analysis and determined by quantitation with a Fuji phosphorimager; results are shown in arbitrary units (dashed line). (B) The presence of U8 RNA-specific binding proteins was determined by gel shift assay. Aliquots eluted in successive fractions from the gel filtration column were assayed for activity. The positions of free U8 RNA and the U complex are indicated at the right.

To determine whether all of the purified proteins were present in U8 RNA-containing complexes, we directly examined the protein composition of the U complex revealed by native gel analysis. Figure 3 (lanes 1 to 4) shows a native gel analysis of a binding assay done under conditions of limiting added protein. The autoradiogram shows the mobility of U8 RNA alone (lane 1) or RNA plus limiting protein (lane 2). Microwave Blue staining for proteins in this native gel is shown in lanes 3 and 4. Note that a single band is detected in the absence of U8 RNA, consistent with the single 75-kDa species detected by size exclusion chromatography (Fig. 2A). Addition of U8 RNA shifted the mobility of the stained protein complex to the position of the U complex detected with labeled U8 RNA (Fig. 3, compare lanes 4 and 2).



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  		FIG. 3. Determination of the protein composition of the U complex bound to U8 RNA. Gel shift binding reaction mixtures with labeled U8 RNA (60 fmol) were incubated with (lane 2) or without (lane 1) highly purified 75-kDa protein (0.1 µg) and resolved on a 4 to 20% native polyacrylamide gel. Lanes 1 and 2 were dried and exposed to X-ray film to determine the mobility of the labeled U8 RNA relative to the U complex. To determine the protein composition of this complex, the 75-kDa protein (0.1 µg) was subjected to native PAGE either alone (lane 3) or preincubated with unlabeled U8 RNA (0.5 µg, lane 4). The gel was stained with Microwave Blue to visualize protein. The single bands from lanes 3 and 4 were cut, incubated in SDS Laemmli buffer for 5 min at 90°C, resolved by SDS-18% PAGE, and visualized by silver staining (lanes 5 and 6, respectively). SDS-PAGE analysis of an unstained region of the gel, lane 3 or 4, yielded no protein bands (data not shown). Lane 7 contained 0.1 µg of the purified 75-kDa protein. Lane 8 contained markers, whose molecular masses are indicated on the right.

The polypeptide composition of the protein bands detected on native gels was examined by SDS-PAGE analysis of excised gel bands. The polypeptide composition of the native gel bands detected in the absence of added RNA (lane 5) and that of the shifted complex in the presence of RNA (lane 6) were equivalent to that of the purified MiniQ fraction (lane 7). This result is consistent with the presence of all of the major polypeptides in the purified fraction in a single complex that binds U8 RNA.

UV cross-linking to U8 snoRNA. To examine which of the subunits of the 75-kDa complex directly contacts U8 RNA, UV cross-linking experiments were performed. 32P-labeled U8 RNA was incubated with an aliquot of the most active binding fraction, irradiated with UV light, and then digested with RNase T1. This sample was then run on a native gel to separate free and degraded RNA from complex-associated RNA. The wet native gel was exposed to film to visualize the protein-RNA complex (Fig. 4, lanes 1 and 2). No cross-linking was detected in the absence of proteins (Fig. 4, lane 1). The primary cross-linked complex (lane 2, indicated by the bracket) was excised from the native gel, and proteins in this complex were resolved by Tris-glycine SDS-PAGE. Autoradiography of this gel revealed that two protein bands were labeled by cross-links to U8. The labeled proteins, migrating at 14 and 18 kDa (lane 3), most probably correspond to bands a and b. These cross-linking results indicate that two proteins in this complex may be in direct contact with U8 RNA.



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  		FIG. 4. UV cross-linking of proteins in the 75-kDa protein to U8 RNA. For UV cross-linking experiments, MiniQ fractions with the highest U8 RNA-binding activity were incubated with [32P]UTP-labeled U8 RNA (lane 2); lane 1 contained labeled U8 RNA incubated under the same conditions without protein. Reaction mixtures were irradiated, treated with RNase T1, and resolved on a 4 to 20% native polyacrylamide gel. The wet gel was exposed to X-ray film, and the radioactive band was cut, incubated in SDS Laemmli buffer for 5 min at 90°C, and subjected to SDS-18% PAGE (lane 3) with 14C-labeled molecular mass markers (lane 4). The radiolabeled cross-linked proteins were visualized by autoradiography. Molecular masses of standard markers are indicated on the right.

Identification of the proteins comprising the 75-kDa complex. To identify polypeptides in the 75-kDa complex, the proteins in the four bands (a, b, c, and d) were sequenced by Edman degradation and MS-MS analysis. The sequences of 19 different peptides were obtained (Fig. 5). BLAST analysis of these peptides indicated that the proteins in bands a, b, c, and d are the Xenopus homologues of human proteins LSm4, LSm8, LSm2, and LSm6, respectively (1). MS/TOF analysis of the 75-kDa complex identified peptides corresponding to LSm3 and -7 (Fig. 5). The sequences of the Xenopus peptides, along with those of the human homologues of these peptides, are shown in Fig. 5; the corresponding peptide in the human protein is indicated in black. With these peptide sequences, a search of the Xenopus expressed sequence tag database (5) identified a putative Xenopus homologue of the LSm8 protein, which is also shown in Fig. 5. This protein is well conserved; there are only five amino acid differences between the human and Xenopus LSm8 sequences.



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  		FIG. 5. Peptides for Xenopus proteins aligned with human Lsm homologues. Shown are the peptide sequences obtained by Edman sequencing (E) or by MS-MS analysis (MS) of the peptides following trypsin digestion of bands a, b, c, and d, as indicated. Some peptides were identified by both methods (E, MS). The peptides obtained are listed in the order in which they appear in the human homologue shown below; the corresponding sequence in the human protein is black, and the sequence not identified is in grey. LSm3 and -7 were identified only by MS/TOF; no peptides were obtained from these proteins by SDS-PAGE. Xenopus LSm8 (xLSm8) is below the human protein (hLSm8). The five positions that vary between the human and Xenopus LSm8 proteins are underlined. Xenopus LSm8 (GenBank accession no. AW641977) is equivalent to #m13e10.w1 in the Blackshear/Soares normalized Xenopus egg library (http://dir.niehs.nih.gov/blast/).

To further examine whether the U complex proteins are Xenopus homologues of the previously characterized human LSms, antibodies against human LSm1 and LSm4 were used in gel shift assays to try to supershift the U complex (Fig. 6). Standard U8 RNA-binding reactions were set up, and a specific anti-LSm4 (lane 3) or anti-LSm1 (lane 6) antibody or an antibody directed against an unrelated protein (lane 4) was added. Lane 5 contains an anti-LSm4 antibody in a binding reaction mixture with no Xenopus proteins. The only reaction mixture that demonstrated a supershift of the complex was in lane 3, which contained both purified protein and the anti-LSm4 antibody, as expected for a complex containing Xenopus homologues of the nuclear LSms. The failure of the anti-LSm1 antibody to supershift this complex is consistent with a nuclear LSm complex in which LSm8, and not LSm1, is present (14, 33).



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  		FIG. 6. Identification of LSm4 protein in the U complex via supershift assay. The presence of specific LSm proteins in the U complex was examined with a gel mobility supershift assay. 32P-labeled U8 RNA was incubated without (lane 1) or with (lanes 2 to 6) protein before addition of antibodies against LSm1 (lane 6), LSm4 (lanes 3 and 5), or an unrelated Xenopus protein that is not specific (NS) for this complex (lane 4). After an additional incubation, the reaction mixtures were resolved by native gel electrophoresis. The migration positions of free U8 RNA, the U complex, and the supershifted band are indicated.

U8 interacts with LSm proteins in vivo. To examine whether U8 snoRNA interactions with LSm proteins could be detected in vivo, a Xenopus cDNA encoding LSm3 was generated via reverse transcription-PCR with primers derived from the human LSm3 sequence (1) and cloned into a vector encoding an amino-terminal nine-amino-acid myc tag epitope (37). RNA was transcribed in vitro from the myc-tagged Xenopus LSm3 constructs (myc-XLSm3) and, as a negative control, from myc-tagged NO38 (37), a nonribosomal, nucleolus-specific protein that does not associate with any snoRNA. The myc-XLSm3 or myc-NO38 RNA was injected into Xenopus oocytes, which were then incubated overnight. Hand-isolated nuclei from 80 injected or uninjected oocytes were used in an immunoprecipitation reaction with protein A-Sepharose resin pretreated with an anti-myc antibody (9E10) or an anti-LSm4 antibody. Northern blot analysis (Fig. 7A) indicated that no U8 RNA coprecipitated with the myc-NO38 protein, but U8 RNA did coprecipitate with the anti-myc antibody in myc-XLSm3-injected oocytes (lane 5) and with the anti-LSm4 in uninjected oocytes (lane 9), demonstrating that the LSm interaction with U8 snoRNA can be detected in vivo. Hybridization with probes specific for the U6 and U1 RNAs confirmed the specificity of the coprecipitation (data not shown). The coimmunoprecipitation does not precipitate all of the U8 RNA present; this may be because the LSms interact only transiently with U8 RNA in vivo, because the incorporation of the tagged subunits into particles is not 100%, or because of the affinity of the antibody for the epitope. These experiments cannot differentiate between these scenarios, but the controls demonstrate specificity.




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  		FIG. 7. U8 associates with LSm proteins in Xenopus nucleoli. (A) Oocytes were left untreated or injected with RNA as indicated. After an overnight incubation, nuclei were hand isolated and used for coimmunoprecipitation. Northern blot analysis was performed on RNA isolated from the supernatant (Supe.) and pellet fractions of oocytes injected with myc-NO38 (lanes 1 and 4) or myc-XLSm3 (lanes 2 and 5) with a 32P-labeled antisense U8 riboprobe. Uninjected oocytes were used to examine RNAs coprecipitating with the endogenous LSm4 protein (lanes 7 and 9) and for the myc antibody control (lanes 6 and 8). The naturally occurring variants of U8 snoRNA differing in length (36) are detected on this high-resolution gel. (B) Lampbrush chromosome preparations stained with 4',6'-diamidino-2-phenylindole (DAPI) and anti-LSm4 antibody from uninjected oocytes showing the localization of endogenous LSm4 protein.

Lampbrush chromosome preparations were made from the nuclei of uninjected oocytes, and the subnuclear localization of the endogenous LSm4 was examined by immunofluorescence assay. LSm4 protein was detected in the extrachromosomal nucleoli and strongly in Cajal bodies of uninjected oocytes (Fig. 7B). The strong nucleolar LSm signal is consistent with a role for the nuclear LSms in rRNA processing, as opposed to only a role in spliceosome formation.

Examination of the specificity of LSm proteins for snoRNAs. Competition binding assays examined the specificity of binding of the LSm-containing complex to U8 snoRNA. Protein from the MiniQ column was added to 32P-labeled U8 snoRNA, and unlabeled competitor snoRNA (U3 or U14), 5S rRNA, or U6 snRNA. The data in Fig. 8 show that while a 25-fold molar excess of unlabeled U8 snoRNA completely competed away binding to labeled U8 (Fig. 8, lane 3), addition of a 200-fold molar excess of nonspecific competitor 5S RNA did not affect U complex formation (lane 9). Likewise, a 200-fold molar excess of either U3 or U14 snoRNA had no effect on the binding of labeled U8 RNA to LSms (lanes 7 and 8). These data provided additional evidence that the 75-kDa complex binds uniquely to U8 with high specificity. This complex does not constitute a common C/D box core protein and does not bind any of the other snoRNAs examined here.



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  		FIG. 8. Binding specificity of purified proteins. Specificity of binding of the 75-kDa complex for U8 RNA was determined by gel shift competition assay. Reaction mixtures contained constant amounts of labeled U8 RNA and purified protein. Unlabeled competitor RNAs were added in molar fold excess, as indicated. Lanes: 1, labeled U8 RNA alone; 2, U8 RNA plus protein. Competitors used included U8 snoRNA (lanes 3 and 4), U6 snRNA (lanes 5 and 6), U3 snoRNA (lane 7), U14 snoRNA (lane 8), and 5S rRNA (lane 9). The U complex and free U8 RNA are indicated.

Since LSms are known to bind U6, we used human U6 snRNA in a gel shift assay and found that it could bind the purified Xenopus proteins (data not shown). Gel shift competition assays compared the relative affinities of this complex for U6 snRNA and U8 snoRNA. Figure 8 (lanes 3 and 4 versus lanes 5 and 6) demonstrated that while this purified Xenopus complex did bind U6 RNA, the affinity of binding was slightly greater for U8 snoRNA than for U6 snRNA. This lower affinity for U6 could be due either to the presence of a nucleolus/U8-specific subunit in this complex, making it more specific for U8, or to species-specific differences: the Xenopus proteins may have a lower affinity for human U6 snRNA.

Identification of the binding site in U8 snoRNA for LSms. To localize the binding site of the LSm complex on U8 RNA, gel shift assays were performed with truncated or mutated U8 RNA transcripts. The schematics at the top of the panels in Fig. 9 show proposed secondary structures of the labeled U8 RNAs or fragments used in the binding reactions shown below. Full-length U8 RNA (Fig. 9, lanes 1 and 9) produces the typical shifted U complex (lanes 2 and 10). Transcripts containing the 5'-most 40 nucleotides of U8 RNA (lanes 3 and 4) failed to form any detectable complex with these proteins. Addition of boxes C and D to the 5'-most 40 nucleotides (but omission of stems 3 and 4) failed to produce the shifted complex (lanes 5 and 6). However, RNA encoding the 3'-most 100 nucleotides did produce a shifted complex that comigrated with the U complex (lanes 7 and 8). These data indicated that the binding site was localized within stems 3 and/or 4 in U8 RNA.



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  		FIG. 9. Localization of the U75 binding site to loop 3 of U8 RNA. 32P-labeled U8 RNA (U8) and truncated U8 RNAs (described in the text and shown schematically above each pair of lanes) were incubated with (+) or without (-) purified MiniQ eluate proteins. Complex formation was analyzed by gel shift assay. Truncated RNAs include U8 5'40 (lanes 3 and 4), 5'40CD (lanes 5 and 6), 3'100 (lanes 7 and 8), C-St3 (lanes 11 and 12), St3-4 (lanes 13 and 14), St4-D (lanes 15 and 16), and Lp3M (lanes 17 and 18). The eight altered nucleotides in the third loop of Lp3M are indicated by the box. The migration positions of the U complex and free U8 RNA are indicated. A weaker shifted complex whose identity is unknown was reproducibly seen with some truncated constructs and is indicated by the asterisk.

In order to further delineate the binding site within U8, three additional subfragments of the 3' end of U8 RNA were generated. Since this region of the RNA contained both boxes C and D, constructs lacking one or both of these elements were generated. Construct St4-D (stem 4 plus box D) failed to form a shifted complex comigrating with the U complex (Fig. 9, lane 16). However, both C-St3 (box C plus stem 3) and St3-4 (stem 3 plus stem 4) formed the U complex (lanes 12 and 14), implying that stem 3 is the U8-binding site for the LSm complex. The faster-migrating band labeled with an asterisk is not the U complex; rather, it may represent an incorrectly or incompletely assembled complex possibly assembled on a weaker binding site.

Next, we examined a previously described U8 cDNA encoding a mutation in the eight-nucleotide sequence in the loop of stem 3 within the context of the full-length U8 RNA (41) (Lp3M; Fig. 9, lanes 17 and 18). U8 Lp3M RNA failed to produce a shifted complex that comigrated with the U complex (Fig. 9, lane 18). These results were consistent with the primary binding site for this complex within the third loop of U8 RNA. Mutation of the sequence of this loop results in a U8 that is not stable in vivo and cannot rescue pre-rRNA processing (41). Interestingly, the sequence of this loop is conserved in the Xenopus, mouse, rat, and human U8 RNAs (36). When used in a gel shift mobility assay, human U8 RNA did form a complex that comigrated with the Xenopus U8 RNA U complex (data not shown). Thus, the interaction described here may be conserved in other species.


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DISCUSSION
 
We describe here the identification and characterization of a protein complex purified from Xenopus ovaries that bound U8 snoRNA with high specificity in a sequence-dependent manner; this purified complex failed to bind in vitro to any of the other snoRNAs examined here. These results demonstrate the specificity of LSm binding to U8 snoRNA but does not preclude the possibility that other snoRNAs may bind the LSms transiently or more stably in vivo. Additional analysis revealed that this complex was comprised of several proteins, was stable in the absence of added RNA, and eluted from a sizing column with a mass of 75 kDa. Peptide sequencing and MS-MS analysis identified four of the proteins in this complex as Xenopus homologues of LSm2, LSm4, LSm6, and LSm8 proteins previously described in yeast and humans (1, 11, 12, 27, 45, 46, 55). Analysis of the purified 75-kDa Xenopus complex with an alternative PAGE buffering system clearly resolved six of the presumed seven members of the Xenopus LSm heteroheptameric complex that binds U8 snoRNA.

Proteins comprising the U complex include subunits of a previously identified heteroheptamer. While there are several known multimeric RNA-binding proteins, the LSms are unusual due to the variety of cellular roles in which they participate. A class of Sm-like proteins, the LSms, was identified in yeast by sequence similarity to the Sm proteins (46). The LSms are an evolutionarily well-conserved protein complex. Homologues have been identified in humans, yeast, and Archaea (14, 16, 33). In Archaea, the SmAP protein forms a homoheptameric ring with a cationic pore (9, 30, 45, 52). There are nine known LSm proteins in eucaryotes that assemble to form heteroheptameric complexes. LSm1 through -7 are present in complexes in the cytoplasm, where they are involved in cytoplasmic mRNA degradation (7, 14, 49). These LSms interact with both the mRNA and the RNA degradative machinery to facilitate decapping and destruction of messages. In yeast, genetic depletion of LSm1 results in a decreased rate of mRNA decay but does not affect pre-mRNA splicing (27).

While the cytoplasmic LSms are involved in mRNA degradation, LSm2 through -8 form the nuclear complex that stabilizes U6 snRNA, which is required for splicing events yielding mRNAs. The nuclear LSms bind the 3' U-rich single-stranded region of U6 and facilitate U6 RNP formation and assembly of U6 with U4 into the di-snRNP and probably the tri-snRNP (1, 12, 27, 45, 55). Genetic depletion of yeast LSm8 showed splicing defects (27) but did not affect the rate of mRNA decay (49). LSm10 assembles with five Sm proteins and possibly one additional protein to form a unique Sm complex that binds the noncanonical Sm site in U7 snRNA (42).

Peptide analysis of the 75-kDa proteins identified LSm2, -3, -4, -6, -7, and -8. The presence of LSm8 would be consistent with the nuclear localization of U8. The nucleolar signal of anti-LSm4 antibodies detected by immunofluorescence assay is consistent with the nucleolar localization of U8. Identification of the remaining protein is required to learn if there is a novel LSm protein designating this as a nucleolus-specific LSm complex. Alternatively, the proteins comprising the U complex may have unique modifications and/or be identical to the U6-binding LSm complex, with the core C/D box proteins directing the nucleolar localization of U8 RNP.

The purified Xenopus LSms bind U8 RNA in a sequence-specific manner. The 3' U-rich single-stranded end of U6 snRNA was previously demonstrated to be essential for binding of the LSm proteins (1, 55); alteration of this U-rich sequence to poly(C) prevented binding in vitro (2). Cross-linking studies indicated additional LSm-binding sites localized to the 3' half of U6 (55). Unlike the binding to U6, binding of the purified 75-kDa complex to U8 RNA did not involve specific recognition of the 3' end. Any truncated form of U8 that contained the third stem-and-loop sequence bound the Xenopus LSms, while full-length U8 RNA with mutations in this sequence failed to bind. U complex binding to U8 requires an octamer (GCUGAUUA) that is well conserved among vertebrate U8 RNAs (36), suggesting that the binding of the LSms to this region of U8 may also be conserved among vertebrates. The same octamer exists at this position in the Xenopus, mouse, and rat sequences; this is a heptamer (the C at position 2 is missing) in the human sequence (36).

As noted above, cross-linking studies have implicated both the 3'-end and internal sequences within the 3' half of U6 in binding of the LSm complex. It is therefore of interest that a sequence similar to the U8 octamer occurs in both U6 snRNA (GaaGAUUA) and U6ATAC (GaagGUUA). This sequence constitutes stem 1 in the U4-U6 interaction, forms helix 1 of the U2-U6 interaction, and is just 3' of the intron base-pairing and reactive sites. It is possible that LSm binding to U8 and U6 involves recognition of conserved internal sequences common to both RNAs.

While the purified Xenopus proteins can bind U6 in vitro, the binding affinity was slightly lower for U6 than for U8. This may be due to (i) the presence of an as yet unidentified U8-specific LSm protein or uniquely modified subunits that provide slightly higher affinity for U8 or (ii) species-specific differences in the proteins that result in lower affinity for the Xenopus proteins binding the human U6 RNA versus the Xenopus U8 RNA. Characterization of the proteins comprising the purified complex and identification of novel posttranscriptional modifications could distinguish between these possibilities.

Functional role for LSms binding U8 RNAs. The mechanistic role of U8 snoRNA binding the LSms is not clear. Mutagenesis of the conserved octamer in the third loop of U8 prevents binding of the LSm complex to the RNA in vitro. In vivo, this mutated RNA is unstable at times longer than 4 h (41), so the functional capacity of this mutant form could not be determined. These results indicate that the conserved octamer is required for LSm binding, which, in turn, stabilizes U8 RNA in vivo and facilitates processing. Another group has examined the functional capacity of two relevant U8 mutations (21). In one construct, {Delta}82-107, the mutant U8 RNA lacks sequences forming the 3' half of stem 3 through the 5' half of stem 4. This deletion variant has the potential to form a modified hairpin stem with the octamer sequence in a terminal loop; it was able to rescue 5.8S/28S processing. In another construct, {Delta}St3,4, both stems 3 and 4 were removed completely. This mutant form shows significantly reduced function relative to that of intact U8 RNA, although a weak rescue phenotype was observed (21). The stability assay performed by Lange et al. was based on a 2-h time point, so RNA stability in vivo due to the potential absence of LSm binding cannot be readily extrapolated from their data. To understand the basis for this apparent weak phenotype, we are examining our truncated constructs, as well as additional mutant forms, to correlate in vitro binding of the LSms with both the in vivo stability and functional capacity of U8 RNA in pre-rRNA processing.

The unique function of LSm particles may be attributable to one specialized subunit (or a few). Across evolution, the Sm and LSm proteins have assembled into complexes with very similar blueprints to generate particles with related but distinct functions. The conserved subunit structure is due to the Sm1 and Sm2 motifs that are present in all Sm and LSm proteins. These two motifs form five antiparallel ß sheets that constitute the subunit interfaces and ultimately determine the dimensions of the particle. That the Sm and LSm proteins form heptamers is a consequence of the shape and composition of the protein subunits. From this point of view, it should be no surprise that some archaeal LSm proteins form heptameric rings while others form hexameric rings (52).

The Sm and LSm particles can be thought of as generic RNA-binding complexes that contain one or more protein subunits providing a highly specialized function. This emphasizes the modular nature of the Sm/LSm proteins: different functional roles are attributable to the presence of specific components. For example, in the nervous system, the SmN protein replaces the B/B' protein in the Sm complex (28) and may facilitate splicing of neuron-specific messages. U7 replaces the D1 and D2 proteins with LSm10 and possibly another, unidentified, protein (42). In the cytoplasm, LSm1 is in the complex that degrades mRNA (49). In the nucleus, the presence of LSm8 in the complex stabilizes U6 (1, 2, 12, 27, 45, 55). Also in the nucleus, pre-RNase P coprecipitates with LSm2 to -7 but does not coprecipitate with an antibody against LSm8, -9, or -1. Eucaryotic mature RNase P does not associate with any of the LSm proteins (45), implying that a novel arrangement of LSms plays a role in a maturation process. In Archaea, however, RNase P does associate with the AP-Sm proteins (AF-Sm1 and AF-Sm2), suggesting that these LSm particles may play a role in tRNA processing (52).

The sequence and structural similarities of the Sm and LSm proteins result in particles with a shared basic function: they bind RNA. Single-stranded RNA binds to one face of a ring-like cationic inner pore and/or threads through this pore (9, 17, 30, 45, 52). LSms bind RNA and modulate RNA-RNA interactions (i.e., between U6 and U4), as well as RNA-protein interactions (i.e., mRNA and the decapping machinery). The role of the LSms binding U8 snoRNA, then, might be expected to be a variation on this same theme. The LSms may be a key factor in the facilitation of various RNA-unwinding events and recruitment of various cleavage factors that would be required for U8 RNA-dependent pre-rRNA processing.

Role for LSm proteins in the U8 snoRNP. One proposed role for the nuclear LSms in binding to U6 snRNA is to facilitate annealing between U6 and U4 snRNAs (1, 27, 55). Thus, binding of the LSms induces modulations in RNA structure to facilitate RNA-RNA interactions. Our identification of an interaction between U8 snoRNA and the LSm proteins provides some intriguing insights into possible additional functions for U8 snoRNP in vivo.

Previously, we presented a model that proposed that U8 is required in vivo to modulate correct folding of pre-rRNA (35). In eucaryotic ribosomes, 5.8S is tethered to 28S via three short base-paired interactions. The longest of these three interactions, the internal transcribed spacer 2 (ITS2) proximal stem, is the first that has the potential to form due to the order of synthesis during transcription by RNA polymerase I (top of Fig. 10). Our model of U8 function proposes that a transient base-paired interaction occurs between the 5' end of U8 snoRNA and the 5' end of 28S rRNA. This interaction facilitates subsequent 28S-5.8S interaction and allows correct formation of the ITS2 proximal stem (Fig. 10), which is required for accumulation of mature 5.8S and 28S rRNAs (10, 38). U8 has base-pairing potential with several additional sites in this region of pre-rRNA (29); mutagenesis studies have demonstrated that not all of these sequences in U8 are required for in vivo function (41). The sequence required for LSm binding in vitro is required for U8 function in vivo (41). We cannot distinguish between the LSms constituting a core component of the U8 RNP or transiently associating with U8 in the nucleolus. Still, the presence of LSm proteins interacting with U8 in the nucleolus would support the proposed role for U8 RNP: the LSms could modulate the structural alterations that would be required not only in U8 snoRNA but also in rRNA to form the base-paired interactions that do exist in the mature ribosome. Finally, identification of these U8-binding proteins and others that may be recruited to the assembling snoRNP-pre-rRNA complex has the potential to reveal much about the molecular mechanisms by which U8 facilitates pre-rRNA processing in vivo.



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  		FIG. 10. Model for the role of U8 and LSms in pre-rRNA processing. The model shows U8 modulating the timing of the interaction between 5.8S and 28S rRNAs and accommodates the basic role of LSms in facilitating RNA-RNA interactions. At the top is a linear schematic of pre-rRNA, transcribed by polymerase I, encoding 18S, ITS1, 5.8S rRNA, ITS2, and 28S rRNA. At the bottom is a schematic of rRNA-rRNA interactions that occur in the mature ribosome, with the precursor sequences looped out. The three intramolecular base-paired interactions (arched arrows at the top and base-paired stems at the bottom) tether 5.8S rRNA to 28S rRNA prior to the cleavages that yield the mature rRNA termini. U8 snoRNA is indicated in its base-paired orientation, with the LSms (a heptameric ring) on the loop of the third stem of U8. Formation of the ITS2 proximal stem is a prerequisite for the later processing steps.


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ACKNOWLEDGMENTS
 
We thank Joan Steitz for the U6 snRNA plasmid and Reinhard Lührmann and Tillman Achsel for helpful discussions, for antibodies against human LSm1 and LSm4, and for communication of results prior to publication. Many thanks go to Joe Gall for the refresher course on lampbrush chromosome preparations, for antibodies, and for loaning necessary equipment and to Lewis Pannell for the MS/TOF analysis. We are grateful to Chris Greer and members of the Peculis lab for encouragement, thought-provoking discussions, and critical reading of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Genetics and Biochemistry Branch, National Institutes of Health, NIDDK, 10 Center Dr., MSC 1766, Bethesda, MD 20892-1766. Phone: (301) 402-8760. Fax: (301) 402-0387. E-mail: bp51h{at}nih.gov. Back


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Molecular and Cellular Biology, June 2002, p. 4101-4112, Vol. 22, No. 12
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.12.4101-4112.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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