Sm core variation in spliceosomal small nuclear ribonucleoproteins from Trypanosoma brucei
2006; Springer Nature; Volume: 25; Issue: 19 Linguagem: Inglês
10.1038/sj.emboj.7601328
ISSN1460-2075
AutoresPingping Wang, Zsófia Pálfi, Christian Preußer, Stephan Lücke, William S. Lane, Christian Kambach, Albrecht Bindereif,
Tópico(s)RNA modifications and cancer
ResumoArticle14 September 2006free access Sm core variation in spliceosomal small nuclear ribonucleoproteins from Trypanosoma brucei Pingping Wang Pingping Wang Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany Search for more papers by this author Zsofia Palfi Zsofia Palfi Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany Search for more papers by this author Christian Preusser Christian Preusser Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany Search for more papers by this author Stephan Lücke Stephan Lücke Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, GermanyPresent address: Howard Hughes Medical Institute, Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA Search for more papers by this author William S Lane William S Lane Harvard Microchemistry and Proteomics Analysis Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Christian Kambach Christian Kambach Structural Biology, Paul Scherrer Institut, Villigen, Switzerland Search for more papers by this author Albrecht Bindereif Corresponding Author Albrecht Bindereif Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany Search for more papers by this author Pingping Wang Pingping Wang Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany Search for more papers by this author Zsofia Palfi Zsofia Palfi Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany Search for more papers by this author Christian Preusser Christian Preusser Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany Search for more papers by this author Stephan Lücke Stephan Lücke Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, GermanyPresent address: Howard Hughes Medical Institute, Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA Search for more papers by this author William S Lane William S Lane Harvard Microchemistry and Proteomics Analysis Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Christian Kambach Christian Kambach Structural Biology, Paul Scherrer Institut, Villigen, Switzerland Search for more papers by this author Albrecht Bindereif Corresponding Author Albrecht Bindereif Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany Search for more papers by this author Author Information Pingping Wang1, Zsofia Palfi1, Christian Preusser1, Stephan Lücke1, William S Lane2, Christian Kambach3 and Albrecht Bindereif 1 1Institut für Biochemie, Justus-Liebig-Universität Giessen, Giessen, Germany 2Harvard Microchemistry and Proteomics Analysis Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA 3Structural Biology, Paul Scherrer Institut, Villigen, Switzerland *Corresponding author. Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, 35392 Giessen, Germany. Tel.: +49 641 99 35 420; Fax: +49 641 99 35 419; E-mail: [email protected] The EMBO Journal (2006)25:4513-4523https://doi.org/10.1038/sj.emboj.7601328 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP-specific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification-tagging and in vitro protein–protein interaction assays. Using in vitro reconstitutions with canonical and U2-specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA-specific Sm sites. Introduction Spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5 are essential factors for splicing of all major, so-called U2-dependent introns. Their functions in splice-site recognition, spliceosome dynamics, and splicing catalysis have been studied in detail, primarily in the mammalian and yeast systems (reviewed by Krämer, 1996; Brow, 2002). Their biochemical composition is characterized very well in the mammalian system (Lührmann et al, 1990; Will and Lührmann, 2001). A highly conserved feature of all known spliceosomal snRNPs is that they share a common core of seven Sm proteins, SmB/B′, D1, D2, D3, E, F, and G, each carrying the characteristic bipartite Sm motif and all seven organized in a ring-like structure (Kambach et al, 1999b; reviewed by Kambach et al, 1999a; Khusial et al, 2005). This heteroheptameric Sm core assembles at the so-called Sm site, which occurs in the U1, U2, U4, and U5 small nuclear RNAs (snRNAs), usually as a single-stranded stretch flanked by stem loops; in mammalian and yeast snRNAs, the Sm site conforms to the consensus sequence 5′-RAU3−6GR-3′ (Branlant et al, 1982; Liautard et al, 1982; Guthrie and Patterson, 1988). The U6 snRNA, in contrast, binds through its 3′ terminal uridine-rich sequence a set of structurally related proteins, the Sm-like (LSm) proteins, LSm2–8 (Séraphin, 1995; Achsel et al, 1999; Mayes et al, 1999; Salgado-Garrido et al, 1999). Interestingly, exchange of a single polypeptide, LSm8 by another LSm protein, LSm1, yields another seven-membered LSm ring, LSm1–7, that is involved in cytoplasmic RNA degradation (reviewed by He and Parker, 2000). Compared with mammals and yeast, only a small subset of protein components of snRNPs have been identified in trypanosomes. Trypanosomes are particularly interesting as they process mRNAs through trans splicing, and in addition—at least in the case of the poly(A) polymerase gene—by cis splicing (reviewed by Liang et al, 2003): The U2, U4/U6, and U5 snRNPs are considered general and essential splicing factors, whereas the spliced leader (SL) RNP and the U1 snRNP represent trans- and cis-splicing-specific components, respectively. An additional point of interest is the fact that trypanosomal snRNAs and their RNPs differ significantly from what we know in other systems, reflecting the large evolutionary distance and trypanosome-specific properties. For example, both the U1 and U5 snRNAs from trypanosomes represent the shortest known orthologs (Dungan et al, 1996; Xu et al, 1997; Schnare and Gray, 1999; Djikeng et al, 2001; Palfi et al, 2002). All five spliceosomal snRNPs have been characterized in trypanosomes to some extent; for example, the Trypanosoma brucei U1 snRNP shows an unusual protein composition (Palfi et al, 2005). From these studies, it is clear that trypanosome snRNPs share a common set of Sm proteins that are organized in the classical heptamer ring (Palfi et al, 2000). However, only five of these polypeptides, SmE/F/G and SmD1/D2, were identified directly by peptide sequences derived from affinity-purified T. brucei U2 snRNP; SmB and D3 came from database searches. Our conclusion that the T. brucei snRNPs share a common Sm core was based mainly on two lines of evidence: first, affinity-purified SL, U2, and U4/U6 snRNPs displayed a common set of at least five polypeptides (Palfi et al, 1991). Second, polyclonal antibodies raised against a mixture of four purified Sm polypeptides (SmE, SmF, SmG, and SmD1; Palfi and Bindereif, 1992) efficiently immunoprecipitated each of the known spliceosomal snRNPs, including the SL RNP (SL, U2, U4/U6: Palfi and Bindereif, 1992; U5: Lücke et al, 1997; U1: Palfi et al, 2002). Here, we report the first evidence for a variation in the Sm core of the spliceosomal snRNPs, specifically in the U2 snRNP of T. brucei. Based on peptide sequences from affinity-purified U2 snRNPs, we have identified two new Sm proteins. Using in vivo tandem affinity purification (TAP) tagging and biochemical protein–protein interaction assays, we have established both of them as U2-specific Sm polypeptides, replacing the standard SmB/D3 dimer in the Sm heptamer ring. In addition, we have set up an in vitro reconstitution system of canonical and U2-specific Sm cores. Based on this, we conclude that variation in the Sm core composition is responsible for the discrimination between snRNA-specific Sm sites. Results T. brucei Sm15K and Sm16.5K, two novel Sm proteins In our initial study (Palfi et al, 2000), we had identified a complete set of seven Sm polypeptides from trypanosomes: five of them, SmE, SmF, SmG, SmD1, and SmD2, were cloned on the basis of peptide sequences derived from affinity-purified T. brucei U2 snRNPs; the two others, SmB and SmD3, had been identified only by database searches. At that time, additional peptide sequences had been obtained from affinity-purified U2 snRNPs: first, from a protein with an apparent molecular mass of 16.5 kDa (16.5K), which appeared to be U2-specific (Palfi et al, 1991); second, from a mixture of proteins in the 15 kDa range, that could not be clearly assigned to any specific snRNP. With the progression of the T. brucei genome project, we were able to assign two of these peptides to the following two respective proteins: Sm16.5K protein (Tb10.70.2250; 14.7 kDa; 131 amino acids) and Sm15K protein (Tb927.6.4340; 12.7 kDa; 117 amino acids; Figure 1). Figure 1.Sequence alignments of two novel Sm proteins from T. brucei: Sm15K and Sm16.5K. (A, B) ClustalW alignment of the Sm15K (A) and Sm16.5K (B) protein sequences from T. brucei (Tb), T. cruzi (Tc), and L. major (Lm). In addition, alignments include sequences of the three trypanosomatid (Tb, Tc, Lm) and the human (Hs) SmB and SmD3 proteins, respectively. For human SmB, the C-terminal extension is not shown; the total numbers of amino acids are given on the right. Below the alignments, a consensus for Sm motifs 1 and 2 is given ($, hydrophobic residue; Séraphin, 1995). GeneDB and NCBI accession numbers: TbSm15K, Tb927.6.4340; TcSm15K, Tc00.1047053506943.114; LmSm15K, LmjF30.3015; TbSmB, Tb927.2.4540; TcSmB, Tc00.1047053507209.10; LmSmB, LmjF27.1970; HsSmB, S10594; TbSm16.5K, Tb10.70.2250; TcSm16.5K, Tc00.1047053506583.10; LmSm16.5K, LmjF36.0535; TbSmD3, Tb927.4.890; TcSmD3, Tc00.1047053508257.150; LmSmD3, LmjF34.3860; HsSmD3, NP_004166. Download figure Download PowerPoint The analysis of their domain structure revealed that both of them carry the bipartite Sm motif. This was surprising, as we had previously reported a complete set of seven canonical Sm proteins in trypanosomes. Therefore, several questions were raised: first, are the two new Sm proteins U2-specific, and, if there is also a heteroheptameric Sm core in the U2 snRNP, which Sm subunits do they replace? Second, what is the snRNA specificity of the SmB and SmD3 proteins identified previously by us through database search (Palfi et al, 2000)? Third, is there at least a common subcore of Sm polypeptides? Initial evidence of the nature of the two new Sm proteins came from Blast searches and sequence alignments. Blast searches yielded SmB and SmD3, which are known to interact with each other (Camasses et al, 1998), as the closest homologs of Sm15K and Sm16.5K, respectively. This suggested that these two new Sm proteins might replace SmB and SmD3 in the U2 Sm core (see Figure 1 for a ClustalW alignment of these two Sm protein sequences from T. brucei, T. cruzi, and Leishmania major, together with SmB and SmD3, respectively, from the three trypanosomatid species and the human system, as well as Supplementary Figure S1 for a phylogenetic tree diagram). T. brucei Sm15K and Sm16.5K are U2-specific Sm proteins To clarify these questions, we used a TAP procedure, followed by the analysis of copurifying snRNAs. Each of the two new Sm proteins, Sm15K and Sm16.5K, as well as all the seven known Sm proteins (SmE, F, G, D1, D2, B, and D3; Palfi et al, 2000) were TAP-tagged at their C-terminus. These constructs were integrated into the T. brucei genome, and cell lines were generated that stably express the tagged Sm proteins (Figure 2A). As shown for Sm16.5K, for which specific antibodies were available (Palfi et al, 1992), the tagged Sm protein and the endogenous protein are incorporated into U2 snRNPs at a ratio of approximately 1:3 (Supplementary Figure S2). Cell lysates were prepared, tagged complexes were purified on IgG-Sepharose, and copurifying snRNAs were detected by Northern blotting (Figure 2B–D). To unequivocally identify each of the known snRNAs, we analyzed on separate Northern blots the U2 snRNA (panel B), U4 and U6 snRNAs (panel C), and SL, U1, and U5 snRNAs (panel D). Figure 2.Both Sm15K and 16.5K proteins are U2-specific. (A) Stable expression of TAP-tagged Sm proteins. Lysates were prepared from the wild-type strain (lane mock) and all T. brucei cell lines stably expressing TAP-tagged Sm proteins (as indicated above the lanes). Protein was analyzed by SDS–polyacrylamide gel electrophoresis and Western blotting with peroxidase anti-peroxidase (PAP) soluble complex (Sigma). The positions of marker proteins are given on the right (sizes in kDa). (B, C, D) Extract was prepared from T. brucei cell lines, which stably express TAP-tagged versions of either of the seven canonical Sm proteins (SmE, SmF, SmG, SmD1, SmD2, SmD3, SmB), or of Sm15K or Sm16.5K proteins (as indicated above the lanes). TAP-tagged complexes were affinity-purified from each cell line, and copurifying RNAs were analyzed by Northern blotting, using a U2-specific probe (B), or mixed probes detecting U4 and U6 snRNAs (C), or SL RNA, U1, and U5 snRNAs (D). The snRNA positions are indicated on the right. The input in panel B represents 5% of the total material, in panels C and D 1% (see lanes I); all of the precipitated material is shown (see lanes P). Note that the probe mixture used for the SmD3, SmB, Sm15K, and Sm16.5K pull-downs contains relatively more SL probe than the probe mixture used for the other Sm polypeptides (e.g., compare input lanes 1 and 11, panel D). M, DIG marker V (Roche). Download figure Download PowerPoint This analysis demonstrated, first, that there is a general subcore of five Sm proteins (SmE/F/G/D1/D2) present in each of the snRNPs U2, U4/U6, U1, and U5, as well as in the SL RNP (Figure 2, panels B–D, lanes 1–10). Comparing the different snRNAs and Sm proteins, the selection efficiencies ranged between 12 and 16% for U2 snRNA, and between 3 and 7% for the other snRNAs, which most likely reflects differential accessibilities of Sm proteins in each of the core complexes. Note that we had previously obtained direct biochemical evidence, based on peptide data, for the presence of these five Sm proteins in purified U2 snRNP (Palfi et al, 2000). Second, in addition to this common five-membered subcore of SmE/F/G/D1/D2, we analyzed the snRNA association of SmD3 and SmB: The Sm proteins SmB and SmD3 are clearly present in the SL, U1, and U5 snRNPs (panel D, lanes 11–14; selection efficiencies: 2–6%). SmB and SmD3 also exist in the U4/U6 snRNP (panel C, lanes 11–14; selection efficiencies: 2–4%), although with SmD3, the pull-down efficiencies of U4 and U6 were consistently lower than with SmB (see Discussion). In contrast, there was no detectable U2 snRNA association with SmD3 and SmB (panel B, lanes 11–14). Third, both the new Sm proteins Sm15K and Sm16.5K showed a highly selective snRNA association. Specifically, selection through either Sm15K or Sm16.5K resulted in high levels of U2 snRNA (10 and 7%, respectively; see panel B, lanes 15–18), but no precipitation of the other snRNAs (except for very minor levels of U6, which most likely reflect coprecipitation with U2 and a low-abundance post-spliceosomal U2/U6 complex; see Figure 2C, lane 16). In sum, this establishes Sm15K and Sm16.5K as two U2-specific Sm proteins, replacing the canonical SmB and D3 subunits. The set of pull-down assays through SmE, F, G, D1, and D2 were performed under low-stringency conditions (150 mM KCl washes), whereas the SmD3, B, 15K, and 16.5K pull-down assays required more stringent conditions (500 mM KCl washes). When the latter set of reactions was performed under low-stringency conditions, low levels of U2 snRNA precipitations were observed also for SmD3 and SmB, as well as low levels of U6 and U5 in the case of Sm15K (data not shown). Most likely this is due to co-precipitation effects caused by larger snRNP complexes, that were disrupted at higher ionic strength. This difference in the stringency of the assays explains also why for SmD3, B, 15K, and 16.5K (lanes 11–18) the pull-down efficiencies were consistently lower than for the other Sm polypeptides (lanes 1–10). We note that the T. brucei Sm15K protein corresponds to a protein recently identified by genome database screening and homology searching (Liu et al, 2004). This protein termed LSm5 (AY551263) had been suggested to be a component of the U6-specific LSm core. In contrast to that study, we have clearly demonstrated here—using a TAP tagging strategy—that Sm15K associates specifically with the U2 snRNA. T. brucei Sm15K and Sm16.5K interact with SmD1 and SmG, respectively The data described above on snRNA associations of the seven canonical and the two U2-specific Sm proteins indicate that in the U2 snRNP SmB and SmD3 are replaced by Sm15K and Sm16.5K, respectively. Next, we needed to establish the overall topology of the newly identified U2-specific Sm core. To obtain biochemical evidence for how the two new U2-specific Sm proteins are arranged in the Sm core, we performed protein–protein interaction assays, using GST pull-down in combination with purified recombinant FLAG-tagged proteins (Figure 3A): GST-Sm15K, GST-Sm16.5K, and as a control, GST alone were immobilized on glutathione-Sepharose, followed by incubation with purified FLAG-tagged SmG or SmD1 proteins, the two potential neighboring Sm proteins. After washing, bound proteins were eluted and analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis and Western blotting, using anti-FLAG antibodies. This provided clear evidence for specific interactions between GST-Sm15K and SmD1, between GST-Sm16.5K and SmG (lanes 3, 6). There was insignificant or no binding in the other combination (lanes 4, 5), and background binding to GST alone and another Sm protein, GST-SmF, was also very low or undetectable (lanes 7, 8, and data not shown). Figure 3.Protein–protein interactions of Sm15K and Sm16.5K proteins. (A) Immobilized proteins GST-Sm15K (lanes 3, 4), GST-Sm16.5K (lanes 5, 6), and as a control, GST (lanes 7, 8) were incubated with purified FLAG-tagged SmG or SmD1 proteins (C-FLAG-SmD1 and N-FLAG-SmG; see 20% of the input, lanes 1 and 2). After washing, bound proteins were eluted and analyzed by SDS-gel electrophoresis and Western blotting, using anti-FLAG antibodies. The positions of the FLAG-tagged SmG and SmD1 proteins are marked on the right. (B) Immobilized proteins GST-Sm15K (lane 3), GST-Sm16.5K (lane 4), and as a control, GST (lane 2) were incubated with purified FLAG-tagged Sm15K protein (C-FLAG-Sm15K; see 20% of the input, lane 1). After washing, bound proteins were eluted and analyzed by SDS-gel electrophoresis and Western blotting, using anti-FLAG antibodies. The position of the FLAG-tagged Sm15K is marked on the right. Download figure Download PowerPoint In addition, we have obtained direct evidence for Sm15K and Sm16.5K interacting with each other (Figure 3B): Immobilized GST protein, GST-Sm15K, and GST-Sm16.5K were incubated with purified FLAG-tagged Sm15K, followed by the elution of bound proteins and their analysis by SDS-gel electrophoresis and Western blotting, using anti-FLAG antibodies. Although there was a low level of self-interaction for Sm15K (lane 3), the interaction was much stronger between GST-Sm16.5K and FLAG-tagged Sm15K (lane 4), and there was no detectable signal for the control combination FLAG-Sm15K and GST (lane 2). Additional support for an Sm15K/16.5K interaction is based on the coexpression of both Sm15K and Sm16.5K proteins in Escherichia coli: first, only by coexpressing both proteins we could obtain high yields of recombinant proteins. Second, recombinant Sm15K and Sm16.5K proteins were purified after coexpression in E. coli, with only one of them, Sm16.5K, carrying a His-tag, yet resulting in Sm15K/16.5K heterodimer in equimolar stoichiometry (as assessed by Coomassie staining; data not shown). In sum, these interaction assays clearly established the topology of the newly identified U2-specific Sm core: Sm15K and Sm16.5K interact with each other and substitute for SmB and SmD3, respectively, bridging between SmD1 and SmG. Sm core variation in the U2 snRNP: model and correlation with special Sm binding site in the U2 snRNA Figure 4A summarizes our model of the U2 snRNP-specific Sm core variation. We note that this remarkable feature of Sm core organization is paralleled only in one other RNP complex, the U7 snRNP, which functions in histone 3′ end processing: in the U7 snRNP, SmD1 and D2 are exchanged by two U7-specific LSm proteins, LSm10 and LSm11 (Pillai et al, 2001, 2003; reviewed by Schümperli and Pillai, 2004). It is noteworthy that in both cases a stable heterodimer unit is replaced, which also acts as an assembly intermediate (Raker et al, 1996): SmD1/D2 in the U7 snRNP, and SmB/D3 in the trypanosome U2 snRNP. We note that replacing two neighboring Sm polypeptides—as opposed to two separate ones—requires fewer switches in specific intersubunit contacts and may therefore be evolutionarily favorable. In contrast, in the LSm heptamer ring only a single subunit is exchanged, LSm1 versus LSm8 (see Introduction for references). Figure 4.Model of Sm core variation in the U2 snRNP (A) and sequence comparison of Sm sites in trypanosomid snRNAs (B). (A) The arrangement of Sm proteins in the standard core (left model) is according to Palfi et al (2000). This composition of Sm polypeptides has been experimentally verified for the SL RNP, and the U1, U5, and U4/U6 snRNPs from T. brucei (this study; see Figure 2). The U2 snRNP, however, contains a variant Sm core, in which the canonical SmB and SmD3 proteins are replaced by Sm15K and Sm16.5K (right model). The relative orientation of the latter two Sm proteins is based on their homology to SmB and SmD3, respectively, and on protein–protein interaction data (see Figure 3). The common subcore of SmD1/D2/F/E/G is marked by heavy lines. (B) Sequence alignment of the Sm sites of U1, U2, U4, U5 snRNAs as well as of the SL RNA from the following trypanosomatid species: T. brucei; T. cruzi, T. congolense, L. major, L. amazonensis, Leptomonas collosoma, Leptomonas seymouri, Crithidia fasciculata, Herpetomonas species. For U1, U4, U5, and SL RNAs, sequences from only a few representative species are shown, for U2 all available sequences. Nucleotides in large letters indicate absolute conservation for the sequences shown, small letters indicate at most one deviation. The consensus sequences of the Sm sites are underlined. R, purine, Y, pyrimidine nucleotide. Download figure Download PowerPoint What are the functional consequences of the altered core in the trypanosome U2 snRNP? Comparing the Sm site sequences in the U2 snRNA with those in SL RNA, U1, U4, and U5 snRNAs revealed a striking difference (Figure 4B): with the exception of U2, a consensus can be derived for each of the other spliceosomal snRNAs that closely resembles the Sm site consensus 5′-RAU3−6GR-3′. In contrast, in the loose consensus, 5′-AAYYrY(U)R-3′, derived from the trypanosomatid U2 snRNAs, an unusual purine position interrupts the central pyrimidine stretch in the middle (see Discussion). In vitro assembly of reconstituted canonical and U2-specific Sm cores: Sm proteins confer specificity for U2 Sm site To address the question of whether the Sm core composition is required and sufficient for snRNA binding specificity, we established an in vitro reconstitution approach. Similarly as previously performed for the mammalian Sm and LSm heptamers (Zaric et al, 2005), each of the three subcomplexes of the trypanosome Sm cores were coexpressed in E. coli and purified, each with a single His6-tag per subcomplex (see Materials and methods for details): for the canonical Sm core, SmD1/D2, SmB/D3, and SmE/F/G; for the U2 Sm core, SmD1/D2, Sm15K/16.5K, and SmE/F/G. In principle, reconstitutions were carried out by incubating equimolar amounts of the three respective subcomplexes together with RNAs to be tested for binding. Reconstituted Sm cores were recovered by His tag pull-down, using Ni-NTA agarose beads, and co-precipitated RNAs were released and analyzed by denaturing polyacrylamide gel electrophoresis. Initially, to test RNA binding specificity, we have used mixtures of in vitro transcribed snRNAs from T. brucei (Figure 5A), then short RNA oligonucleotides covering the U1 and U2 Sm sites (Figure 5B; for quantitations, see Supplementary Figure S3). Figure 5.snRNA specificity of reconstituted canonical and U2 Sm cores. (A) Binding of in vitro transcribed snRNAs. 32P-labeled snRNA from T. brucei were prepared by transcription (20% of U2/U1 and SL/U6/U5 input shown in lanes 1 and 2, respectively). After incubation with recombinant canonical (lanes 3–6) or U2-specific Sm cores (lanes 7–10), co-precipitated RNAs were recovered by His-tag pull-down with Ni-NTA agarose beads and analyzed by denaturing gel electrophoresis (snRNA positions marked on the right; U5 bands denoted by an arrow, U1 bands by asterisks). For each reconstitution reaction, both the pull-down material (P; lanes 3, 5, 7, and 9) and the supernatant fractions (SN; lanes 4, 6, 8, and 10; 20% shown) were analyzed. M, markers (sizes in nucleotides). (B) Binding specificity of Sm site RNA oligonucleotides. 32P-labeled short RNAs were prepared by T7 transcription, containing the U1 and U2 Sm sites, both as wild-type and mutant versions (U1 WT and U1 UA, U2 WT and U2 UA; RNA sequences shown below, with the Sm sites boxed). Each of them was reconstituted in vitro with recombinant canonical (lanes 5–8) or U2-specific Sm cores (lanes 9–12). Co-precipitated RNAs were recovered by His-tag pull-down with Ni-NTA agarose beads and analyzed by denaturing gel electrophoresis. For comparison, 5% of the input RNAs are shown (lanes 1–4). M, markers (sizes in nucleotides). (C) Sequence requirements for binding canonical and U2-specific Sm cores. 32P-labeled RNAs derived from the T. brucei U2 snRNA 3′ half (nucleotides 81–148) were in vitro transcribed: WT, wild type; UA, U93A U95A U96A; ΔG, ΔG94 (sequences and secondary structure model shown on the right; 10% of the input RNAs in lanes 1–3). Following reconstitution in vitro with recombinant canonical (lanes 4–6) or U2-specific Sm cores (lanes 7–9), co-precipitated RNAs were recovered by His-tag pull-down with Ni-NTA agarose beads and analyzed by denaturing gel electrophoresis. M, markers (sizes in nucleotides). Download figure Download PowerPoint First, 32P-labeled U2, SL, U6, U5, and U1 snRNAs from T. brucei were prepared by SP6/T7 transcription and reconstituted as separate mixtures of SL, U6, and U5 snRNAs (Figure 5A, lane 2), as well as U2 and U1 snRNAs (lane 1), followed by heparin treatment. For each reconstitution, both the His-tag pull-down (lanes P) and the supernatant fractions (lanes SN) were analyzed. The canonical Sm core associated with SL, U5, and U1 snRNAs more efficiently than the U2-specific Sm core did (SL: 32 versus 1%; U5: 44 versus 30%; U1: 27 versus 7%; compare input lanes 1–2 with lanes 3–10). U6 snRNA, which is expected to interact with neither of the Sm cores, associated efficiently with the canonical Sm core (12%), but not significantly with the U2-specific Sm core (12 versus 1%; compare lanes 5/6 and 9/10). Finally, U2 snRNA interacted efficiently with both canonical (21%) and U2-specific Sm cores (21 versus 14%; compare lanes 3/4 and 7/8). Based on these in vitro reconstitutions with full-length snRNA transcripts, we conclude that the canonical and U2-specific Sm core show only limited specificity for their respective snRNAs under these conditions. Second, we did reconstitutions with short RNA oligonucleotides comprised of the Sm site sequence of U1 (nucleotides 40–61) or U2 (nucleotides 81–103). To test whether reconstitution depends on an intact Sm site sequence, we also assayed mutant derivatives, where three uridine residues were replaced by adenosines; these three-nucleotide substitutions should inactivate the Sm site (Figure 5B; Sm site sequences below). Each of these 32P-labeled RNA oligonucleotides was generated by in vitro transcription (see lanes 1–4 for 5% input) and reconstituted with the canonical or the U2-specific Sm proteins (lanes 5–8 and 9–12, respectively). In contrast to the previous assays, specificity with these short RNA oligonucleotides was very high: first, U1 and U2 Sm site sequences efficiently assembled only with the canonical
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