Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing

Abstract

A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3' processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3'-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.

Figure 1.

Sequence alignment of Lsm11 proteins of human (Hs; SwissProt P83369; UniGene Hs.187117; genomicsequence AC026407), mouse (Mm; GenBank AF514309; UniGene Mm.45659; LocusLink 72290) and X. laevis (Xl; GenBank AF514310; UniGene Xl.13277). Residues conserved between at least two proteins are shown in inverse print. The conserved Sm motifs 1 and 2 are indicated by shaded boxes. The consensus sequence was adapted from previous sources (Hermann et al. 1995; Achsel et al. 1999). (h) Hydro-phobicamino acids.

Figure 2.

Lsm11 can be cross-linked to U7 snRNA. (A) Polyclonal antibodies to Lsm11 recognize a specific 45-50-kD polypeptide. Samples of a Resource Q U7 peak fraction (RQ; Pillai et al. 2001) were separated by SDS-PAGE, Western blotted, and analyzed with affinity-purified Lsm11 antibody (lane 1). The same band was also detected by the Lsm11 antibody in affinity-purified U7 snRNPs (U7, lane 3), but not in a control precipitation with beads alone (lane 2). (B) Characterization of a UV-cross-link between Lsm11 and U7 snRNA. (Lane 1) Nuclear extract was subjected to UV irradiation and, after incubation with a radiolabeled oligodeoxynucleotide complementary to the 5′ end of U7 snRNA, subjected to SDS-PAGE and autoradiography. The detected bands correspond to noncross-linked U7 snRNA and UV-adducts to Sm G, B/B′ (very faint), and an ∼50-kD polypeptide, respectively (Mital et al. 1993). (Lane 3) Before oligonucleotide annealing and SDS-PAGE, the extract was subjected to immunoprecipitation by Lsm11 antibody; all UV adducts as well as noncross-linked U7 snRNA were precipitated as parts of intact U7 snRNPs. (Lane 2) Precipitation as in lane 3, but without antibody. (Lane 4) Same as in lane 3, except that the extract was boiled prior to immunoprecipitation to denature snRNPs; only the uppermost UV-adduct was precipitated, indicating that it contains Lsm11.

Figure 3.

The N terminus of Lsm11 is essential for histone RNA 3′ end processing. (A) Structure of various constructs containing murine Lsm11. An HA tag introduced at the N terminus and the Sm motifs are shown in dark gray and black, respectively. (B) The chimeric histone-U7 RNA (12/12-U7 RNA; Stefanovic et al. 1995b) used in C contains 49 nt of histone pre-mRNA upstream and 36 nt downstream of the cleavage site (vertical arrow), a connector segment of 28 nt, and 65 nt of U7 RNA sequence. The Sm-binding site is indicated by a black bar. (C) Syntheticm RNAs encoding HA-tagged versions of Lsm11 (see A) or of GFP were injected into the cytoplasm of X. laevis oocytes. After overnight incubation to allow for translation of the recombinant proteins, the oocytes were challenged with radiolabeled, chimeric histone-U7 RNA (see B) and further incubated for 3 h. Cytoplasmic extracts were subjected to immunoprecipitation with either anti-Sm or anti-HA antibodies to enrich for total snRNPs or for particles containing HA-tagged Lsm11, respectively, and the radiolabeled RNA was analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. (Lane 1) Input chimeric RNA. (Lane 3) Precipitation of HA-mLsm11^FL extract with beads lacking antibody. (D) Lsm11 N terminus sequesters histone RNA processing factor(s) from K21 mouse mastocytoma cell nuclear extract (Stauber et al. 1990). Note that processing by the extract (lane 1) is strongly reduced by preincubation of the extract with immobilized GST-tagged N terminus of Lsm11, followed by removal of the bound material (lane 4), but is only slightly affected by a similar treatment with immobilized GST (lane 3). (Lane 2) Incubation in extract inactivated by 15 min preincubation at 50°C. (E) Lsm11 N terminus binds to ZFP100 (Dominski et al. 2002) in vitro. Radiolabeled in vitro translated ZFP100 was incubated with glutathione beads preloaded with GST (lane 2) or GST-mLsm11^N-term (lane 3), and the bound material was analyzed by SDS-PAGE and autoradiography. Lane 1 shows 1/10 volume of input material.

Figure 4.

Association of HA-tagged Lsm11 with U7, but not U1, snRNA. (A) Human 293T cells were transiently transfected with plasmids encoding the various HA-tagged Lsm11 proteins shown in Figure 3A or a fusion between HA tag, the first 136 amino acids of Lsm11, and Sm D2 (N136-D2). Nuclear extracts were incubated with biotinylated oligonucleotides complementary to the 5′ ends of U7 or U1 snRNA and precipitated with magnetic streptavidine beads. The samples were subjected to SDS-PAGE and immunoblotted with anti-HA antibody. (-) Precipitation by beads without oligonucleotide; (input) sample of original nuclear extract. (B) Western blots detecting Lsm11 and Sm B/B′ proteins associated in 293T cells with U7 snRNAs containing either the wild-type Sm-binding site or a consensus Sm-binding sequence derived from spliceosomal snRNAs. The cells were cotransfected with plasmids encoding either Sm WT or Sm OPT U7 RNA (Stefanovic et al. 1995a), modified with a 28-nt sequence tag at the 5′ end (Pillai et al. 2001), and with the plasmid encoding HA-mLsm11^FL. Nuclear extracts were prepared and processed as described for A, except that precipitations were performed with magnetic streptavidine beads with (+) or without (-) biotinylated oligonucleotide complementary to the 28-nt tag. Anti-HA and Y12 anti-Sm antibodies were used to detect HA-mLsm11^FL and Sm B/B′, respectively. (input) Sample of original 28-OPT extract.

Figure 5.

U7 snRNP assembly in Xenopus egg extract is mediated by an SMN complex. (A) Detection of Xenopus Lsm10 in assembled particles. Radioactively labeled Sm WT or Sm OPT U7 snRNAs (Stefanovic et al. 1995a) were incubated with X. laevis egg extract at 20°C for 30 min. Immunoprecipitations were performed with beads lacking antibody (-), polyclonal antiserum against X. laevis Lsm10 (XL10), or monoclonal anti-Sm antibody Y12. RNA extracted from the precipitates was subjected to denaturing polyacrylamide electrophoresis and autoradiography. (B) Detection of Lsm11 photoadduct in assembled particles. Sm WT (W), Sm OPT (O) U7 snRNAs, or U7 snRNA containing a severely mutated Sm-binding site (Sm MUT; M) were subjected to assembly as in A. The products were UV-irradiated and then subjected to SDS-PAGE and autoradiography. The molecular masses (in kilodaltons) of marker proteins and the positions of the free U7 snRNAs and of photoadducts with the Xenopus Sm G, Sm B/B′, and Lsm11 proteins are indicated. (C) Preincubation of extracts with antibodies against SMN complex components inhibits U7 snRNP assembly. Sm WT U7 snRNA was subjected to assembly as in A. The reaction was subsequently analyzed by electrophoresis on a 5% nondenaturing polyacrylamide gel (lane 1). In the other lanes, the egg extracts were preincubated with anti-Gemin2 (lane 2), anti-Gemin4/GIP (lane 3), and anti-HA (lane 4) antibodies. (D) Immunodepletion of extracts for SMN complex components prevents U7 snRNP assembly. The extracts were either mock-depleted (lanes 2-4) or immunodepleted with antibodies against SMN and Gemin2 (lanes 5-7). Assembly reactions were performed as in A, using Sm WT (W), Sm OPT (O), or Sm MUT (M) U7 snRNAs. (Lane 1) U7 Sm WT RNA in the absence of extract.

Figure 6.

Association of Lsm10 and Lsm11 with a specialized SMN complex. (A) Lsm10 and Sm proteins D1/D2 are associated with separate SMN complexes. Cytoplasmic extract from HeLa cells was immunoprecipitated with an antibody reacting with Sm D1/D2 (lanes 1,5) or with Y12 anti-Sm antibody (lanes 3,6). (Lanes 2,4) Samples of the extracts prior to precipitation. The blots were probed with antibodies specific for SMN, D1/D2, or Lsm10 as indicated. (B) HA-tagged Lsm10 and Sm proteins D1/D2 associate with separate SMN complexes. Cytoplasmic extract from HeLa cells stably expressing HA-tagged human Lsm10 was immunoprecipitated with Y12 antibody (lane 2) or with antibody against the HA peptide (lane 3). (Lane 1) Sample of the extract prior to precipitation. The blots were probed with antibodies against D1/D2, HA, Lsm11, SMN, or with Y12 antibody (revealing Sm B/B′) as indicated. (C) Separate SMN complex preparations differ in their Lsm10 and Lsm11 content. Two different preparations of SMN complex (Meister et al. 2000) were analyzed by SDS-PAGE and Coomassie staining (top panels) and by Western blotting (bottom panels) with antibodies against Lsm10, Lsm11, Y12 antibody (revealing Sm B/B′), or with D1/D2 antibody as indicated. Note that preparation SMN-2 does not contain detectable amounts of Lsm10 or Lsm11. The positions of molecular size markers (M), their sizes in kilodaltons, and the positions of prominent protein bands are indicated. (D) Failure of SMN complex lacking Lsm10/11 to assemble U7 snRNA with its wild-type Sm-binding site. SMN complexes 1 and 2 characterized in C were used in assembly reactions with Sm WT (W), Sm OPT (O), or Sm MUT (M) U7 snRNAs. Assembled complexes were detected by immunoprecipitation with Y12 anti-Sm antibody followed by denaturing polyacrylamide gel electrophoresis and autoradiography of the radiolabeled U7 RNAs.