Fig. 1. Purification and polypeptide composition of U7 snRNPs. (A) RNA 3′ end-labelling of fractions enriched for U7 and U1 snRNPs. RNA samples were isolated from U7 and U1 peak fractions of a Resource Q column (see Materials and methods), 3′ end-labelled with α-[³²P]pCp and analysed by denaturing PAGE. The positions and sizes (in bp) of relevant marker bands (HpaII-digested pBR322) are indicated on the left. (B) Polypeptide composition of the Resource Q U7 peak fraction from which the 14 kDa polypeptide (arrow) was identified by tandem mass spectrometry. A sample of the fraction was separated by 15% SDS–PAGE. M, protein marker, sizes in kDa are indicated on the left. (C) Polypeptide composition of affinity-purified U7 snRNPs. U7 (lane 2) or U1 snRNPs (lane 3) were affinity selected from a Resource Q fraction containing both U7 and U1 snRNPs (ResQ; lane 1) using biotinylated RNA-specific antisense oligonucleotides and magnetic streptavidin beads and resolved by 12% SDS–PAGE. The positions of the common Sm proteins present in the U1 snRNP and of streptavidin (SA) released from the magnetic beads are indicated on the right. Unidentified proteins present in the U7 snRNP purification are labelled with asterisks. (D) Identification of Sm proteins present in affinity-purified U7 snRNPs by western probing. ResQ (lane 1) and U7 (lane 3) are labelled as in (C); beads (lane 2) show the background precipitation by beads alone. The antibodies used were (from top to bottom): Y12 anti-Sm antibody, anti-Sm D2, anti-Sm D3, anti-Sm F and anti-Sm G. The Y12 antibody detects a double band corresponding to Sm B/B′, and a weak signal for Sm D1 (Hoch et al., 1999; Brahms et al., 2000).

Fig. 2. Lsm10 is a novel Sm-like protein. (A) Amino acid sequence of Lsm10. Peptides identified by mass spectrometry are underlined. Arrows indicate the region that was deleted in the Lsm10^ΔPstI construct. The cDNA sequence was deposited in the DDBJ/EMBL/GenBank (accession No. AF394685). (B) Alignment of Lsm10 with human Sm D1 and D3 and the related Lsm2 and Lsm4 proteins across the Sm motifs 1 and 2. Highlighted Lsm10 residues are present in some (black) or all (grey) other proteins. The consensus sequence was adapted from previous sources (Hermann et al., 1995; Achsel et al., 1999). h, hydrophobic amino acids.

Fig. 3. Lsm10 is in a complex with U7 snRNA and common Sm proteins. (A) Polyclonal antibodies to Lsm10 recognize a specific 14 kDa polypeptide. Samples of a Resource Q U7 peak fraction similar to that shown in Figure 1B were separated by 15% SDS–PAGE, western blotted and analysed with pre-immune serum (pre, lane 1), immune serum (im, lane 2) and affinity-purified Lsm10^ΔPstI antibody (lane 3). A specific band of 14 kDa was also detected by the Lsm10^ΔPstI antibody in affinity-purified U7 snRNPs (U7, lane 5), but not in a control precipitation with beads alone (bd, lane 4). (B) Native gel analysis of U7 snRNPs. Samples of a Resource Q U7 peak fraction were incubated with a radiolabelled oligonucleotide complementary to the 5′ end of U7 snRNA, followed by incubation with different antibodies as indicated, before separation on a non-denaturing gel (Melin et al., 1992). Only the region between the gel slots and the U7 snRNP complexes is shown. The positions of native U7 snRNPs, as well as that of the antibody-induced supershift, are indicated on the right. (C) Co-immunoprecipitation of U7 snRNA with Lsm10 from HeLa nuclear extracts. Equal amounts of HeLa nuclear extract were incubated with different antibodies as indicated. RNAs were extracted from the immunopellets and analysed by primer extension for U7 and U1 RNA. The migration of the unextended primers and of the U7- and U1-specific extension products are indicated on the right.

Fig. 4. Lsm10 co-localizes with p80 coilin. The top row shows the same HeLa cell viewed by phase contrast (A) and by indirect immunofluorescence microscopy (B) with Lsm10^ΔPstI antibody followed by Oregon Green-labelled goat anti-rabbit antibody. Bottom row: co-staining for (C) p80 coilin, using monoclonal coilin-specific antibody SP10 followed by Texas Red-labelled anti-mouse antibodies and (D) Lsm10 [as in (B)]. In (E), the red and green images from (C) and (D) were merged to demonstrate the co-localization of the brightly stained CBs.

Fig. 5. Importance of the Sm binding site for the association of Lsm10 with U7 snRNA in vivo. (A) Association of HA-tagged proteins with U1 and U7 snRNAs. Human 293T cells were transiently transfected with plasmids encoding HA-tagged Sm proteins or stably transformed with a plasmid encoding HA-tagged Lsm10. Nuclear extracts were incubated with biotinylated oligonucleotides complementary to the 5′-ends of U7 or U1 snRNA and precipitated with magnetic streptavidin 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) RNase mapping of 28-WT and 28-OPT RNAs in nuclear extract from 293T cell clone HA-Lsm10 #2 stably expressing HA-tagged Lsm10. These cells were transfected with plasmids encoding U7 Sm WT and U7 Sm OPT RNA (Grimm et al., 1993; Stefanovic et al., 1995), modified with a 28 nt sequence tag at the 5′ end. The riboprobe, derived from U7 Sm OPT RNA, was complementary to the resulting 28-OPT and 28-WT transcripts over ∼42 and ∼33 nt, respectively. Lanes: tRNA, control hybridization to tRNA; M, HpaII-digested pBR322 DNA (sizes of fragments in bp are indicated on the left). (C) Western blots detecting Lsm10 and Sm proteins associated with 28-WT or 28-OPT RNAs in transfected 293T cells. Nuclear extracts were prepared and processed as described for (A), except that precipitations were performed with magnetic streptavidin beads with (+) or without (–) biotinylated anti-28 oligonucleotide. The top two panels are blots of material precipitated from the same nuclear extracts used in (B). Alternatively, 293T cells were co-transfected with 28-WT or 28-OPT and plasmids for either HA-tagged Sm D2 (two middle panels) or D3 (two bottom panels). Anti-HA and Y12 anti-Sm antibodies were used to detect HA-tagged proteins and Sm B/B′, respectively. Input, sample of original 28-WT extract from each experiment.

Fig. 6. Tentative model for the structure of a U7 snRNP-specific Sm core. A conventional Sm core complex containing all seven Sm proteins as proposed by Kambach et al. (1999) is shown schematically on the left. In the U7 snRNP (shown on the right), the missing D1 and D2 proteins may be replaced by the Lsm10 protein characterized in this paper and by U7-p50, which can be UV-cross-linked to the U7 Sm binding site (Mital et al., 1993; Stefanovic et al., 1995; see text).