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.