snR5 RNA associates with certain Lsm proteins. Lysates from the indicated strains were subjected to immunoprecipitation with anti–c-myc antibodies. RNAs in immunoprecipitates (lanes 3–7) were labeled with [³²P]pCp and fractionated in a 5% polyacrylamide, 8.3 M urea gel. Lanes 1 and 2, total RNA from LSM3(myc)₃ and) LSM3(myc₃ lhp1::LEU2 lysates. The band labeled with the asterisk was identified as snR5. The identities of the U4, U5, and U6 snRNA bands in the immunoprecipitates were confirmed by Northern analyses.

A substantial fraction of snR5 RNA associates with Lsm2–Lsm7 proteins. (A) Anti–c-myc immunoprecipitations were performed from an untagged strain (lane 2), or the indicated c-myc--tagged strains (lanes 3–12). Equal amounts of lysates (as measured by OD₂₆₀) were used for immunoprecipitations. RNAs within immunoprecipitates were subjected to Northern analyses to detect U1, snR5, and U6 RNAs. Anti-Smd1p antibodies (Xue et al., 2000) was used to immunoprecipitate Smd1p (lane 13). The low level of U6 RNA in the LSM7(myc)₃ immunoprecipitate is not reproducible. Lane 1, RNA from an equivalent amount of untagged lysate. (B) Lysates from untagged (lanes 1, 4, and 7), LSM5(myc)₃ (lanes 2, 5, and 8), and LSM8(myc)₃ (lanes 3, 6, and 9) strains were divided in equal aliquots and extracted with phenol (lanes 1–3) or incubated with anti-myc antibodies (lanes 4–6). RNAs in totals (lanes 1–3), immunoprecipitates (4–6), and supernatants (7–9) were subjected to Northern analyses to detect U1, snR5, U6, snR3, RNase P, U3, and

The Lsm-associated snR5 appears partly distinct from the box H/ACA protein-associated snR5. (A) An LSM5(myc)₃ strain was subjected to sequential anti–c-myc and anti-Nhp2p immunoprecipitations. In Set A (lanes 1–3), the lysate was incubated three times with anti–c-myc antibodies. For Set B (lanes 5–7), two anti–c-myc immunoprecipitations were followed by an anti-Nhp2p immunoprecipitation. Set C (lanes 9–11) shows three anti-Nhp2p immunoprecipitations. In Set D (lanes 13–15) two anti-Nhp2p immunoprecipitations were followed by an anti–c-myc immunoprecipitation. Lanes 4, 8, 12, and 16 show the supernatants after the third immunoprecipitation. RNAs from immunoprecipitates and supernatants were subjected to Northern analysis to detect snR5, U6, U1, and snR10 RNAs. Lane 17, RNA from an equivalent amount of lysate. (B) LSM5(myc)₃ GAR1(HA)₃ lysates were fractionated in a 10–36% glycerol gradient. Odd-numbered fractions were divided into three parts and either extracted with phenol (third panel) or incubated with anti–c-myc (top panel) or anti-HA antibodies (second panel). RNAs from immunoprecipitates and totals were subjected to Northern analysis to detect snR5 RNA (top three panels). The third panel was reprobed to detect U1, U5, U4, U6, and U3 snRNAs (bottom four panels).

Lsm2p, Lsm5p, and Lsm6p associate with snR5 RNA in vitro. ³²P-labeled snR5, U6, and U5_L snRNAs were incubated in untagged (lanes 1 and 6), LSM2(myc)₃ (lanes 2 and 7), LSM5(myc)₃ (lanes 3 and 8), LSM6(myc)₃ (lanes 4 and 9), or LSM8(myc)₃ (lanes 5 and 10) extracts. After incubation, samples were divided in half and either extracted with phenol (lanes 1–5) or subjected to immunoprecipitation with anti–c-myc antibodies (lanes 6–10).

Association of Lsm proteins with snR5 RNA requires an intact 3′ end. (A) Top: A secondary structure for snR5 RNA, based on models for H/ACA RNAs (Kiss, 2001), was derived using MFOLD (Mathews et al., 1999). Box H (nts 87–92) and ACA (nts 192–194) sequences are bold. Bottom: snR5 RNA derivatives used in binding. (B) ³²P-labeled snR5 1–197 RNA (lanes 1–6) and truncation mutants snR5 1–86 (lanes 7–12), snR5 60–140 (lanes 13–18), and snR5 110–197 (lanes 19–24) were incubated with extracts from untagged (lanes 3, 9, 15, and 21), LSM5(myc)₃ (lanes 4, 10, 16, and 22), LSM6(myc)₃ (lanes 5, 11, 17, and 23) and LSM8(myc)₃ (lanes 6, 12, 18, and 24) strains. U5_L and U6 snRNAs were included in each reaction. After incubation, reactions were divided in half and extracted with phenol (lanes 2, 8, 14, 20; shown for LSM5(myc)₃ extract) or subjected to immunoprecipitation. Lanes 1, 7, 13, and 19 contain the equivalent amount of labeled RNAs as was added to extracts. Asterisks denote snR5 RNAs. For unknown reasons, less snR5 was bound by Lsm proteins in this experiment compared with Figure 3. (C) ³²P-labeled snR5 mutants, along with U5_L and U6 snRNAs, were incubated with extracts of untagged (lanes 3, 9, 15, and 21), LSM5(myc)₃ (lanes 4, 10, 16, and 22), LSM6(myc)₃ (lanes 5, 11, 17, and 23) and LSM8(myc)₃ (lanes 6, 12, 18, and 24) strains. Each reaction was divided in half and extracted with phenol (lanes 2, 8, 14, 20, shown for LSM5(myc)₃ extract) or subjected to immunoprecipitation. Lanes 1, 7, 13, and 19 contain the equivalent amount of labeled RNAs as was added to the extracts. Asterisks denote snR5 RNAs.

snR5 and Lsm proteins are present in nucleoli. (A) Wild-type, lsm6Δ, and snR5Δ strains were subjected to in situ hybridization using an antisense snR5 probe (a, d, and g, respectively). Nop1p-GFP was used as a nucleolar marker (b, e, and h). DNA was stained with DAPI and is shown in blue in c, f, and i. (B) Live cell imaging of Lsm5p-GFP, Lsm4p-GFP, and Lsm8p-GFP using confocal microscopy. DsRed-Nop1p was used as a nucleolar marker. Only a fraction of cells express detectable DsRed-Nop1.

Lsm proteins are not required for snR5 stability or its function in pseudouridylation. (A) The growth of wild-type and GAL::LSM3 strains is compared during growth in glucose. Strains contain either SNR6 in the high copy plasmid YEplac181 (pSNR6) or YEplac181 (vector). At time 0, strains were switched to medium containing glucose. (B) At intervals after the switch to glucose, RNA was extracted and subjected to Northern analysis to detect snR5, U6, and U1 snRNAs (top three panels). We also examined levels of two 3′ extended U3 precursors (the intron-containing U3-3′ int and the spliced U3-3′) (Kufel et al., 2003b), , and the 7S and 6S precursors to 5.8S rRNA. Although Lsm2–Lsm8 proteins are normally required for stable accumulation of U6 snRNA (Pannone et al., 1998; Mayes et al., 1999; Salgado-Garrido et al., 1999), the overexpressed U6 snRNA is stable. One possibility is that other RNA-binding proteins, whose affinity for U6 is low when U6 is present at wild-type levels, bind and stabilize U6 when the RNA is overexpressed. (C) Total RNA was extracted from GAL::LSM3 strains carrying pSNR6 at intervals after the switch to glucose and assayed for pseudouridines by primer extension. RNA from wild-type and snr5Δ strains was also examined.