Other components of the U11/U12 di-snRNP complex in Arabidopsis. (A) Oligonucleotide affinity selection was performed with protein extracts prepared from protoplasts expressing U1 snRNP-specific proteins U1A and U1C, U2 snRNP-specific proteins U2A′, U2B″, and SF3a60, U2- and U11/U12-specific proteins SF3b49 and p14 tagged with HA, or with GFP and HA tags (U1A). (Left panels) Affinity selection with U11 oligonucleotide. (Right panels) Affinity selection with U12 oligonucleotide. Details are as in Figure 3C. (B) Transiently expressed GFP-tagged U1–70K (lane 3), U1A (lane 4), U2B″ (lane 5), and U2A′ (lane 6) proteins were immunoprecipitated with anti-GFP antibodies and immunoprecipitates were analyzed for the presence of snRNAs by [³²P]pCp ligation. Four spliceosomal snRNAs (immunoprecipitated with anti-m₃G cap antibody; lane 2) and sizes of markers in nucleotides (lane 1) are indicated on the left. 5S and 5.8S RNAs, which appear as background, are indicated on the right. Bands marked with asterisks are of unknown identity.

(A) Arabidopsis U11–35K protein interacts with SR proteins in vitro. Protein extracts from plant protoplasts transformed with plasmids expressing HA-tagged SCL28, SCL30, SCL30a, SCL33, SC35, SRp34, RSp31, and RSp40 proteins or from yeast cells expressing HA-tagged RSZp21, RSZ33, and SRp30 were incubated with glutathione-Sepharose beads coated with Arabidopsis U11–35K proteins. (Lanes 1) One-tenth of the input protein extracts used in pull-downs; (lanes 2 ) protein extracts incubated with glutathione-Sepharose beads only; (lanes 3) pull-down with U11–35K(−Q124); (lanes 4) pull-down with U11–35K(+Q124). Western blotting was performed with anti-HA antibody. (B) Arabidopsis U11–35K protein localizes to the nucleus. Tobacco protoplasts were transiently transformed with plasmids expressing GFP-tagged U11–35K proteins and analyzed by laser scanning confocal microscopy. As control, protoplasts were transformed with plasmids expressing GFP-tagged Arabidopsis U1–70K protein (the leftmost panel). (C) Arabidopsis U11–35K protein (−Q124) colocalizes with SR protein SRp34 in nuclear speckles in both tobacco (upper row; U11–35K and SRp34 were expressed as GFP and RFP fusions, respectively) and Arabidopsis (lower row; U11–35K and SRp34 were expressed as RFP and GFP fusions, respectively) cells. All images in B and C are single confocal sections. Merged images show superimposition of GFP and RFP signals. Arrows in B and C point to nucleoli. Bars, 6 μm (B,C).

Analysis of Arabidopsis U4atac and U6atac-2 snRNAs. (A) Sequence alignment of Arabidopsis and human U4atac snRNAs. Arabidopsis sequence includes a 5′ end upstream promoter region with two conserved regulatory elements (USE and TATA-box) (Waibel and Filipowicz 1990) printed on a gray background. The Sm-binding site is on a green background. Sequence complementary to the GGC triad of U6atac snRNA is printed on a lilac background. Sequence that base-pairs with U6atac in stem II is printed on a blue background. (B) Sequence alignment of human and two Arabidopsis U6atac snRNAs. Arabidopsis sequences include a 5′ upstream promoter sequence with USE and TATA-boxes printed on a gray background. The GGC triad and the region that base-pairs with U4atac snRNA in stem II are printed on lilac and blue backgrounds, respectively. (C) Expression analysis of the Arabidopsis U4atac and U6atac-2 snRNAs as determined by RNaseA/T1 protection assay. For this, 10 μg of total RNA from Arabidopsis seedlings was hybridized with the antisense U4atac and U6atac-2 probes. After RNaseA/T1 protection assay the RNA was run on a 6% denaturing polyacrylamide gel. (Lane 1) U6atac-2 snRNA probe; (lane 2) U6atac-2 snRNA protection assay; (lane 3) U4atac snRNA protection assay; (lane 4) U4atac snRNA probe. The sizes of the ³²P-labeled DNA markers (lane 5) in nucleotides are indicated on the right. On the right side are schematic representations of U4atac and U6atac-2 genomic fragments in pGEM T-easy. (D) Base-pairing interactions between Arabidopsis U4atac and U6atac snRNAs. Potential RNA–RNA base pairs (Watson-Crick) are indicated by dashes and G • U interactions by dots. Sm-binding site of U4atac snRNA is green shaded, and the GGC triad of the U6atac snRNA in stem I is printed on a lilac background.

Arabidopsis U11 snRNP is present in a stable U11/U12 di-snRNP complex. (A,B) Transiently expressed U11–35K protein assembles into snRNP. Arabidopsis cell suspension protoplasts were transiently transformed with plasmids expressing (A) GFP- or (B) HA-tagged U11–35K (both –Q124 and +Q124) and SRp34, and immunoprecipitations were performed with anti-GFP and anti-m₃G antibodies (A), or with anti-HA and anti-m₃G antibodies (B), as described in Materials and Methods. Western blotting was performed with anti-GFP (A) or anti-HA (B) antibodies. Lanes 1 (“input”) in A and B were loaded with 1/10 of the protein extracts used for immunoprecipitations. (Lanes pA) Protein extracts incubated with protein-A Sepharose only; (lanes 3) (α-GFP in A and α-HA in B) immunoprecipitations performed with anti-GFP (A) or anti-HA (B) antibodies; (lanes 4) (α-m₃G) immunoprecipitations performed with anti-m₃G cap antibody. Small arrows point to immunoglobulin heavy and light chains. Large arrows point to immunoprecipitated GFP- or HA-tagged proteins. Note that SRp34 is precipitated with tag-specific antibodies but not with anti-m₃G antibody. (C) Arabidopsis U11–35K protein is speci-fically affinity-selected with U11 snRNA (left panels) and U12 snRNA (right panels) directed antisense oligonucleotides. Protein extracts prepared from Arabidopsis cell suspension protoplasts expressing GFP- or HA-tagged U11–35K(−Q124) or HA-tagged U1–70K and SRp34 were incubated with biotinylated oligonucleotides complementary to either U11 or U12 snRNA (lanes 3) or were mock incubated (lanes 2) and than bound to streptavidin agarose (see Materials and Methods). Proteins pulled down were analyzed by Western blotting with anti-GFP and anti-HA antibodies. Lanes 1 (input) were loaded with 1/20 of the protein extract used for affinity selection. (D) Sedimentation behavior of U11–35K protein on glycerol gradient. Protein extracts from Arabidopsis protoplasts expressing HA-tagged U11–35K(−Q124) were analyzed by 10%–30% glycerol gradient centrifugation. Gradients were fractionated into 20 0.5-mL fractions that were subsequently analyzed by Western blotting with anti-HA antibody. (E) Oligonucleotide affinity selection of HA-tagged U11–35K protein from ~12S (pooled fractions 6 and 7; upper panel) and ~18S (pooled fractions 10–12; lower panel) glycerol gradient peaks. Lanes 1 were loaded with 1/10 of the extract used for affinity selection in lanes 2, 3, and 4. (Lanes 2) Extract incubated with streptavidin beads only; (lanes 3,4) extract incubated with U11 and U12 antisense oligonucleotides, respectively. Western blotting was performed with anti-HA antibody.

Sequence analysis of plant U11/U12 di-snRNP specific proteins. (A) Sequence alignment of U11/U12 di-snRNP 65K proteins from Arabidopsis thaliana (At; At1g09230), Oryza sativa (Os; gi:32992841), Saccharum officinarum (So; gi:35975718), Zea mays (Zm; gi:21206676), Welwitschia mirabilis (Wm; Sgi:42651093), and Homo sapiens (Hs; gi:16553747). The sequences of S. officinarum, Z. mays, and W. mirabilis are deduced from EST clones and are incomplete at their 5′ ends. The two RRMs are overlined, and the RNP1 and RNP2 motifs of RRMs are in red boxes. The proline-rich region is blue overlined, and a short acidic and serine-rich region is in a green box. (B) Sequence alignment of U11 snRNP 59K proteins from A. thaliana (At; At2g46200), O. sativa (Os; gi:32976260), Licopersicon esculentum (Le; gi:47104949), Citrus sinensis (Cs; gi:46213273), Beta vulgaris (Bv; gi:34892003), and H. sapiens (Hs; gi:22027541). The sequences of L. esculentum, C. sinensis, and B. vulgaris are deduced from EST clones, and they are incomplete at their 5′ and 3′ ends. The sequence of O. sativa is incomplete at the 5′ end. The most conserved domains are in red and blue boxes. (C) Sequence alignment of U11 snRNP 48K proteins from A. thaliana (At; At3g04160), S. officinarum (So; gi:34923260) O. sativa (Os; gi:54325546), and H. sapiens (Hs; gi:33457355). The sequences of S. officinarum and O. sativa are deduced from the EST and genomic sequences, respectively, and they are incomplete at both 5′ and 3′ ends. The N-terminal extension of the A. thaliana sequence is not included in the alignment. The most conserved region of the protein is in a red box. Sequences in A–C were aligned by using the ClustalW program and shaded on the BOXSHADE server. The order of sequences is as it appeared in the ClustalW output. Amino acids identical or similar in more than 50% of the analyzed sequences are shown on black or gray background, respectively. The accession numbers of plant proteins refer to their DNA sequences at the NCBI.

Sequence and expression analysis of Arabidopsis U11–35K protein. (A) Schematic representation of exon–intron structure of the gene encoding U11–35K. Exons are represented by gray boxes, and introns by solid lines. Positions of the RRM and RS/RD domains are indicated. The two consecutive YAG consensus 3′ss in intron 4 are indicated (TAGCAG). Below are schematic representations of the three different mRNAs resulting from alternative splicing, with sizes of encoded proteins indicated in amino acids (aa). The amino acid sequence printed in italics is encoded by the included intron 6 of mRNA3. The asterisk denotes the stop codon. (B) Sequence alignment of Arabidopsis thaliana (At2g43370), Oryza sativa (Os; gi:32982022), Solanum tuberosum (St; gi:39823686), Sorghum bicolor (Sb; gi:45965518), Persea americana (Pa; gi:42641885), and Homo sapiens (Hs; gi:5902144) U11–35K proteins. The sequences of S. tuberosum, S. bicolor, and P. americana were deduced from EST clones; note that the C termini of these sequences are not complete. Xs in the S. bicolor sequence denote amino acids of uncertain identity. The RNP1 and RNP2 motifs of RRM are in boxes. Sequences were aligned by using the ClustalW program and shaded on the BOXSHADE server. The order of sequences is as it appeared in the ClustalW output. Amino acids identical or similar in >50% of the analyzed sequences are shown on black or gray backgrounds, respectively. The accession numbers of plant and human proteins refer to their DNA sequences at the NCBI. (C) The aberrant behavior of Arabidopsis U11–35K protein on SDS-PAGE is not due to extensive phosphorylation. Protein extracts from tobacco protoplasts (lanes 1,2) or from yeast cells (lane 3) expressing HA-tagged U11–35K were analyzed by Western blotting with rat monoclonal antibody (Roche) against the HA-tag. A single protein band was detected at ~45 kDa (left panel). Molecular mass standards in kilodaltons are indicated on the left . Protein extracts from tobacco protoplasts (as above) were incubated without (lane –CIP) or with calf intestinal phosphatase (lane +CIP) for 1 h at room temperature and subsequently analyzed by Western blotting with anti-HA antibody (two right panels). SCL30 (lower panel), an SR protein from Arabidopsis that is highly phosphorylated as evident from the shift after CIP treatment, was used as a control (lower panel). U11–35K does not show a significant shift after CIP treatment (upper panel).