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Results: 7

1.
FIG. 1

FIG. 1. From: A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly.

Sequence alignment of S. cerevisiae Snu17p with homologous proteins. (A) Snu17p is aligned with a homolog from H. sapiens (AF078865). Identical residues are boxed in black, and similar residues are boxed in grey. The amino acid positions are indicated by numbers. (B) Snu17p is aligned with an H. sapiens cDNA product from EST186153 AA314241. The position of the Snu17p RRM is indicated above the sequences by a shaded box.

Alexander Gottschalk, et al. Mol Cell Biol. 2001 May;21(9):3037-3046.
2.
FIG. 2

FIG. 2. From: A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly.

Snu17p is a yeast U2 snRNP protein. DNA coding for a tandem repeat of S. aureus protein A was fused 3′ to SNU17. Extracts (50 μl) from the resulting strain (Snu17p-tag), as well as from the parental wild-type strain (No tag), were immunoprecipitated using IgG-agarose and then washed at various NaCl concentrations, as indicated above the lanes. The snRNA content of the precipitates was assayed by Northern blot analysis (snRNAs are indicated). Total refers to the total RNAs from 4 μl of extract (8% of the total input) before immunoprecipitation.

Alexander Gottschalk, et al. Mol Cell Biol. 2001 May;21(9):3037-3046.
3.
FIG. 6

FIG. 6. From: A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly.

RNA composition of complex X. Spliceosomes were assembled on unlabeled actin pre-mRNA. Splicing reactions were performed using wild-type (WT) or snu17Δ extracts at 30°C, either mock treated, treated to destroy U6, or treated with 8 mM EDTA as indicated above each lane. Aliquots were withdrawn after 0, 5, and 20 min, blocked on ice, and treated with a buffer containing heparin. Nondenaturing gels were run, blotted, and sequentially hybridized with probes for the actin precursor (Actin) (a) and subsequently for the U2 (b), U1 (c), U6 (d), U5 (e), and U4 (f) snRNAs. The complexes are indicated on the left. Complex X is an unusual slow-migrating complex seen only in snu17Δ extracts.

Alexander Gottschalk, et al. Mol Cell Biol. 2001 May;21(9):3037-3046.
4.
FIG. 5

FIG. 5. From: A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly.

Deletion of SNU17 leads to accumulation of an unusual spliceosomal complex. (A) Spliceosomes were assembled on a 32P-labeled actin pre-mRNA by incubation in mock-treated wild-type (WT) and snu17Δ extracts at 30°C. Aliquots were withdrawn after 0, 10, and 20 min and treated with a buffer containing heparin. Nondenaturing gels were run for 5 h (7). The identities of the complexes are indicated on the left. The unusual, slow-migrating complex seen only in snu17Δ extracts is designated complex X. (B) Yeast spliceosome assembly, as in reference 7. RNase H-mediated digestion of U6 snRNA (ΔU6) blocks spliceosome assembly at the B complex stage, whereas addition of 5 to 8 mM EDTA blocks spliceosome assembly at the A1 complex stage.

Alexander Gottschalk, et al. Mol Cell Biol. 2001 May;21(9):3037-3046.
5.
FIG. 7

FIG. 7. From: A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly.

Deletion of SNU17 inhibits the dissociation of U1 and U4 from the spliceosome. Splicing-reaction mixtures were incubated with actin pre-mRNA that was transcribed in the presence of biotinylated UTP to incorporate an affinity label. The reaction mixtures were incubated for 20 min at 23°C. Then 5 μl was withdrawn and kept for analysis of total RNAs (Total; lanes 1 and 3). The remaining 120 μl was treated with heparin and then sedimented by centrifugation on a 10 to 30% glycerol gradient. rRNAs from yeast were run on a parallel gradient as sedimentation markers. Fifteen fractions (160 μl each) were collected, and four-fifths of each fraction was affinity purified with streptavidin-agarose (AP = affinity purification of a fraction of the 40S region of the gradient corresponding to fraction 15, the spliceosome [lanes 2 and 4]). After extensive washing, precipitated snRNAs were assayed by Northern blotting.

Alexander Gottschalk, et al. Mol Cell Biol. 2001 May;21(9):3037-3046.
6.
FIG. 4

FIG. 4. From: A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly.

Snu17p is required for efficient splicing in vivo and in vitro. (A) RNA was extracted from wild-type (WT) and snu17Δ cells grown at 25°C (lanes 1 and 3) and after the switch to 37°C for 2 h (lane 4) and 4 h (lanes 2 and 5). Primer extension analysis was performed to measure the levels of unspliced pre-U3A and pre-U3B transcripts, indicated on the right. (B) Splicing reactions were performed at 25°C using the wild-type (lanes 1 to 8) and snu17Δ (lanes 9 to 16) extracts for the time indicated above each lane. To restore splicing, 1.2 pmol of recombinant GST-Snu17p was added to the reaction mixtures prior to addition of the actin precursor (lanes 5 to 8 and 13 to 16). Indicated on the left is the identity of the 32P-labeled RNA species (from top to bottom): intron-lariat-exon 2 intermediate, excised lariat-intron, pre-mRNA, mature mRNA, and cleaved exon 1 intermediate.

Alexander Gottschalk, et al. Mol Cell Biol. 2001 May;21(9):3037-3046.
7.
FIG. 3

FIG. 3. From: A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly.

Pre-mRNA and the lariat-intron intermediate coprecipitate with Snu17p under in vitro splicing conditions. Splicing reactions using either extracts containing Snu17p without tag (lanes 1 to 3 and 7 to 9) or protein-A tagged Snu17p (lanes 4 to 6 and 10 to 12) were incubated under splicing conditions with 32P-labeled actin pre-mRNA (lanes 1 to 6) or U3 pre-mRNA (lanes 7 to 12) for 20 or 40 min at 25°C. Then 93% of the 40-min reaction volume was precipitated with IgG agarose. For each time point, 7% of the total reaction mixtures (lanes 1, 2, 4, 5, 7, 8, 10, and 11) and the precipitate from the remaining 93% of the reaction mixtures (IP; lanes 3, 6, 9 and 12) were assayed for the presence of pre-mRNA, splicing intermediates, and products. Indicated on the left and on the right are the identities of the labeled RNA species: intron-lariat-exon 2 intermediate, excised lariat-intron, pre-mRNA, mature mRNA, and cleaved exon 1 intermediate.

Alexander Gottschalk, et al. Mol Cell Biol. 2001 May;21(9):3037-3046.

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