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1.
FIG. 3.

FIG. 3. From: Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase.

Threonines 60 and 61 and proline 62 are required for SLBP degradation. (A) Mutations in the SFTTP sequence are shown at the left of each blot. The mutant SLBP constructs shown in panel A were stably transfected into HeLa cells. The cells were synchronized by a double-thymidine block, and protein extracts were prepared at 2-h intervals after release from the double-thymidine block. The levels of SLBP were analyzed by Western blotting using the anti-SLBP antibody that detects both the endogenous and His-tagged SLBP (His-SLBP). Lane 1 contains cells blocked at the G1/S border, and lanes 2 to 7 contain cells after release from the block. The time after release is indicated above each lane. (B) Flow cytometry analysis of the cells transfected with the T60/T61 mutant of SLBP. Chromosome content is shown on the x axis, and cell number is shown on the y axis.

Lianxing Zheng, et al. Mol Cell Biol. 2003 March;23(5):1590-1601.
2.
FIG. 6.

FIG. 6. From: Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase.

Histone mRNA is degraded in the presence of a stable SLBP. (A) Cells transfected with the T61A SLBP were synchronized by a double-thymidine block, and the cells were harvested in S phase (4 h after release) and fractionated into nuclear (N) and polyribosomal (P) fractions. The amount of SLBP in each fraction was analyzed by Western blotting using the anti-SLBP antibody to detect both the His-tagged SLBP (His-SLBP) and endogenous SLBP. (B and C) HeLa cells transfected with the S58A mutant SLBP (B) and the T60A/T61A SLBP (C) were synchronized by a double-thymidine block. RNA was prepared from the blocked cells (lanes 1) and from cells at 2-h intervals after release from the block (lanes 2 to 7). Ten micrograms of total cell RNA was resolved by gel electrophoresis, transferred to a Nytran membrane, and probed with either the histone H3 gene (top panel) or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. The time after release and the cell cycle stage is indicated above each lane.

Lianxing Zheng, et al. Mol Cell Biol. 2003 March;23(5):1590-1601.
3.
FIG. 5.

FIG. 5. From: Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase.

LC-MS/MS analysis of His-tagged SLBP phosphorylation. (A) The purified His-tagged SLBP (Fig. 4C, lane 2) was digested with trypsin and subjected to mass spectrometric analysis. Wild-type SLBP (left) and the KRKL mutant SLBP (right) are shown. Extracted ion chromatograms of the product ion at m/z 527.6, which corresponds to the loss of a mass increment of 32.67 or H3PO4 from the triple-charged monophosphorylated peptide T13 of His-tagged SLBP at m/z 560.27. (B) LC-MS/MS spectrum of the tryptic peptide T13 from the His-tagged KRKL mutant SLBP, containing the SFTTP sequence, showing that the phosphoryl group is on either S58 or T60, but not T61. (C) Extracted ion chromatogram of the product ion at m/z 521.63 corresponding to the loss of 65.34 (two HPO3) from the triple-charged diphosphorylated SLBP peptide T13 (m/z 592.94) from the wild-type sample. (D) LC-MS/MS spectrum from the precursor ion at m/z 592.94 obtained at 70.65 min (panel C), confirming the presence of the diphosphorylation of the SLBP peptide T13 in the wild-type sample. The achieved fragment ions together with the MS/MS analysis from a synthetic SLBP T13 peptide phosphorylated at the equivalent position as T60 and T61 (data not shown) demonstrate that both threonine residues in the wild-type sample are phosphorylated. amu, atomic mass unit.

Lianxing Zheng, et al. Mol Cell Biol. 2003 March;23(5):1590-1601.
4.
FIG. 8.

FIG. 8. From: Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase.

G2 extracts from cells expressing a stable SLBP are active in histone pre-mRNA processing. Cells stably expressing the T60A mutant SLBP were synchronized, and nuclear extracts and cytoplasm were prepared from S- and G2-phase cells. Equal amounts of protein were used in the assays for SLBP activity below. (A) Ten micrograms of nuclear (N) and cytoplasmic (C) proteins from S-phase (lanes 1 and 2) or G2-phase (lanes 3 and 4) cells were resolved by electrophoresis on an SDS-8% polyacrylamide gel, and SLBP was detected by Western blotting using antibody against the C-terminal peptide of SLBP. Note that the cytoplasm had 10 times more protein than the nuclear extract, so only 10% as many cell equivalents were analyzed in lanes 2 and 4. His-SLBP, His-tagged SLBP. (B) Equal amounts of nuclear extract from S-phase and G2-phase cells was incubated with a radiolabeled stem-loop (SL) (lane 1), and the complexes were resolved by native gel electrophoresis. (C) Equal amounts of S-phase (lanes 1 and 2) and G2-phase (lanes 3 and 4) cells were incubated with radiolabeled histone pre-mRNA, and the RNA products were analyzed by gel electrophoresis and detected by autoradiography. In lanes 2 and 4, recombinant SLBP was added to the extracts prior to addition of the substrate. The positions of pre-mRNA (Unproc) and processed mRNA (Proc) are shown to the right of the gel.

Lianxing Zheng, et al. Mol Cell Biol. 2003 March;23(5):1590-1601.
5.
FIG. 7.

FIG. 7. From: Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase.

SLBP is the only essential histone pre-mRNA processing factor missing from G1 and G2 nuclear extracts. (A) HeLa cells were synchronized by a double-thymidine block, and equal cell equivalents of nuclear, polyribosomal, and cytosol fractions were prepared from S phase (3 h after release [lanes 2, 5, and 8]), G2 phase (8.5 h after release [lanes 3, 6, and 9]) and G1 phase (14 h after release [lanes 1, 4, and 7]) and analyzed for SLBP by Western blotting. (B) The nuclear extracts prepared from S-, G2-, and G1-phase cells were analyzed for processing activity using the H2a-614 pre-mRNA as a substrate. Recombinant SLBP was added to the extracts in lanes 2, 4, and 6 prior to addition of the pre-mRNA substrate. RNA was prepared from the reaction mixtures, and the pre-mRNA (Unproc) and processed mRNA (Proc) were resolved by gel electrophoresis. The results are representative of three independent preparations of synchronous cells. (C) Flow cytometric analysis of the cells used for preparation of the S-phase (3 h), G2-phase (8.5 h), and G1-phase (14 h) extracts, respectively. Chromosome content is shown on the x axis, and cell number is shown on the y axis.

Lianxing Zheng, et al. Mol Cell Biol. 2003 March;23(5):1590-1601.
6.
FIG. 2.

FIG. 2. From: Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase.

Amino acids 58 to 62 and 95 to 98 are required for SLBP degradation. (A) The sequences of the N-terminal regions of human (H), mouse (M), and Xenopus (X) SLBPs are shown (amino acids 55 to 124 in the human and mouse protein). The three conserved possible phosphorylation sites that were mutated are shown in bold type. The putative cyclin binding site (KRKL) is underlined. Conserved (asterisks) and similar (dashes) amino acids between two sequences are indicated. (B to E) Plasmids encoding the mutant His-tagged SLBPs (His-SLBPs) indicated above each panel were stably transfected into HeLa cells, and the cells were synchronized by a double-thymidine block. Protein extracts were made from the blocked cells (lanes 1) and from cells every 2 h after release from the block (lanes 2 to 7). Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose filters, and the endogenous and His-tagged SLBPs were detected by Western blotting with the anti-SLBP antibody. The time after release and the cell cycle stage is indicated above each lane.

Lianxing Zheng, et al. Mol Cell Biol. 2003 March;23(5):1590-1601.
7.
FIG. 1.

FIG. 1. From: Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase.

Cell cycle regulation of HA-tagged and His-tagged SLBP. SLBP was cloned into the pcDNA3-HA (A) or pcDNA3-his (B) vector and transfected into HeLa cells, and stably transfected cells were selected. The cells were synchronized by a double-thymidine block, arresting the cells at the G1/S border (time zero [lane 1]). Cells were released from the block, and total proteins were prepared at 2-h intervals after release (times shown above the lanes). The proteins were resolved by gel electrophoresis on an SDS-12.5% polyacrylamide gel, and the proteins were transferred to nitrocellulose filters and assayed by Western blotting using an SLBP antibody. The positions of the HA-tagged SLBP (HA-SLBP) or His-tagged SLBP (His-SLBP) and endogenous SLBP are indicated to the left of the gels. (C) Flow cytometry analysis of the synchronized cells in panel B. Chromosome content is shown on the x axis, and cell number is shown on the y axis. (D) Three deletion mutants of SLBP tagged with HA at the amino terminus and missing the first 54 amino acids (HA-Δ54N), the last 72 amino acids (HA-Δ72C), or amino acids 57 to 121 and the last 13 amino acids (HA-Δ65N-Δ13C) were transfected into HeLa cells, and stably transfected cells were selected. The cells were synchronized by a double-thymidine block, arresting the cells at the G1/S border (time zero [lane 1]). Cells were released from the block, and total proteins were prepared at 2-h intervals after release (times shown above the lanes). The proteins were resolved by gel electrophoresis on an SDS-12.5% polyacrylamide gel, transferred to nitrocellulose filters, and assayed by Western blotting using an anti-HA antibody. WT, wild type; N-ter and C-ter, N- and C-terminal regions, respectively; RBD, RNA binding domain.

Lianxing Zheng, et al. Mol Cell Biol. 2003 March;23(5):1590-1601.
8.
FIG. 4.

FIG. 4. From: Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase.

Purification of His-tagged SLBP. (A) HeLa cells were synchronized by a double-thymidine block and released for 4 h, and then MG132 was added for an additional 4 h. Cells were fractionated into nuclear and cytoplasmic fractions, and samples from equal amounts of cells were resolved by gel electrophoresis, and SLBP was detected by Western blotting. Lanes 1 and 2 contain cells harvested 4 h after release from the thymidine block (control). Lanes 3 and 4 contain cells harvested after treatment with MG132 for 6 h, starting 4 h after release. The cytoplasmic lysate from cells treated with MG132 for 6 h (lane 5) was incubated with a biotinylated wild-type (WT) stem-loop RNA (lane 6) or a biotinylated mutant stem-loop RNA with the stem sequence reversed (RS) (lane 7) and then the RNA bound to streptavidin beads. An equivalent amount of total lysate and protein bound to the beads was analyzed by gel electrophoresis, and the SLBP was detected by Western blotting. (B) Cells expressing the His-tagged SLBP (His-SLBP) were synchronized by a double-thymidine block. Four hours after release from the block, MG132 was added for 4 h. Total cell extract was fractionated on Ni-agarose, and the bound proteins were eluted with imidazole. Aliquots of the total lysate (0.1% [lane 1]), unbound protein (0.1% [lane 2]), and bound protein (1% [lane 3]) were analyzed by gel electrophoresis, and the SLBP was detected by Western blotting. The position of the 51-kDa molecular mass marker is indicated to the right of the blot. (C) A biotinylated stem-loop RNA was incubated with the proteins eluted from Ni-agarose, and then streptavidin agarose was added. The resin was washed extensively, and an aliquot of the beads analyzed by SDS-polyacrylamide gel electrophoresis (5% of the total [lane 2]) together with an aliquot of the proteins bound to Ni-agarose (1% of the total [lane 1]). The proteins were detected by staining with Coomassie blue. Note that the His-tagged SLBP (His-SLBP) is not detectable by staining of the total proteins bound to Ni-agarose. The positions of molecular mass markers (in kilodaltons) (lane 3) are shown to the left of the gel.

Lianxing Zheng, et al. Mol Cell Biol. 2003 March;23(5):1590-1601.

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