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1.
FIG. 4.

FIG. 4. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

Xenopus xSLBPs and the chimeric proteins. xSLBP1 and xSLBP2 were arbitrarily divided into three domains, the central 73-amino-acid RBD and the N- and C-terminal domains. These domains were interchanged to give the six chimeric proteins shown at the bottom. The activity of the proteins in processing histone pre-mRNA in vivo and in vitro is indicated (26).

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.
2.
FIG. 2.

FIG. 2. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

SLBP must bind mRNA to stimulate translation. (A) Lysates containing hSLBP or two mutant hSLBPs that do not bind the stem-loop were incubated with the standard polyadenylated CAT mRNA and the Luc-TL or Luc-SL mRNA and assayed for luciferase activity. hSLBPRR/KK has arginines 10 and 11 in the RBD changed to lysines, and hSLBPYY/SS has tyrosines 24 and 27, also in the RBD, changed to serines (12). (B) Lysates containing xSLBP1 were incubated with the standard polyadenylated CAT mRNA and the Luc-TL or Luc-SL mRNA in the presence of either excess 30-nt RNA containing the histone stem-loop (SL) or with the reverse stem-loop (RS) and then assayed for luciferase activity.

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.
3.
FIG. 6.

FIG. 6. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

Effect of xSLBP1 deletion mutant forms on translation activation in vitro. (A) Deletions in the amino- and carboxy-terminal regions of xSLBP1. (B) Lysates containing the xSLBP1 deletion mutant forms shown in panel A were incubated with the standard polyadenylated CAT mRNA and the Luc-TL or Luc-SL mRNA. Protein synthesis (top) and luciferase activity (bottom) were assayed as described in Fig. 1C and D, respectively. The bar graph shows the averages of two experiments with two different batches of RRL, and the error bars represent the standard deviations.

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.
4.
FIG. 5.

FIG. 5. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

Assay of the effects of xSLBP chimeras on translation in vitro. Lysates containing the xSLBP chimeras shown in Fig. 4 were incubated with the standard polyadenylated CAT mRNA and the Luc-TL or Luc-SL mRNA and assayed for luciferase activity. The reaction mixtures were analyzed by gel electrophoresis, and the proteins were detected by autoradiography (top). Luciferase activity was assayed by luminometry, and the fold activation was expressed as the ratio of Luc-SL activity to Luc-TL activity (bottom). The bar graph shows the averages of two experiments with two different batches of RRL, and the error bars represent the standard deviations.

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.
5.
FIG. 9.

FIG. 9. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

Translation activation by MS2-hSLBP fusion protein in vitro. Lysates containing MS2 (lanes 1 to 3), MS2-hSLBP fusion protein (lanes 4 to 6), or hSLBP (lanes 7 to 9) were incubated with the standard polyadenylated CAT mRNA and the Luc-TL, Luc-SL, or Luc-MS2 mRNA. Protein synthesis (top) and luciferase activity (bottom) were assayed as described in Fig. 3C and D, respectively. The bar graph shows the averages of three experiments with two different batches of RRL, and the error bars represent the standard deviations.

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.
6.
FIG. 8.

FIG. 8. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

Alanine scanning of the xSLBP1 translation activation domain. (A) Amino acids 65 to 89 in the N-terminal region of xSLBP1 were replaced sequentially, five residues at a time, with five alanines (underlined). WT, wild type. (B) Lysates containing the five-alanine substitutions shown in panel A were incubated with the standard polyadenylated CAT mRNA and the Luc-TL or Luc-SL mRNA. Protein synthesis (top) and luciferase activity (bottom) were assayed as described in Fig. 1C and D, respectively. The bar graph shows the averages of two experiments with two different batches of RRL, and the error bars represent the standard deviations. (C) Oocytes injected at T0 with the xSLBP1 five-alanine substitution mRNAs and then again at T1 = 16 h with Luc-SL or Luc-TL mRNA in combination with R-Luc-SL mRNA were harvested at T2 = 32 h, processed, and analyzed for luciferase activity as described in Fig. 1C. The averages of two experiments with two different batches of oocytes are shown. The error bars represent the standard deviations. (D) A fraction corresponding to one oocyte worth of the same lysate used for the luciferase assay of panel C was used for detection of the xSLBP1 five-alanine substitution mutant proteins. The Western blot shown was performed as described previously. Oocytes injected at T0 and T1 with buffer show the level of endogenous xSLBP1 protein (lane 13).

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.
7.
FIG. 7.

FIG. 7. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

Effect of xSLBP1 deletion mutant forms on translation activation in vivo. (A) Oocytes were injected with xSLBP1 deletion mutant mRNAs (Fig. 6A) or buffer at T0. At T1, they were injected with the R-Luc-SL mRNA together with the Luc-SL or Luc-TL mRNA. Oocytes were harvested at T2 and assayed for Renilla and firefly luciferase activities as described in Fig. 3C. The results shown are the averages of two experiments with two different batches of oocytes, with the error bars representing the standard deviations. (B) One oocyte equivalent of lysate from oocytes injected with mRNAs encoding the mutant SLBPs described in panel A was mixed with 5 ng of radiolabeled stem-loop RNA and analyzed by gel electrophoresis. The xSLBP/SL complex was detected by autoradiography. (A) Oocytes injected with buffer (lanes 9 to 11) or mRNAs encoding mutant SLBPs containing the intact C-terminal region of xSLBP1 (lanes 3 to 8) were analyzed by Western blotting as described in Fig. 3B. (B) Total RNA from oocytes injected as described in panel A and harvested at T2 = 32 h was hybridized to 3′-end-radiolabeled DNA complementary to the 3′ end of Luc-SL mRNA and subjected to S1 nuclease treatment. The protected DNA fragment was resolved by PAGE and detected by autoradiography. The Luc-SL mRNA protects a fragment 523 nt long that maps to the expected 3′ end, whereas Luc-TL mRNA protects a 508-nt fragment that maps to the start of the loop, where the sequences of Luc-SL and Luc-TL diverge.

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.
8.
FIG. 3.

FIG. 3. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

SLBP stimulates translation of Luc-SL mRNA in vivo. (A) Schematic of the in vivo assay. Xenopus oocytes were injected either with buffer or with a synthetic mRNA encoding an SLBP (T0). At 12 to 16 h later (T1), the oocytes were injected with the reporter firefly luciferase mRNA (Luc-test) mixed with a synthetic mRNA encoding Renilla luciferase ending in the histone stem-loop (R-Luc-SL). After incubation for an additional 12 to 16 h (T2), the oocytes were harvested and assayed for both luciferase activity and SLBP expression by Western blotting and by mobility shift assay with a radiolabeled stem-loop as the probe. The stability of reporter mRNAs was determined by S1 nuclease protection with a radiolabeled DNA fragment complementary to the 3′ end of the Luc-SL mRNA. (B) Oocytes were injected at T0 with buffer (lanes 3, 4, 7, and 8) or with xSLBP1 (lanes 1and 2) or xSLBP2 (lanes 5 and 6) and then at T1 with either the Luc-SL mRNA (lanes 1, 3, 5, and 7) or Luc-TL mRNA (lanes 2, 4, 6, and 8), together with R-Luc-SL mRNA. Oocytes were collected and lysed 8, 16, and 24 h later (T2 = 8, 16, and 24 h). Total oocyte protein was resolved by gel electrophoresis, transferred to nitrocellulose, and xSLBP1 (lanes 1 to 4) or xSLBP2 (lanes 5 to 8) was detected with appropriate antibodies. Panel B shows the Western blots of samples collected at T2 = 16 h. (C) Oocytes were injected with buffer (top), xSLBP1 mRNA (middle), or xSLBP2 mRNA (bottom). After 16 h, the oocytes were injected with the R-Luc-SL mRNA together with the Luc-SL, Luc-TL, or Luc-polyA mRNA. At 8, 16, or 24 h later, oocytes were harvested and assayed for Renilla and firefly luciferase activities. The ratio of Luc-TL luciferase activity to Renilla luciferase activity was determined, and this value was set at 1. The activity of the Luc-SL and Luc-polyA mRNAs was expressed relative to that of the Luc-TL mRNA.

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.
9.
FIG. 1.

FIG. 1. From: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro.

SLBP stimulates histone mRNA translation in vitro. (A) Structure of the 3′ end of the luciferase reporter mRNAs. The Luc-SL mRNA ends in the histone stem-loop that binds SLBP, the Luc-TL mRNA ends in a stem-loop that does not bind SLBP, and the Luc-polyA mRNA ends in a poly(A) tail 50 nt long. ORF, open reading frame. (B) Schematic of the in vitro translation assay. An aliquot of RRL was incubated either with buffer or with SLBP DNA in the presence of T7 RNA polymerase and [35S]methionine. After incubation for 90 min, an aliquot of the lysate was mixed with a fresh aliquot of reticulocyte lysate together with a luciferase uncapped reporter mRNA and a polyadenylated uncapped CAT mRNA as an internal standard. After incubation for 90 min at 30°C, the reaction products were analyzed by SDS-PAGE and the in vitro-synthesized proteins were detected by autoradiography. An aliquot of the assay was analyzed for luciferase activity by luminometry. (C) Lysates containing no SLBP (lanes 1 to 3), xSLBP2 (lanes 4 to 6), or xSLBP1 (lanes 7 to 9) were incubated with the standard polyadenylated CAT mRNA and the Luc-TL (lanes 1, 4, and 7), Luc-SL (lanes 2, 5, and 8), or Luc-polyA (lanes 3, 6, and 9) mRNA. The reaction products were analyzed by gel electrophoresis, and the proteins were detected by autoradiography. (D) The autoradiogram (panel C) was quantified, and relative luciferase activity was calculated. In addition, an aliquot of each reaction mixture was analyzed for luciferase activity with a luminometer. Quantification of the results by PhosphorImager and by luciferase assay yielded identical results. Panel D shows the results of an average of five experiments with five different batches of RRL, and the error bars represent the standard deviation. The fold activation is the result of luciferase activity from the Luc-SL and Luc-polyA mRNAs divided by the luciferase activity from the Luc-TL mRNA.

Ricardo Sànchez, et al. Mol Cell Biol. 2002 October;22(20):7093-7104.

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