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1.

Figure. From: A co-repressor assembly nucleated by Sex-lethal in the 3?UTR mediates translational control of Drosophila msl-2 mRNA.

Fig. 8. Molecular surface representation of dSXL RRMs bound to RNA. Two different orientations, vertically rotated by 180°, are shown (Handa et al., 1999). Residues that differ between Drosophila and Musca SXL are highlighted in yellow (conservative changes) and orange (non-conservative changes). A bound RNA oligomer derived from the tra mRNA is depicted using a stick representation. The solvent-exposed surface of RRM1, containing a high proportion of residues that differ from Musca SXL, is suggested to interact with translational co-repressors.

Marica Grskovic, et al. EMBO J. 2003 October 15;22(20):5571-5581.
2.

Figure. From: A co-repressor assembly nucleated by Sex-lethal in the 3?UTR mediates translational control of Drosophila msl-2 mRNA.

Fig. 6. dSXL binding to the msl-2 3′UTR promotes the assembly of high molecular weight polypeptides on sequences adjacent to the regulatory SXL-binding sites. (A) Sequence of the 3′UTR msl-2 segment relevant for translational repression (3′). SXL-binding site E, site F or sequences adjacent to those sites were substituted by CU repeats (mut1, mut3 and mut2456, respectively). (B) Binding of high molecular weight polypeptides to the sequences shown in (A), as determined by UV-crosslink/co-immunoprecipitation assays similar to those described in Figures 4 and 5.

Marica Grskovic, et al. EMBO J. 2003 October 15;22(20):5571-5581.
3.

Figure. From: A co-repressor assembly nucleated by Sex-lethal in the 3?UTR mediates translational control of Drosophila msl-2 mRNA.

Fig. 4. High molecular weight proteins associate with the msl-2 3′UTR in a dSXL-dependent manner. The 32P-labelled msl-2 3′probe was incubated in a typical translation reaction in the absence (lanes 1–5) or presence (lanes 6–10) of recombinant dSXL. After UV-crosslinking and digestion with RNase T1, complexes were immunoprecipitated with α-SXL antibody or pre-immune serum (ctrl). T, total set of crosslinked proteins before immunoprecipitation; S, supernatant after immunoprecipitation; P, pellet. The position of crosslinked recombinant dSXL is indicated. Asterisks mark proteins that crosslink consistently, bands marked with a dot are not always seen.

Marica Grskovic, et al. EMBO J. 2003 October 15;22(20):5571-5581.
4.

Figure. From: A co-repressor assembly nucleated by Sex-lethal in the 3?UTR mediates translational control of Drosophila msl-2 mRNA.

Fig. 3. Tethered function analysis of dSXL. (A) Schematic diagram of the experimental approach. The RNA-binding peptide λ was fused to the N-terminus of dSXL to yield λSXL. λSXL binds to mRNAs containing dSXL-binding sites (black ovals) via its RRMs, and to mRNAs harbouring boxB/λ-binding sites (black squares) via the λ peptide. λLλ mRNA is a derivative of BLEF mRNA, in which the dSXL-binding sites are replaced by boxB elements. The open rectangles denote the luciferase open reading frame, and the thin lines represent msl-2 UTR sequences. (B) Translational inhibition of indicated mRNAs by λSXL and λU1A at a 20-fold molar excess of protein to RNA. Consistent results were obtained when the same range of protein concentrations as that used for Figures 1 and 2 was tested (data not shown).

Marica Grskovic, et al. EMBO J. 2003 October 15;22(20):5571-5581.
5.

Figure. From: A co-repressor assembly nucleated by Sex-lethal in the 3?UTR mediates translational control of Drosophila msl-2 mRNA.

Fig. 2. RRM1 of dSXL contributes to translational repression. (A) Schematic representations of the Drosophila and Musca SXL proteins (dSXL and mSXL, respectively) and their RRMs (dRBD3 and mRBD), as well as of hybrid proteins containing combinations of these (hRBD1, hRBD2 and hRBD2c7). The corresponding amino acid positions of dSXL and mSXL included in the derivatives are indicated. (B) WT and BLEF mRNAs were assayed for repression by dSXL and mSXL, and similar results were obtained for both mRNAs. Shown here is the integration of those results. The translation inhibition curves are normalized for the binding activity of the mSXL and dSXL preparations, as measured by gel mobility-shift assay using the minimal 5′ and 3′ probes (C). (D) Translational repression by the RRM derivatives. Translation reactions were incubated with increasing amounts of dRBD3 (green line), mRBD (purple line), hRBD1 (red line), hRBD2 (light blue line) or hRBD2c7 (orange line) and analysed as described in Figure 1C. dRBD4 (dark blue line) is shown for reference. (E) RNA binding of the RRM derivatives. Gel mobility-shift assays were performed with increasing concentrations of mRBD, dRBD3, hRBD1, hRBD2 and hRBD2c7 and a probe containing 215 nt of the msl-2 5′UTR (positions 189–403).

Marica Grskovic, et al. EMBO J. 2003 October 15;22(20):5571-5581.
6.

Figure. From: A co-repressor assembly nucleated by Sex-lethal in the 3?UTR mediates translational control of Drosophila msl-2 mRNA.

Fig. 7. Factors present in the Drosophila embryo extracts are required for translational repression of msl-2 mRNA. (A) Binding of dSXL to the 3′ probe or its derivatives (see Figure 6A), measured by gel mobility-shift assay. A probe containing the NRE of maternal hunchback mRNA was used as a specificity control (ctrl). (B) Functional titration of dSXL co-repressors. Drosophila embryo extracts were pre-incubated with 3′, mut2456, mut1, mut3, 5′ or control (multiple cloning site from pBluescript vector) RNAs, and typical translation reactions were subsequently assembled. The molar amount of competitor RNA was 10-, 100- or 200-fold higher than that of reporter mRNA. The translation assay was performed as described in Figure 1C. For simplicity, only the data obtained at 50-fold molar excess of dSXL over reporter mRNA are shown. Similar results were obtained with WT and BLEF mRNAs. (C) Specific titration of co-repressor crosslinking. Typical translation reactions containing Drosophila embryo extracts, 3′ probe and 50-fold molar excess of dRBD4 over probe were incubated with unlabelled 3′ or mut2456 competitor RNAs. The molar amount of competitor RNA was 10-, 50-, 100- or 200-fold higher than that of the probe. The UV-crosslink/co-immunoprecipitation assays were performed as described in Figure 4.

Marica Grskovic, et al. EMBO J. 2003 October 15;22(20):5571-5581.
7.

Figure. From: A co-repressor assembly nucleated by Sex-lethal in the 3?UTR mediates translational control of Drosophila msl-2 mRNA.

Fig. 5. (A) The association of high molecular weight polypeptides with the msl-2 3′UTR correlates tightly with translational repression. 3′ (lanes 1–6) or 5′ (lanes 7–12) RNA probes were incubated in Drosophila translation extracts including recombinant dSXL or SXL derivatives that either repress translation efficiently (dRBD2 and dRBD4) or not (mSXL, dRBD3, mRBD). The ability of each derivative to repress translation is indicated. After UV-crosslinking and immunoprecipitation, proteins present in the pellet were separated in a denaturing 8% acrylamide gel and visualized by autoradiography. Asterisks mark polypeptides of high intensity that specifically co-immunoprecipitate with dSXL (see also Figure 4). (B) The slow mobility band (∼215 kDa) contains SXL. dRBD4 lacking GST (lane 1), or dRBD4 and dSXL (both containing a GST tag, lanes 2 and 3) were incubated in Drosophila translation extracts containing 3′ RNA probe. In lanes 4 and 5, the sample was treated with TEV protease after UV-crosslinking and immunoprecipitation. Proteins were separated in a 6–15% acrylamide gel and visualized by autoradiography. The right panel shows an extended exposure of the upper portion of the gel shown on the left after adjustment of brightness and contrast to reduce background noise.

Marica Grskovic, et al. EMBO J. 2003 October 15;22(20):5571-5581.
8.

Figure. From: A co-repressor assembly nucleated by Sex-lethal in the 3?UTR mediates translational control of Drosophila msl-2 mRNA.

Fig. 1. Mapping the translational repressor domain of dSXL. (A) Schematic representation and domain organization of dSXL and its deletion derivatives. The amino acid numbers included in each derivative are indicated. (B) Scheme of the RNA constructs used in this study. WT mRNA contains the full-length 5′ (626 nt) and 3′ (1047 nt) UTRs of msl-2 fused to the firefly luciferase open reading frame (Gebauer et al., 1999). The SXL-binding sites are denoted A to F (black ovals). BLEF mRNA contains the minimal msl-2 sequences required for translational repression, which consist of 69 nt in the 5′UTR including site B, and 46 nt in the 3′UTR including sites E and F (Gebauer et al., 2003). Probes used for gel mobility-shift and UV-crosslink assays are also depicted. (C) WT mRNA was translated in Drosophila embryo extracts in the presence of increasing amounts of dSXL (pink line), dRBD1 (yellow line), dRBD2 (light blue line), dRBD3 (green line) or dRBD4 (dark blue line). Renilla luciferase mRNA was co-translated as an internal control (black line). Firefly luciferase values were corrected for Renilla expression and plotted as the percentage of the activity obtained in the absence of recombinant protein against the molar ratio of protein to mRNA.

Marica Grskovic, et al. EMBO J. 2003 October 15;22(20):5571-5581.

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