1. Drosophila Sex-Lethal Inhibits the Stable Association of the 40S Ribosomal Subunit with msl-2 mRNA 
Fátima Gebauer, Marica Grskovic, Matthias W Hentze 
Molecular Cell 
Volume 11, Issue 5, Pages (May 2003)
DOI: /S (03)00176-X

2. Figure 1
Translational Repression of msl-2 mRNA Is Independent of the Cap Structure
(A) Schematic representation of the WT msl-2 construct. The full-length 5′ (626 nt) and 3′ (1047 nt) UTRs of msl-2 were fused to the firefly luciferase coding region. The SXL binding sites are denoted A to F (filled ovals).
(B) Translation of msl-2 is stimulated by the m7GpppG-cap structure. In vitro translation extracts derived from Drosophila embryos were primed with WT mRNA modified with either a canonical m7GpppG cap or an ApppG cap. The translation output was monitored by measuring luciferase activity and is represented as a percentage of that obtained with the m7GpppG-containing mRNA.
(C) Translation inhibition by SXL is independent of the cap structure. Increasing amounts of purified recombinant SXL were added to translation reactions containing the mRNAs described in (B). The quantities of SXL used were 2, 4, 8, 20, 40, and 80 ng per 10 μl reaction. Renilla luciferase mRNA translation was monitored as an internal control. The firefly luciferase values were corrected for Renilla expression and were plotted as the percentage of the luc activity obtained in the absence of SXL against the molar excess of SXL to template mRNA used in the reaction.
Molecular Cell  , DOI: ( /S (03)00176-X)

3. Figure 2 msl-2 Sequences Involved in SXL-Mediated Regulation
(A) Mutational analysis of msl-2 mRNA. The different constructs tested are schematically represented with the corresponding name indicated on the left. Deletions are denoted as dotted lines while substitutions are denoted in red color. The open rectangle represents the luciferase open reading frame. The length of the UTRs is also indicated. Translation of these mRNAs was tested in the same way as described in the legend to Figure 1C. For simplicity, only the data obtained at a 20-fold molar excess of SXL over mRNA are shown. The other tested concentrations yielded consistent results.
(B) Translation inhibition of the BLEF and WTS mRNAs (left and right panels, respectively) and mutants containing substituted SXL binding sites (denoted as empty ovals). Translation of BLEF and derivatives was assessed as described in the legend to Figure 1C. Translation reactions containing WTS and derivatives were performed in the presence of 35S-methionine. SXL was added at 0, 6, 20, and 40 molar excess with respect to mRNA, and CAT mRNA was cotranslated as an internal control. Translation products were selected by immunoprecipitation with α-FLAG and α-CAT antibodies, resolved in a 15% acrylamide gel, and quantitated in the phosphorimager. For WTS (A−) and WTS (cap−) mRNAs, the average of two experiments is shown.
(C) Representative examples of the translation reactions containing WTS mRNA and mutants quantitated in (B). In the case of WTS (cap−) mRNA, the reaction was linearly scaled up 5-fold to increase signal.
(D) Sequences flanking the SXL binding sites EF in the 3′ UTR are important for regulation. Segments of 8 (mut 1 to mut 5) or 6 (mut 6) nucleotides in the EF 3′ UTR were substituted by (CT)4 or (CT)3, respectively. Translation of mRNAs containing these mutations was tested as described in the legend to Figure 1C. These mRNAs contained a wild-type 5′ UTR including 354 nt of msl-2 sequence (same as min mRNA in Figure 2A). Translation of the wild-type mRNA (EF) and a deletion mutant lacking msl-2 sequences in the 3′ UTR (Δ3′) were carried out as positive and negative controls, respectively. Only the data points corresponding to a 20-fold molar excess of SXL to template mRNA are shown.
(E) The 3′ UTR msl-2 sequences provide a function in addition to SXL binding. Translational repression profiles of BLEF mRNA, containing the normal configuration of SXL binding sites, and BLB mRNA, containing the 5′ UTR sequence of B2 mRNA both in the 5′ and 3′ UTRs.
Molecular Cell  , DOI: ( /S (03)00176-X)

4. Figure 3 SXL Regulates an Early Step in Translation Initiation
(A) SXL blocks 80S ribosome formation. WTS (left panel) or BEFmutS (right panel) mRNAs, containing a cap and a poly(A) tail of 73 residues, were 32P labeled and incubated in a translation reaction containing cycloheximide in the absence (blue line) or presence (pink line) of SXL. The translation reactions were loaded on 5%–25% linear sucrose gradients, and complexes were resolved by centrifugation. After fractionation from the bottom of the gradient, the radioactivity was monitored, expressed as the percentage of total counts recovered, and plotted against the fraction number. The position of ribosomal complexes was determined by monitoring the absorbance at 254 nm.
(B) SXL controls small ribosomal subunit association with the mRNA. Radiolabeled WTS (left panel) and BEFmutS (right panel) mRNAs were incubated in the absence (blue line) or presence (pink line) of SXL in a translation reaction containing cycloheximide and GMP-PNP, and processed as described in (A). The sedimentation of subpolysomal mRNPs was determined by inclusion of EDTA (green line).
Molecular Cell  , DOI: ( /S (03)00176-X)