DEV002212 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Binding of GST-Pum and Nos to the CycB 3′ UTR. Above, gel mobility shift experiments using CycB RNA fragments A-F and GST-Pum at concentrations of 0, 0.15, 0.62 and 2.5 μM in lanes 1-4 of each titration, respectively. Note that dimeric GST-Pum binds to fragment D, in contrast to either the purified monomeric Pum RBD or the native Pum in embryonic extracts. Below, a western blot showing recruitment of His-tagged Nos to RNPs formed with GST-Pum and the indicated CycB fragments. The mutant hb site is UG21AC (Sonoda and Wharton, 1999). Note the lower molecular weight Nos degradation product in the input lane that does not bind Pum-RNA complexes, as described (Sonoda and Wharton, 1999).

• Supplemental Figure 2 -

  Fig. S2. Identification of a functional NRE. (A) Regulation of various maternal CycB mRNAs assayed in embryos from CycB²/+ females that also bear the indicated CycB transgene. (B) Deletion of the CycB NRE in the ΔI+II mRNA has no effect on its localization to the pole plasm, as assayed by in situ hybridization. (C) Confocal images of CycB (green) and Vasa (red) in various embryos. Note that repression is restored to pum⁻ embryos upon expression of the C-terminal portion of Pum containing the RBD (Δ2′ in Wharton et al., 1998), demonstrating that the large N-terminal domain of Pum is dispensable for regulation of CycB as is the case for regulation of hb.

• Supplemental Figure 3 -

  Fig. S3. Binding of Pum and Nos to the CycB NRE. (A) Sequence of the wild-type 50 nt CycB NRE (nt 594-643 of the 3′UTR) and various mutant (mut) sites. The positions of binding sites for Pum (red) and Nos (green) inferred from the experiments in B-D are indicated. (B) Gel mobility shift experiments showing binding of the Pum RBD to various RNAs, as indicated. For the titration on the left, lane 1 contains no protein and lanes 2-16 contain 0.004-1.3 μM Pum, increasing in 1.5-fold steps in successive lanes. In the titrations on the right, Pum (0, 0.13, 0.25, 0.65, 1.29, 2.59, 3.88, 5.18, 7.77, and 10 μM in lanes 1 to 10) was incubated with various RNAs, as indicated. The low-mobility complex that we assume contains (at least) two molecules of Pum is indicated with an arrowhead here and in C. (C) Gel mobility shift experiments showing the binding of Pum RBD (0, 0.13, 1.29, 5.17 μM in lanes 1 to 4 of each titration) to the indicated CycB NREs. Binding of Pum is affected by substitutions in the NREs highlighted in red. (D) Recruitment of His6-tagged Nos to complexes of GST-Pum and the CycB NREs indicated above. Essentially, no His6-Nos is recruited in control experiments using the mut1+2 RNA that does not detectably bind Pum (not shown). (E) Recruitment of His6-tagged Brat to GST-Pum-RNA-His6-Nos complexes assembled on various RNA fragments. The eIF-4E RNA binds Pum with high affinity (Menon et al., 2004), but neither Nos nor Brat is recruited above background levels to the GST-Pum-eIF-4E RNA complex.