4. RESULTS
4.1.4 Characterization of La binding to the cyclin D1 translational start site context embedded in
-site is part of the D1-FL RNA and was deleted in all terminal B-deletion mutants (ΔB2, ΔB2.1, and ΔB2.2), thus it cannot be excluded that the La protein directly binds the AUG translational start site.
4.1.4 Characterization of La binding to the cyclin D1 translational start site context
-compared to the 1st La-RNP. Further, those two complexes are formed at the expense of the primary complex, which is represented by a fading signal intensity of the 1st La-RNP. Note the additional higher mobility band, marked by a pound sign in figure 4.1.7B, which may represent by hLa refolded free RNA rather than a La-RNP. These multiple complex formation is discussed in detail in section 5.5.2. The dissociation constant for the formation of all three complexes is KD ≈ 80 nM, thus the affinity to the synthesized RNA is less than to the D1-FL RNA (KD ≈ 45 nM).
In order to demonstrate that the complex formation in the EMSAs is mediated specifically by human recombinant La and not by contaminants from the protein purification, an antibody supershift assay was performed. This was done by utilizing 300 ng of recombinant La protein pre-incubated with 5 μl of the specific monoclonal La antibody SW5 or the isotype control mouse IgG2α,κ antibody as described in methods 3.3.6.4. Both La antibody mixtures were subsequently used for binding studies using D1-ATG RNA. Due to the higher mass of the La:SW5 complex a very low mobility La:SW5:D1-ATG complex was formed (figure 4.1.8).
The La:control antibody mixture had no effect on the mobility of the La:D1-ATG RNP.
Hence, this experiment demonstrates that the recombinant hLa protein binds the D1-ATG RNA and not any contaminating protein from the protein purification process. The signal
Figure 4.1.7: La binds to the translational start site context of cyclin D1 mRNA. A) Sequence of the D1-ATG, the AUG start codon is underlined and the most important positions -3 and +4 of the Kozak sequence are indicated. B) 10 nM of labeled D1-ATG RNA was incubated with increasing amounts of recombinant human La protein, 10, 60, 200, 400, 800, 1000, 1600, and 3000 nM and separated by a native EMSA. The free RNA and the La-RNP complexes are indicated on the left. # = free restructured D1-ATG RNA. C) Quantitative EMSA analysis. The La-RNP complexes are plotted against the molar La protein concentration. The dissociation constant was determined by non-linear regression as 80.3 nM.
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-intensity and the mobility 1st La-RNP is slightly affected in the presence of either antibody, SW5 or IgG2α,κ, which is likely due to different buffer conditions.
In order to study the importance of the cyclin D1 AUG start codon and the authentic Kozak sequence for La binding mutations were introduced in the D1-ATG sequence. In the mu2 RNA mutant the AUG was changed to AGG in order to test the importance of the uridine [62] for La recognition, whereas in the mu3 RNA mutant the Kozak sequence was changed from the strong authentic D1 sequence (5’-GCCAUGGAA-3’) to a weak Kozak sequence (5’-CAGAUGCAC-3’) [214] [215] [216].
The RNA oligoribonucleotides, mu2 (34 nts, -17 to +18) and mu3 (34 nts; -17 to +18), were synthesized by Integrated DNA Technologies Inc., for sequence information refer to figure 4.1.9A and 2.11. These RNAs contain the original cyclin D1 sequence from the 5’-UTR as well as from the ORF. The changes in the sequence are indicated in bold and the start codon is underlined (figure 4.1.9A).
The effects of these mutations on La binding to D1-ATG were studied by competitive EMSAs using mu2 and mu3 as cold competitor RNAs and labeled D1-ATG (figure. 4.1.9B).
The RNA oligoribonucleotide quality was assessed on a 10% denaturing PAGE with subsequent ethidium bromide staining (figure 4.1.9C), the RNAs were pure and of similar concentrations. The faster mobility of the mu2 and mu3 RNAs was caused by the smaller size of those oligoribonucleotides, which were 13 nucleotides smaller than the D1-ATG RNA.
The unlabeled D1-ATG RNA oligoribonucleotide served as a positive competition control, a control reaction for La binding to D1-ATG in the absence of competitor was included as well.
The D1-ATG RNA is shifted into three complexes with the La protein, the fastest mobility complex (1st La-RNP) is the major RNP complex, and the intensities of the other two
Figure 4.1.8: Supershift assay identifies human La as D1-ATG RNA-binding partner. Monoclonal anti-La antibody SW5 and control antibody IgG2α,κ were incubated with recombinant Ni-NTA purified La protein before subjected to a standard EMSA with radiolabeled D1-ATG RNA as substrate. A control reaction without protein and one without antibody was included in the EMSA study (left two lanes). The free RNA, La-RNP complexes, and the supershift are indicated on the left.
The D1-ATG RNA is shifted specifically into a complex with hLa.
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-complexes, 2nd and 3rd La-RNP are weaker. As expected unlabeled D1-ATG RNA was a strong competitor for the La:[32P]-D1-ATG complex formation (figure 4.1.9B). Increasing concentrations (100 nM, 250 nM, and 500 nM) of the start codon RNA mutant mu2 out-competes the binding of La to the [32P]-D1-ATG very efficiently (figure 4.1.9B). This suggests that the AUG to AGG substitution had no effect on La binding. In contrast the mu3 competitor RNA was not able to compete for La: [32P]-D1-ATG RNA-binding when added in excess amounts (100 nM, 250 nM, and 500 nM) suggesting that the cyclin D1 Kozak sequence surrounding the authentic CCND1 translational start site is critical for La binding.
In summary, these studies identified for the first time the La binding site within the cyclin D1 5’-UTR. This binding site is located between nts -8 and + 3 and efficient binding depends mainly on a strong Kozak consensus sequence context.
Figure 4.1.9: The binding of the hLa protein to D1-ATG RNA depends on a strong Kozak consensus sequence. Competitive EMSAs were performed to identify the role of the translational start site codon and its context. A) Sequences of the synthesized RNA oligoribonucleotides. The start codon is underlined;
changes in the nucleotide sequence are in bold. The most important positions of the consensus Kozak consensus sequence are indicated. B) Competitive EMSA were performed using 10-, 50-, 100-fold excess amounts of unlabeled RNA and 10 nM [32P]-labeled cyclin D1 ATG RNA. As negative binding and competition control served a reaction without hLa and competitor RNA, respectively. Cold D1-ATG RNA and mu2 RNA, but not mu3 RNA, are out-competing binding of La to radiolabeled D1-ATG RNA. C) Ethidium bromide staining of 400 ng cold D1-ATG, mu2, and mu3 RNAs separated by denaturing PAGE.
The purity and concentrations of all RNAs are similar.
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