La binds the 5’-UTR of cyclin D1 at the 3’-terminus

-merely one primary La-RNP (1^st La-RNP) is formed, but the D1-FL RNA is shifted into an additional secondary La-RNP (2^nd RNP) in the presence of higher La concentrations (160, 320, 640, and 960 nM). This additional secondary La-RNP is of lower mobility. The formation of both La-RNPs by hLa was considered as one La:D1-FL RNA complex for the EMSA quantification, which is displayed in figure 4.1.2D. A dissociation constant of K_D≈ 45 nM was calculated. The dissociation constant of 45 nM is in the lower nanomolar range, suggesting an affine binding of La to the cyclin D1 5’-UTR.

-DNA:RNA hybrid and 320 nM of recombinant La and analyzed by native PAGE figure 4.1.4B). The hybridization of DNA oligoribonucleotides to the RNA results in different RNA folding represented by the aberrant running behavior of free DNA:D1-FL RNA hybrids (figure 4.1.4B: 2 AS, 3 AS, 5 AS, 6 AS) in the native gel. Further, some hybrids, e.g.

3AS:D1-FL and 6AS:D1-FL, display more than one hybrid species due to alternative folding events.

Figure 4.1.3: La binds the 3’-terminus of the cyclin D1 5’ UTR. To map the binding site of La the protein’s ability to bind terminal deletion mutants of the cyclin D1 RNA was studied by EMSA. A) Cartoon of the RNA deletion mutants created, part A consists of the 99 nucleotides of the 5’ terminus of the cyclin D1 5’-UTR and lack the 3’- terminal. Part B lacks the 5’- terminal part, but contains the 110 nucleotides of the 3’- terminus. B) 320 nM of recombinant human La was incubated with 10 nM [³²P]-labeled cyclin D1 transcripts and separated by native EMSA. The free full length RNA and protein FL complex are indicated on the left. Free part A and part B RNA positions are indicated on the right, the part B and protein complex is indicated on the right as well. The hyphen represents reactions without protein. C) Denaturing PAGE of [³²P]-labeled in vitro transcribed RNAs. All transcripts were of the expected size and of high purity. FL = cyclin D1 5’ UTR full length, A = part A, B = part B, nts = nucleotides

- ⁷⁴

-The complex formation of the hLa with DNA:D1-FL hybrid (figure 4.1.4B) was not as efficient as the EMSA described above and shown for the D1-FL RNA. Because of the different mobilities of the DNA:D1-FL hybrids it is difficult to identify La:DNA:D1-FL hybrid complexes. An asterisk in figure 4.1.4B indicates those potential complexes, which are formed with 2AS:D1-FL, 5AS:D1-FL, and 6AS:D1-FL RNA hybrids. As described before the annealing of the 3AS oligoribonucleotide to the D1-FL RNA resulted in an additional low mobility RNA species in the absence of hLa. Upon La addition the distinct band representing this slower mobility hybrid RNA species is more diffused and of weaker signal intensity, which does not allow for a conclusion regarding La-binding to this region (nts -103 to -53) in the FL RNA. Hybridizing the oligoribonucleotides 4AS or 7AS to the radiolabeled D1-FL RNA resulted in one DNA:D1-D1-FL species. In addition, those two oligoribonucleotides were able to disrupt the La:D1-FL complex formation, suggesting nucleotide -79 to +3 are critical for La binding to the cyclin D1 5’-UTR.

Figure 4.1.4: La probably binds between nts -79 to +3 of the CCND1 5’-UTR. A) Cartoon of the location of the DNA oligoribonucleotides complimentary regions to the cyclin D1 5’-UTR RNA. The DNA oligoribonucleotide names are indicated on the left and the nucleotide positions are left and right of the oligoribonucleotides. B) For binding studies 320 nM recombinant hLa protein was incubated with the RNA alone and with RNA:DNA hybrids and separated by native EMSAs. Asterisks indicate possible complexes of La and the RNA:DNA hybrid. Reactions without the protein served as negative control (indicated by a hyphen). FL = full length, AS = antisense

- ⁷⁵

-Competitive EMSAs were performed to further map the binding site of La in the cyclin D1 5’-UTR more precisely. The RNP formation of La and the D1-FL RNA can be challenged in the presence of competitors, thus an unlabeled (cold) competitor RNA containing a binding site for the La protein can out-compete the La:[³²P]-D1-FL complex formation when added in excess amounts. This out-competition is then represented by a reduction or loss of the EMSA shift. Unlabeled RNAs (D1-FL, part A and B) were synthesized (3.3.1 and 3.3.2) and used as competitors.

Figure 4.1.5: Recombinant hLa binds to the 3’-terminus of the cyclin D1 5’-UTR. Cold, unlabeled RNA competitors were used to map the binding site of La in the CCND1 5’-UTR. A) Cartoon of the cold competitors RNAs, D1-FL, part A, and part B with the location of their deletion indicated. B) Ethidium bromide stained denaturing PAGE gel of unlabeled RNA transcripts. All transcripts were of expected size and high purity. C) 160 nM recombinant human La were pre-incubated with increasing concentrations of cold transcripts (10-, 25-, and 50-fold excess to labeled probe) before 10 nM [³²P]-labeled cyclin D1 full length transcript was added and separated by native EMSA. The free full RNA and La:D1-FL RNP complexes are indicated on the left. Cold FL and part B probes are competing for La binding with the radioactive labeled FL transcript. Cold part A transcript competes weakly when the highest concentration is used.

- ⁷⁶

-Figure 4.1.6: The La binding site is in close proximity to the translational start site. A) Cartoon of analyzed cold competitor RNAs D1-FL and part B deletion transcripts ΔB1, ΔB2, ΔB2.1, and ΔB2.1.

Nucleotide deletions are indicated above each transcript. B) 400 ng of each unlabeled transcript was separated by denaturing PAGE and stained with ethidium bromide. All transcripts were of expected size and high purity. C) and D) Cold RNA probes in excess amounts of 10-, 25-, and 50-fold, and additional 2.5- and 5-fold for D1-FL C) pre-incubated with 160 nM recombinant La protein before binding to D1-FL.

- ⁷⁷

-The cold transcripts were allowed to interact with 160 nM recombinant human La for 10 min before 10 nM of [³²P]-labeled D1-FL was added (refer to 3.3.6.4 for details). According to the binding curve in figure 4.1.2D the La binding site was not fully saturated at a concentration of 160 nM La, thus this La concentration allows binding of more RNA as either competitor RNA or labeled D1-FL RNA. As positive binding control served the interaction of La with labeled D1-FL RNA in absence of competitors, the unlabeled cold D1-FL served as a positive competition control in the experiments. The La:[³²P]-D1-FL interaction was analyzed on native PAGE and displayed in figure 4.1.5C. La is shown to form a complex with the labeled D1-FL in the absence of competitor RNAs (figure 4.1.5C). As expected, the interaction of hLa with the labeled D1-FL probe is lost upon the addition of increasing amounts (100 nM, 250 nM, and 500 nM; 10-, 25-, and 50-fold) of cold D1-FL RNA (figure 4.1.5C). Similarly, adding increasing amounts of part B competitor RNA reduces the complex formation of La with the labeled RNA dramatically (figure 4.1.5C). However, the part A competitor RNA is only slightly reducing the complex La:D1-FL RNP formation.

Note that only one La:D1-FL complex is formed and no other slower mobility complexes are detected. These EMSA findings and observations utilizing DNA:RNA hybrids as targets for the hLa protein (figure 4.1.4B) strongly suggest that the main La binding site is located in the 3’-terminal half of the cyclin D1 5’-UTR.

With this information as a starting point, cold competitors with deletions in part B were transcribed to again further map the La binding site more precisely by competitive EMSA.

Figure 4.1.6A shows the deletions within part B utilized for additional mapping experiments.

The primer sequences for the amplification of different DNA templates from the pRCD1F vector are listed in 2.12.1. The internal deletion in ΔB1 (nts -209 to -110 and -37 to +3) was achieved by a fusion PCR strategy as described in 3.3.1. As described above and in 3.3.2, the DNA templates were used for in vitro transcription and the RNA quality was assessed on a denaturing PAGE with subsequent ethidium bromide staining (figure 4.1.6B). The competitive EMSA was carried out as described above, as positive binding control served a La:D1-FL reaction without cold competitors. The radiolabeled D1-FL RNA was shifted into one complex with recombinant La in the absence of competitor RNAs and was successfully competed with increasing amounts of cold D1-FL RNA (25, 50, 100, 250, and 500 nM) or ΔB1 (100, 250, and 500 nM) (figure 4.1.6C). In contrast increasing concentrations (100 nM, 250 nM, and 500 nM) of competitor ΔB2, ΔB2.1, or ΔB2.2 were not able to efficiently compete for binding (figure 4.1.6C). These findings suggest that hLa binds to nts -8 and +3 of the cyclin D1 5’-UTR containing the start codon AUG. As mentioned, the translational start

- ⁷⁸

-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