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The organization of RNA contacts by PTB for regulation of FAS splicing.

Mickleburgh I, Kafasla P, Cherny D, Llorian M, Curry S, Jackson RJ, Smith CW - Nucleic Acids Res. (2014)

Bottom Line: RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA.The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker.Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK.

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Effects of mutations altering the PTB RRM3-4 di-domain on its splicing and RNA-binding functions. (A) FAS WT RNA was spliced in vitro in HeLa nuclear extract in the absence (−) or presence of recombinant PTB mutants as indicated. PTB mutants were added to 10 and 30 ng/μl. Specific splicing products were detected by primer extension with end-labeled primers. The percent exon skipping shown below each lane is the average ± SD of three replicates. (B) Fe(II)-BABE Cys PTB mutants were used in directed hydroxyl radical probing assays. 0.1 μM of FAS WT RNA were incubated with 0.9, 1.2 μM or 1.5 μM Fe(II)-BABE-PTB mutants. Analysis was performed as described in Figure 2B. Cleavage sites are indicated by vertical lines on the left of the corresponding bands on the gel. Cuts produced by the lower amount of derivatized protein added are indicated. The colour-coding introduced in Figure 2 is also applied here to depict cleavages produced by cysteines in the different RRMs of PTB. Lane 1 (T) depicts a sequencing ladder generated by the same primer. (C) and (D) Fe(II)-BABE Cys PTB mutants were used in directed hydroxyl radical probing assays as described above. 0.1 μM of FAS WT RNA were incubated with 0.9 and 1.2 μM (blue wedges) or 0.9, 1.2 μM and 1.5 μM (red wedges) of Fe(II)-BABE-PTB mutants. Analysis was performed as described above. Cleavage sites are indicated by vertical lines on the left of the corresponding bands on the gel, as described for panel B. The graphs presented within each panel have been produced from quantification of the relevant gel by SAFA software. Peaks corresponding to the bands indicated by vertical lines in the gels are shown by arrows. The exact nucleotide positions of the cleavage sites are indicated by the numbers above the arrows. The asterisks at the gel and the graphs in panel D show artefact-signals produced during the processing of the gel.
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Figure 6: Effects of mutations altering the PTB RRM3-4 di-domain on its splicing and RNA-binding functions. (A) FAS WT RNA was spliced in vitro in HeLa nuclear extract in the absence (−) or presence of recombinant PTB mutants as indicated. PTB mutants were added to 10 and 30 ng/μl. Specific splicing products were detected by primer extension with end-labeled primers. The percent exon skipping shown below each lane is the average ± SD of three replicates. (B) Fe(II)-BABE Cys PTB mutants were used in directed hydroxyl radical probing assays. 0.1 μM of FAS WT RNA were incubated with 0.9, 1.2 μM or 1.5 μM Fe(II)-BABE-PTB mutants. Analysis was performed as described in Figure 2B. Cleavage sites are indicated by vertical lines on the left of the corresponding bands on the gel. Cuts produced by the lower amount of derivatized protein added are indicated. The colour-coding introduced in Figure 2 is also applied here to depict cleavages produced by cysteines in the different RRMs of PTB. Lane 1 (T) depicts a sequencing ladder generated by the same primer. (C) and (D) Fe(II)-BABE Cys PTB mutants were used in directed hydroxyl radical probing assays as described above. 0.1 μM of FAS WT RNA were incubated with 0.9 and 1.2 μM (blue wedges) or 0.9, 1.2 μM and 1.5 μM (red wedges) of Fe(II)-BABE-PTB mutants. Analysis was performed as described above. Cleavage sites are indicated by vertical lines on the left of the corresponding bands on the gel, as described for panel B. The graphs presented within each panel have been produced from quantification of the relevant gel by SAFA software. Peaks corresponding to the bands indicated by vertical lines in the gels are shown by arrows. The exact nucleotide positions of the cleavage sites are indicated by the numbers above the arrows. The asterisks at the gel and the graphs in panel D show artefact-signals produced during the processing of the gel.

Mentions: The preceding data indicated the importance of RNA contacts by RRM3 and 4 for PTB repressor activity, despite the low specificity of RRM4 contacts. RRM3 and 4 form a stable di-domain with back-to-back packing of the two RRMs involving the highly conserved linker (11,40). Mutation of the packing interface decreased affinity of recombinant RRM34 di-domain for RNA, and reduced repressor activity of full length PTB upon the CSRC N1 exon in a co-transfection assay (13). We therefore decided to test the packing mutant, C4pack (E502K; V505E; I509K, all in helix 2 of RRM4) for its effects upon FAS splicing (Figure 6A). The packing mutant showed a substantial reduction in repressor activity, with 46% of WT activity at 30 ng/μl (Figure 6A lanes 6, 7), while the RRM4 binding mutant had 39% of WT activity in this experiment (Figure 6A, lanes 8, 9). We tested the effects of the packing mutation upon RNA contacts by RRMs 3 and 4 by tethered OH radical probing. All contacts by RRMs 3 and 4 were abolished by the packing mutant (Figure 6B), indicating that stable RNA contacts by both RRMs are dependent upon their back-to-back packing. In contrast, no deleterious effects of the packing mutation were observed in a filter binding assay (Table 1).


The organization of RNA contacts by PTB for regulation of FAS splicing.

Mickleburgh I, Kafasla P, Cherny D, Llorian M, Curry S, Jackson RJ, Smith CW - Nucleic Acids Res. (2014)

Effects of mutations altering the PTB RRM3-4 di-domain on its splicing and RNA-binding functions. (A) FAS WT RNA was spliced in vitro in HeLa nuclear extract in the absence (−) or presence of recombinant PTB mutants as indicated. PTB mutants were added to 10 and 30 ng/μl. Specific splicing products were detected by primer extension with end-labeled primers. The percent exon skipping shown below each lane is the average ± SD of three replicates. (B) Fe(II)-BABE Cys PTB mutants were used in directed hydroxyl radical probing assays. 0.1 μM of FAS WT RNA were incubated with 0.9, 1.2 μM or 1.5 μM Fe(II)-BABE-PTB mutants. Analysis was performed as described in Figure 2B. Cleavage sites are indicated by vertical lines on the left of the corresponding bands on the gel. Cuts produced by the lower amount of derivatized protein added are indicated. The colour-coding introduced in Figure 2 is also applied here to depict cleavages produced by cysteines in the different RRMs of PTB. Lane 1 (T) depicts a sequencing ladder generated by the same primer. (C) and (D) Fe(II)-BABE Cys PTB mutants were used in directed hydroxyl radical probing assays as described above. 0.1 μM of FAS WT RNA were incubated with 0.9 and 1.2 μM (blue wedges) or 0.9, 1.2 μM and 1.5 μM (red wedges) of Fe(II)-BABE-PTB mutants. Analysis was performed as described above. Cleavage sites are indicated by vertical lines on the left of the corresponding bands on the gel, as described for panel B. The graphs presented within each panel have been produced from quantification of the relevant gel by SAFA software. Peaks corresponding to the bands indicated by vertical lines in the gels are shown by arrows. The exact nucleotide positions of the cleavage sites are indicated by the numbers above the arrows. The asterisks at the gel and the graphs in panel D show artefact-signals produced during the processing of the gel.
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Figure 6: Effects of mutations altering the PTB RRM3-4 di-domain on its splicing and RNA-binding functions. (A) FAS WT RNA was spliced in vitro in HeLa nuclear extract in the absence (−) or presence of recombinant PTB mutants as indicated. PTB mutants were added to 10 and 30 ng/μl. Specific splicing products were detected by primer extension with end-labeled primers. The percent exon skipping shown below each lane is the average ± SD of three replicates. (B) Fe(II)-BABE Cys PTB mutants were used in directed hydroxyl radical probing assays. 0.1 μM of FAS WT RNA were incubated with 0.9, 1.2 μM or 1.5 μM Fe(II)-BABE-PTB mutants. Analysis was performed as described in Figure 2B. Cleavage sites are indicated by vertical lines on the left of the corresponding bands on the gel. Cuts produced by the lower amount of derivatized protein added are indicated. The colour-coding introduced in Figure 2 is also applied here to depict cleavages produced by cysteines in the different RRMs of PTB. Lane 1 (T) depicts a sequencing ladder generated by the same primer. (C) and (D) Fe(II)-BABE Cys PTB mutants were used in directed hydroxyl radical probing assays as described above. 0.1 μM of FAS WT RNA were incubated with 0.9 and 1.2 μM (blue wedges) or 0.9, 1.2 μM and 1.5 μM (red wedges) of Fe(II)-BABE-PTB mutants. Analysis was performed as described above. Cleavage sites are indicated by vertical lines on the left of the corresponding bands on the gel, as described for panel B. The graphs presented within each panel have been produced from quantification of the relevant gel by SAFA software. Peaks corresponding to the bands indicated by vertical lines in the gels are shown by arrows. The exact nucleotide positions of the cleavage sites are indicated by the numbers above the arrows. The asterisks at the gel and the graphs in panel D show artefact-signals produced during the processing of the gel.
Mentions: The preceding data indicated the importance of RNA contacts by RRM3 and 4 for PTB repressor activity, despite the low specificity of RRM4 contacts. RRM3 and 4 form a stable di-domain with back-to-back packing of the two RRMs involving the highly conserved linker (11,40). Mutation of the packing interface decreased affinity of recombinant RRM34 di-domain for RNA, and reduced repressor activity of full length PTB upon the CSRC N1 exon in a co-transfection assay (13). We therefore decided to test the packing mutant, C4pack (E502K; V505E; I509K, all in helix 2 of RRM4) for its effects upon FAS splicing (Figure 6A). The packing mutant showed a substantial reduction in repressor activity, with 46% of WT activity at 30 ng/μl (Figure 6A lanes 6, 7), while the RRM4 binding mutant had 39% of WT activity in this experiment (Figure 6A, lanes 8, 9). We tested the effects of the packing mutation upon RNA contacts by RRMs 3 and 4 by tethered OH radical probing. All contacts by RRMs 3 and 4 were abolished by the packing mutant (Figure 6B), indicating that stable RNA contacts by both RRMs are dependent upon their back-to-back packing. In contrast, no deleterious effects of the packing mutation were observed in a filter binding assay (Table 1).

Bottom Line: RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA.The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker.Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK.

Show MeSH
Related in: MedlinePlus