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Post-Translational Modifications in Polypyrimidine Tract Binding Proteins PTBP1 and PTBP2 - PubMed

  • ️Mon Jan 01 2018

Post-Translational Modifications in Polypyrimidine Tract Binding Proteins PTBP1 and PTBP2

Jeffrey M Pina et al. Biochemistry. 2018.

Abstract

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Many of these RNA binding proteins occur as gene families with members sharing a high degree of primary structure identity and domain organization yet have tissue-specific expression patterns and regulate different sets of target exons. How highly similar members in a gene family can exert different splicing outcomes is not well understood. We conducted mass spectrometry analysis of post-translational phosphorylation and acetylation modifications for two paralogs of the polypyrimidine tract binding protein family, PTBP1 and PTBP2, to discover modifications that occur in splicing reaction mixtures and to identify discrete modifications that may direct their different splicing activities. We find that PTBP1 and PTBP2 have many distinct phosphate modifications located in the unstructured N-terminal, linker 1, and linker 2 regions. We find that the two proteins have many overlapping acetate modifications in the RNA recognition motifs (RRMs) with a few distinct sites in PTBP1 RRM2 and RRM3. Our data also reveal that lysine residues in the nuclear localization sequence of PTBP2 are acetylated. Collectively, our results highlight important differences in post-translational modifications between the paralogs and suggest a role for them in the differential splicing activity of PTBP1 and PTBP2.

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Conflict of interest statement

Notes

The authors declare no competing financial interest

Figures

Figure 1.
Figure 1.

(A and B) Purification of His6−tagged PTBP1 and PTBP2. Aliquots (10 μL) of the indicated fractions were analyzed by SDS− PAGE. The gel was stained with Coomassie blue dye. The positions and sizes (kilodaltons) of marker polypeptides are shown at the left. (C) Purification of His6−tagged PTBP from 50 μL splicing reaction mixtures containing HeLa nuclear extract, 2.2 mM MgCl2, 0.4 mM ATP, and 20 mM creatine phosphate. Aliquots (10 μL) of the indicated fractions were analyzed by SDS−PAGE. The gel was stained with Gel Code blue safe stain. The positions and sizes (kilodaltons) of marker polypeptides are shown at the left.

Figure 2.
Figure 2.

Mechanisms of reversible covalent modifications. (A) Lysine acetylation−deacetylation. Acetyl groups are added by lysine acetyltransferases (KATs) and removed by deacetylases (KDACs). (B) Phosphorylation. Phosphate groups are added by kinases to the side chain hydroxyl groups of serine, threonine, and tyrosine residues. Phosphate groups are removed by phosphatases via hydrolysis.

Figure 3.
Figure 3.

Domain structure (top) of the PTB proteins indicating the segments defined in this study. The percent amino acid sequence identity between PTBP1 isoform 4 and PTBP2 is indicated below. Aligned amino acid sequences (bottom) of human PTBP1 isoform 4 and PTBP2. Gaps in the alignment are indicated as dashes. Residues identical to those of PTBP1 are shown as dots. RNA recognition motifs (RRMs) are highlighted in gray.38 Vertical lines above the sequence indicate PTBP1 residues that interact with RNA.38 Arrowheads below the sequence indicate RNA-interacting residues that are different in PTBP2. The bipartite nuclear localization sequence is indicated as NLS. PTBP1-Raver1-interacting motifs are marked with pink bars.

Figure 4.
Figure 4.

(A−C) Cartoon representation of NMR solution structures of PTBP1 RRM1 (PDB entry 2AD9), RRM2 (PDB entry 2ADB), and RRM3 and −4 (PDB entry 2ADC) bound to a CUCUCU hexamer.38 The main chain cartoon traces are colored gray. Post-translationally modified residues identified by mass spectrometry and relevant cytosine and uracil bases of the hexamer are labeled. Side chains of modified residues and the CUCUCU RNA hexamer are shown as sticks and colored by element.

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