Understanding the Role of Post‐Translational Modifications on the Splicing Activity of RNA Binding Proteins

Abstract

The Polypyrimidine Tract Binding Protein 1 (PTBP1) is an RNA binding protein that controls the alternative splicing of many gene transcripts. PTBP1 belongs to a gene family with paralogs expressed in varying cell types and stages of cell growth. PTPB1 is expressed in most cell types but is absent in differentiating neurons and muscle. Paralog PTBP2 is highly expressed in differentiating neurons. The amino acid sequence of PTBP1 is 74% identical to PTBP2 and the two proteins have a similar domain organization with 4 RNA binding domains (RBDs) connected by 3 linker regions and an N‐terminal region. The two proteins have over‐lapping and non‐overlapping target exons; PTBP1 functions as a splicing repressor of many neuronal exons while PTBP2 does not. PTBP1 is expressed in neuronal progenitor cells, but is down‐regulated during neuronal differentiation while the level of PTBP2 is up‐regulated. The switch in the levels of the two proteins effects the splicing of many exons that are sensitive to PTBP1, thus causing a change in the neuronal splicing program that is critical for development and maturation of neurons. How the two proteins exert different splicing outcomes is not well understood. Recent studies using PTBP1‐PTBP2 chimeric proteins highlighted regions of PTBP1 that change the splicing activity of PTBP2, and revealed the two proteins have different protein‐protein interactions. Thus, we hypothesize that additional factors such as post‐translational modifications (PTMs) dictate PTBP splicing activity. To test this, we used in vitro splicing reaction conditions, in which PTBP1 and 2 showed different splicing activity. To capture splicing relevant PTM's bacterial expressed recombinant PTB proteins were incubated in splicing reaction mixtures containing HeLa nuclear extract, pull‐down using Ni2+‐NTA beads, boiled and separated on SDS‐PAGE. The corresponding PTBP gel bands were analyzed by mass spectrometry for PTM's. Our Results highlight that PTBP1 and 2 have many PTM's including phosphate, acetate and ubiquitin groups. The N‐terminal and linker 1 region are modified primarily by phosphate groups while the RRM domains are modified by acetate and ubiquitin groups. We also note that some residues contain more than one type of modification such as acetate and ubiquitin. Percent modified for each residue‐modification was calculated by counting the modified residue and the total number of peptide fragments containing that residue. Our results highlight that the type and degree of modification vary between the two proteins, in regions including the N‐terminal, linker 1 and linker 2. These regions were identified by the chimera study to alter PTBP2 activity. Currently, we are conducting a mutational analysis of these modified residues that are different between the two proteins, to determine the role of PTM's in PTBP splicing activity.Support or Funding InformationThis work was supported by a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [5T34GM008612‐21]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.