Multiple determinants of splicing repression activity in the polypyrimidine tract binding proteins, PTBP1 and PTBP2.
Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.
California State University Fullerton, Department of Chemistry and Biochemistry, Fullerton, California 92831, USA.
Niroshika M
NM
Keppetipola
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095, USA.
Kyu-Hyeon
KH
Yeom
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095, USA.
Adrian L
AL
Hernandez
California State University Fullerton, Department of Chemistry and Biochemistry, Fullerton, California 92831, USA.
Tessa
T
Bui
Department of Basic Medical Sciences, University of Arizona, College of Medicine-Phoenix, Phoenix, Arizona 85004, USA.
Shalini
S
Sharma
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095, USA.
Douglas L
0000-0002-2705-8187
DL
Black
D000818:Animals; D002478:Cells, Cultured; D005091:Exons; D006801:Humans; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012326:RNA Splicing
D038941:Polypyrimidine Tract-Binding Protein
RNA binding proteins; alternative splicing