Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain

A. L.M. Bothwell, D. W. Ballard, W. M. Philbrick, G. Lindwall, S. E. Maher, M. M. Bridgett, S. F. Jamison, M. A. Garcia-Blanco

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes and Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes and Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes and Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a ~60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed.

Original languageEnglish (US)
Pages (from-to)24657-24663
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number36
StatePublished - Dec 1 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain'. Together they form a unique fingerprint.

  • Cite this

    Bothwell, A. L. M., Ballard, D. W., Philbrick, W. M., Lindwall, G., Maher, S. E., Bridgett, M. M., Jamison, S. F., & Garcia-Blanco, M. A. (1991). Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain. Journal of Biological Chemistry, 266(36), 24657-24663.