Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing.
alpha-Tropomyosin exons 2 and 3 are spliced in a mutually exclusive manner. Exon 3 is included as the default exon in the mRNA of most cell types, whereas exon 2 is only included in the mRNA of smooth muscle cells. The primary determinant for the default selection of exon 3 is the branchpoint/polypyrimidine tract. This element upstream of exon 3 clearly and effectively outcompetes the corresponding element upstream of exon 2. To identify trans-acting factors that bind to this important cis element, we used UV cross-linking to identify a 57-kD protein whose binding characteristics directly correlate with 3'-splice-site selection in cis-competition splicing assays. This protein appears to be identical to polypyrimidine tract-binding protein. In this report we have used oligonucleotides derived from peptide sequences to isolate and sequence cDNA clones encoding this 57.2-kD protein. The primary sequence reveals a novel protein with significant homology to other RNA-binding proteins. Expression of the mRNA is detected in all tissues and cells examined, although its levels exhibit tissue-specific and developmental regulation. Using a biochemical complementation assay, we have found that this protein, along with a 100-kD protein, exists as part of a large complex that is required to rescue splicing from depleted nuclear extracts.
Patton
J G
JG
Howard Hughes Medical Institute, Department of Cardiology, Children's Hospital, Boston, Massachusetts.
Mayer
S A
SA
Tempst
P
P
Nadal-Ginard
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D038941:Polypyrimidine Tract-Binding Protein; D011131:Polyribonucleotides / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D012332:RNA, Heterogeneous Nuclear / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins; D011817:Rabbits; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D002352:Carrier Proteins; D011131:Polyribonucleotides; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038941:Polypyrimidine Tract-Binding Protein