Multiple transcription start sites in the rat insulin-like growth factor-I gene give rise to IGF-I mRNAs that encode different IGF-I precursors and are processed differently in vitro.
Two distinct class 1 and class 2 rat liver IGF-I mRNAs contain different 5' leader exons, 1 and 2. RNase protection, primer extension, RACE PCR and ribonuclease H mapping established the complete structure of the 5' end of class 1 and class 2 IGF-I mRNAs. Two major transcription start sites in exon 1 yield class 1 IGF-I mRNAs, including 345 or 245 bases of exon 1. Multiple, clustered transcription start sites in exon 2 yield class 2 IGF-I mRNAs with 84-50 bases of exon 2. Cell-free translation of in vitro transcribed IGF-I mRNAs suggests that class 1 and class 2 mRNAs preferentially initiate translation at distinct AUG codons to result in IGF-I precursors with either 48 residue class 1 pre-peptides or 32 residue class 2 pre-peptides. Some translation initiation also occurs at a downstream AUG common to class 1 and 2 mRNAs to yield IGF-I precursors with a 22 residue pre-peptide. Inclusion of microsomal membranes in translations suggests that the three different pre-peptides each function as co-translationally cleaved signal peptides. However, treatment of processed precursors with endoglycosidase H indicates that co-translational processing of precursors with 22 and 32 residue pre-peptides leads to glycosylation of downstream IGF-I precursor sequences whereas co-translational processing of precursors with 48 residue pre-peptide is not associated with glycosylation.
Simmons
J G
JG
Department of Physiology, University of North Carolina, Chapel Hill 27599.
Van Wyk
J J
JJ
Hoyt
E C
EC
Lund
P K
PK
D000818:Animals; D001483:Base Sequence; D004247:DNA; D004285:Dogs; D005091:Exons; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D015183:Restriction Mapping; D016914:Ribonuclease H; D014158:Transcription, Genetic
D011498:Protein Precursors; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D004247:DNA; D016914:Ribonuclease H