Protamine-1 and -2 mRNA in round spermatids is associated with RNA-binding proteins.
RNA-binding proteins in round spermatids have previously been assigned to the coding sequence of Prm1- and Prm2-mRNA. To further characterize this protein-RNA interaction, prior to cDNA synthesis, microdissected cell profiles were digested with different proteases exhibiting a specific cleavage site followed by both conventional and real-time quantitative PCR. Best results were obtained with proteinase K and A followed by factor Xa protease, genenase I, and proteases V8. While enterokinase revealed PCR signals solely for Prm2, no amplification signal was obtained using chymotrypsin. These data suggest a protein segment rich in basic amino acids to be important for the binding to Prm1- and Prm2-mRNA. The fact that phenanthroline treatment instead of protease digestion also resulted in amplification signals suggests the involvement of zinc-finger-like protein-RNA interactions. Employing different primer pairs, RNA-binding proteins were shown to be localized at the 5' end of Prm1- and Prm2-mRNA. Since protein-RNA interactions are a common principle of posttranscriptional regulation of gene expression, the combination of microdissection, protease digestion, and real-time quantitative PCR provides a suitable tool for its investigation in a cell type-specific manner. Furthermore, the presence of RNA-binding proteins within the coding sequence of mRNAs demands proteinase K treatment prior to cDNA synthesis, a compelling necessity for the study of gene expression.
Steger
Klaus
K
Institute of Veterinary Anatomy, Frankfurter Strasse 98, 35392 Giessen, Germany. Klaus.Steger@vetmed.uni-giessen.de
Fink
Ludger
L
Klonisch
Thomas
T
Bohle
Rainer M
RM
Bergmann
Martin
M
D002469:Cell Separation / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007834:Lasers; D008297:Male; D008875:Middle Aged; D011479:Protamines / Q000235:genetics*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013087:Spermatids / Q000378:metabolism*
D018076:DNA, Complementary; C082073:PRM1 protein, human; D011479:Protamines; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C068621:protamine 2; D010450:Endopeptidases