Isolation of hnRNP complexes from Drosophila melanogaster.
Nascent RNA polymerase II transcripts, heterogeneous nuclear RNAs (hnRNAs), become associated with nuclear proteins (hnRNP Proteins), and their processing into mRNAs takes place in these hnRNP complexes. hnRNP complexes have previously been purified from vertebrate cells. Here we report the isolation of hnRNP complexes from an invertebrate organism, the fruitfly Drosophila melanogaster. Candidate hnRNP proteins were purified from D. melanogaster embryos by ssDNA affinity chromatography, and mAbs were produced to many of the major proteins. Genuine hnRNP proteins were identified by several criteria, including nucleoplasmic localization, association with nascent transcripts, crosslinking to poly(A)-containing RNA in living cells, and amino acid sequence. In addition, mAbs that cross-react between the fruitfly and human hnRNP proteins were obtained. Most importantly, using hnRNP-specific mAbs we have purified the hnRNP complexes from D. melanogaster cells. These RNAase-sensitive complexes contain at least 10 major proteins designated hrps, the most abundant proteins having apparent molecular masses of 36, 38, 39, 40, 44, 48, 54, 62, 70, and 75 kD. cDNAs and complete sequences for several of these proteins have been obtained and are presented in the accompanying paper (Matunis, E. L., M. J. Matunis, and G. Dreyfuss. 1992. J. Cell Biol. 116:257-269). The purification of D. melanogaster hnRNP complexes will facilitate genetic and cytological studies on the function of hnRNA-binding proteins and on the posttranscriptional regulation of gene expression.
Matunis
M J
MJ
Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104-6148.
Matunis
E L
EL
Dreyfuss
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D002875:Chromosomes / Q000648:ultrastructure; D004331:Drosophila melanogaster / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007526:Isoelectric Point; D008970:Molecular Weight; D012323:RNA Processing, Post-Transcriptional; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012332:RNA, Heterogeneous Nuclear; D012261:Ribonucleoproteins