Genome-wide analysis of mRNAs bound to the histone stem-loop binding protein.
The replication-dependent histone mRNAs are cell-cycle-regulated and expressed only during S phase. In contrast to all other eukaryotic mRNAs, the histone mRNAs end in a highly conserved 16-nucleotide stem-loop rather than a poly(A) tail. The stem-loop is necessary and sufficient for the post-transcriptional regulation of histone mRNA during the cell cycle. The histone mRNA 3' stem-loop is bound by the stem-loop binding protein (SLBP) that is involved in pre-mRNA processing, translation, and stability of histone mRNA. Immunoprecipitation (IP) of RNA-binding proteins (RBPs) followed by microarray analysis has been used to identify the targets of RNA-binding proteins. This method is sometimes referred to as RIP-Chip (RNA IP followed by microarray analysis). Here we introduce a variation on the RIP-Chip method that uses a recombinant RBP to identify mRNA targets in a pool of total RNA; we call this method recombinant, or rRIP-Chip. Using this method, we show that recombinant SLBP binds exclusively to all five classes of histone mRNA. We also analyze the messages bound to the endogenous SLBP on polyribosomes by immunoprecipitation. We use two different microarray platforms to identify enriched mRNAs. Both platforms demonstrate remarkable specificity and consistency of results. Our data suggest that the replication-dependent histone mRNAs are likely to be the sole target of SLBP.
Townley-Tilson
W H Davin
WH
Department of Genetics, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Pendergrass
Sarah A
SA
Marzluff
William F
WF
Whitfield
Michael L
ML
D001483:Base Sequence; D015870:Gene Expression; D015894:Genome, Human; D006367:HeLa Cells; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D006657:Histones; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C092663:SLBP protein, human; D039221:mRNA Cleavage and Polyadenylation Factors; C026452:messenger ribonucleoprotein