This proposal attempts to develop methods for the purification and characterization of individual nuclear pre-mRNA-protein complexes. Because binding of eukaryotic RNA binding proteins to nascent transcripts occurs in the nucleus during transcription, it is believed that the particular constellation of RNA binding proteins that a given transcript acquires to form a distinct ribonucleoprotein (RNP) complex will control its RNA processing, RNA export and RNA stability. These post-transcriptional pathways are critically important for gene expression. Moreover, because >95% of human genes generate multiple transcript isoforms it is also important to find out if differentially spliced mRNA isoforms have distinct patterns of RNA binding proteins. The hypothesis being tested is that different nuclear pre-mRNAs/RNPs have distinct protein compositions which contribute to their fate. If successful, this approach would have a major impact in our understanding of how nuclear RNP structure controls the RNA processing and fates of newly transcribed messenger RNA molecules. In order to approach this question, we will: 1. Develop technology to purify individual Drosophila nuclear pre-mRNPs using biotinylated anti-sense chimeric LNA-DNA oligonucleotides targeted to specific pre-mRNAs and analyze their protein composition by mass spectrometry. This proposal will outline one specific aim that builds on the expertise of my lab in evaluating genome-wide patterns of alternative pre-mRNA splicing and the distribution of RNA splicing factors on nuclear pre-mRNAs.
This proposal attempts to develop methods for the purification and characterization of individual nuclear pre-mRNA-protein complexes. Because binding of eukaryotic RNA binding proteins to nascent transcripts occurs in the nucleus during transcription, it is believed that the particular constellation of RNA binding proteins that a given transcript acquires to form a distinct ribonucleoprotein (RNP) complex will control its RNA processing, RNA export and RNA stability. These post-transcriptional pathways are critically important for gene expression and can be perturbed in disease states.
