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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM094890-03
Application #
8324309
Study Section
Special Emphasis Panel (ZGM1-GDB-7 (EU))
Program Officer
Bender, Michael T
Project Start
2010-09-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$295,652
Indirect Cost
$97,652
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Majumdar, Sharmistha; Singh, Anita; Rio, Donald C (2013) The human THAP9 gene encodes an active P-element DNA transposase. Science 339:446-8
Taliaferro, J Matthew; Aspden, Julie L; Bradley, Todd et al. (2013) Two new and distinct roles for Drosophila Argonaute-2 in the nucleus: alternative pre-mRNA splicing and transcriptional repression. Genes Dev 27:378-89
Taliaferro, J Matthew; Marwha, Dhruv; Aspden, Julie L et al. (2013) The Drosophila splicing factor PSI is phosphorylated by casein kinase II and tousled-like kinase. PLoS One 8:e56401