This is a proposal to study very early steps in the formation of U1 small nuclear RNA (U1 RNA). Precursors of U1 (pre- U1RNA) and other snRNAs that are made by RNA polymerase II are exported from the nucleus to the cytoplasm where they undergo incorporation into small nuclear ribonucleo- protein particles (snRNPs), base modification and trimming at the 3' end. These snRNPs are then imported into the nucleus, where final modification of the RNA occurs and where they participate in splicing of messenger RNA precursors. Without appropriate formation of snRNPs, cells would die since they would be unable to process messenger RNA precursors. The three goals of this project are: 1) To define mechanisms by which cells control the types and amounts of U1 RNAs. We will do this in mouse tissue culture cells that have been transfected either stably or transiently with mouse chimeric U1RNA genes. These experiments are designed to test whether the cells control U1RNA levels by limiting synthesis or degradation of RNA and to learn if these amounts are monitored by sensing the levels accumulated products. 2) To define complexes that contain pre-U1 RNA prior to export of the RNA to the cytoplasm. We plan to do this in Xenopus laevis oocytes or oocyte nuclei (germinal vesicles). We will analyze the proteins with which pre- U1RNA interacts prior to export by purification of the 10-11S particles containing pre-U1RNA, photo cross lining of RNA and proteins, and by analysis of particles containing mutated RNA. 3) To learn how pre-U1RNA is exported. For this, we will exploit our ability to isolate intact, functioning nuclei (germinal vesicles) to develop a defined experimental system for the analysis of export. This will allow us to test if cytoplasm is required and if nuclear proteins accompany the RNA through the nuclear pore complex. The participation of various nuclear and cytoplasmic structures will be studied by cell fractionation, electron microscopy and isolation of RNA- protein complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM030220-12
Application #
2175725
Study Section
Molecular Biology Study Section (MBY)
Project Start
1982-04-01
Project End
1997-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
12
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Lund, Elsebet; Liu, Mingzhu; Hartley, Rebecca S et al. (2009) Deadenylation of maternal mRNAs mediated by miR-427 in Xenopus laevis embryos. RNA 15:2351-63
Eis, Peggy S; Garcia-Blanco, Mariano A (2008) Quantification of microRNAs, splicing isoforms, and homologous mRNAs with the invader assay. Methods Mol Biol 488:279-318
Lund, E; Dahlberg, J E (2006) Substrate selectivity of exportin 5 and Dicer in the biogenesis of microRNAs. Cold Spring Harb Symp Quant Biol 71:59-66
Eis, Peggy S; Tam, Wayne; Sun, Liping et al. (2005) Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci U S A 102:3627-32
Lund, Elsebet; Guttinger, Stephan; Calado, Angelo et al. (2004) Nuclear export of microRNA precursors. Science 303:95-8
Trotta, Christopher R; Lund, Elsebet; Kahan, Lawrence et al. (2003) Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates. EMBO J 22:2841-51
Dahlberg, James E; Lund, Elsebet; Goodwin, Elizabeth B (2003) Nuclear translation: what is the evidence? RNA 9:1-8
Glodowski, Doreen R; Petersen, Jeannine M; Dahlberg, James E (2002) Complex nuclear localization signals in the matrix protein of vesicular stomatitis virus. J Biol Chem 277:46864-70
Johnson, Arlen W; Lund, Elsebet; Dahlberg, James (2002) Nuclear export of ribosomal subunits. Trends Biochem Sci 27:580-5
Rutjes, S A; Lund, E; van der Heijden, A et al. (2001) Identification of a novel cis-acting RNA element involved in nuclear export of hY RNAs. RNA 7:741-52

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