We propose to study the mechanisms responsible for synthesis of small nuclear RNAs such as Ul (or U2) snRNAs. The unique character of vertebrate snRNA transcription units, is revealed in the obligatory coupling of promoter function to 3' end formation and in the rapid export of the transcript from the nucleus. To understand these processes and the factors that control them, we will focus our efforts on the synthesis and control of one particular snRNA, Ul RNA. These experiments will be carried out using both in vitro and in vivo transcription systems derived from Xenopus, mouse or human cells. We have succeeded in establishing an efficient and accurate system for transcription of snRNA genes in vitro. The system, based on careful preparation of intact or disrupted nuclei from Xenopus oocytes, will be optimized for its ability to retain activity during extraction and for the ability to respond to added templates. With an understanding of this Xenopus germinal vesicle system, we will attempt to develop a comparable system from mammalian (HeLa) cell nuclei. These transcription systems will be fractionated for isolation and characterization of trasnscription factors that are required for snRNA gene transcription; of particular interest will be those factors that activate snRNA genes specifically. Because of the coupling of snRNA synthesis to 3' end formation and export, we shall study the mechanisms by which newly synthesized snRNAs are transported out of the nucleus in intact oocytes. In a related project, we will also study requirements for incorporation of snRNAs into snRNPs and the transport of the snRNPs back to the nucleus. Accumulation of UI RNA ln vivo is responsive to at least two controls which 1) fix the total amount of the RNA in cells and 2) modulate the type of Ul RNA, in a developmentally controlled way. By stable transfection of mouse cells with chimeric Ul or Ul/U2 genes, we will determine how cells are able to sense the level of snRNAs and what mechanisms are used to modulate snRNA transcription. We will determine when during the cell cycle Ul RNA is made and when during S phase various types of Ul genes are replicated. Models for mechanisms of control will be tested in transgenic mice. Finally, as our ability to make defined transcription extracts from Xenopus progresses, we will attempt to reproduce the developmental controls of Xenopus Ul gene transcription in vitro.
| 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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