James E. Dahlberg - US grants

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.

DESCRIPTION: Trafficking of RNA and protein between the nucleus and cytoplasm is essential to cell viability and it must be both efficient and well-controlled. The process of RNA export presents an opportunity to check th integrity of the products of transcription and processing, prior to their export to the cytoplasm. The applicant proposes that proofreading is a key feature in the export of RNAs. RNAs or RNPs that are present in excessive quantities, or that are defective as a consequence of mutation or incomplete o incorrect processing, are hypothesized to be retained and then degraded in the nucleus. To learn how various types of RNAs are exported from the nucleus and what features contributes to their proofreading, three specific aims will be pursued. These are: (1) to study the mechanisms by which tRNAs are exported an to determine what features of tRNAs are necessary and sufficient for their export; (2) to study the mechanisms by which the constitutive transport elemen (CTE) of MPMV is able to promote the release of incompletely spliced pre-mRNAs from spliceosomes and to relate export of that element to the process of norma mRNA export; and (3) to study the structure of a small RNA export element identified in a screen for exportable RNAs and determine what factors lead to its export or retention. For most of the experiments, Xenopus laevis oocytes will be used as a model system to identify and study transport factors, but some studies involve genetic selections in yeast. The focus will be on the structure and functions of cis-acting signals in RNA, their interactions with specific RNA export factors, and the mechanisms by which these factors promote export.