Altering RNA stability is the major mechanism regulating the expression of genes encoding oncogenes, homeobox proteins, lymphokines, cytokines, cytoskeletal proteins, and growth factor receptors to name a few. Posttranscriptional processes also control the expression of a number of hormone-regulated genes [eg. growth hormone, casein, apolipoproteins]. The availability of a hormone-regulated RNA is particularly useful for study of fundamental mechanisms of RNA stability, since the addition or removal of the stimulus provides a well-defined molecular switch. To date the sequences that impart stability or instability to a number of mRNAs have been described. However no eucaryotic ribonuclease that catalyzes RNA metabolism has been identified. The subject of the present proposal is the regulation of serum albumin mRNA stability by estrogen (E) in Xenopus laevis liver. Administration of E in vivo or to liver explant cultures causes the mRNAs encoding the major serum proteins to disappear from the cytoplasm. The destabilized RNAs are unique in that they have very short (17 residue), discrete poly(A) tails. Poly(A) length is unaffected by E. During the preceding grant period an E-induced nuclease [termed xln for Xenopus liver nuclease) was identified that has the expected properties for an enzyme that catalyzes regulated RNA degradation.
The Specific Aims for this proposal are: 1) To isolate and clone xln, prepare antibodies and expression vectors, and use these tools to study the nature of RNA selectivity and structural requirements of RNA cleavage sites; 2) To map the sites of albumin RNA cleavage introduced in vivo following E by Sl protection and primer extension, and determine their structural context; 3) To use antibody and cDNA clones developed in Aim 1 to study the regulation of the nuclease by E; 4) To transfect constructs that will produce hybrid globin-albumin mRNA into primary hepatocyte cultures to map functional instability determinants and examine the role of poly(A) length in regulated mRNA instability; and 5) To reconstitute albumin RNA degradation in vitro. The long term goals of this research project are to identify and characterize all of the molecular components involved in regulated mRNA instability [ie. nuclease, RNP proteins, RNA targeting and cleavage sites, other interacting RNAs] in order to reconstitute the process of regulated RNA degradation in vitro.
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