The process of polyadenylation is essential in the biogenesis of eukaryotic mRNA. The poly(A) tall is thought to participate in mRNA translation and stability. Defects in the formation of the poly(A) tail decrease the amount of mRNA available for translation into protein, and thus interfere with normal cell function. In addition, polyadenylation can play a role in the regulation of gene expression, especially in cases with alternative selection of poly(A) sites. In this way, it becomes part of a cell's response to stimuli governing growth, differentiation, and tissue-specific gene expression. The goals of this proposal are to determine the factors responsible for this processing and how they interact with each other to give an active processing complex. Understanding the basic mechanism of polyadenylation will make it feasible to ask how the process is regulated as the physiological. state of the cell changes, and how this regulation affects mRNA levels globally or specifically. This research will characterize the factors which recognize the signal sequences on polyadenylation precursor. It will focus on two proteins, pl55 and p68, which require the AAUAAA sequence to bind to precursor RNA, are found in polyadenylation-specific complexes, and dissociate from the RNA once it is cleaved and polyadenylated. These observations are consistent with a role for these proteins in the polyadenylation process, possibly in signal recognition and assembly of the processing complex. Their relationship to the enzymes responsible for cleavage and poly(A) addition will be determined by whether they chromatographically cofractionate. Other experiments using an in vitro system will explore how polyadenylation specific factors interact with each other and with precursor RNA and products during the polyadenylation reaction. Three approaches will be taken to clone polyadenylation-specific factors: a) screening of lambda gt11 human cDNA expression libraries with RNA probes containing polyadenylation signal sequences; b) use of ultraviolet-crosslinked ribonucleoprotein complexes as immunogens to produce antibodies to polyadenylation-specific proteins; and c) sufficient purification of these proteins so as to use them either as antigens for specific antibody production or to obtain protein sequence to generate DNA probes. Either reagent would then be used to screen expression libraries. Using an in vitro system, the 3' end processing of mRNAs from bovine leukemia virus, an oncogenic retravirus, and the role of viral specific factors in this processing will be examined. Finally, the role of polyadenylation in transcription termination will be investigated. Transcriptional templates which encode a self-cleaving RNA sequence and a termination site will be used to determine the role of cleavage of the nascent transcript on termination of RNA polymerase 11 transcription. This will be studied in vivo using transient expression assays, and if possible, with an in vitro system capable of transcription initiation and elongation, polyadenylation, and termination.
| Hyman, L E; Moore, C L (1993) Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites. Mol Cell Biol 13:5159-67 |
