The aim of this investigation is to examine the inter-relationships among several RNA processing events in vivo by introducing antibodies directed against specific cellular components into living cells and observing their effects. The antibodies will be introduced into the cells by fusing cells to liposomes (small phospholipid vesicles), which can be manufactured to contain antibodies using technology we developed in our lab. The liposome-entrapped antibodies will be delivered into the cells by mixing the cells and liposomes under conditions which facilitate fusion. We will introduce antibodies that recognize small nuclear RNA containing ribonucleoprotein particles (snRNPs), particles that have been implicated in RNA processing. We will use antibodies from patients with lupus erythematosus as well as monoclonal antibodies directed against the same components. As controls we will use lupus antibodies that recognize ribosomes, and rabbit antibodies that recognize tubulin in addition to antibodies from non-immune serum. The intracellular effects of the liposome-introduced antibodies will be assessed using three intron-containing gene transcripts synthesized by RNA polymerase II: 1) an actin gene (A2), 2) a gene coding for the 83,000 dalton heat shock protein (pLambda6.1), and 3) a chimeric gene comprised of the hsp70 promoter linked to an alcohol dehydrogenase gene (pHAP). The products of these genes will be examined at the level of transcription, capping, polyadenylation, splicing, and transport to the cytoplasm. If the antibodies inhibit one or more of these processes, then the processing intermediates which result from the blockage will be analyzed and an attempt will be made to isolate a processing complex. We will also examine the cells in the electron microscope to precisely define the location of the antibodies within the cell (chromatin) using a colloidal gold labeled second antibody. This approach will enable us to study RNA processing in vivo and gain an understanding of the molecular mechanisms involved in RNA processing and will provide information on the interrelationships among the various RNA processing steps in the living cell.
| Corell, R A; Gross, R H (1992) Splicing thermotolerance maintains Pre-mRNA transcripts in the splicing pathway during severe heat shock. Exp Cell Res 202:233-42 |
