45.1 Control of Alternative Pre-mRNA Splicing by RNA Pol II Elongation: Faster Is Not Always Better A. R. Kornblihtt 1 , M. de la Mata 1 , C. R. Alonso 2 , S. Kadener 1 , J. P. Fededa 1 , and G. Nogue ´s 1 1 Universidad de Buenos Aires, Buenos Aires, Argentina; and 2 University of Cambridge, Cambridge, UK The realisation that the mammalian proteomic complexity is achieved with a limited number of genes demands a better understanding of alternative splicing regulation. Our group in studies performed on the fibronectin gene originally described promoter control of alternative splicing. Recently, other labs extended our findings to the cystic fibrosis, CD44 and CGRP genes strongly supporting a coupling between transcription and pre-mRNA processing. A possible model for promoter control of alterna- tive splicing involves modulation of RNA pol II (pol II) processivity. In fact, transcriptional activators such as VP-16 and HIV-Tat, that enhance pol II elongation, stimulate exon skipping while chromatin compaction elicited by template replication provokes lower pol II processivities which result in higher exon inclusion rates. Chromatin immunoprecipitation with antibod- ies to pol II showed that exon inclusion correlates with higher pol II densities in upstream gene regions. Furthermore, we proved the proces- sivity model with a direct tool: the C4 mutation (R741H) of the Drosophila pol II largest subunit that confers the enzyme a lower elongation rate. Expression of a human equivalent (R749H) of the Drosophila C4 pol II in cultured cells inhibits skipping of the fibronectin EDI exon and favours the use of distal 5' sites in adenovirus E1a splicing. In addition, re-splicing of the Hox gene Ultrabithorax is stimulated in fly embryos mutant for C4. Interestingly the C4 mutation has been described to enhance the ultra- bithorax phenotype. These results support the processivity mechanism in vivo and demonstrate the physiological importance of the transcriptional control of splicing. 46.1 Amino Acid Sequences within the 1 Domain of Human Apolipoprotein B Mediate Rapid Intracellular Degradation. L. Lapierre 1 , D. L. Currie 1 , Z. Yao 3 , J. Wang 2 , and R. S. McLeod 1 1 Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, Canada; 2 Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Ottawa, ON, Canada; and 3 Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL, USA Apolipoprotein (apo) B contains a 1 domain (between the carboxyl ter- mini of apo-B22 to -B43) that is predicted to be composed of extensive amphipathic -strands. Pulse-chase analysis of truncated apoB variants has revealed that sequences between the carboxyl termini of apo-B37 and -B42 governed the secretion efficiency and intracellular stability of apoB. Thus, while apo-B37, -B34 and -B29 were stable and secreted efficiently, -B42 and –B100 were secreted poorly and were sensitive to degradation. Degradation of –B42 and B100 could be partially inhibited by the protea- some inhibitors ALLN and MG132. Amino acid sequence analysis sug- gested that a segment encompassing amino acid residues 1724 to 1921 of human apoB (i.e. between the carboxyl termini of apo-B38.0 and -B42.4) was 34% identical and 63% homologous to fatty acid binding proteins, for which crystal structures reveal orthogonal -sheet structures. To test the hypothesis that -structures are involved in apoB degradation, fusion proteins were created that contained apoB29 (i.e. the N-terminal 1305 amino acids of apoB100) linked either to fragments derived from the 1 domain of apoB, namely B34 – 42, B34 –37, or B37– 42, or to the liver fatty acid binding protein. In transfected cells, the fusion proteins containing the 1 domain segments B34 – 42 or B37– 42 exhibited rapid intracellular degradation, but the fusion proteins containing liver fatty acid binding protein or B34 –37 were stable and secreted efficiently. Degradation of fusion proteins containing B34 – 42 or B37– 42 could be blocked by ALLN. Thus, the presence of specific sequences in the 1 domain of human apoB increases the susceptibility of the protein to rapid intracellular degrada- tion. We conclude that sequences within the 1 domain are not function- ally equivalent. Abstracts © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. 796 Molecular & Cellular Proteomics 2.9 This paper is available on line at http://www.mcponline.org