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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. 900 TIIIRD AVENUE NEW YORK. N.Y. 10022 Memorandum To: Dr. McAllister and Staff From: D.H.Ford Re: Site visit with Dr. D.Helfman, Cold Springs Harbor Laboratory, NY., Sept.8, 1992 . Visitors: D.H.Ford and A.Eisenberg Grant No. 3008 entitled "Molecular basis for tissue-specific alternative RNA splicing." Goals: To determine the molecular basis for tissue-specific alternative sp'1 cing, using the rat p-tropomyocin gene as a model to determine the molecular basis of alternative RNA splicing. Aims: To identify and isolate the cellular factor which inhibits the expression of the skeletal muscle-specific exon in non-muscle cells, and to biochemically identify the-cellular factors involved in tissue-specific RNA processing. As expressed by Dr. J. Watson in the Annual Report for the laboratory, Dr. Helfman is focussing on the ability of multi-parted genes to be put together in different ways to make a variety of related proteins (isoforms) Thus, the explanation of how splicing of neighboring stret_9hes of RNA (exons) into different combinations might explain structural difference between heart and skeletal muscle, etc. Results: During the first half year of this program Dr. Helfman has identified a,factor responsible for preventing the use of skeletal muscle specif~exon 7 in non-musdle cells. He and his colleagues have identified and purified at least one protein from non-muscle cells which interacts with specific sequences in the,d -tropomyosin pre-mRNA which are involved in blocking the use of skeletal muscle exon codes in non- muscle cells. His results demonstrated that intron sequences upstream of the 3' slice site of exon 7 were critical for complex formation and expression of exons for the synthesis of muscle protein. This factor has been purified to homogeneity. He is currently obtaining antibodies to this protein and isolating cDNA clones. A question which arises is that if there are intron-associated factors which negatively regulate RNA splicing, are there also factors which promote one sequence over another. The protein factor which Dr. Helfman has isolated which appears to block expression of the muscle exon 7 is identical to the polypyrimidine tract bbnding protein which other studies have suggested is important in the recognition and efficient use of 3'-splice sites. Details of his recently accepted publication on this protein will be occurring in JBC, "The polypyrimidine tract binding protein (PTB) interacts with sequences involved in alternative splicing of 6-tropomyosin pre-mRNA," Mulligan, et al. This publication will ACK CTR. Comment: An exciting fast-moving program directed by an enthusiastic young nvestigator who appears to be on the 'cutting edge' of this type of research. From his presentation to Dr. Eisenberg and myself, which was beautifully done, I would think he would make an excellent teacher. He certainly artfully educated us on what is a complex subject. What appeared of considerable significance was that his results are suggesting that introns may not simply be spacers between the effector exons, but may play a role in their expression by either negative or positive regulation. DHF