912 Fairchild Center, M.C. 2433
New York, N.Y. 10027
Several aspects of the mechanisms of gene expression in cultured mammalian cells are being investigated. The common thread is to use a genetic approach, isolating mutant or transfected cells that are altered in one or another step in gene expression. A molecular analysis of the altered phenotypes provides new information about the normal processes.
Much of our current efforts are in the area of RNA processing. We are investigating the following major unresolved questions in RNA processing are: 1) How are splice sites recognized among the thousands of bases in a typical transcript? Many consensus splice site sequences are present, but only the "real" ones are used; 2) How is mRNA transported out of the nucleus?
A major current effort is to understand how the splicing machinery is able to identify internal exons (~100 nt) in a sea of introns (~10000 nt). Large mammalian genes contain many false exons, identified as having the splice site consensus sequences typical of real exons, yet are not spliced. One possible explanation is that the many false splice sites are repressed by neighboring cis-acting sequences. In a search for such splicing silencers, we have found that such sequences are surprisingly common in the human genome, and thus probably in introns, occurring with a frequency of one every few hundred nucleotides. In contrast, the frequency of silencing sequences derived from E. coli DNA is much lower. Many of these sequences contain polypyrimidine tracts, and we are pursuing the idea that they act as non-productive sites for spliceosome assembly.
As one strategy we have chosen a model false exon (pseudo exon) from the hprt gene, a sequence that looks as if it should be an exon, but is not. We have asked what mutational perturbation is necessary to turn this pseudo exon into a functional exon. We found that the pseudo exon was defective in both its 3' and 5' splice sites and in addition was bounded by a silencing sequence. It was necessary to replace both the 3' and 5' splice sites and to remove the inhibitory sequence to activate splicing. On the other hand, no apparent enhancer sequence was necessary once these changes had been made. Using in vitro splicing and spliceosome assembly assays, we have found that these pseudo spice sites are individually defective at an early stage in recognition.
Our results as well those of others point a major role of the surrounding context in defining the use of a consensus sequence as a splice site. Our present experiments are designed to define this context effect in molecular detail. One approach is to insert a functional exon at many different positions in a large intron to examine how the resulting marginal context differences affect splicing. Using genetic selection techniques, we plan to map functional contexts to ~10 nt resolution. Another plan is to mutate a splice site at all non-conserved positions to obtain the spectrum of sequences that support splicing within a given context. The experiment would then be repeated in the face of systematic context changes (splice mating sequence, exon sequence, intron sequences).
We are also using a computational approach to search for signals that differentiate true exons from false ones. In collaboration with Christina Leslie in Computer Science, we are comparing the sequence context surrounding true exons with those of false exons in an effort to pinpoint consistent differences that are may be sites of molecular recognition. This strategy may allow us to identify repressive signals as well as possible enhancers of splicing. We would welcome students (undergraduates and Masters students) with programming ability to participate in this bioinformatics project.
Another area of our interest is the role of nuclear organization on RNA processing. We have found that transcripts of a gene placed in different positions within the genome are processed with reduced efficiency compared to the gene at its wild type position. Splicing and polyadenylation do not seem to be the steps affected, leaving mRNA transport and/or nuclear mRNA degradation as possible sensitive steps.
Research project pages (private)
|Genomic sequences that inhibit splicing||Pseudo exons abound|
Xiang Zhang, graduate student (on left)
Representative Publications (go to new site for updated list)
Zhang, X. H-F., J. Lee, and L.A. Chasin. (2003) The effect of nonsense codons on splicing: a genomic analysis. RNA, 9: 637-9. Manuscript
Fairbrother, W. and Chasin, L.A. (2000) Human genomic sequences that inhibit splicing . Mol. Cell. Biol. Mol. Cell. Biol. 20: 6816-6825 Abstract Manuscript
Sun, H. and Chasin, L.A. (2000) Defective splice signals in an intronic false exon. Mol. Cell. Biol. 20:6414-6425. Abstract Manuscript
Noe, V., C.J. Ciudad, and L.A. Chasin. (1999) Effect of differential polyadenylation and cell growth phase on dihydrofolate reductase mRNA stability. J. Biol. Chem. 274:27807-27814. Abstract
Bai, Y. , D. Lee, T. Tu, and L.A. Chasin. (1999) Control of 3' splice site selection in vivo by ASF/SF2 and hnRNP A1. Nucleic Acids Res. 27:1126-1134Abstract
Chen, Chao and Lawrence A. Chasin. (1999) Cointegration of DNA molecules introduced into mammalian cells by electroporation, Somat. Cell Molec. Genet. 24: 249-256..Abstract
Noe, V., C. Chen, C. Alemany, M. Nicolas, I. Caragol, L.A. Chasin, and C. J. Ciudad. (1997). Cell growth regulation of the hamster dihydrofolate reductase gene promoter by transcription factor Sp1. Eur. J. Biochem. 249: 13-20
Kessler, O. & Chasin, L.A. (1996) The Effect of Nonsense Mutations on Nuclear and Cytoplasmic Adenine Phosphoribosyltransferase RNA. Mol Cell Biol 16:4426-4435. Abstract
Chen, I-T., and Chasin, L.A. (1994) Large exon size does not limit splicing in vivo. Mol. Cell. Biol. 14:2140-2146.Abstract
Carothers, A.M., Urlaub, G., Grunberger, D., and Chasin, L.A. (1993) Splicing mutations and their second-site suppressors at the dhfr locus in Chinese hamster cells. Mol. Cell. Biol. 13:5085- 5098.Abstract
Chen, I-T. and Chasin, L.A. (1993) Direct selection for splicing mutations in a dhfr minigene. Mol. Cell. Biol. 13:289-300.Abstract
Kessler, O., Jiang, Y., and Chasin, L.A. (1993) The order of intron removal during the splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA. Mol. Cell. Biol. 13:6211-6222.Abstract
Ciudad, C.J., Morris, A.E., and Chasin, L.A. (1992) Point mutational analysis of the hamster dihydrofolate reductase minimum promoter. J. Biol. Chem. 263:16274-16282.Abstract
|C2005||Introduction to Molecular and Cellular Biology, I|
|F2401||Contemporary Biology, I|
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