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cases. The branch-point adenosine, also invariant, usually is 20-50 bases from the 3' splice site. The central region of the intron, which may range from 40 bases to 50 kilobases in length, generally is unnecessary for splicing to occur. [See R. A. Padgett et al., 1986, Ann. Rev. Biochem. 55:1119, and E. B. Keller and W. A. Noon, 1984, Proc. Nat'l. Acad. Sci. USA 81:7417.]

containing multiple exons, splicing of exons in the nascent RNA usually begins before transcription of the gene is complete.

Early evidence that introns are removed during splicing came from electron microscopy of RNA-DNA hybrids between adenovirus DNA and the mRNA encoding hexon, a major virion capsid protein (Figure 12-5). Other studies revealed nuclear viral RNAs that were colinear with the viral DNA (primary transcripts) and RNAs with one or two of the introns removed (processing intermediates). These results, together with the findings that the 5' cap and 3' poly(A) tail at each end of long mRNA precursors are retained in shorter mature cytoplasmic mRNAs, led to the realization that in-trons are removed from primary transcripts as exons are spliced together.

The location of splice sites—that is, exon-intron junctions—in a pre-mRNA can be determined by comparing the sequence of genomic DNA with that of the cDNA prepared from the corresponding mRNA. Sequences that are present in the genomic DNA but absent from the cDNA represent in-trons and indicate the positions of splice sites. Such analysis of a large number of different mRNAs revealed moderately conserved, short consensus sequences at the splice sites flanking introns in eukaryotic pre-mRNAs; in higher organisms, a pyrimidine-rich region just upstream of the 3' splice site also is common (Figure 12-6). Studies with deletion mutants have shown that much of the center portion of introns can be removed without affecting splicing; generally only 30-40 nucleotides at each end of an intron are necessary for splicing to occur at normal rates.

Analysis of the intermediates formed during splicing of pre-mRNAs in vitro led to the discovery that splicing of exons proceeds via two sequential transesterification reactions (Figure 12-7). Introns are removed as a lariat-like structure in which the 5' G of the intron is joined in an unusual 2',5'-phos-phodiester bond to an adenosine near the 3' end of the intron. This A residue is called the branch point because it forms an RNA branch in the lariat structure. In each transesterification reaction, one phosphoester bond is exchanged for another. Since the number of phosphoester bonds in the molecule is not changed in either reaction, no energy is consumed. The net result of these two reactions is that two exons are ligated and the intervening intron is released as a branched lariat structure.

Intron

Intron

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