| Popis: |
In order to establish the structural features of the cis-elements involved in splicing and in its regulation, we have analyzed a synthetic premessenger RNA, derived form the E3 transcript unit of adenovirus-2 and previously shown to be a good substrate for in vitro splicing. The transcript was probed by enzymatic and chemical methods and we present the structure in solution of the upstream exon and of the 5′ part of the intron. This 417 nucleotide long fragment, which overlaps the exon-intron junction harbors the natural 5′ splice site D1 and an intronic cryptic site, Dcr1, used when D1 is suppressed. The 5′ exon is folded in three stem-loop structures and D1 is located in a free single-stranded region close to the foot of the most stable of these structures (ex1-HP1). The 5′ part of the intron also contains a stable hairpin structure (IVS1-hp1), which sequesters Dcr1. The different structural context of the two 5′ splice sites may partly explain the selection of D1 and the silencing of Dcr1. We also found a long-range, 20 base-pair, exon-intron interaction, which agrees with the enzymatic and chemical probings and was further demonstrated by the study of the colinear messenger RNA, lacking the intron and of 5′ deletion transcripts, lacking the 5′ part of the exon. This folding creates a three-branched structure, including IVS1-hp1 and divides the 5′ part of the transcript into two domains. Finally, only a few sequences are not involved in folded structures. Free single-stranded fragments are found between the exonic hairpins and at the beginning of the intron and are mostly U-rich. All the structural features of the adenovirus-2 transcript are conserved in adenovirus-5, in spite of 37 nucleotide substitutions. |
