Origin of intervening sequences within mammalian genes and the universal signal for their removal.

The mammalian genome is enormous (approximately 3.2 × 109 base pairs) and contains multitudes of senseless base sequences. Consequently, any two very closely linked genes are still separated by a spacer a few thousand base pairs long. Therefore, tandem fusions of neighboring coding sequences can only be accomplished by the posttranscriptional removal of former spacers from polycistronic nuclear transcripts. In this manner, the splicing mechanism to remove intervening sequences originated. Pairs of base sequences, which occupy both the junctions of individual intervening sequences and serve as splicing signals, are neither species-specific nor cell-type specific, since they all have apparently descended from the single, ancestral sequence 14 bases long: CCCACAG/GUGAGAG. The prototype upstream signal can be drawn as CCC.ACAG/GUGAGAG; the codon-straddling AG marking the 3′coding sequence end. Its prototype downstream partner can be drawn as CCCACAG/GU.GAG.AG (bases of an intervening sequence in italics). As soon as individual transcripts began to contain two or more intervening sequences, the immediate need arose to diversify splicing signal base sequences, for the prototype splicing signal CCCACAG/GUGAGAG remains as such after the act of each splicing, thus, splicing continues until all the internal coding sequences have been removed. Furthermore, an inadvertent splicing involving the first upstream signal and the second or third downstream signal cannot be avoided. Due to these necessary diversifications, coding halves of most of the splice partners now bear little resemblance to the prototype upstream and downstream sequences; CCCACAG and GUGAGAG. As far as coding halves of the splicing pair are concerned, the only stringent requirement that remains is the need for upstream coding sequences to end in the straddling AG. However, straddling AG base doublets can now be found within the codon. This is because triplet base counting units by which a straddling AG is defined can become out of phase with codons. By contrast, intervening halves of the splicing partner deviated relatively little from the prototype GUGAGAG ––––– CCCACAG. This is due to the persistent requirement for the 5′ and 3′ ends of each intervening sequence to maintain a reasonable base sequence complementarity that permits base pairing. Of all the coding sequences examined, AG/GU base quadruplets were never found in the coding sequence interior, and herein lies the significance of the triplet base counting rule. Without this restriction in the choice of AG base doublets, a number of apparent upstream splice signals can be found within the coding sequence. For example, the first coding sequence of mouse Hb βmaj contains the sequence which can be drawn as AAG/GUGAACUCC specifying Lys. Val. Asn. Ser. at the 17th to 20th position. In the case of intervening sequences, however, the rule that their 3′ends should be marked by straddling AG is not enforced when the prototype sequence CCCACAG or its immediate derivatives such as CUUAG occupy their 3′ ends. Accordingly, those intervening sequences endowed with the pair of prototype sequences GUGAGAG ––––– CCCACAG or their immediate derivatives can tolerate numerous internal base changes. Thus, the possession of long intervening sequences does not unduly burden the mammalian genome with too large an additional mutation load.