In vitroprocessing of the human growth hormone primary transcript

To study the sequence of events during processing of primary RNA transcripts and to gain more insight into the mechanism of splice site selection, the in vitro processing of a 2.5 kb human growth hormone (hGH) pre-mRNA containing four introns and an alternative 3' splice site for intron B was analysed. In order to process the hGH pre-mRNA the preparation of the HeLa cell nuclear extract had to be modified, indicating differences in factor requirement for processing this pre-mRNA. After an unusual long lag phase of one hour splicing intermediates begin to accumulate. Intron A and D are removed with correct ligation of exons 1/2 and 4/5. Most splice sites are used--albeit with variable efficiencies--except the splice sites surrounding exon 3 and the 3' alternative splice site within exon 3; as a consequence "exon skipping" events take place. Using a pre-mRNA containing only intron B neither the 5' nor the 3' splice site is cleaved, indicating that the 3' splice site of intron B is not recognized. The results show that splice sites can differ considerably in their requirement for splicing factors.