Alternative splicing of transcripts of the transposon Restless is maintained in the foreign host Neurospora crassa and can be modified by introducing mutations at the 5′ and 3′ splice sites

Abstract

The primary transcript of the transposon Restless from Tolypocladium inflatum undergoes an unusual mechanism of alternative splicing by employing either of two 3′ “CAG” splice sites. These are separated by only four nucleotides, thus generating two different splice products, which differ in their coding capacity. To analyse whether this alternative splicing occurs in its natural host exclusively, we introduced the transposon into the heterologous host Neurospora crassa. In addition to the wild-type transposon sequence, transposon sequences mutagenised in vitro with modified 5′ and 3′ intron splice sites were generated. RNA was isolated from transformants and RT-PCR was performed with specific oligonucleotides flanking the intron sequence. Alternative splicing was analysed, employing a simple test procedure based on the convenient presence of a BamHI restriction site between both splice sites. The ratio of alternative splicing seems to be influenced by both the 5′ and the 3′ splice site, as mutations at either position influence the ratio of alternative splice products. At the 5′ splice site, mutating the first “C” has a strong effect on the ratio of alternative splicing, while mutating the second “C” has little or no effect. Similarly, at the 3′ splice site, only mutations at the first 3′ “CAG” change the ratio of alternative splicing. It appears that alternative splicing of the Restless intron is not host-specific, but is influenced by the intron splice site sequences themselves.