Group II introns are ribozymes that are encoded within all domains of life. They are also capable of mobility through an RNA intermediate. Due to similarities in RNA structure and splicing mechanisms, group II introns are thought to have been the ancestors of nuclear pre-mRNA introns and snRNAs.
In this dissertation I report the discovery of a unique ORF-less group II intron, C.te.I1, in the human pathogen Clostridium tetani. The intron is encoded within a surface layer protein region of the C. tetani chromosome and possesses an unusual genomic organization such that a full-length copy of the intron is followed downstream by three copies of the RNA structural domains 5 and 6 (D5/6). This arrangement led to the hypothesis that C.te.I1 is capable of alternative splicing utilizing the downstream copies of D5/6 as alternate 3ʹ splice sites. RNA extractions and RT-PCR support the hypothesis and revealed that the splicing reaction of C.te.I1 links a surface layer protein ORF (CTC00465) in the upstream exon to one of four downstream ORFs that encode transglutaminase-related or protease-related reading frames (CTC00467-CTC00470). Including unspliced transcript, five mRNAs are produced.
Sequencing of the exon junctions showed that the 5ʹ splice site utilized by C.te.I1 is shifted 8 nt upstream both in vivo and in vitro. Use of this splice site is critical to alternative splicing as it results in the elimination of the stop codon at the end of CTC00465 and results in the correct ligation of 5ʹ and 3ʹ exon sequences. Site-directed mutagenesis and self-splicing assays for C.te.I1 revealed that the shifted splice site is due to a novel EBS1-IBS1 pairing. Although C.te.I1 is thought to be derived from a mobile Class B (IIB) intron that lost its ORF, the intron was found to have evolved to utilize a IIA-like mechanism of 3ʹ splice site recognition. These changes represent structural adaptations of the intron to its role in alternative splicing. The structural adaptations and splicing of C.te.I1 illustrate the plasticity of group II introns in that they can adapt new RNA structural and catalytic properties which can be utilized to affect gene expression.