Multiple control mechanisms intersect to ensure cellular need for RNA is matched by expression of the transcriptome. The alternative formation of functional and aberrant transcripts is a point of gene expression control, which can be implemented through changing the mRNAs cleavage site, the amount of functional mRNA produced can be reduced in a process known as transcriptional attenuation. GAA repetitive regions are associated with transcriptional defects and formation of DNA:RNA hybrid structures, and highly expanded repeats cause the neurodegenerative human disease Friedreich ataxia. Here we explore a link between GAA repetitive regions, transcriptional defects and transcriptional attenuation in Saccharomyces cerevisiae.
Using 3’-focussed RNA-seq we and others noticed that a surprising number of transcripts contained what appeared to be abundant truncated isoforms that would result in aberrant protein products. We identified that many of the genes that have such unannotated 3‘ends, overlapped with GAA repetitive regions in whole transcriptome RNA-seq data. To validate truncations in candidate genes we characterised transcript ends and poly(A) tail lengths of transcripts using targeted amplicon sequencing of 3’-ends. We could detect uncharacterised prematurely terminated transcripts at GAA repetitive regions, which are possible sites of gene expression regulation though transcriptional attenuation. Furthermore, they have atypical poly(A) tail length distributions, and a proportion of unadenylated transcripts, suggesting that they are not produced by canonical cleavage and polyadenylation. Paradoxically, the apparently abundant internal truncations that first drew our attention, were a product of mis-priming by oligo(dT) based cDNA synthesis methods prone to mispriming at A rich GAA repeats, masking real transcript ends.
We are currently exploring how these transcripts are terminated, and if the transcription cleavage site choice is stress responsive. That is, if transcriptional attenuation and nascent transcript cleavage at GAA repeats is a mechanism to toggle the transcriptional output at specific genetic loci.