Extensive transcriptional heterogeneity revealed by isoform profiling

Abstract

Variation among RNA transcript isoforms can be generated from alternative start and polyadenylation sites, and results in RNAs and proteins with different properties being generated from the same genomic sequence; here a new method termed transcript isoform sequencing is described in yeast, and the method allows a fuller exploration of transcriptome diversity across the compact yeast genome. The expression of eukaryotic genomes is a complicated matter, a long way from the old picture of a series of distinct protein-coding genes separated by less-important tracts of DNA. Lars Steinmetz and colleagues have used a novel technique termed TIF-Seq to demonstrate that the yeast genome containing around 6,000 protein-coding genes produces more than 1.88 million unique transcript isoforms (TIFs), defined as unique combinations of start (5′) and end (3′) RNA sequences. This work demonstrates that the complexity of overlapping transcript isoforms has been greatly underestimated previously. Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect messenger RNA stability, localization and translation1, or produce truncated proteins that differ in localization2 or function3. Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed4,5. Here, by jointly determining both transcript ends for millions of RNA molecules, we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries seems to be the rule rather than the exception, even within a single population of yeast cells. Over 26 major transcript isoforms per protein-coding gene were expressed in yeast. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, approximately 70% of genes express alternative isoforms that vary in post-transcriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution and phenotypic diversity between single cells. These data also indicate that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes.