Introns are key regulators of eukaryotic gene expression and present a potentially powerful tool for the design of synthetic eukaryotic gene expression systems. However, intronic control over gene expression is governed by a multitude of complex, incompletely understood, regulatory mechanisms. Despite this lack of detailed mechanistic understanding, here we show how a relatively simple model enables accurate and predictable tuning of synthetic gene expression system in yeast using several predictive intron features such as transcript folding and sequence motifs. Using only natural Saccharomyces cerevisiae introns as regulators, we demonstrate fine and accurate control over gene expression spanning a 100 fold expression range. These results broaden the engineering toolbox of synthetic gene expression systems and provide a framework in which precise and robust tuning of gene expression is accomplished.

RNA splicing is the central process of intron removal in eukaryotes known to regulate various cellular functions such as growth, development, and response to external signals. The canonical sequences indicating the splicing sites needed for intronic boundary recognition are well known. However, the roles and evolution of the local folding of intronic and exonic sequence features adjacent to splice sites has yet to be thoroughly studied. Here, focusing on four fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, and Candida albicans), we performed for the first time a comprehensive high-resolution study aimed at characterizing the encoding of intronic splicing efficiency in pre-mRNA transcripts and its effect on intron evolution. Our analysis supports the conjecture that pre-mRNA local folding strength at intronic boundaries is under selective pressure, as it significantly affects splicing efficiency. Specifically, we show that in the immediate region of 12-30 nucleotides (nt) surrounding the intronic donor site there is a preference for weak pre-mRNA folding; similarly, in the region of 15-33 nt surrounding the acceptor and branch sites there is a preference for weak pre-mRNA folding. We also show that in most cases there is a preference for strong pre-mRNA folding further away from intronic splice sites. In addition, we demonstrate that these signals are not associated with gene-specific functions, and they correlate with splicing efficiency measurements (r = 0.77, P = 2.98 × 10 −21) and with expression levels of the corresponding genes (P = 1.24 × 10 −19 ). We suggest that pre-mRNA folding strength in the above-mentioned regions has a direct effect on splicing efficiency by improving the recognition of intronic boundaries. These new discoveries are contributory steps toward a broader understanding of splicing regulation and intronic/transcript evolution.

The two major steps of gene expression are transcription and translation. While hundreds of studies regarding the effect of sequence features on the translation elongation process have been published, very few connect sequence features to the transcription elongation rate. We suggest, for the first time, that short transcript sub-sequences have a typical effect on RNA polymerase (RNAP) speed: we show that nucleotide 5-mers tend to have typical RNAP speed (or transcription rate), which is consistent along different parts of genes and among different groups of genes with high correlation. We also demonstrate that relative RNAP speed correlates with mRNA levels of endogenous and heterologous genes. Furthermore, we show that the estimated transcription and translation elongation rates correlate in endogenous genes. Finally, we demonstrate that our results are consistent for different high resolution experimental measurements of RNAP densities. These results suggest for the first time that transcription elongation is partly encoded in the transcript, affected by the codon-usage, and optimized by evolution with a significant effect on gene expression and organismal fitness.

Supplementary data are available at Bioinformatics online.

It is generally believed that introns are not translated; therefore, the potential intronic features that may be related to the translation step (occurring after splicing) have yet to be thoroughly studied. Here, focusing on four fungi, we performed for the first time a comprehensive study aimed at characterizing how translation efficiency is encoded in introns and affects their evolution. By analysing their intronome we provide evidence of selection for STOP codons close to the intronic 5′ end, and show that the beginning of introns are selected for significantly high translation, presumably to reduce translation and metabolic costs in cases of non-spliced introns. Ribosomal profiling data analysis in Saccharomyces cerevisiae supports the conjecture that in this organism intron retention frequently occurs, introns are partially translated, and their translation efficiency affects organismal fitness. We show that the reported results are more significant in highly translated and highly spliced genes, but are not associated only with genes with a specific function. We also discuss the potential relation of the reported signals to efficient nonsense-mediated decay due to splicing errors. These new discoveries are supported by population-genetics considerations. In addition, they are contributory steps towards a broader understanding of intron evolution and the effect of silent mutations on gene expression and organismal fitness.