Systematic functional and mechanistic analysis of gene regulation by antisense transcription in Saccharomyces cerevisiae
Final Report Abstract
Tiling arrays and RNA sequencing have revealed that pervasive transcription is widespread in the eukaryotic genome, mainly due to the bidirectional nature of most promoters. Currently it is a major topic of research to understand which of these transcripts are functional, and how they function. The transcripts include antisense RNAs (asRNA), a class of non-coding transcripts that overlap protein-coding genes in opposing direction. Several asRNAs have been implicated in the regulation of specific sense genes, but a global mechanism and mutual antisense transcript characteristics are still elusive. The goal of this study was to investigate the global mechanisms of antisense dependent gene regulation in yeast by employing a new method to disrupt a large number of these transcripts specifically and in high throughput. For this we developed and applied a seamless gene manipulation strategy based on an sfGFP tag in combination with a unidirectional transcriptional terminator to block the expression of 162 antisense transcripts in Saccharomyces cerevisiae. We then investigated the effect of this genespecific antisense abrogation using different assays and growth conditions. Using high throughput microscopy and quantitative PCR, we could show that the expression of an asRNA alone is not enough to influence sense gene expression, as only around 25% of the asRNAs had a significant effect on the corresponding sense gene’s protein abundance. Other factors including the degree of sequence overlap at the sense transcription start site are important contributors to regulatory functionality. Also, genes that are regulated by asRNAs have characteristic chromatin methylation marks, specifically increased H3K4me di- and trimethylation. In general, asRNAs appear to have a weak effect on sense expression. The abundance of some proteins, however, seems to be regulated by asRNAs in an ‘on/off-switch’ manner, indicating that there are important gene-dependent specifications. Future studies are warranted to characterize these mechanisms in more detail. The work furthermore led to the identification of a few genes that exhibited very strong and highly condition-specific antisense gene regulation. A detailed functional analysis revealed a series of new factors involved in this process that are part of an ongoing investigation. We also identified how transcript boundary variation and alternative polyadenylation can diversify post-transcriptional gene regulation by selective RNA-protein interactions. We revealed that changes in single nucleotides within a gene’s 3’UTR can strongly affect the resulting transcript’s stability. These findings provide additional information about the dynamics and mechanisms of gene regulation. Finally, we developed 5PSeq, a new genome-wide technology to map mRNA degradation in yeast. Using this method we revealed widespread co-translational mRNA decay. While this part of our work strays a little from our originally specified specific aims, it provides valuable information about the mechanisms governing gene expression, as well as a technological development. Overall, this DFG grant has resulted in many meaningful insights to advance the field of gene regulation. We anticipate continuing to collaborate beyond this grant and expect to prepare a new grant application in the near future.
Publications
- (2014). Alternative polyadenylation diversifies post-transcriptional regulation by selective RNA-protein interactions. Mol Syst Biol. Feb 25;10:719
Gupta, I., Clauder-Münster, S., Klaus, B., Järvelin, A.I., Aiyar, R.S., Benes, V., Wilkening, S., Huber, W., Pelechano, V. & Steinmetz, L.M.
(See online at https://doi.org/10.1002/msb.135068) - (2014). PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast. PLoS ONE. Dec 10;9(12):e114590
Huber, F., Meurer, M., Bunina, D., Kats, I., Maeder, C.I., Stefl, M., Mongis, C., and Knop, M.
(See online at https://doi.org/10.1371/journal.pone.0114590) - (2014). Protein quality control at the inner nuclear membrane. Nature. Dec 18;516(7531):410-3
Khmelinskii, A., Blaszczak, E., Pantazopoulou, M., Fischer, B., Omnus, D.J., Le Dez, G., Brossard, A., Gunnarsson, A., Barry, J.D., Meurer, M., Kirrmaier, D., Boone, C., Rabout, G., Ljungdahl, P. and Knop, M.
(See online at https://doi.org/10.1038/nature14096) - (2015). High-throughput ChIP-Seq for large-scale chromatin studies. Mol Syst Biol. Jan 12;11(1):777
Chabbert, C.D., Adjalley, S.H., Klaus, B., Fritsch, E.S., Gupta, I., Pelechano, V. & Steinmetz, L.M.
(See online at https://doi.org/10.15252/msb.20145776) - (2015). Widespread co-translational RNA decay reveals ribosome dynamics. Cell. Jun 4;161(6):1400-12
Pelechano, V., Wei, W. & Steinmetz, L.M.
(See online at https://doi.org/10.1016/j.cell.2015.05.008) - (2016). Genome-wide quantification of 5′-phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics. Nat Protoc. Feb;11(2):359-76
Pelechano, V., Wei, W. & Steinmetz, L.M.
(See online at https://doi.org/10.1038/nprot.2016.026) - Protein Abundance Control by Non-coding Antisense Transcription. Cell Reports Volume 15, Issue 12, 21 June 2016, Pages 2625-2636
Huber, F., Bunina, D., Gupta, I., Khmelinskii, A., Meurer, M., Theer, P., Steinmetz, L.M. & Knop, M.
(See online at https://doi.org/10.1016/j.celrep.2016.05.043)
