Poster abstracts
Poster number 84 submitted by Hiranthi Kulathungage
Rif-seq reveals C. crescentus mRNA decay is globally coordinated with transcription and translation
Hiranthi Kulathungage (Department of Biological Sciences, Wayne State University, Detroit, MI, USA), James R. Aretakis (Department of Biological Sciences, Wayne State University, Detroit, MI, USA), Kavya Vaidya (Department of Physics, University of Illinois, Urbana-Champaign, IL, USA), Nadra Al-Husini, Nisansala S. Muthunayake (Department of Biological Sciences, Wayne State University, Detroit, MI, USA), Sangjin J. Kim (Department of Physics, University of Illinois, Urbana-Champaign, IL, USA), Jared M. Schrader (Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI, USA)
Abstract:
mRNA decay is an essential and highly regulated step of gene expression that acts as an off switch1,2. To examine the global regulation of mRNA decay in the bacterium C. crescentus, we utilized Rif-seq, a method which allows for global measurement of mRNA lifetimes. We observed about 1/3 of the mRNAs are cotranscriptionally degraded and many mRNAs in different gene functional categories have variable turnover rates. To examine the role of translation on mRNA stability, we mapped the global RNA decay sites containing a 5’ P end sequencing. Across the transcriptome, we identified a majority of mRNA cut sites in 5’ UTRs, the translation initiation regions, and in coding sequences. Using ribosome profiling, we observed that half-life is correlated with translation efficiency and mRNA 5’ P cleavage sites are enriched in regions that are poorly occupied by ribosomes. To distinguish the individual roles of translation initiation and elongation on mRNA half-life, we conducted Rif-seq experiments on cells treated with initiation inhibitor retapamulin and elongation inhibitor chloramphenicol, which showed that retapamulin led to a slight stabilization of mRNA half-life, while chloramphenicol led to a near total stabilization of the entire transcriptome. In line with this observation that mRNA decay rates are strongly influenced by the translation elongation rate, we examined the codon adaptation index (CAI) and surprisingly, observed a strong negative correlation between CAI and global mRNA half-lives (R2 = 0.66). To systematically alter the rates of translation initiation and elongation and test their impact on mRNA decay, we designed synthetic YFP mRNAs with weak and strong initiation regions (TIRs) and combined these with codon usage ranging from highly suboptimal to highly optimal. Across the synthetic mRNAs we observed an inverse correlation between mRNA half-lives and CAI, while we observed a stabilizing impact from the TIR strength. While initiation and elongation influence the overall translation efficiency, TIR and CAI appear to work in an antagonistic manner to determine the overall mRNA half-life.
References:
1Presnyak, V., Alhusaini, N., Chen, Y.-H., Martin, S., Morris, N., Kline, N., Olson, S., Weinberg, D., Baker, K. E., Graveley, B. R., & Coller, J. (2015). Codon optimality is a major determinant of mrna stability. Cell, 160(6), 1111–1124.
2Chan, L. Y., Mugler, C. F., Heinrich, S., Vallotton, P., & Weis, K. (2018). Non-invasive measurement of mrna decay reveals translation initiation as the major determinant of mrna stability. eLife, 7.
Keywords: mRNA decay, translation initiation and elongation, Rif-seq, Ribosome profiling