Regulation of co- and post-transcriptional pre-mRNA processing in response to environmental stress in green algae

Yale University, New Haven CT

Investigators

Abstract

Co-transcriptional processing of precursor messenger RNA (pre-mRNA) through 5’ capping, splicing, and 3’ end formation regulates gene expression by controlling transcript diversity and stability. Aspects of these processes are sensitive to environmental stress. In mammals and plants, alternative isoforms are generated under stress conditions through alternative splicing (AS) and alternative polyadenylation (APA). A variety of genes in human tissue culture cells also exhibit repression of 3’ end cleavage under stress, leading to the production of downstream-of-gene (DoG) RNAs. Human activity contributes to abiotic stresses in aquatic environments, such as changes in water temperature and purity, with the introduction of toxic heavy metals like arsenic. These factors threaten water quality and expose aquatic organisms to more frequent stress events. How environmental stressors, such as heat and water pollution, affect pre-mRNA processing in aquatic organisms like algae is unknown. To address this gap in knowledge, I propose the use of the freshwater green alga Chlamydomonas reinhardtii as a model organism due to its ease of laboratory cultivation and its fully-sequenced, intron-rich genome. I hypothesize that environmental stressors – changes in temperature and exposure to arsenite – induce specific changes in pre-mRNA processing in C. reinhardtii that can be quantified and evaluated for biological significance. To address this hypothesis, I propose three aims. In Aim 1, I will determine the effects of environmental stress on global pre-mRNA processing by identifying changes in AS and APA using next generation sequencing (NGS) and long-read sequencing (LRS) of mature RNA. LRS is a relatively recent sequencing strategy that identifies all of the sequence in single transcripts. This associates all possible changes, such as alternative exons and alternative polyadenylation sites, enabling me to predict the protein isoforms produced in every case. This can critically impact the interpretation regarding function. My preliminary results have identified over 300 cases of alternative cassette exon usage induced by high temperature; this includes an exon skipping event that is predicted to introduce a premature termination codon within the sequence coding for the kinase domain of a serine-threonine protein kinase. In Aim 2, I will characterize transcriptional defects induced by environmental stress by quantifying DoG induction using NGS and LRS of nascent RNA, a specialty of the Neugebauer lab that has previously led to mechanistic insights. For both Aims 1 and 2, I will validate the biological significance of these RNA processing events by generating mutant strains with CRISPR and by assessing their performance in a stress survival assay. Finally, in Aim 3, I will analyze the conservation of gene sequences and RNA processing changes among freshwater algae using comparative genomics and reverse transcription polymerase chain reaction (RT-PCR). Together, these results will provide valuable insights into the ways aquatic organisms cope with environmental stress at a molecular level. These results will also enable the development of bioassays and/or bioindicators that relate molecular phenotypes, such as changes in AS, APA, or DoG induction, with water quality.