A matter of time: assessing the biological functions of oscillating natural antisense long non-coding RNA transcripts

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van Veen, Elizabeth
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University College Cork
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Circadian rhythms allow organisms to coordinate their metabolic and physiological responsesin accordance with Earth’s predictable daily, and yearly fluctuations in environmentalconditions. The roles of oscillating protein-coding transcripts have been well studied in theplant circadian clock, however recent research has identified that the transcription of long nonprotein coding RNAs can also oscillate in a diurnal manner. Here we document two circadianregulated lncRNAs that are natural antisense transcripts of two CDFs in the model plantArabidopsis thaliana. The first lncRNA “FLORE” forms a natural antisense transcript pair with CDF5, and these transcripts were shown to display antiphasic expression, reflecting a mutual inhibitory relationship. By resorting to ChIRP-seq, a multitude of putative FLORE genomic targets were identified, and through the implementation of molecular and bioinformatics techniques, four genes of interested were selected. Three of these genes; CCL, XPB1, and SR45A, exhibited upregulated transcription in a FLORE over-expressor under LD conditions. The fourth gene, RH27, displayed altered expression patterns between biological replicates. Finally, SR45A and RH27 were shortlisted for in-depth analysis. Histochemical GUS staining revealed that RH27 is transcriptionally active in newly developing tissues, as well as root tips and reproductivetissues. Finally, we found that a depletion in a gene related to SR45A (SR45) was associatedwith marginal changes in FLORE alternative splicing patterns. The second lncRNA “lncCDF1” is a natural antisense transcript of CDF1. Recent data have identified the CDFs as regulators of abiotic stress responses, and previous unpublished experiments have shown both CDF1 and lncCDF1 to be induced by drought. Here, we recorded a decrease in ABA sensitivity during seed germination, in a lnccdf1 promoter TDNA insertion mutant. Additionally lncCDF1 promoter activity appeared to be induced by ABA in two day old seedlings. Contrastingly, modulation of lncCDF1 transcript levels was not associated with altered ABA-mediated responses in older seedlings. Finally, we observed that CDF1 transcription is reduced in response to lncCDF1 over-expression, suggesting lncCDF1 may act in cis to repress lncCDF1. Taken together, these findings highlight the diverse regulatory functions carried out by lncRNAs in plants, and our findings provide a basis for further study on these transcripts.
lncRNA , Circadian clock
van Veen, E. 2021. A matter of time: assessing the biological functions of oscillating natural antisense long non-coding RNA transcripts. MRes Thesis, University College Cork.