Figure 1.
Incorporation of 4sU and 4tU into K. brevis RNA.
A. Slot blots of biotinylated total RNA (1 µg) show that K. brevis specifically incorporates 0.2 mM 4-thiouracil (4tU), but not 0.2 mM 4-thiouridine (4sU), during a 20 h exposure. B. Following exposure to 0.2 mM 4tU, incorporation into RNA is detected in as little as 5 minutes (10 µg RNA loaded per slot). A representative blot is shown from multiple studies. An equal amount total RNA from cells treated with DMSO (carrier), processed similarly, served as a negative control to measure non-specific binding of biotin. Detection was with streptavidin-HRP.
Figure 2.
Representative bioanalyzer profiles of pre-existing and newly synthesized RNA fractions.
A. Following 4-thiouracil (4tU) incorporation, biotinylation, and streptavidin-magnetic bead purification, newly synthesized RNA fractions are comparable to purified mRNA samples from K. brevis. With increasing labeling time, synthesis of ribosomal RNA becomes discernible, with peaks at approximately 41 and 44 s. The peak at 23 s is the manufacturer supplied internal marker (visually absent in B because of differences in the Y-axis scale). B. Bioanalyzer profiles of pre-existing RNA are similar to typical K. brevis total RNA profiles with dominant ribosomal peaks at approximately 41 and 44 s.
Figure 3.
Karenia brevis mRNA half-lives.
Following 2 h incubation with 0.2 mM 4-thiouracil, total RNA was biotinylated and bead purified. Total, pre-existing, and newly synthesized RNA fractions were hybridized to a custom oligonucleotide microarray and mRNA half-lives were calculated for 7086 features using HALO. Following normalization by linear regression, newly synthesized RNA to pre-existing RNA ratios were used to calculate half-lives.
Figure 4.
mRNA half-life distributions for selected GO terms.
Gene ontology terms were assigned using Blast2GO Yeast GO slim. mRNA half-lives were binned on time and distributions are shown for selected GO terms. A. Biological Process GO terms. GO:0006950, response to stress; GO:0019725, cellular homeostasis; GO:0006091, generation of precursor metabolites and energy; GO:0045333, cellular respiration. B. Molecular Function GO terms. GO:0030528, transcription regulator activity; GO:0045182, translation regulator activity.
Table 1.
Significantly over-enriched gene ontology (GO) categories, assigned using the standard GO vocabulary, among features with calculated half-lives of at least 3 days.
Figure 5.
mRNA half-live distributions for pentatricopeptide repeat (PPR) protein transcripts.
mRNA half-lives were calculated for 47 of the 93 features annotated as PPRs on the array. mRNA half-lives were binned on time and distributions are shown for these PPR transcripts. The distribution of 7086 features of the K. brevis transcriptome are re-plotted from figure 3 for ease of comparison.
Figure 6.
Stability of selected mRNAs following transcription inhibition assessed by qPCR.
Triplicate cultures were treated with 2 µg·mL−1 actinomycin D for 12, 24, or 48 h and the transcript abundance determined relative to DMSO carrier controls at each time point. Means ±SD (n = 3) are plotted. The PPR transcript was undetected in two of three replicates at 24 and 48 h following 40 PCR cycles, therefore SDs were not calculated. Dashed lines represent a 2-fold change.
Table 2.
Array features with significantly different abundance during the first hour post-nitrate addition, relative to N-depleted cultures.
Table 3.
Array features with significantly different abundance during the fourth hour post-nitrate addition, relative to N-depleted cultures.