(A) Northern blot showing the presence of truncated frq mRNA species in both de-optimized strains using an RNA probe targeted to 5′ end of frq mRNA (indicated in ; Figure 2-figure supplement 1E). * indicates a non-specific band. (B) Northern blot showing both full-length and truncated frq mRNA are enriched in poly(A)-containing RNAs. Equal amounts of total RNA or poly(A) RNA were loaded in each lane. Probe specific for 5′ end of frq was used. (C) Poly(A) sites mapped by 3′ RACE. Arrows indicate the mapped poly(A) sites, the red arrows indicate the major poly(A) site that was found in both frq-deopt1 and frq-deopt2 strains, and the black line indicates potential PAS motif (AUAAAU in frq-deopt1 and AAUAAA in frq-deopt2). Nucleotides that are mutated are shown in red. (D) ChIP assay showing RNA pol II levels at the frq transgene loci in the wt-frq-aq and frq-deopt2-aq strains. The ChIP results were normalized by input DNA and represented as Input%. The promoter of qrf was replaced by a qa-2 promoter and tissue were cultured in the absence quinic acid to block qrf transcription. The triangle on the top indicates the location of mapped poly(A) sites. The previously known heterochromatin region ?63 in Neurospora was used as the negative control. Error bars shown are standard deviations (n = 3). *p<0.05. (E) Northern blot analysis showing premature transcription termination of qrf. f-frq is an frq codon-optimized strain (Zhou et al., 2013a).
There are two main options for how this type of truncated polyadenylated frq mRNAs can be produced: PAS-dependent untimely transcription cancellation or partial destruction away from full-size frq mRNAs followed by polyadenylation (van Hoof ainsi que al., 2002; Frischmeyer ainsi que al., 2002; West mais aussi al., 2006; LaCava mais aussi al., 2005). Regarding untimely transcription termination, RNA polymerase II (pol II) terminates after synthesis of the 5′ section of the pre-mRNA , which is following put-out regarding chromatin (Proudfoot, 2016). , 2003; Xue mais aussi al., 2014) (Contour dos-figure enhance 1E), that can complicate brand new translation of Chip performance. To get over it side effects, we composed a couple of additional frq constructs, wt-frq-aq, and frq-deopt2-aq, where the promoter out-of qrf is replaced of the quinic acid (QA) inducible qa-dos supporter. During the frq null strains switched with this constructs, expression of your own full-size and you can truncated frq wasn’t dependent on QA, but qrf was just indicated in the presence from QA (Figure dos-shape complement 1F). For this reason, i cultured wt-frq-aq and you may frq-deopt2-aq challenges throughout the lack of QA and you may performed a chip assay using an antibody against pol II phosphorylated within serine 2. The pol II membership on frq supporter and you will 5′ UTR have been comparable regarding wt-frq-aq and you can frq-deopt2-aq stresses, but pol II accounts between and 3′ prevent out-of frq ORF were atically on frq-deopt2-aq filters as compared to wt-frq-aq filter systems (Figure 2D). Together, this type of abilities demonstrate that codon deoptimization off frq abolished the phrase due to untimely transcription cancellation.
Codon deoptimization out of frq as well as lead to the brand new untimely transcription cancellation from qrf because the shown by the loss of complete-size qrf and look of truncated qrf mRNA regarding frq-deopt1 and you may frq-deopt2 strains (Profile 2E and Profile dos-figure supplement 1F). 3′ Battle result revealed that this new 3′ concludes of the truncated qrf mRNAs from the frq-deopt1 stresses and nearby throughout the deoptimized area that have a possible Pas (AUAAAA) motif 21-nt upstream of one’s 3′ closes (Contour dos-contour complement 1G). It must be noted that wt-frq gene has also an equivalent putative Jamais theme, indicating that nucleotide series close Jamais theme is additionally expected to own transcription cancellation.