SummaryThe carbon storage regulatory system of Escherichia coli controls the expression of genes involved in carbohydrate metabolism and cell motility. CsrA binding to glgCAP transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay. CsrB RNA functions as an antagonist of CsrA by sequestering this protein and preventing its action. In this paper, we elucidate further the mechanism of CsrA-mediated glgC regulation. Results from gel shift assays demonstrate that several molecules of CsrA can bind to each glgC transcript. RNA footprinting studies indicate that CsrA binds to the glgCAP leader transcript at two positions. One of these sites overlaps the glgC Shine-Dalgarno sequence, whereas the other CsrA target is located further upstream in an RNA hairpin. Results from toeprint and cell-free translation experiments indicate that bound CsrA prevents ribosome binding to the glgC Shine-Dalgarno sequence and that this reduces GlgC synthesis. The effect of two deletions in the upstream binding site was examined. Both of these deletions reduced, but did not eliminate, CsrA binding in vitro and CsrA-dependent regulation in vivo. Our findings establish that bound CsrA inhibits initiation of glgC translation, thereby reducing glycogen biosynthesis. This inhibition of translation probably contributes to destabilization of the glgC transcript that was observed previously.
Introduction
In eukaryotes, mRNA decay is generally initiated by the shortening of the poly(A) tail mediated by the major deadenylase complex Ccr4-Caf1-Not. The deadenylated transcript is then rapidly degraded, primarily via the decapping-dependent pathway. Here we report that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases in vivo: Caf1 is required for the regulated degradation of specific transcripts, and Ccr4 is responsible for basal degradation. Intriguingly disruption of the Ccr4-Caf1-Not complex leads to deadenylation-independent decapping. Additionally, decapping is correlated with a novel transcript modification, addition of a CUCU sequence. A member of the nucleotidyltransferase superfamily, CutA, is required for this modification, and its disruption leads to a reduced rate of decapping and subsequent transcript degradation. We propose that 3 modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for decapping and efficient degradation.
Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors. One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available. This signalling mechanism has been shown to require a region of 218 nucleotides within the 3′ untranslated region of areA mRNA. We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium. areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition. We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.
SummaryThe GATA factor AreA is a wide-domain regulator in Aspergillus nidulans with transcriptional activation and chromatin remodelling functions. AreA interacts with the nitrate-specific Zn 2 -C 6 cluster protein NirA and both proteins cooperate to synergistically activate nitrate-responsive genes. We have previously established that NirA in vivo DNA binding site occupancy is AreA dependent and in this report we provide a mechanistic explanation for our previous findings. We now show that AreA regulates NirA at two levels: (i) through the regulation of nitrate transporters, AreA affects indirectly the subcellular distribution of NirA which rapidly accumulates in the nucleus following induction; (ii) AreA directly stimulates NirA in vivo target DNA occupancy and does not act indirectly by chromatin remodelling. Simultaneous overexpression of NirA and the nitrate transporter CrnA bypasses the AreA requirement for NirA binding, permits utilization of nitrate and nitrite as sole N-sources in an areA null strain and leads to an AreA-independent nucleosome loss of positioning.
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