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. 2013 Aug;33(15):3026-35.
doi: 10.1128/MCB.00349-13. Epub 2013 Jun 3.

A stress-activated, p38 mitogen-activated protein kinase-ATF/CREB pathway regulates posttranscriptional, sequence-dependent decay of target RNAs

Jun Gao  1 , Jacy L Wagnon, Reine M Protacio, Galina V Glazko, Marjorie Beggs, Vinay Raj, Mari K Davidson, Wayne P Wahls
Affiliations

A stress-activated, p38 mitogen-activated protein kinase-ATF/CREB pathway regulates posttranscriptional, sequence-dependent decay of target RNAs

Jun Gao et al. Mol Cell Biol. 2013 Aug.

Abstract

Broadly conserved, mitogen-activated/stress-activated protein kinases (MAPK/SAPK) of the p38 family regulate multiple cellular processes. They transduce signals via dimeric, basic leucine zipper (bZIP) transcription factors of the ATF/CREB family (such as Atf2, Fos, and Jun) to regulate the transcription of target genes. We report additional mechanisms for gene regulation by such pathways exerted through RNA stability controls. The Spc1 (Sty1/Phh1) kinase-regulated Atf1-Pcr1 (Mts1-Mts2) heterodimer of the fission yeast Schizosaccharomyces pombe controls the stress-induced, posttranscriptional stability and decay of sets of target RNAs. Whole transcriptome RNA sequencing data revealed that decay is associated nonrandomly with transcripts that contain an M26 sequence motif. Moreover, the ablation of an M26 sequence motif in a target mRNA is sufficient to block its stress-induced loss. Conversely, engineered M26 motifs can render a stable mRNA into one that is targeted for decay. This stress-activated RNA decay (SARD) provides a mechanism for reducing the expression of target genes without shutting off transcription itself. Thus, a single p38-ATF/CREB signal transduction pathway can coordinately induce (promote transcription and RNA stability) and repress (promote RNA decay) transcript levels for distinct sets of genes, as is required for developmental decisions in response to stress and other stimuli.

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Figures

Fig 1
Fig 1
Stress induces the posttranscriptional stability and decay of specific target RNAs. (A) De novo transcription was blocked by addition of phenanthroline, cells were treated without or with osmotic stress (0.5 M KCl) for 60 min, and then RNAs were analyzed by deep sequencing. Plotted are the effects of stress on the abundance of each transcript (y axis) and total RNA-seq counts per transcript (x axis) (note log scales). Red indicates that the change in transcript stability was significant (FDR ≤ 0.001); yellow indicates that the change was not meaningful due to low counts and high variance between replicates; the blue lines are for visual reference (4-fold change in stability). (B) List of top functional gene ontology (GO) categories for stress-stabilized and stress-destabilized transcripts.
Fig 2
Fig 2
The M26 sequence motif and Atf1 protein regulate stress-induced RNA loss. The diagram shows the fbp1 locus and longest transcript, the position of an M26 sequence motif (boxed), and a two-nucleotide substitution (lowercase) that removes the M26 motif (M26Δ). The transcript was analyzed by Northern blotting of total RNA following removal of glucose (stress) for the indicated times. The data are representative of independent biological replicates. The rRNA serves as an internal loading control. Note that stress-induced loss of the long fbp1 transcript (*) requires Atf1 and the M26 sequence motif.
Fig 3
Fig 3
An engineered M26 sequence motif can trigger stress-induced decay of an otherwise stable mRNA. (A) A single nucleotide substitution (lowercase) creates an M26 sequence motif (boxed) in the ade6 mRNA. The M375 mRNA serves as a nucleotide substitution type-matched, codon type-matched negative control. (B) Stress induces M26-dependent mRNA decay. Cultures were treated without or with phenanthroline (to block transcription) and then with osmotic stress (0.5 M KCl) for the indicated times. Total RNA samples were analyzed by Northern blotting for the ctt1 and ade6 mRNAs. Note that even when de novo transcription is blocked (as shown by ctt1 mRNA), stress triggers M26-dependent decay of the long ade6 mRNA and its conversion into a shorter form. (C to F) The mRNA decay product is shortened at its 5′ end, lacks a cap, and has a free 5′ phosphate indicative of nucleolytic decay. Samples containing equal amounts of long and short mRNA (C) were subjected to 5′-RLM-RACE (D) in the presence or absence of decapping enzyme TAP, and then the RLM-RACE products (E) were visualized by agarose gel analysis (F). Note that the long M26 transcript is capped (requires cap removal to produce product), whereas the short transcript is not.
Fig 4
Fig 4
The position of the M26 sequence motif directs the position of mRNA truncation. (A) Diagram indicating the positions and orientation of the M26 sequence motif in the different ade6 alleles analyzed. (B) Following nutritional stress (growth to late log/stationary phase), the ends of mRNAs were mapped by reverse transcription from radiolabeled primers. Autoradiographs contain parallel DNA sequencing reactions from a plasmid template. Note that the 5′ ends of control (M375) transcripts map to the promoter region, but the processed 5′ ends map near M26 for the original M26 sequence motif and for an inverted M26 sequence motif (M26-B). (C) The positions of M26 sequences and RNA scission events (mapped as in panel B) are shown relative to predicted secondary structures for each M26-bearing mRNA (nucleotide positions are relative to the start codon). Note that strand scission can occur 5′ or 3′ of M26, but it always occurs in a ssRNA loop that is very close to M26 in dsRNA (≤45 nucleotides away).
Fig 5
Fig 5
The protein kinase Spc1, Atf1-Pcr1 heterodimer, and M26 motif are essential for SARD of ade6 mRNA. Relevant genotypes were as indicated, cells were cultured with or without nitrogen source (stress) for 60 min, and total RNA was analyzed by Northern blotting with a probe for the ade6 ORF. The ade6Δ (deletion) allele serves as a negative control for hybridization specificity. The 5′-truncated mRNA is indicated (*). (A) Nitrogen stress triggers M26-dependent decay (shortening) of mRNA. (B) All transcripts (including M26 short) require an intact promoter region, so the M26 site in DNA does not create a new promoter in its vicinity. All samples were run on the same gel, and extraneous lanes were removed (line) (C) Core components of the p38-ATF/CREB pathway are essential for the stress-induced, M26-dependent mRNA decay (loss of the upper band).
Fig 6
Fig 6
Graphical summary and model. (A) In addition to regulating the transcription of specific target genes upon stress (not depicted), the p38-ATF/CREB pathway targets specific transcripts for decay. (B) Hypothetically, binding of Atf1-Pcr1 heterodimer to M26 (or M26-like) sequences in dsRNA directs a yet-unidentified nuclease to cleave ssRNA within a nearby loop, which can be upstream or downstream of M26. RNA strand scission, decay of 5′ portions of the transcript (rapid), and decay of 3′ portions (slower) are portrayed as independent steps, although they might be coupled.
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