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. 2015 Aug;200(4):1133-48.
doi: 10.1534/genetics.115.176743. Epub 2015 Jun 4.

Control of mRNA Stability in Fungi by NMD, EJC and CBC Factors Through 3'UTR Introns

Ying Zhang  1 , Matthew S Sachs  2
Affiliations

Control of mRNA Stability in Fungi by NMD, EJC and CBC Factors Through 3'UTR Introns

Ying Zhang et al. Genetics. 2015 Aug.

Abstract

In higher eukaryotes the accelerated degradation of mRNAs harboring premature termination codons is controlled by nonsense-mediated mRNA decay (NMD), exon junction complex (EJC), and nuclear cap-binding complex (CBC) factors, but the mechanistic basis for this quality-control system and the specific roles of the individual factors remain unclear. Using Neurospora crassa as a model system, we analyzed the mechanisms by which NMD is induced by spliced 3'-UTR introns or upstream open reading frames and observed that the former requires NMD, EJC, and CBC factors whereas the latter requires only the NMD factors. The transcripts for EJC components eIF4A3 and Y14, and translation termination factor eRF1, contain spliced 3'-UTR introns and each was stabilized in NMD, EJC, and CBC mutants. Reporter mRNAs containing spliced 3'-UTR introns, but not matched intronless controls, were stabilized in these mutants and were enriched in mRNPs immunopurified from wild-type cells with antibody directed against human Y14, demonstrating a direct role for spliced 3'-UTR introns in triggering EJC-mediated NMD. These results demonstrate conclusively that NMD, EJC, and CBC factors have essential roles in controlling mRNA stability and that, based on differential requirements for these factors, there are branched mechanisms for NMD. They demonstrate for the first time autoregulatory control of expression at the level of mRNA stability through the EJC/CBC branch of NMD for EJC core components, eIF4A3 and Y14, and for eRF1, which recognizes termination codons. Finally, these results show that EJC-mediated NMD occurs in fungi and thus is an evolutionarily conserved quality-control mechanism.

Keywords: Neurospora crassa; RNA stability; cap-binding complex (CBC); exon junction complex (EJC); nonsense-mediated mRNA decay (NMD); post-transcriptional control; ribosome; spliced 3′-UTR intron; translation.

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Figures

Figure 1
Figure 1
Analyses of uORF-containing and spliced 3′-UTR-containing transcripts. (A) Structures of mRNAs specifying arg-2, eif5, upf1, eif4a3, erf1, y14, and cox-5. The solid bars indicate the genic coding regions; the bars with gradients depict uORFs; the triangles indicate the positions and sizes of 3′-UTR introns (no other intron positions are indicated). The distance between the ORF stop codon and the splice site, the size of the spliced intron, and the distance between the splice site and the major poly(A) site are given [e.g., for eif4a3, 139, (62), 93 nt]. Here and throughout, shading indicates mRNA features: blue, uORFs that trigger NMD; yellow, spliced 3′-UTRs that trigger NMD; gray, no NMD-triggering features. An extended form of the spliced eif4a3 mRNA (Figure S4C) contains an additional 87 nt (indicated by dashed lines between parentheses). (B) Northern analyses of wt, Δupf1, Δupf1 his-3::upf1, Δupf1 his-3::upf1- 3XFLAG, Δupf2, Δupf2 his-3::upf2-3XFLAG, Δupf2, his-3::upf2-HAT-FLAG, and Δupf3. Total RNA (3 μg/lane) was analyzed with radiolabeled probes for arg-2, eif4a3, erf1, eif5, and cox-5. Levels of mRNA were quantified and normalized to the level of cox-5 mRNA. The quantification shown below the bands represents the average and standard deviation from three independent growth experiments. 25S and 18S rRNA bands stained with ethidium bromide from representative gels are shown below the Northerns. The doublet for the intronless eif5 transcript likely represents mRNA isoforms. (C) eif4a3, erf1, and y14 3′-UTR introns are efficiently spliced in all strains examined. M, markers; gDNA, PCR products of genomic DNA template; cDNA, PCR products from cDNA templates. The predicted sizes of PCR products from different templates are indicated. The Δy14 strain lacks y14 mRNA. See also Figure S1 and Figure S2.
Figure 2
Figure 2
mRNA levels and mRNA stability are affected by growth in cycloheximide and by Δupf1. (A) Strategy for pulse–chase analyses of mRNA stability. N. crassa conidia were germinated and grown for 6 hr; 0.2 mM 4TU was then added. After 15 min incubation with 4TU, 10 mM uracil was added to the culture, and total RNA and 4TU–RNA was purified from samples taken at 0, 3, and 10 min. In one experiment, cycloheximide (CYH) was added after the 15-min 4TU-pulse; following 5 min of incubation with CYH, the uracil-chase was then performed. (B) Total RNA from wt, Δupf1, and wt cells treated with 200 μg/ml CYH and subjected to 4TU pulse–chase were analyzed by Northern blotting as in Figure 1B; the arg-2 and cox-5 probes were added together. (C) Pulse–chase analyses of 4TU RNA from wt, Δupf1, and CYH-treated wt. 4TU RNA was purified and quantified by RT–qPCR and levels of 4TU mRNAs for arg-2, eif5, eif4a3, erf1, and cox-5 were normalized to levels of 4TU-labeled 25S rRNA in each sample (4TU-labeled 25S rRNA is stable during a 10-min chase) and then normalized to the level at time 0. Differences in expression are shown in relative units (RUs). The results are the average of three independent experiments. The mRNA half-lives (in minutes), calculated by fitting data to first order-decay parameters, are shown, as are R2 values for each calculation (in parentheses). ND, not determined. See also Figure S3.
Figure 3
Figure 3
Effects of Δupf1, Δupf2 mutations and correction of these mutants on the stability of selected mRNAs. 4TU RNA from wt, Δupf1, Δupf1 his-3::upf1, Δupf1 his-3::upf1-3XFLAG strains (A) or Δupf2, Δupf2 his-3::upf2-3XFLAG and Δupf2, his-3::upf2-HAT-FLAG strains (B) were analyzed by pulse–chase for arg-2, cox-5, upf1, upf2, eif4a3, erf1, and eif5 mRNAs using the procedures described in Figure 2. See also Figure S6.
Figure 4
Figure 4
Effects of Δxrn1 and correction of the mutant on the stability of selected mRNAs. (A) 4TU RNA from wt, Δxrn1, and Δxrn1 xrn1[Barr] strains were analyzed by pulse–chase for arg-2, eif5, eif4a3, erf1, and cox-5 mRNAs using the procedures described in Figure 2. (B) Levels of mRNA in total RNA from wt, Δxrn1, and Δxrn1 xrn1[Barr] strains was quantified by RT–qPCR and mRNA levels were normalized to the level of 25S rRNA.
Figure 5
Figure 5
Effects of the arg-2 uORF-encoded AAP and the eif4a3 and erf1 3′-UTR introns on the level and stability of luciferase (luc) reporter mRNA in wt and Δupf1 strains. Measurements to analyze the effects on reporter genes containing the arg-2 uORF (A), the eif4a3 3′-UTR intron (B), and the erf1 3′-UTR intron (C) and controls are shown as follows for each: levels of mRNA in total RNA (left), half-lives of mRNA measured by 4TU RNA pulse–chase (middle), and levels of luciferase enzyme activity/mRNA (right). wt (solid bars) or Δupf1 (open bars) strains contained the luc reporters shown in Figure S4A. Measurements of RNA were accomplished as in Figure 4. Luciferase enzyme activity was measured and normalized to luc mRNA levels. See also Figure S4.
Figure 6
Figure 6
Effects of Δy14, Δmago, Δcbp20, and Δcbp80, and correction of these mutants, on the stability of selected mRNAs. (A) Aliquots (3 μg) of total RNA extracted from wt, Δy14, Δy14 y14-Barr, Δmago, and Δmago mago[Barr] were separated on formaldehyde denaturing gels and analyzed as described in Figure 1B with the indicated probes. 4TU RNA from wt, Δy14, Δy14 y14[Barr], Δmago, Δmago mago[Barr] (B) or wt, Δcbp20, Δcbp20 cbp20[Barr], Δcbp80, Δcbp80 cbp80[Barr] (C) was analyzed by pulse–chase for arg-2, eif5, eif4a3, erf1, and cox-5 mRNAs using the procedures described in Figure 2. See also Figure S6.
Figure 7
Figure 7
Effects of the eif4a3 3′-UTR intron on the level and stability of luc reporter mRNA in wt, Δy14 and Δcpb80 strains. Measurements to analyze the effects on reporter genes containing the eif4a3 3′-UTR intron and controls are shown as follows for each: levels of mRNA in total RNA (left), half-lives of mRNA measured by 4TU RNA pulse–chase (middle), and levels of luciferase enzyme activity/mRNA (right). (A) wt (solid bars) and Δy14 (shaded bars) strains contained the luc reporters shown in Figure S4B. (B) wt (solid bars) and Δcbp80 (hatched bars) strains contained the luc reporters shown in Figure S4B. Measurements were obtained as described in Figure 5. (C) RT–qPCR analysis of eif4a3, erf1, y14, arg-2, eif5, and cox-5 mRNAs purified from wt, Δy14, and Δmago strains by immunopurification of mRNPs with anti-Y14 antibody as described in Materials and Methods. The level of each transcript was normalized to the total amount of purified RNA and then normalized to the level of input transcript. Mock immunopurification from wt without antibody (wt-AB) served as the control. (D) RT–qPCR analyses of luc, eif4a3, and arg-2 mRNAs purified from wt and luc reporter strains by immunopurification of mRNPs with anti-Y14 antibody. mRNA enrichment is shown normalized to the enrichment of eif4a3 in immunopurification from wt cells. See also Figure S5 and Figure S6.
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