Degradation of YRA1 Pre-mRNA in the cytoplasm requires translational repression, multiple modular intronic elements, Edc3p, and Mex67p

Abstract

Intron-containing pre-mRNAs are normally retained and processed in the nucleus but are sometimes exported to the cytoplasm and degraded by the nonsense-mediated mRNA decay (NMD) pathway as a consequence of their inclusion of intronic in-frame termination codons. When shunted to the cytoplasm by autoregulated nuclear export, the intron-containing yeast YRA1 pre-mRNA evades NMD and is targeted by a cytoplasmic decay pathway mediated by the decapping activator Edc3p. Here, we have elucidated this transcript-specific decay mechanism, showing that Edc3p-mediated YRA1 pre-mRNA degradation occurs independently of translation and is controlled through five structurally distinct but functionally interdependent modular elements in the YRA1 intron. Two of these elements target the pre-mRNA as an Edc3p substrate and the other three mediate transcript-specific translational repression. Translational repression of YRA1 pre-mRNA also requires the heterodimeric Mex67p/Mtr2p general mRNA export receptor, but not Edc3p, and serves to enhance Edc3p substrate specificity by inhibiting the susceptibility of this pre-mRNA to NMD. Collectively, our data indicate that YRA1 pre-mRNA degradation is a highly regulated process that proceeds through translational repression, substrate recognition by Edc3p, recruitment of the Dcp1p/Dcp2p decapping enzyme, and activation of decapping.

Figure 1. YRA1 pre-mRNA is translationally repressed and Edc3p does not play a significant role in the repression mechanism.

The polyribosomal association of YRA1 pre-mRNA and mRNA in wild-type (A) and edc3Δ (B) cells was analyzed by sucrose gradient fractionation and Northern blotting. Upper panels: absorbance tracings at 254 nm; lower panels: Northern blots of individual gradient fractions. Blots were hybridized with a probe complementary to YRA1 transcripts. The percentages of the YRA1 pre-mRNA and mRNA in the mRNP and the polyribosomal fractions are indicated. Overexposed blots for enhanced YRA1 pre-mRNA signals are indicated by an asterisk.

Figure 2. Trans- and cis-inhibition of translation have no effect on Edc3p-mediated YRA1 pre-mRNA degradation.

(A) Effects of trans-inhibition of translation on the steady-state levels of YRA1 pre-mRNA and mRNA. Initiation was inhibited by inactivation of Prt1p, termination was inhibited by inactivation of Sup45p, and elongation was inhibited by treating cells with cycloheximide. At the indicated times post-inhibition, RNA was isolated from culture aliquots and subjected to Northern analysis. Blots were hybridized with probes complementary to the YRA1, ADE2, or SCR1 transcripts, with the latter serving as a loading control. (B) The effects of cis-inhibition of translation initiation. A stem-loop structure was inserted into the 5′-UTRs of the YRA1 gene or its C-773 allele and the relative steady-state levels of the respective pre-mRNA and mRNA transcripts in wild-type (1), upf1Δ (2), edc3Δ (3), and upf1Δedc3Δ (4) cells were determined by Northern blotting as in (A). A schematic diagram of full-length YRA1 pre-mRNA and the related transcripts derived from the SL31-YRA1, C-773, and SL31-C-773 alleles is shown above the Northern blot. Smaller rectangles denote the 5′- and 3′-UTRs and larger rectangles denote the exons and the intron. The relative position of the 5′-UTR stem-loop structure is indicated, as are the nucleotides comprising the A of the initiator AUG (1), the 5′ (285) and 3′ (1052) boundaries of the intron, and the terminal nucleotide of the termination codon (1447).

Figure 3. Effects of 5′ and 3′ deletions of the YRA1 intron on Edc3p-mediated YRA1 pre-mRNA decay.

A set of yra1 alleles containing 3′ or 5′ deletions of the YRA1 intron was constructed and the steady-state levels of transcripts encoded by each of these alleles in wild-type (1), upf1Δ (2), edc3Δ (3), and upf1Δedc3Δ (4) cells were determined by Northern blotting. Blots were hybridized with probes complementary to the YRA1 or SCR1 transcripts, with the latter serving as a loading control. The positions of YRA1 pre-mRNAs encoded by the endogenous and all the exogenous YRA1 alleles are marked by a triangle and by diamonds, respectively. A schematic diagram of the yra1 alleles analyzed is shown above the Northern blot, with the relative position of each deletion indicated. Pre-mRNAs encoded by each of the YRA1 mutant alleles cannot be spliced to produce mRNAs, as the 5′ or 3′ splicing signals were deleted from these pre-mRNAs.

Figure 4. YRA1 intron modules exhibit synergistic and partially redundant activities.

A set of yra1 alleles containing different combinations of YRA1 intron modules was constructed and the steady-state levels of the transcripts encoded by each of these alleles in wild-type (1), upf1Δ (2), edc3Δ (3), and upf1Δedc3Δ (4) cells were determined by Northern blotting. Blots were hybridized with probes complementary to the YRA1 or SCR1 transcripts, with the latter serving as a loading control. The positions of YRA1 pre-mRNAs encoded by the endogenous and all the exogenous YRA1 alleles are marked by a triangle and by diamonds, respectively. A schematic diagram of the analyzed yra1 alleles is shown above the Northern blot, with the relative positions and the implicated functions of modules A, B, C, D, and E indicated. Pre-mRNAs encoded by each of these recombinant YRA1 alleles cannot be spliced to produce mRNAs, as they lack either the 5′ or the 3′ splicing signals, or both of these signals.

Figure 5. Intron modules C, D, and E mediate translational repression of YRA1 pre-mRNA.

(A–B) The polyribosomal association of the YRA1 transcripts encoded by the C-672 allele in wild-type cells (A) or upf1Δ cells (B) was analyzed by sucrose gradient fractionation and Northern blotting. Upper panels: absorbance tracings at 254 nm; middle panels: Northern blots of individual gradient fractions; lower panels: schematic diagrams of the C-677 allele. Blots were hybridized with a probe complementary to YRA1 transcripts. The percentages of the C-672 YRA1 pre-mRNA present in the mRNP and polyribosomal fractions are indicated. (C–D) The polyribosomal association of the YRA1 transcripts encoded by the C-773 and SL31-C-773 alleles in upf1Δ cells was analyzed by sucrose gradient fractionation and Northern blotting. Upper panels: absorbance tracings at 254 nm; middle panels: Northern blots of individual gradient fractions; lower panels: schematic diagrams of the C-773 and SL31-C-773 alleles. Blots were hybridized with a probe complementary to YRA1 transcripts. The percentages of the C-773 or SL31-C-773 YRA1 pre-mRNAs present in the mRNP and polyribosomal fractions are indicated. (E) Analyses of steady-state RNA and protein expression from the HA-C-672 and HA-C-773 alleles in wild-type (1), upf1Δ (2), edc3Δ (3), and upf1Δedc3Δ (4) cells by Northern and Western blotting. Northern blots were hybridized with probes complementary to the YRA1 or PGK1 transcripts, with the latter serving as a loading control. The positions of the endogenous and exogenous YRA1 pre-mRNAs are indicated by a triangle and by diamonds, respectively. Western blots of whole-cell extracts were probed with monoclonal antibodies against HA or Pgk1p, with the latter serving as a loading control. A schematic diagram of HA-C-672 and HA-C-773 YRA1 alleles is shown above the Northern and Western blots. The relative positions of the triple HA tag, the intron modules, and the intron deletions are indicated. Pre-mRNAs encoded by the HA-C-672 and HA-C-773 YRA1 alleles cannot be spliced to produce mRNAs, as they lack the 5′ splicing signals. (F) Analyses of the steady-state levels of YRA1 pre-mRNAs encoded by the N-400 and SL31-N-400 alleles in wild-type (1), upf1Δ (2), edc3Δ (3), and upf1Δedc3Δ (4) cells by Northern blotting. Blots were hybridized with probes complementary to the YRA1 or SCR1 transcripts, with the latter serving as a loading control. The positions of the endogenous and exogenous YRA1 pre-mRNAs are indicated. A schematic diagram of the wild-type, N-400, and SL31-N-400 YRA1 alleles is shown above the Northern blot. The relative positions of the 5′-UTR stem-loop structure, the intron modules, and the intron deletions are indicated.

Figure 6. Mex67p is a component of the cytoplasmic YRA1 pre-mRNP that functions in repressing YRA1 pre-mRNA translation.

(A) Analysis of the effect of inactivation of Mex67p on the translation of YRA1 pre-mRNA. upf1Δedc3Δmex67-5 cells were grown at 25°C and then shifted to 37°C for 6 min. The polyribosomal association of YRA1 pre-mRNA and mRNA in these cells before or after the temperature shift was analyzed by sucrose gradient fractionation and Northern blotting. Upper panels: absorbance tracings at 254 nm; lower panels: Northern blots of individual gradient fractions. Blots were hybridized with a probe complementary to YRA1 transcripts. The percentages of YRA1 pre-mRNA and mRNA in the mRNP and the polyribosomal fractions are indicated. (B) Analysis of the association of Mex67p with YRA1 pre-mRNA. Whole cell extracts from upf1Δ edc3Δ strains harboring either the MEX67 or the HA-MEX67 allele were incubated with anti-HA antibodies. Proteins and RNAs precipitated by the antibodies were analyzed by Western blotting (left panel) and RT-PCR (right panel). I, input; S, supernatant fraction; P, pellet fraction. HA-Mex67p and specific RT-PCR products for YRA1 and CYH2 pre-mRNAs were detected in the pellet fraction. RT, reverse transcriptase. (C) Analysis of the effect of tethering Mex67p on YRA1 pre-mRNA decay. A DNA fragment containing two MS2-coat protein binding sites was inserted into the intronic region of the F7, R1-F7, F12, and N-400 alleles of YRA1. The steady-state levels of the YRA1 pre-mRNA transcripts encoded by the resulting F7-MS2, R1-F7-MS2, F12-MS2, and N-400-MS2 alleles in wild-type (1), upf1Δ (2), edc3Δ (3), and upf1Δedc3Δ (4) cells that do or do not express the MS2-coat- Mex67p or Sub2p fusion proteins were determined by Northern blotting. Blots were hybridized with probes complementary to the YRA1 or SCR1 transcripts, with the latter serving as a loading control. The positions of the endogenous and exogenous YRA1 pre-mRNAs and YRA1 mRNA are indicated. A schematic diagram of the analyzed alleles is shown above the Northern blot, with the relative positions of the MS2-binding sites, the intron modules, and the intron deletions indicated.

Figure 7. Inactivation of Mrt2p causes rapid degradation of YRA1 pre-mRNA by NMD.

edc3Δ and edc3Δupf1Δ cells harboring the fully functional GFP-MTR2 (A) allele or the temperature-sensitive mtr2–9 (B), mtr2–21 (C), or mtr2–26 (D) alleles were grown at the permissive temperature (25°C), then shifted to the restrictive temperature (37°C) for indicated times. Cells from each time point were collected and the levels of YRA1 or PGK1 transcripts were analyzed by Northern blotting. Blots were hybridized with probes complementary to the YRA1, PGK1, or SCR1 transcripts, with the latter serving as a loading control. The positions of the normal YRA1 mRNA species and the atypical longer YRA1 mRNA species that accumulated in cells harboring the mrt2–9, mtr2–21, or mtr2–26 alleles at late time points are indicated by a triangle and by diamonds, respectively. Graphs to the right of the figure depict YRA1 pre-mRNA levels for each allele +/− Upf1p normalized to the corresponding “0” time point.