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

Dong S, Jacobson A, He F - PLoS Biol. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2864733&req=5

pbio-1000360-g007: 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.

Mentions: The results described in Figure 6 show that Mex67p plays a role in repressing YRA1 pre-mRNA translation, inhibiting YRA1 pre-mRNA degradation by NMD, and promoting the transcript's degradation by Edc3p. Since Mex67p and Mtr2p function as a complex in mRNA export [25], we sought to assess whether Mtr2p also plays a role in YRA1 pre-mRNA decay. Accordingly, we constructed a set of edc3Δ and upf1Δedc3Δ strains harboring temperature-sensitive mtr2 alleles and analyzed the effect of Mtr2p inactivation on the accumulation of YRA1 pre-mRNA. We analyzed three different temperature-sensitive alleles (mtr2–9, mtr2–21, and mtr2–26) and included the fully functional GFP-tagged MTR2 allele as a wild-type control. Previous studies had shown that the proteins encoded by these three temperature-sensitive alleles no longer interacted with Mex67p and at the restrictive temperature, cells harboring each of these alleles all manifested inhibition of nuclear mRNA export and mislocalization of Mex67p to cytoplasmic foci [25]. Our analyses indicated that, at the permissive temperature (25°C), edc3Δ cells harboring the GFP-MTR2, mtr2–9, mtr2–21, or mtr2–26 alleles all accumulated comparably high levels of YRA1 pre-mRNA (compare “0” time points, left panels in Figure 7A–D). However, when shifted to the restrictive temperature (37°C), edc3Δ cells harboring the GFP-MTR2 allele behaved dramatically different from edc3Δ cells harboring the mtr2–9, mtr2–21, or mtr2–26 alleles. During the 24-min time course, edc3ΔGFP-MTR2 cells maintained relatively high levels of YRA1 pre-mRNA at each time point (Figure 7A, left panel). In contrast, edc3Δ cells harboring the mtr2–9, mtr2–21, or mtr2–26 alleles exhibited significant decreases in YRA1 pre-mRNA level for each time point (compare the 3 6 12 and 24-min time points, Figure 7B–D left panels to that of Figure 7A). These decreases were transcript-specific since, during the 24-minute time course, PGK1 mRNA levels remained unchanged in edc3Δ and edc3Δupf1Δ cells harboring the GFP-MTR2, mtr2–9, mtr2–21, or mtr2–26 alleles (Figures 7A–D). Moreover, the dramatic decrease of YRA1 pre-mRNA levels in edc3Δ mtr2 cells was likely caused by rapid degradation of YRA1 pre-mRNA by NMD, especially at the early time points (3 and 6 min), because deletion of UPF1 mitigated these decreases (Figure 7B–D, right panels). Deletion of UPF1 from edc3Δmtr2–9, edc3Δmtr2–21, and edc3Δ mtr2–26 cells did not result in increased YRA1 pre-mRNA accumulation at late time points (12 and 24 min, Figure 7B–D, right panels), suggesting that inactivation of Mtr2p may have also blocked YRA1 pre-mRNA nuclear export, causing almost complete depletion of the cytoplasmic pool of YRA1 pre-mRNA. Consistent with this interpretation, mtr2–9, mtr2–21, or mtr2–26 cells in both edc3Δ and edc3Δupf1Δ backgrounds all accumulated a new, longer YRA1 mRNA species at late time points of the temperature shift (12 and 24 min, Figure 7B–D). The accumulation of this new YRA1 mRNA species is reminiscent of our previous observations in cells subject to Mex67p inactivation [15]. Taken together, the consequences of Mtr2p inactivation suggest that, similar to its role in general mRNA export, Mtr2p likely functions in a complex with Mex67p to repress YRA1 pre-mRNA translation.


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

Dong S, Jacobson A, He F - PLoS Biol. (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864733&req=5

pbio-1000360-g007: 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.
Mentions: The results described in Figure 6 show that Mex67p plays a role in repressing YRA1 pre-mRNA translation, inhibiting YRA1 pre-mRNA degradation by NMD, and promoting the transcript's degradation by Edc3p. Since Mex67p and Mtr2p function as a complex in mRNA export [25], we sought to assess whether Mtr2p also plays a role in YRA1 pre-mRNA decay. Accordingly, we constructed a set of edc3Δ and upf1Δedc3Δ strains harboring temperature-sensitive mtr2 alleles and analyzed the effect of Mtr2p inactivation on the accumulation of YRA1 pre-mRNA. We analyzed three different temperature-sensitive alleles (mtr2–9, mtr2–21, and mtr2–26) and included the fully functional GFP-tagged MTR2 allele as a wild-type control. Previous studies had shown that the proteins encoded by these three temperature-sensitive alleles no longer interacted with Mex67p and at the restrictive temperature, cells harboring each of these alleles all manifested inhibition of nuclear mRNA export and mislocalization of Mex67p to cytoplasmic foci [25]. Our analyses indicated that, at the permissive temperature (25°C), edc3Δ cells harboring the GFP-MTR2, mtr2–9, mtr2–21, or mtr2–26 alleles all accumulated comparably high levels of YRA1 pre-mRNA (compare “0” time points, left panels in Figure 7A–D). However, when shifted to the restrictive temperature (37°C), edc3Δ cells harboring the GFP-MTR2 allele behaved dramatically different from edc3Δ cells harboring the mtr2–9, mtr2–21, or mtr2–26 alleles. During the 24-min time course, edc3ΔGFP-MTR2 cells maintained relatively high levels of YRA1 pre-mRNA at each time point (Figure 7A, left panel). In contrast, edc3Δ cells harboring the mtr2–9, mtr2–21, or mtr2–26 alleles exhibited significant decreases in YRA1 pre-mRNA level for each time point (compare the 3 6 12 and 24-min time points, Figure 7B–D left panels to that of Figure 7A). These decreases were transcript-specific since, during the 24-minute time course, PGK1 mRNA levels remained unchanged in edc3Δ and edc3Δupf1Δ cells harboring the GFP-MTR2, mtr2–9, mtr2–21, or mtr2–26 alleles (Figures 7A–D). Moreover, the dramatic decrease of YRA1 pre-mRNA levels in edc3Δ mtr2 cells was likely caused by rapid degradation of YRA1 pre-mRNA by NMD, especially at the early time points (3 and 6 min), because deletion of UPF1 mitigated these decreases (Figure 7B–D, right panels). Deletion of UPF1 from edc3Δmtr2–9, edc3Δmtr2–21, and edc3Δ mtr2–26 cells did not result in increased YRA1 pre-mRNA accumulation at late time points (12 and 24 min, Figure 7B–D, right panels), suggesting that inactivation of Mtr2p may have also blocked YRA1 pre-mRNA nuclear export, causing almost complete depletion of the cytoplasmic pool of YRA1 pre-mRNA. Consistent with this interpretation, mtr2–9, mtr2–21, or mtr2–26 cells in both edc3Δ and edc3Δupf1Δ backgrounds all accumulated a new, longer YRA1 mRNA species at late time points of the temperature shift (12 and 24 min, Figure 7B–D). The accumulation of this new YRA1 mRNA species is reminiscent of our previous observations in cells subject to Mex67p inactivation [15]. Taken together, the consequences of Mtr2p inactivation suggest that, similar to its role in general mRNA export, Mtr2p likely functions in a complex with Mex67p to repress YRA1 pre-mRNA translation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

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.

Show MeSH
Related in: MedlinePlus