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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.

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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.
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pbio-1000360-g004: 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.

Mentions: The results obtained with 3′ deletions of the intron implied that there is a functional dependency of ERE modules A and B on TRE module C, as a YRA1 pre-mRNA containing ERE modules A and B but lacking TRE module C is partially susceptible to NMD (allele N-712, Figure 3). Interestingly, the two ERE modules do not share significant sequence homology and appear to have some functional difference. YRA1 pre-mRNA containing module A alone is susceptible to both NMD and Edc3p-mediated decay (allele N-372, Figure 3). In contrast, YRA1 pre-mRNA containing module B alone exhibits exclusive substrate specificity for NMD (allele I-R4-NR2, Figure S3). These observations raise the possibility that ERE modules A and B may have different requirements for TRE elements and perform at least partially redundant functions in Edc3p-mediated YRA1 pre-mRNA decay. To assess this possibility, we generated YRA1 pre-mRNAs containing different combinations of intron modules A, B, C, or additional intronic sequences, and analyzed the steady-state levels of the transcripts encoded by these alleles in wild-type, upf1Δ, edc3Δ, and upf1Δedc3Δ cells. Transcripts containing modules A and C exhibited the same specificity for the Edc3p-mediated pathway as did full-length YRA1 pre-mRNA (allele R-AC, Figure 4). Transcripts containing modules B and C exhibited specificity for NMD but not for Edc3p-mediated decay (allele R-BC, Figure 4). Notably, transcripts containing modules B and C plus downstream sequences up to nt 942 exhibited specificity for Edc3p-mediated decay comparable to that manifested by full-length YRA1 pre-mRNA (allele R-BCD, Figure 4). This result suggests that the segment downstream of module C, from nt 743 to nt 942, is also involved in Edc3p-mediated YRA1 pre-mRNA degradation and we, therefore, designated this region as module D. Altogether, these data show that the ERE modules A and B indeed collaborate with different TRE modules and have at least partially redundant activities in Edc3p-mediated YRA1 pre-mRNA decay.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864733&req=5

pbio-1000360-g004: 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.
Mentions: The results obtained with 3′ deletions of the intron implied that there is a functional dependency of ERE modules A and B on TRE module C, as a YRA1 pre-mRNA containing ERE modules A and B but lacking TRE module C is partially susceptible to NMD (allele N-712, Figure 3). Interestingly, the two ERE modules do not share significant sequence homology and appear to have some functional difference. YRA1 pre-mRNA containing module A alone is susceptible to both NMD and Edc3p-mediated decay (allele N-372, Figure 3). In contrast, YRA1 pre-mRNA containing module B alone exhibits exclusive substrate specificity for NMD (allele I-R4-NR2, Figure S3). These observations raise the possibility that ERE modules A and B may have different requirements for TRE elements and perform at least partially redundant functions in Edc3p-mediated YRA1 pre-mRNA decay. To assess this possibility, we generated YRA1 pre-mRNAs containing different combinations of intron modules A, B, C, or additional intronic sequences, and analyzed the steady-state levels of the transcripts encoded by these alleles in wild-type, upf1Δ, edc3Δ, and upf1Δedc3Δ cells. Transcripts containing modules A and C exhibited the same specificity for the Edc3p-mediated pathway as did full-length YRA1 pre-mRNA (allele R-AC, Figure 4). Transcripts containing modules B and C exhibited specificity for NMD but not for Edc3p-mediated decay (allele R-BC, Figure 4). Notably, transcripts containing modules B and C plus downstream sequences up to nt 942 exhibited specificity for Edc3p-mediated decay comparable to that manifested by full-length YRA1 pre-mRNA (allele R-BCD, Figure 4). This result suggests that the segment downstream of module C, from nt 743 to nt 942, is also involved in Edc3p-mediated YRA1 pre-mRNA degradation and we, therefore, designated this region as module D. Altogether, these data show that the ERE modules A and B indeed collaborate with different TRE modules and have at least partially redundant activities in Edc3p-mediated YRA1 pre-mRNA decay.

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