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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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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).
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pbio-1000360-g002: 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).

Mentions: The observed translational repression of YRA1 pre-mRNA suggests that Edc3p-mediated YRA1 pre-mRNA degradation occurs independently of ongoing protein synthesis. To test this notion, we carried out two sets of experiments. First, we examined the effects of trans-inhibition of translation initiation, elongation, or termination on YRA1 pre-mRNA accumulation. In these experiments, initiation was inhibited by using the temperature-sensitive prt1-1 allele to inactivate Prt1p, a component of the translation initiation factor eIF3 complex [21]; elongation was inhibited by treating cultures with the drug cycloheximide [22]; and termination was inhibited by using the temperature-sensitive sup45-2 allele to inactivate Sup45p, the yeast eukaryotic release factor 1 (eRF1) [23]. We evaluated the effects of each of these three translation blocks in both EDC3 and edc3Δ backgrounds and found that none of them affected the accumulation of YRA1 pre-mRNA. While subjected to any of these three translation blocks, EDC3 cells all accumulated low levels of YRA1 pre-mRNA and edc3Δ cells all accumulated high levels of YRA1 pre-mRNA (Figure 2A). As controls, we found that each of the three translation blocks caused 2- to 5-fold increases in the levels of nonsense-containing ade2-1 mRNA (Figure 2A). These results show that inhibition of any of the three basic steps of translation has no effect on the degradation of YRA1 pre-mRNA, i.e., even when general translation is inhibited YRA1 pre-mRNA is still degraded in an Edc3p-dependent manner.


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)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864733&req=5

pbio-1000360-g002: 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).
Mentions: The observed translational repression of YRA1 pre-mRNA suggests that Edc3p-mediated YRA1 pre-mRNA degradation occurs independently of ongoing protein synthesis. To test this notion, we carried out two sets of experiments. First, we examined the effects of trans-inhibition of translation initiation, elongation, or termination on YRA1 pre-mRNA accumulation. In these experiments, initiation was inhibited by using the temperature-sensitive prt1-1 allele to inactivate Prt1p, a component of the translation initiation factor eIF3 complex [21]; elongation was inhibited by treating cultures with the drug cycloheximide [22]; and termination was inhibited by using the temperature-sensitive sup45-2 allele to inactivate Sup45p, the yeast eukaryotic release factor 1 (eRF1) [23]. We evaluated the effects of each of these three translation blocks in both EDC3 and edc3Δ backgrounds and found that none of them affected the accumulation of YRA1 pre-mRNA. While subjected to any of these three translation blocks, EDC3 cells all accumulated low levels of YRA1 pre-mRNA and edc3Δ cells all accumulated high levels of YRA1 pre-mRNA (Figure 2A). As controls, we found that each of the three translation blocks caused 2- to 5-fold increases in the levels of nonsense-containing ade2-1 mRNA (Figure 2A). These results show that inhibition of any of the three basic steps of translation has no effect on the degradation of YRA1 pre-mRNA, i.e., even when general translation is inhibited YRA1 pre-mRNA is still degraded in an Edc3p-dependent manner.

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