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The consensus 5' splice site motif inhibits mRNA nuclear export.

Lee ES, Akef A, Mahadevan K, Palazzo AF - PLoS ONE (2015)

Bottom Line: The motif, however, does not disrupt splicing or the recruitment of UAP56 or TAP/Nxf1 to the RNA, which are normally required for nuclear export.This motif is also depleted from the beginning and ends of the 3'terminal exons of spliced mRNAs, but less so for lncRNAs.Our data suggests that the presence of the 5'splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, MSB Room 5336, Toronto, ON, M5S 1A8, Canada.

ABSTRACT
In eukaryotes, mRNAs are synthesized in the nucleus and then exported to the cytoplasm where they are translated into proteins. We have mapped an element, which when present in the 3'terminal exon or in an unspliced mRNA, inhibits mRNA nuclear export. This element has the same sequence as the consensus 5'splice site motif that is used to define the start of introns. Previously it was shown that when this motif is retained in the mRNA, it causes defects in 3'cleavage and polyadenylation and promotes mRNA decay. Our new data indicates that this motif also inhibits nuclear export and promotes the targeting of transcripts to nuclear speckles, foci within the nucleus which have been linked to splicing. The motif, however, does not disrupt splicing or the recruitment of UAP56 or TAP/Nxf1 to the RNA, which are normally required for nuclear export. Genome wide analysis of human mRNAs, lncRNA and eRNAs indicates that this motif is depleted from naturally intronless mRNAs and eRNAs, but less so in lncRNAs. This motif is also depleted from the beginning and ends of the 3'terminal exons of spliced mRNAs, but less so for lncRNAs. Our data suggests that the presence of the 5'splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise.

No MeSH data available.


Related in: MedlinePlus

Characterizing the nuclear retained mRNP state.(A) Plasmids containing the indicated genes were microinjected into nuclei. After 2 hrs, cells were fixed and stained for β-globin mRNA by FISH and SC35 by immunofluorescence. Each row represents a single field of view. The color overlay shows mRNA in red and SC35 in green. Speckles containing mRNA are indicated by0020arrows (Scale bar = 10μm). (B) Microinjected cells were analyzed for the distribution of mRNAs into nuclear speckles. Individual nuclear speckles, as determined by SC35 staining were analyzed for mRNA content by Pearson Correlation Analysis. For details on the analysis please see the methods section. Each bar is the average and standard error of the mean of three experiments, each of which consist of 150–200 speckles analyzed from 15–20 cells. Note that nuclear-speckle association is enhanced by the V5-His element even in the presence of splicing. (C) The amount of various mRNA present in nuclear speckles (as defined by the brightest 10% pixels in the nucleus, using SC35 immunofluorescence—see methods section and Akef et al., 2013 for details) as a percentage of either the total nuclear (“spec/nuc”) or total cellular (“spec/tot”) mRNA level in cells 1 hr post-microinjection. Each data point represents the average and standard error of the mean of 10–20 cells. Note that β-globin-Δi was not enriched in speckles as ~10% of the nuclear RNA fluorescence was present in these regions, which represents 10% of the total nuclear area. (D-E) Similar to (A-B), except that the co-localisation of c-ftz-Δi +/- V5-His RNA with nuclear speckles was analysed. As a control, the colocalization of SC35-positive speckles with microinjected 70kD dextran conjugated to oregon green dye (“OG”) was analyzed.
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pone.0122743.g004: Characterizing the nuclear retained mRNP state.(A) Plasmids containing the indicated genes were microinjected into nuclei. After 2 hrs, cells were fixed and stained for β-globin mRNA by FISH and SC35 by immunofluorescence. Each row represents a single field of view. The color overlay shows mRNA in red and SC35 in green. Speckles containing mRNA are indicated by0020arrows (Scale bar = 10μm). (B) Microinjected cells were analyzed for the distribution of mRNAs into nuclear speckles. Individual nuclear speckles, as determined by SC35 staining were analyzed for mRNA content by Pearson Correlation Analysis. For details on the analysis please see the methods section. Each bar is the average and standard error of the mean of three experiments, each of which consist of 150–200 speckles analyzed from 15–20 cells. Note that nuclear-speckle association is enhanced by the V5-His element even in the presence of splicing. (C) The amount of various mRNA present in nuclear speckles (as defined by the brightest 10% pixels in the nucleus, using SC35 immunofluorescence—see methods section and Akef et al., 2013 for details) as a percentage of either the total nuclear (“spec/nuc”) or total cellular (“spec/tot”) mRNA level in cells 1 hr post-microinjection. Each data point represents the average and standard error of the mean of 10–20 cells. Note that β-globin-Δi was not enriched in speckles as ~10% of the nuclear RNA fluorescence was present in these regions, which represents 10% of the total nuclear area. (D-E) Similar to (A-B), except that the co-localisation of c-ftz-Δi +/- V5-His RNA with nuclear speckles was analysed. As a control, the colocalization of SC35-positive speckles with microinjected 70kD dextran conjugated to oregon green dye (“OG”) was analyzed.

Mentions: We noticed that nuclear retained c-ftz-Δi-V5-His and β-globin-i-V5-His mRNAs formed distinct foci in the nucleus and we wondered if these colocalized with nuclear speckles. Nuclear speckles are foci that contain RNA polymerase II, splicesomal components, various splicing factors (such as SR proteins) and mRNA export factors [31]. Certain mRNAs can be targeted to speckles, either by post-transcriptional splicing events [14,32] or by yet-to-be identified motifs present in many mRNAs [13]. Since splicing promotes the efficient nuclear-speckle targeting of β-globin [13], we decided to investigate the localization of newly synthesized β-globin-Δi with or without the V5-His element. We microinjected plasmids containing the various genes into the nuclei of U2OS cells and the newly synthesized RNA was visualised by FISH staining at various time points post-injection. Nuclear speckles were imaged using immunofluorescence directed towards the protein SC35, a speckle marker [33,34]. We then performed Pearson Correlation Analysis, as previously described (see [13]), and found that the V5-His fragment promoted nuclear speckle-targeting of β-globin-Δi (Fig 4A and 4B). When the V5-His sequence was incorporated into β-globin-i it enhanced nuclear speckle-targeting above what splicing alone promoted (Fig 4A and 4B). Association of the various mRNAs with nuclear speckles happened within the first hour of expression (Fig 4B) and persisted over the next hour. The amount of total mRNA in the speckles also increased in the presence of the V5-His element, regardless of whether the mRNA was spliced (i) or not (Δi) (Fig 4C). The targeting of β-globin-Δi to nuclear speckles was also seen in transfected cells (S1 Fig). We then repeated these experiments with c-ftz-Δi and c-ftz-Δi-V5-His. As reported previously (see [13]), c-ftz-Δi has a natural propensity to associate with nuclear speckles, but this increased with the presence of the V5-His element (Fig 4C–4E).


The consensus 5' splice site motif inhibits mRNA nuclear export.

Lee ES, Akef A, Mahadevan K, Palazzo AF - PLoS ONE (2015)

Characterizing the nuclear retained mRNP state.(A) Plasmids containing the indicated genes were microinjected into nuclei. After 2 hrs, cells were fixed and stained for β-globin mRNA by FISH and SC35 by immunofluorescence. Each row represents a single field of view. The color overlay shows mRNA in red and SC35 in green. Speckles containing mRNA are indicated by0020arrows (Scale bar = 10μm). (B) Microinjected cells were analyzed for the distribution of mRNAs into nuclear speckles. Individual nuclear speckles, as determined by SC35 staining were analyzed for mRNA content by Pearson Correlation Analysis. For details on the analysis please see the methods section. Each bar is the average and standard error of the mean of three experiments, each of which consist of 150–200 speckles analyzed from 15–20 cells. Note that nuclear-speckle association is enhanced by the V5-His element even in the presence of splicing. (C) The amount of various mRNA present in nuclear speckles (as defined by the brightest 10% pixels in the nucleus, using SC35 immunofluorescence—see methods section and Akef et al., 2013 for details) as a percentage of either the total nuclear (“spec/nuc”) or total cellular (“spec/tot”) mRNA level in cells 1 hr post-microinjection. Each data point represents the average and standard error of the mean of 10–20 cells. Note that β-globin-Δi was not enriched in speckles as ~10% of the nuclear RNA fluorescence was present in these regions, which represents 10% of the total nuclear area. (D-E) Similar to (A-B), except that the co-localisation of c-ftz-Δi +/- V5-His RNA with nuclear speckles was analysed. As a control, the colocalization of SC35-positive speckles with microinjected 70kD dextran conjugated to oregon green dye (“OG”) was analyzed.
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Related In: Results  -  Collection

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pone.0122743.g004: Characterizing the nuclear retained mRNP state.(A) Plasmids containing the indicated genes were microinjected into nuclei. After 2 hrs, cells were fixed and stained for β-globin mRNA by FISH and SC35 by immunofluorescence. Each row represents a single field of view. The color overlay shows mRNA in red and SC35 in green. Speckles containing mRNA are indicated by0020arrows (Scale bar = 10μm). (B) Microinjected cells were analyzed for the distribution of mRNAs into nuclear speckles. Individual nuclear speckles, as determined by SC35 staining were analyzed for mRNA content by Pearson Correlation Analysis. For details on the analysis please see the methods section. Each bar is the average and standard error of the mean of three experiments, each of which consist of 150–200 speckles analyzed from 15–20 cells. Note that nuclear-speckle association is enhanced by the V5-His element even in the presence of splicing. (C) The amount of various mRNA present in nuclear speckles (as defined by the brightest 10% pixels in the nucleus, using SC35 immunofluorescence—see methods section and Akef et al., 2013 for details) as a percentage of either the total nuclear (“spec/nuc”) or total cellular (“spec/tot”) mRNA level in cells 1 hr post-microinjection. Each data point represents the average and standard error of the mean of 10–20 cells. Note that β-globin-Δi was not enriched in speckles as ~10% of the nuclear RNA fluorescence was present in these regions, which represents 10% of the total nuclear area. (D-E) Similar to (A-B), except that the co-localisation of c-ftz-Δi +/- V5-His RNA with nuclear speckles was analysed. As a control, the colocalization of SC35-positive speckles with microinjected 70kD dextran conjugated to oregon green dye (“OG”) was analyzed.
Mentions: We noticed that nuclear retained c-ftz-Δi-V5-His and β-globin-i-V5-His mRNAs formed distinct foci in the nucleus and we wondered if these colocalized with nuclear speckles. Nuclear speckles are foci that contain RNA polymerase II, splicesomal components, various splicing factors (such as SR proteins) and mRNA export factors [31]. Certain mRNAs can be targeted to speckles, either by post-transcriptional splicing events [14,32] or by yet-to-be identified motifs present in many mRNAs [13]. Since splicing promotes the efficient nuclear-speckle targeting of β-globin [13], we decided to investigate the localization of newly synthesized β-globin-Δi with or without the V5-His element. We microinjected plasmids containing the various genes into the nuclei of U2OS cells and the newly synthesized RNA was visualised by FISH staining at various time points post-injection. Nuclear speckles were imaged using immunofluorescence directed towards the protein SC35, a speckle marker [33,34]. We then performed Pearson Correlation Analysis, as previously described (see [13]), and found that the V5-His fragment promoted nuclear speckle-targeting of β-globin-Δi (Fig 4A and 4B). When the V5-His sequence was incorporated into β-globin-i it enhanced nuclear speckle-targeting above what splicing alone promoted (Fig 4A and 4B). Association of the various mRNAs with nuclear speckles happened within the first hour of expression (Fig 4B) and persisted over the next hour. The amount of total mRNA in the speckles also increased in the presence of the V5-His element, regardless of whether the mRNA was spliced (i) or not (Δi) (Fig 4C). The targeting of β-globin-Δi to nuclear speckles was also seen in transfected cells (S1 Fig). We then repeated these experiments with c-ftz-Δi and c-ftz-Δi-V5-His. As reported previously (see [13]), c-ftz-Δi has a natural propensity to associate with nuclear speckles, but this increased with the presence of the V5-His element (Fig 4C–4E).

Bottom Line: The motif, however, does not disrupt splicing or the recruitment of UAP56 or TAP/Nxf1 to the RNA, which are normally required for nuclear export.This motif is also depleted from the beginning and ends of the 3'terminal exons of spliced mRNAs, but less so for lncRNAs.Our data suggests that the presence of the 5'splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, MSB Room 5336, Toronto, ON, M5S 1A8, Canada.

ABSTRACT
In eukaryotes, mRNAs are synthesized in the nucleus and then exported to the cytoplasm where they are translated into proteins. We have mapped an element, which when present in the 3'terminal exon or in an unspliced mRNA, inhibits mRNA nuclear export. This element has the same sequence as the consensus 5'splice site motif that is used to define the start of introns. Previously it was shown that when this motif is retained in the mRNA, it causes defects in 3'cleavage and polyadenylation and promotes mRNA decay. Our new data indicates that this motif also inhibits nuclear export and promotes the targeting of transcripts to nuclear speckles, foci within the nucleus which have been linked to splicing. The motif, however, does not disrupt splicing or the recruitment of UAP56 or TAP/Nxf1 to the RNA, which are normally required for nuclear export. Genome wide analysis of human mRNAs, lncRNA and eRNAs indicates that this motif is depleted from naturally intronless mRNAs and eRNAs, but less so in lncRNAs. This motif is also depleted from the beginning and ends of the 3'terminal exons of spliced mRNAs, but less so for lncRNAs. Our data suggests that the presence of the 5'splice site motif in mature RNAs promotes their nuclear retention and may help to distinguish mRNAs from misprocessed transcripts and transcriptional noise.

No MeSH data available.


Related in: MedlinePlus