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Spliceosomal introns in the 5' untranslated region of plant BTL RING-H2 ubiquitin ligases are evolutionary conserved and required for gene expression.

Aguilar-Hernández V, Guzmán P - BMC Plant Biol. (2013)

Bottom Line: Promoter-GUS fusion lines were used to confirm the IME effect of these 5'UTR introns on gene expression.IMEter scores of BTLs were compared with the 5'UTR introns of two gene families MHX and polyubiquitin genes.Our results indicated that gene expression dependent on a 5'UTR intron is an efficient regulatory mechanism in BTL E3 ligases that has been preserved throughout plant evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados, Unidad Irapuato, Apartado Postal 629, Irapuato, Gto 36821, Mexico. pguzman@ira.cinvestav.mx.

ABSTRACT

Background: Introns located close to the 5' end of a gene or in the 5' untranslated region often exert positive effects on gene expression. This effect, known as intron-mediated enhancement (IME), has been observed in diverse eukaryotic organisms, including plants. The sequences involved in IME seem to be spread across the intron and function in an additive manner. The IMEter algorithm was developed to predict plant introns that may enhance gene expression. We have identified several plant members of the BTL class of E3s, which may have orthologs across eukaryotes, that contain a 5'UTR intron. The RING finger E3 ligases are key enzymes of the ubiquitination system that mediate the transfer of ubiquitin to substrates.

Results: In this study, we retrieved BTL sequences from several angiosperm species and found that 5'UTR introns showing a strong IMEter score were predicted, suggesting that they may be conserved by lineage. Promoter-GUS fusion lines were used to confirm the IME effect of these 5'UTR introns on gene expression. IMEter scores of BTLs were compared with the 5'UTR introns of two gene families MHX and polyubiquitin genes.

Conclusions: Analysis performed in two Arabidopsis BTL E3 ligases genes indicated that the 5'UTR introns were essential for gene expression in all the tissues tested. Comparison of the average 5'UTR intron size on three gene families in ten angiosperm species suggests that a prevalent size for a 5'UTR intron is in the range of 600 nucleotides, and that the overall IMEter score within a gene family is preserved across several angiosperms. Our results indicated that gene expression dependent on a 5'UTR intron is an efficient regulatory mechanism in BTL E3 ligases that has been preserved throughout plant evolution.

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Histochemical GUS assays of transgenic lines containing AthBTL1 and AthBTL4 promoters. Representative samples of three-day-old dark-grown seedlings, 6- and 18-day-old light-grown seedlings, adult rosette and cauline leaves, and inflorescence from each of the transgenic lines are shown.
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Figure 4: Histochemical GUS assays of transgenic lines containing AthBTL1 and AthBTL4 promoters. Representative samples of three-day-old dark-grown seedlings, 6- and 18-day-old light-grown seedlings, adult rosette and cauline leaves, and inflorescence from each of the transgenic lines are shown.

Mentions: To determine the effect of the 5′UTR introns of BTLs on gene expression, we examined promoter-GUS fusions with or without the 5′UTR intron in two A. thaliana BTL genes, AthBTL1 and AthBTL4. Histochemical analysis was performed in several tissues and growth conditions from etiolated and young light-grown seedlings to adult tissues and inflorescences (Figure 4). The AthBTL1 and AthBTL4 promoters displayed GUS expression from the native promoters throughout etiolated or light-grown young seedlings. In 18-day-old seedlings, GUS expression was mainly detected in the petiole of the leaves and in the cotyledons. Expression was highly reduced throughout adult leaves, where it was detected primary in trichomes. GUS staining was readily observed in inflorescences in lines with either promoter construct (Figure 4). GUS activity was abolished in the intronless constructs of both promoters, suggesting that the 5′UTR introns in AthBTL1 and AthBTL4 harbor elements involved in enhancing the level of transcription. The specific GUS activity in lines carrying the wild-type promoter constructs was between 70-100 times higher in seedlings (roots and green tissue) and inflorescences compared to the intronless lines, and between 15-30 times higher in leaves where expression from these two promoters was much less intense (Figure 5).


Spliceosomal introns in the 5' untranslated region of plant BTL RING-H2 ubiquitin ligases are evolutionary conserved and required for gene expression.

Aguilar-Hernández V, Guzmán P - BMC Plant Biol. (2013)

Histochemical GUS assays of transgenic lines containing AthBTL1 and AthBTL4 promoters. Representative samples of three-day-old dark-grown seedlings, 6- and 18-day-old light-grown seedlings, adult rosette and cauline leaves, and inflorescence from each of the transgenic lines are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225707&req=5

Figure 4: Histochemical GUS assays of transgenic lines containing AthBTL1 and AthBTL4 promoters. Representative samples of three-day-old dark-grown seedlings, 6- and 18-day-old light-grown seedlings, adult rosette and cauline leaves, and inflorescence from each of the transgenic lines are shown.
Mentions: To determine the effect of the 5′UTR introns of BTLs on gene expression, we examined promoter-GUS fusions with or without the 5′UTR intron in two A. thaliana BTL genes, AthBTL1 and AthBTL4. Histochemical analysis was performed in several tissues and growth conditions from etiolated and young light-grown seedlings to adult tissues and inflorescences (Figure 4). The AthBTL1 and AthBTL4 promoters displayed GUS expression from the native promoters throughout etiolated or light-grown young seedlings. In 18-day-old seedlings, GUS expression was mainly detected in the petiole of the leaves and in the cotyledons. Expression was highly reduced throughout adult leaves, where it was detected primary in trichomes. GUS staining was readily observed in inflorescences in lines with either promoter construct (Figure 4). GUS activity was abolished in the intronless constructs of both promoters, suggesting that the 5′UTR introns in AthBTL1 and AthBTL4 harbor elements involved in enhancing the level of transcription. The specific GUS activity in lines carrying the wild-type promoter constructs was between 70-100 times higher in seedlings (roots and green tissue) and inflorescences compared to the intronless lines, and between 15-30 times higher in leaves where expression from these two promoters was much less intense (Figure 5).

Bottom Line: Promoter-GUS fusion lines were used to confirm the IME effect of these 5'UTR introns on gene expression.IMEter scores of BTLs were compared with the 5'UTR introns of two gene families MHX and polyubiquitin genes.Our results indicated that gene expression dependent on a 5'UTR intron is an efficient regulatory mechanism in BTL E3 ligases that has been preserved throughout plant evolution.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados, Unidad Irapuato, Apartado Postal 629, Irapuato, Gto 36821, Mexico. pguzman@ira.cinvestav.mx.

ABSTRACT

Background: Introns located close to the 5' end of a gene or in the 5' untranslated region often exert positive effects on gene expression. This effect, known as intron-mediated enhancement (IME), has been observed in diverse eukaryotic organisms, including plants. The sequences involved in IME seem to be spread across the intron and function in an additive manner. The IMEter algorithm was developed to predict plant introns that may enhance gene expression. We have identified several plant members of the BTL class of E3s, which may have orthologs across eukaryotes, that contain a 5'UTR intron. The RING finger E3 ligases are key enzymes of the ubiquitination system that mediate the transfer of ubiquitin to substrates.

Results: In this study, we retrieved BTL sequences from several angiosperm species and found that 5'UTR introns showing a strong IMEter score were predicted, suggesting that they may be conserved by lineage. Promoter-GUS fusion lines were used to confirm the IME effect of these 5'UTR introns on gene expression. IMEter scores of BTLs were compared with the 5'UTR introns of two gene families MHX and polyubiquitin genes.

Conclusions: Analysis performed in two Arabidopsis BTL E3 ligases genes indicated that the 5'UTR introns were essential for gene expression in all the tissues tested. Comparison of the average 5'UTR intron size on three gene families in ten angiosperm species suggests that a prevalent size for a 5'UTR intron is in the range of 600 nucleotides, and that the overall IMEter score within a gene family is preserved across several angiosperms. Our results indicated that gene expression dependent on a 5'UTR intron is an efficient regulatory mechanism in BTL E3 ligases that has been preserved throughout plant evolution.

Show MeSH