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Exploring mRNA 3'-UTR G-quadruplexes: evidence of roles in both alternative polyadenylation and mRNA shortening.

Beaudoin JD, Perreault JP - Nucleic Acids Res. (2013)

Bottom Line: To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo.The 3'-UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA.Clearly, G-quadruplexes located in the 3'-UTRs of mRNAs are cis-regulatory elements that have a significant impact on gene expression.

View Article: PubMed Central - PubMed

Affiliation: RNA Group/Groupe ARN, Département de biochimie, Faculté de médecine et des sciences de santé, Pavillon de recherche appliquée au cancer, Université de Sherbrooke, QC J1E 4K8, Canada.

ABSTRACT
Guanine-rich RNA sequences can fold into non-canonical, four stranded helical structures called G-quadruplexes that have been shown to be widely distributed within the mammalian transcriptome, as well as being key regulatory elements in various biological mechanisms. That said, their role within the 3'-untranslated region (UTR) of mRNA remains to be elucidated and appreciated. A bioinformatic analysis of the 3'-UTRs of mRNAs revealed enrichment in G-quadruplexes. To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo. The 3'-UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA. Clearly, G-quadruplexes located in the 3'-UTRs of mRNAs are cis-regulatory elements that have a significant impact on gene expression.

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The FXR1 3′-UTR G4 structure in cellulo. (a) Schematic representation of the Fluc–FXR1 transcripts resulting from the RNase H hydrolysis. The upper numbers correspond to the numbering from the 5′-end of the hydrolyzed product, whereas lower ones refer to the start of the FXR1 3′-UTR (black part). The arrows map the different PA sites as determined by the 3′-RACE experiments [alternative (APA) and canonical (Can PA) sites]. The short and long mRNA isoforms produced are shown. (b, c) Northern blot hybridizations of the RNA samples previously subjected to RNase H hydrolysis in either the absence (−) or the presence (+) of oligo-dT. The numbers on the left refer to the sizes of a molecular RNA ladder, whereas those on the right are the estimated sizes of the two isoforms. 7SL RNA was probed as an internal control. (d–f) Gene expression levels of constructs either at the mRNA level as determined by northern blot hybridization (for FXR1 n = 5, whereas for FXR1 AltPAS-mut n = 3; nd indicates not detectable) (d), by RNase protection assay (FXR1 and FXR1 AltPAS-mut n = 3) (e) (gray) or at the protein level as determined by luciferase assay (FXR1 n = 7, FXR1 AltPAS-mut n = 3) (f) (black). The x-axis identifies the constructions used and the y-axis the fold difference (wt result divided by G/A-mutated result). (g) Luciferase assays in the presence of various concentrations of PhenDC3 (0–50 µM; n = 3). Error bars, mean ± SD, **P < 0.01 and ****P < 0.0001.
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gkt265-F3: The FXR1 3′-UTR G4 structure in cellulo. (a) Schematic representation of the Fluc–FXR1 transcripts resulting from the RNase H hydrolysis. The upper numbers correspond to the numbering from the 5′-end of the hydrolyzed product, whereas lower ones refer to the start of the FXR1 3′-UTR (black part). The arrows map the different PA sites as determined by the 3′-RACE experiments [alternative (APA) and canonical (Can PA) sites]. The short and long mRNA isoforms produced are shown. (b, c) Northern blot hybridizations of the RNA samples previously subjected to RNase H hydrolysis in either the absence (−) or the presence (+) of oligo-dT. The numbers on the left refer to the sizes of a molecular RNA ladder, whereas those on the right are the estimated sizes of the two isoforms. 7SL RNA was probed as an internal control. (d–f) Gene expression levels of constructs either at the mRNA level as determined by northern blot hybridization (for FXR1 n = 5, whereas for FXR1 AltPAS-mut n = 3; nd indicates not detectable) (d), by RNase protection assay (FXR1 and FXR1 AltPAS-mut n = 3) (e) (gray) or at the protein level as determined by luciferase assay (FXR1 n = 7, FXR1 AltPAS-mut n = 3) (f) (black). The x-axis identifies the constructions used and the y-axis the fold difference (wt result divided by G/A-mutated result). (g) Luciferase assays in the presence of various concentrations of PhenDC3 (0–50 µM; n = 3). Error bars, mean ± SD, **P < 0.01 and ****P < 0.0001.

Mentions: To further evaluate the role of G-quadruplexes as positive regulatory elements for APA units, a second candidate was studied. The fragile X-related mental retardation autosomal homolog 1 (FXR1) gene produces an mRNA with a 3′-UTR 870 nt in length that possesses both a PG4 sequence and a putative internal APA unit located around position 250 (Figure 3a; note that the numbering from the positions of the FXR1 3′-UTR differs because the 102 upstream nucleotides of the Fluc-coding sequence and the restriction site are also considered). Initially, the ability of the FXR1 3′-UTR PG4 sequence to fold into a G-quadruplex in vitro was assessed. The same three methods described earlier in the text were used, and all agreed that it adopts a G4 structure in the presence of a physiological concentration of KCl (Supplementary Figure S1).Figure 3.


Exploring mRNA 3'-UTR G-quadruplexes: evidence of roles in both alternative polyadenylation and mRNA shortening.

Beaudoin JD, Perreault JP - Nucleic Acids Res. (2013)

The FXR1 3′-UTR G4 structure in cellulo. (a) Schematic representation of the Fluc–FXR1 transcripts resulting from the RNase H hydrolysis. The upper numbers correspond to the numbering from the 5′-end of the hydrolyzed product, whereas lower ones refer to the start of the FXR1 3′-UTR (black part). The arrows map the different PA sites as determined by the 3′-RACE experiments [alternative (APA) and canonical (Can PA) sites]. The short and long mRNA isoforms produced are shown. (b, c) Northern blot hybridizations of the RNA samples previously subjected to RNase H hydrolysis in either the absence (−) or the presence (+) of oligo-dT. The numbers on the left refer to the sizes of a molecular RNA ladder, whereas those on the right are the estimated sizes of the two isoforms. 7SL RNA was probed as an internal control. (d–f) Gene expression levels of constructs either at the mRNA level as determined by northern blot hybridization (for FXR1 n = 5, whereas for FXR1 AltPAS-mut n = 3; nd indicates not detectable) (d), by RNase protection assay (FXR1 and FXR1 AltPAS-mut n = 3) (e) (gray) or at the protein level as determined by luciferase assay (FXR1 n = 7, FXR1 AltPAS-mut n = 3) (f) (black). The x-axis identifies the constructions used and the y-axis the fold difference (wt result divided by G/A-mutated result). (g) Luciferase assays in the presence of various concentrations of PhenDC3 (0–50 µM; n = 3). Error bars, mean ± SD, **P < 0.01 and ****P < 0.0001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkt265-F3: The FXR1 3′-UTR G4 structure in cellulo. (a) Schematic representation of the Fluc–FXR1 transcripts resulting from the RNase H hydrolysis. The upper numbers correspond to the numbering from the 5′-end of the hydrolyzed product, whereas lower ones refer to the start of the FXR1 3′-UTR (black part). The arrows map the different PA sites as determined by the 3′-RACE experiments [alternative (APA) and canonical (Can PA) sites]. The short and long mRNA isoforms produced are shown. (b, c) Northern blot hybridizations of the RNA samples previously subjected to RNase H hydrolysis in either the absence (−) or the presence (+) of oligo-dT. The numbers on the left refer to the sizes of a molecular RNA ladder, whereas those on the right are the estimated sizes of the two isoforms. 7SL RNA was probed as an internal control. (d–f) Gene expression levels of constructs either at the mRNA level as determined by northern blot hybridization (for FXR1 n = 5, whereas for FXR1 AltPAS-mut n = 3; nd indicates not detectable) (d), by RNase protection assay (FXR1 and FXR1 AltPAS-mut n = 3) (e) (gray) or at the protein level as determined by luciferase assay (FXR1 n = 7, FXR1 AltPAS-mut n = 3) (f) (black). The x-axis identifies the constructions used and the y-axis the fold difference (wt result divided by G/A-mutated result). (g) Luciferase assays in the presence of various concentrations of PhenDC3 (0–50 µM; n = 3). Error bars, mean ± SD, **P < 0.01 and ****P < 0.0001.
Mentions: To further evaluate the role of G-quadruplexes as positive regulatory elements for APA units, a second candidate was studied. The fragile X-related mental retardation autosomal homolog 1 (FXR1) gene produces an mRNA with a 3′-UTR 870 nt in length that possesses both a PG4 sequence and a putative internal APA unit located around position 250 (Figure 3a; note that the numbering from the positions of the FXR1 3′-UTR differs because the 102 upstream nucleotides of the Fluc-coding sequence and the restriction site are also considered). Initially, the ability of the FXR1 3′-UTR PG4 sequence to fold into a G-quadruplex in vitro was assessed. The same three methods described earlier in the text were used, and all agreed that it adopts a G4 structure in the presence of a physiological concentration of KCl (Supplementary Figure S1).Figure 3.

Bottom Line: To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo.The 3'-UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA.Clearly, G-quadruplexes located in the 3'-UTRs of mRNAs are cis-regulatory elements that have a significant impact on gene expression.

View Article: PubMed Central - PubMed

Affiliation: RNA Group/Groupe ARN, Département de biochimie, Faculté de médecine et des sciences de santé, Pavillon de recherche appliquée au cancer, Université de Sherbrooke, QC J1E 4K8, Canada.

ABSTRACT
Guanine-rich RNA sequences can fold into non-canonical, four stranded helical structures called G-quadruplexes that have been shown to be widely distributed within the mammalian transcriptome, as well as being key regulatory elements in various biological mechanisms. That said, their role within the 3'-untranslated region (UTR) of mRNA remains to be elucidated and appreciated. A bioinformatic analysis of the 3'-UTRs of mRNAs revealed enrichment in G-quadruplexes. To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo. The 3'-UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA. Clearly, G-quadruplexes located in the 3'-UTRs of mRNAs are cis-regulatory elements that have a significant impact on gene expression.

Show MeSH
Related in: MedlinePlus