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A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

Kolev NG, Hartland EI, Huber PW - Nucleic Acids Res. (2008)

Bottom Line: The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage.Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts.Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

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A manganese-dependent ribozyme in the 3′-UTR of Vg1 mRNA. (A) The secondary structure of the region encompassing residues 210–350 of the Vg1 localization element (VLE). An arrow indicates the site of cleavage and boxes enclose a consensus polyadenylation signal at position 334 and a nonconsensus signal at 272. The structure was generated using mfold version 3.1 (51). (B) Vg1 RNA (radiolabeled at the 3′-end) was incubated overnight in cleavage buffer in the presence (lane 3) or absence (lane 4) of 10 mM manganese and then analyzed on a denaturing polyacrylamide gel alongside ribonuclease T1 and alkaline hydrolysates (lanes 1 and 2, respectively). The doublet in lane 3 is also apparent in the T1 digest (lane 1) and is due to length heterogeneity in the substrate RNA. (C) A 5′ hydroxyl group at the cleavage site. The 3′ cleavage product was incubated with polynucleotide kinase and ATP and compared to untreated fragment and an alkaline hydrolysate. (D) A 3′ phosphate at the cleavage site. A portion of a cleavage reaction containing an equal mixture of substrate and product RNA was incubated with poly(A) polymerase and compared to an untreated sample. Only the substrate RNA is polyadenylated.
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Figure 1: A manganese-dependent ribozyme in the 3′-UTR of Vg1 mRNA. (A) The secondary structure of the region encompassing residues 210–350 of the Vg1 localization element (VLE). An arrow indicates the site of cleavage and boxes enclose a consensus polyadenylation signal at position 334 and a nonconsensus signal at 272. The structure was generated using mfold version 3.1 (51). (B) Vg1 RNA (radiolabeled at the 3′-end) was incubated overnight in cleavage buffer in the presence (lane 3) or absence (lane 4) of 10 mM manganese and then analyzed on a denaturing polyacrylamide gel alongside ribonuclease T1 and alkaline hydrolysates (lanes 1 and 2, respectively). The doublet in lane 3 is also apparent in the T1 digest (lane 1) and is due to length heterogeneity in the substrate RNA. (C) A 5′ hydroxyl group at the cleavage site. The 3′ cleavage product was incubated with polynucleotide kinase and ATP and compared to untreated fragment and an alkaline hydrolysate. (D) A 3′ phosphate at the cleavage site. A portion of a cleavage reaction containing an equal mixture of substrate and product RNA was incubated with poly(A) polymerase and compared to an untreated sample. Only the substrate RNA is polyadenylated.

Mentions: The predicted secondary structure at the 3′-end of the localization element in Vg1 mRNA includes a short helix having UUU paired with GAAA (Figure 1A), which is the minimum structural motif required for activity in the manganese-dependent ribozyme (9). We tested an RNA fragment, encompassing nucleotides 210–350 of the VLE, for cleavage in the presence of 10 mM manganese. Cleavage occurs specifically between G331 and A332 (Figure 1B), which is the expected site based upon previous studies of this ribozyme (8,9). In initial assays for cleavage activity, we detected an additional product that exhibited considerably lower mobility relative to the starting substrate on denaturing polyacrylamide gels (data not shown). The appearance of this species was eliminated by the inclusion of cap analog (m7GpppA) in the reaction mixture. This alternative reaction and the identity of the secondary product are being investigated.Figure 1.


A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

Kolev NG, Hartland EI, Huber PW - Nucleic Acids Res. (2008)

A manganese-dependent ribozyme in the 3′-UTR of Vg1 mRNA. (A) The secondary structure of the region encompassing residues 210–350 of the Vg1 localization element (VLE). An arrow indicates the site of cleavage and boxes enclose a consensus polyadenylation signal at position 334 and a nonconsensus signal at 272. The structure was generated using mfold version 3.1 (51). (B) Vg1 RNA (radiolabeled at the 3′-end) was incubated overnight in cleavage buffer in the presence (lane 3) or absence (lane 4) of 10 mM manganese and then analyzed on a denaturing polyacrylamide gel alongside ribonuclease T1 and alkaline hydrolysates (lanes 1 and 2, respectively). The doublet in lane 3 is also apparent in the T1 digest (lane 1) and is due to length heterogeneity in the substrate RNA. (C) A 5′ hydroxyl group at the cleavage site. The 3′ cleavage product was incubated with polynucleotide kinase and ATP and compared to untreated fragment and an alkaline hydrolysate. (D) A 3′ phosphate at the cleavage site. A portion of a cleavage reaction containing an equal mixture of substrate and product RNA was incubated with poly(A) polymerase and compared to an untreated sample. Only the substrate RNA is polyadenylated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553595&req=5

Figure 1: A manganese-dependent ribozyme in the 3′-UTR of Vg1 mRNA. (A) The secondary structure of the region encompassing residues 210–350 of the Vg1 localization element (VLE). An arrow indicates the site of cleavage and boxes enclose a consensus polyadenylation signal at position 334 and a nonconsensus signal at 272. The structure was generated using mfold version 3.1 (51). (B) Vg1 RNA (radiolabeled at the 3′-end) was incubated overnight in cleavage buffer in the presence (lane 3) or absence (lane 4) of 10 mM manganese and then analyzed on a denaturing polyacrylamide gel alongside ribonuclease T1 and alkaline hydrolysates (lanes 1 and 2, respectively). The doublet in lane 3 is also apparent in the T1 digest (lane 1) and is due to length heterogeneity in the substrate RNA. (C) A 5′ hydroxyl group at the cleavage site. The 3′ cleavage product was incubated with polynucleotide kinase and ATP and compared to untreated fragment and an alkaline hydrolysate. (D) A 3′ phosphate at the cleavage site. A portion of a cleavage reaction containing an equal mixture of substrate and product RNA was incubated with poly(A) polymerase and compared to an untreated sample. Only the substrate RNA is polyadenylated.
Mentions: The predicted secondary structure at the 3′-end of the localization element in Vg1 mRNA includes a short helix having UUU paired with GAAA (Figure 1A), which is the minimum structural motif required for activity in the manganese-dependent ribozyme (9). We tested an RNA fragment, encompassing nucleotides 210–350 of the VLE, for cleavage in the presence of 10 mM manganese. Cleavage occurs specifically between G331 and A332 (Figure 1B), which is the expected site based upon previous studies of this ribozyme (8,9). In initial assays for cleavage activity, we detected an additional product that exhibited considerably lower mobility relative to the starting substrate on denaturing polyacrylamide gels (data not shown). The appearance of this species was eliminated by the inclusion of cap analog (m7GpppA) in the reaction mixture. This alternative reaction and the identity of the secondary product are being investigated.Figure 1.

Bottom Line: The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage.Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts.Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

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Related in: MedlinePlus