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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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Characterization of the self-cleavage reaction. All reactions were carried out in standard conditions, excepting the indicated variable. The metal dependence of cleavage was tested with the indicated divalent cation at a concentration of 10 mM.
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Figure 2: Characterization of the self-cleavage reaction. All reactions were carried out in standard conditions, excepting the indicated variable. The metal dependence of cleavage was tested with the indicated divalent cation at a concentration of 10 mM.

Mentions: Cleavage of the Vg1 3′-UTR was characterized further by determining the metal, temperature and pH dependence of the reaction (Figure 2). In addition to manganese, the only other divalent metal that supports this activity is cadmium. The amount of cleavage increases with temperature between 16°C and 30°C, above which there is an appreciable degradation of the RNA. The pH optimum of the reaction is centered near 7.5. The concentration of Tris buffer has no effect on the extent of the reaction, eliminating the possibility that it is participating in the reaction as a general base. The inclusion of magnesium has little effect on the activity of the ribozyme, indicating that formation of the catalytically competent structure in the RNA does not depend on this divalent cation, nor is there any apparent competitive binding of magnesium to the manganese site. The cleavage activity in Vg1 mRNA is strikingly similar to that first described for the manganese ribozyme in the Tetrahymena group I intron (8,9).Figure 2.


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

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

Characterization of the self-cleavage reaction. All reactions were carried out in standard conditions, excepting the indicated variable. The metal dependence of cleavage was tested with the indicated divalent cation at a concentration of 10 mM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Characterization of the self-cleavage reaction. All reactions were carried out in standard conditions, excepting the indicated variable. The metal dependence of cleavage was tested with the indicated divalent cation at a concentration of 10 mM.
Mentions: Cleavage of the Vg1 3′-UTR was characterized further by determining the metal, temperature and pH dependence of the reaction (Figure 2). In addition to manganese, the only other divalent metal that supports this activity is cadmium. The amount of cleavage increases with temperature between 16°C and 30°C, above which there is an appreciable degradation of the RNA. The pH optimum of the reaction is centered near 7.5. The concentration of Tris buffer has no effect on the extent of the reaction, eliminating the possibility that it is participating in the reaction as a general base. The inclusion of magnesium has little effect on the activity of the ribozyme, indicating that formation of the catalytically competent structure in the RNA does not depend on this divalent cation, nor is there any apparent competitive binding of magnesium to the manganese site. The cleavage activity in Vg1 mRNA is strikingly similar to that first described for the manganese ribozyme in the Tetrahymena group I intron (8,9).Figure 2.

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.

Show MeSH
Related in: MedlinePlus