Limits...
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.

Show MeSH

Related in: MedlinePlus

A manganese-dependent ribozyme in the zipcode of β-actin mRNA. (A) The predicted secondary structure of the zipcode element in the 3′-UTR of β-actin mRNA (52). An arrow marks the site of manganese-dependent cleavage. (B) Zipcode 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 alkaline and ribonuclease T1 hydrolysates (lanes 1 and 2, respectively). (C) The metal dependence of self-cleavage in zipcode RNA. RNA was incubated overnight in the presence of the indicated metal. Weak cleavage with Cu2+ has been reported previously for the manganese ribozyme (53); whereas, Zn2+ triggers nonspecific degradation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553595&req=5

Figure 5: A manganese-dependent ribozyme in the zipcode of β-actin mRNA. (A) The predicted secondary structure of the zipcode element in the 3′-UTR of β-actin mRNA (52). An arrow marks the site of manganese-dependent cleavage. (B) Zipcode 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 alkaline and ribonuclease T1 hydrolysates (lanes 1 and 2, respectively). (C) The metal dependence of self-cleavage in zipcode RNA. RNA was incubated overnight in the presence of the indicated metal. Weak cleavage with Cu2+ has been reported previously for the manganese ribozyme (53); whereas, Zn2+ triggers nonspecific degradation.

Mentions: β-Actin mRNA is localized to the leading edge of fibroblasts or the growth cones of developing neurites (19). A 54-nt element in the 3′-UTR, called the zipcode, mediates localization (Figure 5A). We have detected manganese-dependent cleavage of an oligoribonucleotide that contains the zipcode hairpin (Figure 5B). As with all other GAAA ribozymes, cleavage occurs specifically between the G and A residues. Similarly, cadmium is the only other metal that supports appreciable site-specific cleavage of this RNA. There is one notable difference between the zipcode RNA and the canonical manganese-dependent ribozyme. In the former, the consensus A:U base pair immediately flanking the cleavage site is apparently an A:C or possibly an A:A pair. Other examples of manganese-dependent ribozymes in which the GAAA is not paired with UUU have been detected within the group I intron of Chlamydomonas reinhardti chloroplast 23S rRNA (15). The structure of a manganese-dependent ribozyme has not been determined; however, in a theoretical model, the uracil residue in question is not involved in either the chemistry of the cleavage reaction or as a ligand to the metal (9). The structure of the ribozyme in the zipcode RNA supports an earlier proposal that the UUU segment serves only as a guide sequence to orient the metal-binding adenosine residues (9).Figure 5.


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 zipcode of β-actin mRNA. (A) The predicted secondary structure of the zipcode element in the 3′-UTR of β-actin mRNA (52). An arrow marks the site of manganese-dependent cleavage. (B) Zipcode 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 alkaline and ribonuclease T1 hydrolysates (lanes 1 and 2, respectively). (C) The metal dependence of self-cleavage in zipcode RNA. RNA was incubated overnight in the presence of the indicated metal. Weak cleavage with Cu2+ has been reported previously for the manganese ribozyme (53); whereas, Zn2+ triggers nonspecific degradation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: A manganese-dependent ribozyme in the zipcode of β-actin mRNA. (A) The predicted secondary structure of the zipcode element in the 3′-UTR of β-actin mRNA (52). An arrow marks the site of manganese-dependent cleavage. (B) Zipcode 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 alkaline and ribonuclease T1 hydrolysates (lanes 1 and 2, respectively). (C) The metal dependence of self-cleavage in zipcode RNA. RNA was incubated overnight in the presence of the indicated metal. Weak cleavage with Cu2+ has been reported previously for the manganese ribozyme (53); whereas, Zn2+ triggers nonspecific degradation.
Mentions: β-Actin mRNA is localized to the leading edge of fibroblasts or the growth cones of developing neurites (19). A 54-nt element in the 3′-UTR, called the zipcode, mediates localization (Figure 5A). We have detected manganese-dependent cleavage of an oligoribonucleotide that contains the zipcode hairpin (Figure 5B). As with all other GAAA ribozymes, cleavage occurs specifically between the G and A residues. Similarly, cadmium is the only other metal that supports appreciable site-specific cleavage of this RNA. There is one notable difference between the zipcode RNA and the canonical manganese-dependent ribozyme. In the former, the consensus A:U base pair immediately flanking the cleavage site is apparently an A:C or possibly an A:A pair. Other examples of manganese-dependent ribozymes in which the GAAA is not paired with UUU have been detected within the group I intron of Chlamydomonas reinhardti chloroplast 23S rRNA (15). The structure of a manganese-dependent ribozyme has not been determined; however, in a theoretical model, the uracil residue in question is not involved in either the chemistry of the cleavage reaction or as a ligand to the metal (9). The structure of the ribozyme in the zipcode RNA supports an earlier proposal that the UUU segment serves only as a guide sequence to orient the metal-binding adenosine residues (9).Figure 5.

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