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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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In vivo translation of wild-type and mutant Vg1 mRNA lacking the GAAA ribozyme sequence. Stage VI oocytes were injected with capped mRNA and incubated overnight in OR2 buffer containing [35S]methionine/cysteine. Vg1 protein was immunoprecipitated from whole-cell extract prepared from 20 oocytes and analyzed by SDS–PAGE followed by autoradiography. Lane 1, oocytes injected with water only; lane 2, oocytes injected with wild-type mRNA; lane 3, oocytes injected with mRNA in which the GAAA ribozyme sequence was changed to CCCA.
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Figure 4: In vivo translation of wild-type and mutant Vg1 mRNA lacking the GAAA ribozyme sequence. Stage VI oocytes were injected with capped mRNA and incubated overnight in OR2 buffer containing [35S]methionine/cysteine. Vg1 protein was immunoprecipitated from whole-cell extract prepared from 20 oocytes and analyzed by SDS–PAGE followed by autoradiography. Lane 1, oocytes injected with water only; lane 2, oocytes injected with wild-type mRNA; lane 3, oocytes injected with mRNA in which the GAAA ribozyme sequence was changed to CCCA.

Mentions: In order to test whether the manganese ribozyme has any effect on the expression of Vg1 mRNA in vivo, we mutated the GAAA sequence to CCCA and then compared translation of this and wild-type mRNAs in Xenopus oocytes. Capped transcripts were injected into stage VI oocytes, which were kept overnight in OR2 solution containing [35S]-labeled amino acids. The amount of injected RNA (8 ng) represents an 80-fold excess compared to the amount of endogenous Vg1 mRNA (16). Equal numbers of oocytes were disrupted and Vg1 protein was retrieved by immunoprecipitation and analyzed by SDS–PAGE/autoradiography (Figure 4). The two mRNAs exhibit comparable levels of translation, indicating that the ribozyme sequence has no physiological activity. Inclusion of manganese in the culture medium had no effect on the relative levels of translation of wild-type and mutant Vg1 mRNAs (data not shown). This situation with Vg1 mRNA appears similar to the processing of Xenopus U16 snoRNA. This small RNA, which is located within the third intron of mRNA encoding ribosomal protein L1, is generated by endonucleolytic cleavage of the mRNA as opposed to splicing-dependent excision. Two functional manganese-dependent ribozymes are found close to the actual in vivo processing sites that are, nonetheless, cleaved by an endoribonuclease which, remarkably, requires manganese as a cofactor (17,18).Figure 4.


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

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

In vivo translation of wild-type and mutant Vg1 mRNA lacking the GAAA ribozyme sequence. Stage VI oocytes were injected with capped mRNA and incubated overnight in OR2 buffer containing [35S]methionine/cysteine. Vg1 protein was immunoprecipitated from whole-cell extract prepared from 20 oocytes and analyzed by SDS–PAGE followed by autoradiography. Lane 1, oocytes injected with water only; lane 2, oocytes injected with wild-type mRNA; lane 3, oocytes injected with mRNA in which the GAAA ribozyme sequence was changed to CCCA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: In vivo translation of wild-type and mutant Vg1 mRNA lacking the GAAA ribozyme sequence. Stage VI oocytes were injected with capped mRNA and incubated overnight in OR2 buffer containing [35S]methionine/cysteine. Vg1 protein was immunoprecipitated from whole-cell extract prepared from 20 oocytes and analyzed by SDS–PAGE followed by autoradiography. Lane 1, oocytes injected with water only; lane 2, oocytes injected with wild-type mRNA; lane 3, oocytes injected with mRNA in which the GAAA ribozyme sequence was changed to CCCA.
Mentions: In order to test whether the manganese ribozyme has any effect on the expression of Vg1 mRNA in vivo, we mutated the GAAA sequence to CCCA and then compared translation of this and wild-type mRNAs in Xenopus oocytes. Capped transcripts were injected into stage VI oocytes, which were kept overnight in OR2 solution containing [35S]-labeled amino acids. The amount of injected RNA (8 ng) represents an 80-fold excess compared to the amount of endogenous Vg1 mRNA (16). Equal numbers of oocytes were disrupted and Vg1 protein was retrieved by immunoprecipitation and analyzed by SDS–PAGE/autoradiography (Figure 4). The two mRNAs exhibit comparable levels of translation, indicating that the ribozyme sequence has no physiological activity. Inclusion of manganese in the culture medium had no effect on the relative levels of translation of wild-type and mutant Vg1 mRNAs (data not shown). This situation with Vg1 mRNA appears similar to the processing of Xenopus U16 snoRNA. This small RNA, which is located within the third intron of mRNA encoding ribosomal protein L1, is generated by endonucleolytic cleavage of the mRNA as opposed to splicing-dependent excision. Two functional manganese-dependent ribozymes are found close to the actual in vivo processing sites that are, nonetheless, cleaved by an endoribonuclease which, remarkably, requires manganese as a cofactor (17,18).Figure 4.

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