Cleavage of Inhibin a Subunit mRNA by Engineered Ribozyme L. GE AND A. W. SEITZ Department of Veterinaryand Animal Sciences University of Massachusetts Amherst, Massachusetts 01003
Ribozymes are small RNA molecules that cleave specific RNA molecules enzymatically. Haseloff and Gerlachl constructed ribozymes based on the hammerhead model of self-cleaving RNAs, in which most of the conserved nucleotides were arranged on the ribozyme, leaving only three required nucleotides, GUC, GUA, or GUU, on the RNA substrate. Such engineered hammerhead ribozymes have been shown to cleave chloramphenicol acetyltransferase (CAT) mRNA and HIV-1 gag mRNA in both cell-free systems and cell c~ltures.l-~ We designed and constructed a ribozyme to regulate the expression of inhibin. Inhibin is a peptide hormone that suppresses synthesis and release of folliclestimulating hormone (FSH). When inhibin levels in ewes and rats were decreased using immunization methods, FSH levels were elevated and ovulation rates increased. We predict that a decrease in inhibin synthesis will increase FSH levels and oocyte production. Inhibin consists of two subunits: a and p. The a subunit is unique to inhibin. Two p subunits form another peptide hormone, activin, which stimulates FSH release. Our ribozyme was designed to cleave inhibin a subunit mRNA. The three required nucleotides, GUA, GUC, and GUU, are present in several sites in inhibin (Y subunit mRNA, allowing a choice of several cleavage sites. Comparison of known sequences in rat, bovine, porcine, and human inhibin a subunit mRNA showed a short identical or highly homologous sequence of about 20 nucleotides flanking a GUC sequence. Our ribozyme was chosen to recognize this site in order to cleave the inhibin a subunit mRNA in other species. The ribozyme was synthesized as double-stranded oligodeoxynucleotides and cloned into the plasmid pGEM-4Z. In vitro transcripts of this cloned ribozyme and the cloned rat inhibin a subunit cDNA were incubated together at 50°C. Northern blot analysis of the incubations revealed two extra bands representing cleavage products of the expected size (FIG. 1). Cleavage was specific, as no cleavage of antisense inhibin a subunit transcript was observed (FIG.1). The results demonstrate that the flanking sequences of the cleavage site are accessible to the ribozyme. This ribozyme is expected to be active in vivo, as the same cleavage was observed also at 37°C (data not shown). The ribozyme was designed before mouse inhibin a subunit cDNA was sequenced. The results also showed that this ribozyme cleaves mouse inhibin a subunit mRNA in ovarian mRNA in vitro (data not shown). It suggests that it is possible to design a ribozyme to target conserved sequences of a given gene in order to cleave the transcripts of the same gene in other species in which the DNA sequence is unknown. 263
ANNALS NEW YORK ACADEMY OF SCIENCES
264
B
R
FIGURE 1. Northern blot of in vitro cleavage. The in vitro transcript of Mlu I linearized plasmid rINA-13: a cDNA clone containing rat inhibin a cDNA (a kind gift from Dr. Kelly Mayo), is about 1.3 kb. The expected cleavage products are approximately 0.8 and 0.5 kb. The ribozyme and inhibin a subunit in vitro transcripts were incubated in 50 mM Tris.CI (pH 7.5) and 20 mM MgCl2 at both 50°C and 37°C for 1 hour. The cleavage products were separated on 3.5% Nusieve GTG agarose/formaldehyde gel, transferred to nylon membranes, and hybridized with 32P-labeled rat inhibin a subunit cDNA. (A) Lanes 1 and 2: substrate incubated without the ribozyme; lanes 3,4, and 5: substrate incubated with the ribozyme. The molar ratio of ribozyme to the substrate was 30:l. (B)Lane I: antisense inhibin a subunit mRNA incubated without the ribozyme; lane 2: antisense inhibin a mRNA incubated with the ribozyme. All samples were incubated at 50°C for 1 hour. REFERENCES
1. HASELOFF, J. & W. L. GERLACH. 1988. Nature 334 585-591. 2. CAMERON, F. H. & P. A. JENNINGS.1989. Proc. Natl. Acad. Sci. USA 8 6 9139-9143. 3. SARVER,N., E. M. CANTIN,P. S . CHANG,J. A. ZAIA,P. A. LADNE,D. A. STEPHENS & J. J. R o w . 1990. Science 247: 1222-1225. 4. WOODRUFF, T. K., G. MEUNIER, P. B. JONES, A. J. W. HSUEH& K. E. MAYO.1987. Mol. Endocrinol. k 561-568.
Ribozymes are small RNA molecules that cleave specific RNA molecules enzymatically. Haseloff and Gerlachl constructed ribozymes based on the hammerhead model of self-cleaving RNAs, in which most of the conserved nucleotides were arranged on the ribozyme, leaving only three required nucleotides, GUC, GUA, or GUU, on the RNA substrate. Such engineered hammerhead ribozymes have been shown to cleave chloramphenicol acetyltransferase (CAT) mRNA and HIV-1 gag mRNA in both cell-free systems and cell c~ltures.l-~ We designed and constructed a ribozyme to regulate the expression of inhibin. Inhibin is a peptide hormone that suppresses synthesis and release of folliclestimulating hormone (FSH). When inhibin levels in ewes and rats were decreased using immunization methods, FSH levels were elevated and ovulation rates increased. We predict that a decrease in inhibin synthesis will increase FSH levels and oocyte production. Inhibin consists of two subunits: a and p. The a subunit is unique to inhibin. Two p subunits form another peptide hormone, activin, which stimulates FSH release. Our ribozyme was designed to cleave inhibin a subunit mRNA. The three required nucleotides, GUA, GUC, and GUU, are present in several sites in inhibin (Y subunit mRNA, allowing a choice of several cleavage sites. Comparison of known sequences in rat, bovine, porcine, and human inhibin a subunit mRNA showed a short identical or highly homologous sequence of about 20 nucleotides flanking a GUC sequence. Our ribozyme was chosen to recognize this site in order to cleave the inhibin a subunit mRNA in other species. The ribozyme was synthesized as double-stranded oligodeoxynucleotides and cloned into the plasmid pGEM-4Z. In vitro transcripts of this cloned ribozyme and the cloned rat inhibin a subunit cDNA were incubated together at 50°C. Northern blot analysis of the incubations revealed two extra bands representing cleavage products of the expected size (FIG. 1). Cleavage was specific, as no cleavage of antisense inhibin a subunit transcript was observed (FIG.1). The results demonstrate that the flanking sequences of the cleavage site are accessible to the ribozyme. This ribozyme is expected to be active in vivo, as the same cleavage was observed also at 37°C (data not shown). The ribozyme was designed before mouse inhibin a subunit cDNA was sequenced. The results also showed that this ribozyme cleaves mouse inhibin a subunit mRNA in ovarian mRNA in vitro (data not shown). It suggests that it is possible to design a ribozyme to target conserved sequences of a given gene in order to cleave the transcripts of the same gene in other species in which the DNA sequence is unknown. 263
ANNALS NEW YORK ACADEMY OF SCIENCES
264
B
R
FIGURE 1. Northern blot of in vitro cleavage. The in vitro transcript of Mlu I linearized plasmid rINA-13: a cDNA clone containing rat inhibin a cDNA (a kind gift from Dr. Kelly Mayo), is about 1.3 kb. The expected cleavage products are approximately 0.8 and 0.5 kb. The ribozyme and inhibin a subunit in vitro transcripts were incubated in 50 mM Tris.CI (pH 7.5) and 20 mM MgCl2 at both 50°C and 37°C for 1 hour. The cleavage products were separated on 3.5% Nusieve GTG agarose/formaldehyde gel, transferred to nylon membranes, and hybridized with 32P-labeled rat inhibin a subunit cDNA. (A) Lanes 1 and 2: substrate incubated without the ribozyme; lanes 3,4, and 5: substrate incubated with the ribozyme. The molar ratio of ribozyme to the substrate was 30:l. (B)Lane I: antisense inhibin a subunit mRNA incubated without the ribozyme; lane 2: antisense inhibin a mRNA incubated with the ribozyme. All samples were incubated at 50°C for 1 hour. REFERENCES
1. HASELOFF, J. & W. L. GERLACH. 1988. Nature 334 585-591. 2. CAMERON, F. H. & P. A. JENNINGS.1989. Proc. Natl. Acad. Sci. USA 8 6 9139-9143. 3. SARVER,N., E. M. CANTIN,P. S . CHANG,J. A. ZAIA,P. A. LADNE,D. A. STEPHENS & J. J. R o w . 1990. Science 247: 1222-1225. 4. WOODRUFF, T. K., G. MEUNIER, P. B. JONES, A. J. W. HSUEH& K. E. MAYO.1987. Mol. Endocrinol. k 561-568.
