Intermolecular Cleavage by the Newt Ribozyme

Abstract

To analyse the trans-cleavage activity of the hammerhead ribozyme occurring in the ovary of the newt (Notophthalmus, Triturus) in more detail, six synthetic ribozymes representing natural and modified hammerhead sequences were tested with both short oligoribonucleotides and long transcripts as substrates. The same analysis was also performed with the monomer (330 nucleotides) newt ribozyme and variants thereof. None of the ribozymes comprising the newt natural sequence showed activity under multiple turnover conditions, regardless of sequence changes in stem and loop 11. With excess of ribozyme, the same ribozymes cleaved only to a limited extent a short substrate and extremely poorly a target site embedded within a long transcript. The addition of whole ovary cell extract had little influence on cleavage activity of short substrates. However, sequence changes in stems I and 111 to target different sequences considerably improved cleavage ability of the ribozymes under all conditions used. An RNA secondarystructure folding program showed that ribozymes with the natural newt sequence did not fold in a hammerhead structure whereas those with the changes in stem I and 111 did. These results suggest that the sequence of the stems I and 111 impairs the assembly of the newt ribozyme into a bimolecular hammerhead complex in vitro and that proteins present in the ovaries do not facilitate activity.Keywords: catalytic RNA ; hammerhead ; trans-cleavage; RNA-binding protein.A family of highly repetitive DNA sequences, dispersed in small clusters in the newt (Notophthalmus, Triturus) genome, codes for stable, strand-specific transcripts found in both somatic and germinal tissues ([l] and Cremisi, F., personal communication). The transcripts correspond in size precisely to monomeric (330 bp) and multimeric DNA repeat units. Evidence that synthetic dimer-rize transcripts undergo site-specific, selfcatalyzed cleavage in vitro suggested that the same reaction might be involved in the processing of large multimeric transcripts in vivo 12, 31. The self-cleavage reaction requires Mg" and occurs within a conserved structure named the hammerhead, similar to that of infectious plant RNAs [4].The ovarian ribozyme transcripts differ from the analogous transcripts in other newt tissues in having an intact self-cleavage site, located about SO nucleotides from the 5' end [2]. Therefore, it seems that monomer length transcripts of the ovarian newt ribozyme ,are more likely to arise rather from trancription than from self-processing by cleavage. It has indeed been shown that the same promoter elements that regulate polymerase-11-dependent transcription of small nuclear RNAs are involved in transcription of the newt ribozyme [5, 61. The presence of an apparently functional gene that codes for the small RNA with a hammerhead domain suggests that a biological function of this particular ribozyme may be trans-cleavage of a specific, but still unknown RNA target in vivo [5, 61. Here we report an in vitro study of the trans-cleaving properties o...

The newt ribozyme is part of a riboprotein complex

“…A recent study on the kinetics of in vitro cleavage (28) shows that the 330-nt monomeric ribozyme is not able to catalytically trans-cleave a 12-mer RNA substrate; no cleavage is observed at 25°C in 10 mM MgCl 2 under single or multiple turnover conditions, whereas, in agreement with previous results (3), cleavage is observed at 45°C in 50 mM MgCl 2 , using an excess of ribozyme over substrate (Fig. 4A, lane 3).…”

Selection of tetracycline inducible self-cleaving ribozymes as synthetic devices for gene regulation in yeast

“…Biphasic hammerhead cleavage has been previously reported for the S. mansoni and the newt hammerhead ribozyme. 44,45,48 Both the full-length ribozyme N79 and the selected tetracycline-dependent ribozymes resulted in similar biphasic cleavage behaviour. These data indicate that the biphasic cleavage is not caused by the attached aptamer or our construct design.…”

“…Biphasic hammerhead cleavage has been previously reported and regarded as misfolding of in vitro transcribed RNA. [44][45][46] To investigate this, we performed cleavage kinetics of the full-length ribozyme N79 with different MgCl 2 concentrations (0.1 to 10 mM, data not shown). Cleavage efficiency of N79 did not pass 30% whereas the cleavage rate increased with elevated concentrations of magnesium.…”

Characterization of a native hammerhead ribozyme derived from schistosomes

“…A native gel mobility study suggests the presence of at least two conformations. Alternative conformations have also been proposed to cause complications in the newt extended HHRz (Marusic et al 1997). An observed rate of ligation that is much less than that of cleavage as well as a chase experiment indicate that increased ligation is not a significant contributor to the observed biphasic kinetics.…”