Precise branch point mapping and quantification of splicing intermediates
Precise branch point mapping and quantification of splicing intermediatesJörg Vogel*, Wolfgang R. Hess and Thomas Börner
Department of Biology, Humboldt University, Chausseestraße 117, 10115 Berlin, Germany
Received February 24, 1997;Revised and Accepted March 18, 1997
ABSTRACT
Lariat intermediates of a group II intron were investigated via RT-PCR. Several reverse transcriptases appeared capable of reading through a branched nucleotide. A new method has been established that yields precise information about the location of the branch point within an intron. As an extension of our approach, antisense transcripts of the previously cloned PCR products were successfully used in RNase Protection Assays, providing a tool for quantification of splicing intermediates. Application of the method presented to other self-splicing introns as well as introns in nuclear pre-mRNAs is envisaged.
Excision of most introns proceeds via two successive transesterifications followed by release of a branched lariat-shaped RNA molecule, in which the 5' end of the intron is linked to an internal adenosine residue (branch point) by a 2'-5' phosphodiester.
Unsatisfactory results are often obtained if primer extension analysis is applied to map the location of the branch point within an intron. Based upon reverse transcriptase ceasing synthesis as it encounters a branched nucleotide (9 ), artefacts are likely to arise as reverse transcription is also blocked by stable secondary structures of RNA and certain base modifications (10 and references therein). Furthermore, no direct evidence is obtained that the nucleotide determined is covalently linked to the 5' end of the intron.
Recently, the ability of MMLV RNase H- and AMV reverse transcriptases to read through a 2'-5' linkage in a template has been reported (6 ). Both enzymes appear to stop as they encounter the backbone modification, but only pause and continue to synthesise after a few minutes. We wondered whether the major stop observed with reverse transcriptase at a branched nucleotide is analogous to this pausing. If so, first strand cDNA synthesis initiating at a primer antisense to the 5' part of the intron should yield products that are extended beyond the branch point (Fig. 1 A). Amplification of these molecules would subsequently be achieved by PCR, using the same oligonucleotide and a second primer (sense) binding to the more 3' part of the intron.
There is a growing number of plant mutants that exibit splice-defects of individual group II introns (3 ,4 ). An example of this, intron excision from chloroplast transcripts encoding tRNALys is completely blocked in white tissues of the barley mutant line, albostrians, even though precursor RNAs accumulate to high level. Since splicing proceeds normally in green tissue, this offers an excellent system to test the proposed method.
First strand cDNA synthesis was carried out at 42oC for 1 h using 1 [mu]g of total cellular RNA, antisense primer TRNKT7AS and Superscript II RNase H- reverse transcriptase (Gibco BRL), strictly following the manufacturer's protocol (for details see ref. 3 and Fig. 1 B). After heat inactivation of the enzyme, 3 U of RNase H (Gibco BRL) were added and digestion was carried out for 20 min at 37oC. One twentieth of the mixture was taken as a template in a 50 [mu]l PCR, containing 50 pmol of sense primer TRNKINS1, 50 pmol of nested antisense primer TRNKIAS1, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 200 [mu]M dNTPs and 2.5 U Ampli-Taq Gold (Perkin-Elmer). Preincubation for 12 min at 93oC was followed by 38 cycles of 1 min at 93oC, 45 s at 58.5oC, 30 s at 72oC and a final step of 10 min at 72oC. As shown in Figure 1 C, RT-PCR of RNA from green tissue yielded a single band of 126 bp, being exactly of the size expected for a lariat-derived product extended from TRNKT7AS past the branch point into the upstream intron sequence (lane 3). Amplification of the 126 bp product was also successful if templates had been generated by tTh polymerase at 60oC (Eurogentec) or RAV2 reverse transcriptase at 42oC (Amersham) according to the manufacturer's protocols. A slightly increased amount of PCR product was obtained if the RNase H digestion step in MMLV experiments was omitted (lane 6). Subsequent analysis of the 126 bp bands on an ABI DNA Sequencer placed the branching nucleotide 7 nt upstream of the intron/3' exon border, being exactly the position of the adenosine residue that was deduced from secondary structure analysis. As predicted, all sequences determined started with the binding site of TRNKINS1 and stretched to the branch point, at which they continued with the first nucleotide of the intron to the 5' end of primer TRNKIAS1. Intriguingly, with all reverse transcriptases used, misincorporation of adenosine instead of thymidine occured at the branch point, which was further confirmed by cloning of PCR products into plasmid vector pMOS (Amersham) and sequencing of several inserts.
REFERENCES
1 Copertino,D.W., Hall,E.T., Van Hook,F.W., Jenkins,K.P. and Hallick,R.B. (1994) Nucleic Acids Res., 22, 1029-1036.MEDLINE Abstract
