Detection of the 5 '-cap structure of messenger RNAs with the use of the cap-jumping approach

An effective procedure for specific determination of the cap structure at t he 5'-terminus of mRNA and for isolation of the corresponding full-length c DNA has been developed. The procedure involves covalent, attachment of an o ligonucleotide template extender to the 5'-cap structure of mRNA followed b y RT-PCR using M-MLV SuperScript II reverse transcriptase. In the course of reverse transcription, the enzyme 'jumps over' the cap structure and inclu des the sequence complementary to the oligonucleotide template extender int o the X-end of the first cDNA, strand. The cap-jumping method was successfu lly tested using some mammalian cellular mRNAs, genomic RNAs of tobacco mos aic virus (TMV) U1 and the recently isolated crucifer-infecting tobamovirus . Moreover, cDNA products corresponding to the, genomic tobamovirus RNA wer e obtained from total RNA extracted from tobacco plants infected by crucife r-infecting tobamovirus or tobacco mosaic virus. Using the cap-jumping meth od, we have shown for the first time that genomic crucifer-infecting tobamo virus (crTMV) RNA contains a 5'-cap structure. This improved method can be recommended for the construction of full-length and 5'-end enriched cDNA li braries, identification of capped RNAs and determination of their 5'-termin al sequences.