IDENTIFICATION OF A 67 KDA PROTEIN THAT BINDS SPECIFICALLY TO THE PRE-RRNA PRIMARY PROCESSING SITE IN A HIGHER PLANT

摘要

In radish pre-rRNA primary processing cleavage occurs at a UUUUCGCGC element (motif P) mapped in the 5'-external transcribed spacer (Delcasso-Tremousaygue et al., 1988), Significantly, motif P is part of a cluster of homologous elements including three UUUUCCGG elements (motifs A(123)) and a single UUUUGCCCC element (motif B), Here we used the EMSA to identify in radish extracts an RNA-binding activity, NF C, that specifically interacts with the pre-rRNA A(123)Bp sequence, Using different RNA probes and competitors we show that NF C recognises a 38 base RNA sequence including the 3'-end of motif A(3) and motifs B and P. NF C binds to poly U, but not to poly A, poly C or poly G, Therefore we used poly (U) Sepharose chromatography as a final step to obtain pure NF C fractions, These, analysed by SDS-PAGE, revealed two major polypeptides of 67 and 60 kDa, According to UV cross-linking analysis the 67 kDa polypeptide corresponds to NF C activity, while the 60 kDa species is a proteolysed form of this protein. We also showed that NF C is enriched in nuclear extracts, Based on its stringent RNA substrate specificity and its nuclear localisation we propose that NF C is involved in pre-rRNA primary processing in plants.