The Zea mays glycine-rich RNA-binding protein MA16 is bound to a ribonucleotide(s) by a stable linkage

Abstract

Expression of the gene encoding the maize glycine-rich RNA-binding protein MA16 is developmentally regulated and it is involved in environmental stress responses. The MA16 protein shows a wide spectrum of RNA-binding activities. On the basis of in vivo labelling, where a [32P]phosphate label was linked to the MA16 protein, Freire and Pages (Plant Mol Biol 29:797–807, 1995) suggested that the protein may be post-translationally modified by phosphorylation. However, further analysis showed that the [32P]phosphate label was sensitive to different treatments, suggesting that modification distinct from protein phosphorylation might occur in the MA16 protein. Biochemical analysis revealed that this [32P]phosphate labelling was resistant to phenol extraction and denaturing SDS-PAGE but sensitive to micrococcal nuclease, RNase A and RNase T1 treatments. The mobility of [35S] labelled MA16 protein on SDS-PAGE did not significantly changed after the nuclease treatments suggesting that the [32P]phosphate label associated to MA16 protein could be a ribonucleotide or a very short ribonucleotide chain. In addition, immunoprecipitation of labelled extracts showed that the ribonucleotide(s) linked to the MA16 protein was removed by phosphorolytic activity. This activity could be catalysed by a phosphate-dependent ribonuclease. The C-terminus of MA16 protein harbouring a glycine-rich domain was predicted to be an intrinsically disordered region.