The heterogeneous nuclear ribonucleoproteins I and K interact with a subset of the Ro ribonucleoprotein-associated Y RNAs in vitro and in vivo

The hY RNAs are a group of four small cytoplasmic RNAs of unknown function that are stably associated with at least two proteins, Ro60 and La, to form Ro ribonucleoprotein complexes. Here we show that the heterogeneous nuclea r ribonucleoproteins (hnRNP) I and K are able to associate with a subset of hY RNAs in vitro and demonstrate these interactions to occur also in vivo in a yeast three-hybrid system. Experiments performed in vitro and in vivo with deletion mutants of hY1 RNA revealed its pyrimidine-rich central loop to be involved in interactions with both hnRNP I and K and clearly showed t heir binding sites to be different from the Ro60 binding site. Both hY1 and hY3 RNAs coprecipitated with hnRNP I in immunoprecipitation experiments pe rformed with HeLa S100 extracts and cell extracts from COS-l cells transien tly transfected with VSV-G-tagged hnRNP-I, respectively. Furthermore, both anti-Ro60 and anti-La antibodies coprecipitated hnRNP I, whereas coprecipit ation of hnRNP K was not observed. Taken together, these data strongly sugg est that hnRNP I is a stable component of a subpopulation of Ro RNPs, where as hnRNP K may be transiently bound or interact only with (rare) Y RNAs tha t are devoid of Ro60 and La. Given that functions related to translation re gulation have been assigned to both proteins and also to La, our findings m ay provide novel clues toward understanding the role of Y RNAs and their re spective RNP complexes.