To rule out the possibility that RNA or a tertiary factor present in cell extracts mediates the protein–protein interaction, we carried out the pulldown assay using purified proteins such as Sbp1 and Pab1 (Supplemental Fig. S2A). Heparin column and stringent multiple washing procedures were used to eliminate residual RNAs associated with these RNA-binding proteins (see Materials and Methods). Under our experimental conditions, Sbp1 directly binds to initiation factors Pab1 via its central RGG domain. Importantly, the RGG domain of Sbp1, not the RRM domains, is required for the protein–protein interactions, as demonstrated by results obtained from the pulldown assay using domain mutants of Sbp1 (Fig. 3B,C). We further showed that four RRM domains in Pab1 (RRM1-4) are required for the binding with Sbp1. Consistent with our results, Sbp1 was identified to associate with Pab1 in a recent study (Richardson et al. 2012).