We show that the two RRMs of Sbp1 bind to the poly(A) region of 5′UTRPab1 specifically, and this protein–RNA interaction is important for the inhibition of translation initiation of Pab1 mRNA. Four observations support this conclusion. First, direct binding of Sbp1 to 5′UTRPab1 was shown by a TRAP assay where Sbp1 was pulled down from cell lysates using 5′UTRPab1 alone and 5′UTRPab1 assembled with a preformed 43S PIC (Supplemental Fig. S1B). Consistent with this result, a recent genome-wide, cross-linking study shows that Sbp1 preferentially binds to the 5′UTRs of mRNAs (Mitchell et al. 2013). Second, 5′UTRPab1, but not its sequence complement, 5′UTRPab1-complement, competes for the binding of Sbp1 (Fig. 1). Third, binding analyses based on EMSAs reveal that the individual domains of Sbp1, while folded properly, have much weaker affinities for the 5′UTRPab1 than the full-length protein and, therefore, suggests that the domains in the protein act cooperatively for RNA binding.