Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation

Abstract

The cellular polypyrimidine tract-binding protein (PTB) is recruited by the genomic RNAs of picornaviruses to stimulate translation initiation at their internal ribosome entry site (IRES) elements. We investigated the contribution of the individual RNA recognition motif (RRM) domains of PTB to its interaction with the IRES of foot-and-mouth disease virus (FMDV). Using a native gel system, we found that PTB is a monomer, confirming recent reports that challenged the previous view that PTB is a dimer. Mapping the spatial orientation of PTB relative to the bound IRES RNA, we found that the two C-terminal RRM domains III and IV of PTB bind in an oriented way to the IRES. Domain III contacts the IRES stem-loop 2, while domain IV contacts the separate IRES 3¢ region. PTB domain I appears not to be involved directly in RNA binding, but domain II stabilizes the RNA binding conferred by domains III and IV. A PTB protein containing only these two C-terminal PTB domains is sufficient to enhance the entry of initiation factor eIF4G to the IRES and stimulate IRES activity, and the long-lived PTB-IRES interaction stabilized by domain II is not a prerequisite for this function. Thus, PTB most likely acts as an RNA chaperone to stabilize IRES structure and, in that way, augment IRES activity.

Activation of picornaviral IRESs by PTB shows differential dependence on each PTB RNA-binding domain

“…On the other hand, deletion of RBD3 or RBD4 almost eliminated all stimulation, particularly the RBD4 deletion. The authors concluded that RBDs3 and 4 were sufficient for stimulation (Song et al 2005), in contrast to our conclusions for the EMCV IRES.…”

“…A previous report on the stimulation of the FMDV IRES (which is structurally similar to the EMCV core IRES) by PTB deletion mutants found that deletion of RBD1 had no discernible effect (Song et al 2005), and deletion of RBD2, or both RBDs1 and 2, reduced stimulation by <50%, far less than observed here with point mutations inactivating RBD2. On the other hand, deletion of RBD3 or RBD4 almost eliminated all stimulation, particularly the RBD4 deletion.…”

“…In sharp contrast to the FMDV results (Song et al 2005), a deletion mutant which retained just RBDs3 and 4 failed to stimulate the EMCV IRES (with a 7A-bulge at the Domain J-K three-way junction) but did bind to this IRES, according to the results of UV-cross-linking assays, and this was confirmed by the fact that it inhibited stimulation by fulllength wild-type GST-PTB. Precisely the same results were found for a mutant that retained just RBDs1 and 2.…”

Structure-function relationships of the polypyrimidine tract binding protein

“…2). Several footprinting studies have mapped PTB binding sites on different IRESs and identified several pyrimidine stretches embedded in stem loops and single-stranded regions (in domain K and H of the picornaviruses type II IRESs [50,51], in domain 3 of the hepatitis C virus [52] and also in cellular RNAs [53 -55], see Fig. 2).…”

“…2). Moreover, reports have shown that interactions between PTB (or the neuronal nPTB) and cellular [56] or viral IRESs [51] are essential for the IRESs to attain their correct functional conformation. Hence, in its interaction with IRES RNAs, PTB acts as an RNA chaperone.…”